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Abstracts -
Oral Presentations
Alireza Tehrani
Supervisor: Dr. Heidar-Zadeh
Compact, Property-Specific Representation of Molecular Surfaces
Molecular surfaces are a common representation of a molecule as certain chemical concepts are thought to occur on the boundaries of a molecule. However, constructing quantitative representations on the surface alongside its properties, for example its electrostatic potential, remains a difficult challenge. Motivated by advances in shape analysis, and the use of a spectrum to correlate chemical structure to an interpretable signal, we represent these surfaces with their properties as a "spectrum" that encodes its shape, symmetry, its potential intermolecular reaction and is invariant under translations, rotations and reflections of the molecule. This presentation shares our ideas and results for developing alignment-free and property-specific molecular similarity measures for predicting molecular properties.
Anastasia Messina
Supervisor: Dr. Cathleen Crudden
Electrochemical Characterization of NHC-TLR based Au Biosensors for Pathogen Detection
Anastasia Messina, Dianne Lee, and Cathleen Crudden
Biosensors are analytical devices able to convert a biological response into a detectable electrical signal. Biosensors hold potential for exciting applications across industrial, agricultural, medical, and scientific fields, however, this potential remains theoretical as current biosensors lack balance between cost, efficacy, and stability. Current biosensors with high recognition sensitivity are time-invasive and expensive. On the other hand, most time-efficient biosensors lack stability in ambient conditions rendering them impractical for application. Novel bioanalytical approaches look to enhance the quality of biosensors by decreasing response time and optimizing sensitivity, selectivity, and sensor stability. Addressing these concerns, we turn to self-assembled monolayers (SAMs) of N-heterocyclic carbenes (NHCs) to serve as the base of our Au biosensors. Compared to a literature favourite, SAMs of thiols, SAMs of NHCs hold a longer half-life and remain inert for longer periods in air, water, and extreme pH conditions. Building our sensors from the bottom-up, we functionalized our NHC-based Au biosensors with toll-like receptors (TLRs), via bioconjugation, to bind to and signify the presence of pathogenic domains in solution. The structural conservation at the TLRs’ base, yet variety observed within their ectodomains, make them attractive functional units for a biosensor. Using the same sensing platform and click chemistry, we continue to experiment ways to functionalize our sensors with IgG1 antibodies. Herein, I aim to showcase specific binding characteristics of NHC-based gold biosensors functionalized by TLRs. The sensitivity and specificity offered by TLRs on NHC-based gold biosensors show promise for an affordable, stable, and efficient modular pathogen detection platform.
Fabiola De Leon Gonzalez
Supervisor: Dr. Chantelle Capicciotti
Expanding the Glycoengineering Biochemical Toolkit to Study Cell-Surface Glycoconjugates through Selective Exo-Enzymatic Labeling (SEEL)
Fabiola V. De Leon Gonzalez & Dr. Chantelle J. Capicciotti
Cell-surface glycoconjugates play essential roles in several physiological processes, which often occur through highly specific recognition events mediated by glycan-binding proteins (GBPs). However, glycan-protein interactions are typically transient and low-affinity binding events, making them challenging to capture and study. Metabolic oligosaccharide engineering (MOE) is currently a common glycan labeling strategy used to study cell-surface glycans; sugars bearing a bio-orthogonal detectable probe, such as azides, alkynes, or photo-crosslinkers, are metabolically incorporated into cellular glycans to interrogate glycan-dependent physiological processes. MOE, however, is limited by native cellular metabolic machinery, which can result in low probe incorporation, and non-selective display of the sugar probe on cell-surface glycans. For this reason, our group is developing a selective exogenous (exo)-enzymatic labeling (SEEL) strategy for cell-surface glycoengineering using exogenously introduced glycosyltransferases (GTs) and unnatural nucleotide-(NT-) sugar bio-orthogonal probes. Photo-crosslinking NT-sugar probes (ex. diazirine (DAz)) are especially advantageous for SEEL as they can covalently capture glycan-protein binding complexes, allowing for isolation and identification of the binding partners. This presentation will discuss the development of a chemo-enzymatic synthetic strategy used to produce unnatural NT-sugar probes to be used for SEEL. The synthesis of UDP-GlcNAc and UDP-GalNAc analogs containing azide, alkyne, diazirine, and trifluoroacetate moieties on the C2 N-acyl side chain will be discussed, along with preliminary cell-surface glycoengineering experiments using an azido UDP-GlcNAc derivative and a GlcNAc-transferase, demonstrating the promise of our SEEL strategy. We hypothesize SEEL approach will overcome limitations of MOE due to the specificity of the GTs employed, allowing for selective interrogation of glycan-protein interactions.
Jennifer Kolwich
Supervisor: Dr. Avena Ross
The Live Microbial Cage-Match: Monitoring real-time microbe interactions with direct mass spectrometry imaging
Jennifer L. Kolwich, Jian Yu, Haidy Metwally, Hailey A. Tomm, Richard D. Oleschuk, and Avena C. Ross.
When penicillin was discovered and developed into a widely available antibiotic drug, it revolutionized modern medicine and increased global life expectancy. However, the current rate of new antibiotic discovery and approval is being outpaced by the emergence of antimicrobial resistance. If the antibiotic discovery rate remains the same, in just 30 years, deaths caused by drug-resistant microbes will reach an estimated 10 million annually. Historically, microbes have been a critical source of clinically relevant antibiotics, but the discovery of new microbially-derived natural products has plummeted in recent decades. Two major challenges slowing down natural product discoveries are the laborious nature of standard culture-to-compound techniques and the risk of rediscovering known compounds after such a lengthy process. This research aims to tackle both of these issues by using mass spectrometry imaging (MSI) to directly sample the metabolites of microbes in unique and stressful conditions.
Using direct MSI allows us to easily sample and map small changes in the metabolite profile of bacteria without the need for extraction. With these images, we can identify changes in the molecular profile over time along intersecting regions of co-cultured microbes, a unique competitive environment known for inducing ecologically important natural products. This low-preparation detection method will not only allow us to dynamically probe a variety of interactions, but also let us correlate it with real-time visual growth data, while simultaneously dereplicating hits caused by known molecules using the mass data, thus increasing potential for new, biologically active natural product discoveries.
Jennifer McLeod
Supervisor: Dr. Zhe She
Label-Free Pathogen Detection using Broad Spectrum Biorecognition Element
Jennifer F. McLeod, R. Stephen Brown, Zhe She
The 2021 UN World Water Development Report highlights the undervaluation of water, impacting water accessibility and climate action. Despite Canada’s relative wealth, there is a serious water crisis amongst many First Nations communities, where several short- and long-term drinking water advisories are in effect. The SARS-CoV-2 pandemic highlighted the necessity for access to clean water to guarantee appropriate sanitation. Ensuring the potability of water for basic human needs requires time consuming and costly analytical methods, highly qualified personnel, and equipment. Point-of-care (POC) devices eliminate the need for highly qualified persons and equipment, allowing tests to be performed on site.
The demand for rapid detection with high sensitivity and low cost has increased attention toward electrochemical biosensors. A biosensor contains three key parts: a biorecognition element (BRE), a sensor chip, and a linker molecule which connects the two. Using a gold sensor chip, whole cell pathogen detection by toll-like receptor BREs is demonstrated.1,2 The result demonstrates the versatility in the biosensor scaffold, with implications for multi-analyte detection.
[1] McLeod, J. et al. Analyst 2020, 145 (18), 6024-6031
[2] Singh, I. et al. Chem Commun 2021, 57 (68), 8421-8424
Joshua Kofsky
Supervisor: Dr. Chantelle Capicciotti
New Synthetic Approaches to Access O-GalNAc Glycosides
Joshua M. Kofsky, Chantelle J. Capicciotti
The surface of all cells is decorated by a diverse mixture of complex carbohydrate structures called glycans. One class of these cell-surface glycans are the mucin-type O-GalNAc (N-acetylgalactosamine; GalNAc) glycans, which are implicated in many biological functions, such as inflammation and homing of hematopoietic progenitor stem cells to bone marrow. Despite their abundance and importance, the functions of specific O-glycan sequences are not well-understood. O-glycans are highly heterogeneous with a wide variety of linear and branched structures containing a diversity of epitopes. This structural complexity has made it challenging to access defined O-glycan structures to understand their biological roles. Chemoenzymatic synthesis of complex O-glycans represents a promising route to access these structures.
This presentation will discuss a new chemoenzymatic strategy to access O-glycan starting from an α-linkered GalNAc substrate that was prepared chemically. A new diazotransfer protocol is described for the introduction of a non-participating C2-azido moiety to facilitate the installation of a 1,2-cis glycosidic linkage found in O-glycans. These ɑ-GalNAc substrates are suitable for enzymatic extension by glycosyltransferases to access defined O-glycan structures. Key to our strategy is installing branching points enzymatically to obtain Core 1 and Core 2 O-glycans. Core 1 glycans are obtained using the bacterial enzyme BiGalHexNAcP, which can be branched to a trisaccharide Core 2 glycan using the mammalian enzyme GCNT4. From these substrates, we can selectively extend each branch of the oligosaccharide by harnessing the specificity of glycosyltransferases, accessing a broad range of symmetrical and previously-inaccessible asymmetrical structures.
Kailey Browne
Supervisor: Dr. Zhe She
Ultrasensitive Electrochemical Phosphate Detection by Pyridine-zinc(II) Complex
Kailey Browne, Yu Pei, Ishwar Singh, Sarah Jane Payne, and Zhe She
The presence of phosphate within water systems is a driving force in the prevalence of harmful algal blooms or cyanobacteria blooms. Accordingly, phosphates are important environmental health parameters that are monitored using analytical techniques. Currently, phosphates are monitored using chromatographic and colorimetric techniques. Major disadvantages of these techniques include their poor selectivity towards specific anions, as well as the high cost of analysis. This work explores a new, electrochemical detection method, which can provide a cheaper yet sensitive technique. By implementing self-assembled monolayers through the modification of a gold working electrode with pyridine-zinc(II) complex, we were able to improve the selectivity of the sensor towards phosphate anions. . This modification allows for an indirect and ultrasensitive method of detection of the H2PO4- species, ranging in concentrations between 0.0 and 1.2 fM phosphate. Electrochemical techniques such as cyclic voltammetry (CV) and square-wave voltammetry (SWV) were used to explore phosphate detection abilities. These techniques allowed for detection limits of 4.0 x 10-16 and 4.0 x 10-17 M H2PO4- to be obtained with CV and SWV, respectively. Selectivity of this sensor was also successfully explored for phosphate detection in the presence of potential interfering agents such as sulfate, carbonate and chlorine. Trace levels of phosphate within a tap water sample were also successfully detected with the use of this ultrasensitive sensor. We anticipate that this sensitive and reproducible technique will have applications for low-level phosphate detection in environment and treated waters.
Kristen Harrington
Supervisor: Dr. Kevin Stamplecoskie
Assessing the Properties of Nanoclusters for Photodynamic Therapy
Kristen Harrington and Kevin Stamplecoskie
Every year more than 200,000 Canadians are diagnosed with cancer. Despite many advances in cancer research, there is still need for improved cancer treatment options. This project involves the synthesis and characterization of metal nanoclusters, which have been proposed as photodynamic therapy (PDT) agents, which is a type of cancer therapy. As clusters are being explored for their use in PDT, it important to identify clusters that are both stable in physiological conditions and able to produce reactive oxygen species, the active compounds in PDT. Here, we use absorbance and emission spectroscopy to monitor the stability of clusters in conditions that mimic physiological conditions. We also quantify the production of reactive oxygen species from different clusters to determine if they will be effective PDT agents. Overall, cluster stability and reactive oxygen species production varies with cluster structure and ligand. This work provides useful information in selecting nanoclusters that will be suitable for PDT.
Kristin Partanen
Supervisor: Dr. Zhe She
Fabricating Electrodes using Conductive 3-D Printing Materials and Electrochemical Gold Deposition
Kristin Partanen, Yu Pei, Phillip Hillen, Malek Hassan, Kevin McEleney, Sarah Jane Payne, Richard Oleschuk*, Zhe She*
Bacterial and viral detections are important in environmental monitoring and clinical diagnostics. Current methods, such as bacteria culture tests and polymerase chain reactions, are successful when detecting bacteria and viruses, but are costly, require trained personnel, can take up to 5 days to receive results, and are inaccessible in resource poor settings.1 Therefore, it is important to move towards point-of-care testing (POCT) to decrease the detection time of bacterial species. There is a growing interest towards electrochemical (EC) sensors, such as the glucose meter, as they are based on compact potentiostats which offer portable and low-cost solutions for POCT, further decreasing the need of trained personnel.2 For an effective EC sensor, an electrode chip needs to be fabricated for sensing. Techniques, such as electron beam lithography and photolithography, have been used to design features on chips but they are costly, can be time-consuming, and may not be accessible.3 Therefore, there is a need to investigate new methods of fabricating chips that are capable for sensing applications. By using 3-Dimensional (3-D) printing technology, this can reduce the costs of chip fabrication due to the low-cost of the printer and allow for rapid prototyping capability. In this study, we explore enhancing the conductivity of 3-D printed electrodes along with EC gold deposition using two commercial conductive filament brands. These electrodes were then able to demonstrate a practical application by detecting Cu2+ using anodic stripping voltammetry at drinking water concentration thresholds.
Matthew Webster
Investigating Metal Contacts for New Semiconductor Radiation Detectors
New compound semiconductors are being grown and optimized for use as room temperature radiation detectors for essential industries such as security and medical imaging. While we are improving the chemical purity and crystallinity of various compounds, we are concurrently optimizing the electrical contacts required to get signal out of the crystal. I have investigated several nano-deposited metals with different contact geometries in order to achieve the best properties.
Neil Grenade
Supervisors: Dr. Avena Ross and Dr. Graeme Howe
Uncovering convergent evolution in the biosynthesis of tambjamines
Neil Lawrence Grenade, Graeme W. Howe and Avena C. Ross
Bacterial natural products (NPs) are an immensely valuable source of therapeutics. As modern DNA sequencing provides access to an increasing array of microbial genomes, the molecules produced by most NP biosynthetic gene clusters (BGCs) remain unknown. In this work, we identify an orphan BGC that is responsible for the production of the antitumor tambjamine, BE-18591. Although BE-18591 is a close structural analogue of tambjamine YP1 produced by Pseudoalteromonas tunicata, the biosynthetic routes to produce these molecules differ significantly. Notably, the C12-alkylamine tail that is appended to the bipyrrole core of tambjamine YP1 is derived from fatty acids shunted from the primary metabolism of the P. tunicata, whereas the S. albus BGC encodes a dedicated system for the in situ biosynthesis of the alkylamine portion of tambjamine BE-18591. The proposed steps to formation of the fatty amine in the BE-18591 pathway are also unprecedented in natural product biosynthesis. These remarkably different biosynthetic strategies represent a rare example of convergent evolution between phyla of bacteria in the production of natural products.
Phung Nguyen
Supervisor: Dr. Amanda Bongers
Exploring how encoding modality affects recognition memory of chemical names and structures
Phung Nguyen, Amanda Bongers
Using and interpreting scientific models requires the development of representational competence—the ability to understand and make connections between information in multiple modalities—and is necessary for visual reasoning. However, there is limited research into how encoding modality influences how learners process information from scientific models, and how applicable psychological research is in contexts where representational competence cannot be assumed. With the emphasis on visual reasoning in the sciences and particularly in chemistry, we were interested in whether the well-documented drawing effect—where drawing the referent of a word produces superior memory of that word over other encoding modes—holds true in the context of learning chemical names and structures. We conducted an online encoding and recognition experiment with chemistry undergraduate students, graduate students, and faculty as participants testing four encoding conditions: drawing, viewing, imagining, and writing. The results show that writing produced the best recognition memory for chemical names (Experiment 1) but the worst memory for chemical structures (Experiment 2). The absence of the drawing effect in these experiments can be attributed to the students’ lack of representational competence which was confirmed from the results for chemistry graduate students and faculty (Experiment 3) where we observed a gradation in memory performance in line with chemistry expertise. We conclude that, when representational competence is lacking, writing enhances recognition of the chemical name but undermines memory of the structure.
Rebecca Chen
Supervisors: Dr. Zhe She & Dr. Stephen Brown
Electrochemical Detection of Antibiotic-resistant Bacteria
Rachel Chen, Zhe She, Stephen Brown
A contaminant of emerging concern with significant impact on the environment and in clinical settings is antibiotic-resistant bacteria (ARB). Increasing antibiotic pollution in water and soil from activities such as pharmaceutical use in humans and farming livestock correlates with an increase in ARB. In 2019, the CDC reported more than 2.8 million infections and 35,000 deaths caused by antibiotic-resistant bacteria and fungi in the USA alone. Current methods for detecting ARB are not suitable for real-time monitoring due to expensive procedures that require lab facilities, analytical instrumentation with highly trained personnel, and can take up to 48 hours or more to obtain results due to sample preparation time and backlog. These methods cannot be used for rapid, routine testing of ARB nor for on-site monitoring, especially in remote areas.
Development of a point-of-care testing device eliminates the needs for expensive analytical instruments and personnel. The recent SARS-CoV-2 pandemic demonstrated the efficiency and accessibility of point-of-care biosensors comparable to the classic examples of the blood glucose meter and the home pregnancy test. This research addresses the demand for rapid, sensitive, and specific in-situ detection of ARB at a low cost. Development of an electrochemical biosensor for ARB detection can be done with a direct method where a biorecognition element like an antibody will bind directly to ARB, or via an indirect method by detecting an indicator biomolecule that is made specifically by ARB. These diverse sensing methods offer versatility to the biosensor and the ability for detection of multiple ARB analytes.
Meet The Judges
Dr. Kevin Stamplecoskie
Assistant Professor, Materials and Physical Research Interest: Nanomaterials for Photonics; Light-Harvesting, Optoelectronics, Chemical Sensing Spectroscopy/Transient Spectroscopy; Fluorescence Spectroscopy, Ultrafast Transient Absorption Spectroscopy, Excited-State Behaviour of Materials
Dr. Chantelle Capicciotti
Assistant Professor, Biological & Organic Research Interests: Chemical Glycobiology, Chemoenzymatic Glycan Synthesis, Carbohydrate-Protein Interactions and Function, Cell-Surface Glyco-Engineering and Display of Biomolecules
Dr. Graeme Howe
Assistant Professor, Biological & Organic Research Interests: Origins of enzymatic catalysis; Elucidation of enzyme mechanisms, Understanding natural and directed evolution of enzymes, Evaluating the role of protein dynamics in catalysis Organic reaction mechanisms: Kinetic isotope effects as mechanistic probes, Computational modelling of transition states, Interplay of solvent effects and transition state structures
Abstracts -
Poster Presentations
Alessia Rizzello
Supervisor: Dr. David Zechel
Identification of Genes Essential for Sulfamate and Fluorine Incorporation During Nucleocidin Biosynthesis
Ola Pasternak, Andreas Bechthold, and David L. Zechel
Nucleocidin is an adenosine derivative containing 4’-fluoro and 5’-O-sulfamoyl substituents. In this study, nucleocidin biosyn- thesis is examined in two newly discovered producers, Streptomyces virens B-24331 and Streptomyces aureorectus B- 24301, which produce nucleocidin and related derivatives at titers 30-fold greater than S. calvus. This enabled the identi- fication of two new O-acetylated nucleocidin derivatives, and a potential glycosyl-O-acetyltransferase. Disruption of nucJ, nucG, and nucI, within S. virens B-24331, specifying a radical SAM/Fe-S dependent enzyme, sulfatase, and arylsulfatase, respectively, led to loss of 5’-O-sulfamoyl biosynthesis, but not fluoronucleo- side production. Disruption of nucN, nucK, and nucO specifying an amidinotransferase, and two sulfotransferases respectively, led to loss of fluoronucleoside production. Identification of S. virens B-24331 as a genetically tractable and high producing strain sets the stage for understanding nucleocidin biosynthesis and highlights the utility of using 16S-RNA sequences to identify alternative producers of valuable compounds in the absence of genome sequence data.
Alex MacDonald
Supervisor: Dr. Philip Jessop
Andre Castillo
Supervisors: Dr. Diane Beauchemin, Dr. Iris Koch, Dr. Michael Palmer
Investigation of arsenic and other mine related contaminants in garden produce from Yellowknife
Andre Isaac Castillo, Hannah Fortin, David Patch, Debora Motta Meira, Diane Beauchemin, Iris Koch, Michael Palmer
The legacy of mining in Yellowknife, Northwest Territories has resulted in elevated arsenic (As) contamination in soils which has resulted in concentration much higher than the maximum recommended by Canadian guidelines. As agricultural initiatives in Yellowknife increase, which include community members growing and selling produce from local gardens, a better understanding of As levels and other metals in garden produce helps to support these initiatives. It is also imperative to investigate any potential health impacts of the consumption of garden produce grown in Yellowknife. Previous studies have shown that while levels of As were higher than the national average, consumption of garden produce was still safe. Twenty years later, the study is being revisited to determine if there are any changes in concentrations and risk of As, along with those of other mine related contaminants, such as cadmium (Cd) and lead (Pb), in garden produce. The work presented will look to measure arsenic in the Yellowknife area garden produce and soils. In vitro bioaccessibility assay (IVBA) measurements on various garden produce such as kale, carrots, beetroots, and potatoes will be presented. Speciation analysis of As (III) and As (V) using X-ray absorption near edge structure (XANES) analysis of garden produce will be presented to show various forms of As present in kale, carrot and beetroot vegetables.
Angus Sullivan
Supervisor: Dr. Cathleen Crudden
Synthesis and characterization of a monodentate NHC-protected Au11-nanocluster via reduction with KC8
Angus I. Sullivan,[a] Joseph F. DeJesus,[a]† Sami Malola,[b] Shinjiro Takano,[c] Tatsuya Tsukuda*,[c] Hannu Häkkinen*,[b] and Cathleen M. Crudden*[a][d]
Atomically precise gold nanoclusters are an exciting and growing class of nanomaterial. While normally protected with ligands such as thiols or phosphines, gold nanoclusters protected with N-heterocyclic carbenes (NHCs) have recently garnered attention due to potential improvements in stability and optical properties of the resulting clusters. However, as this field is in its infancy, little work has been done with reducing agents beyond sodium borohydride (NaBH4), a reagent that dominated synthetic efforts in other clusters as well. Herein, we report the use of potassium intercalated graphite (KC8) in the synthesis of nanoclusters, and the novel Au11-nanocluster protected with monodentate NHC ligands it produces, [Au11(NHC)8Br2]Br. The cluster is characterized by ESI-MS, UV-Vis spectroscopy, 1H and 13C{1H} NMR. DFT calculations enable us to propose a structure for the nanocluster, which is in agreement with our experimental data.
Daniel Reddy
Supervisor: Dr. Richard Oleschuk
Micro Hyper-Channels Based on Laser Refined Nanostructured Cellulose
Lishen Zhang, Daniel O. Reddy, Timothy T. Salomons, and Richard Oleschuk
David Kunar
Supervisor: Dr. Peng Wang
Solution Synthesis and Growth of Single Crystal CsPbBr3 for Radiation Detection
David Kunar, Yu Wu, Ramjee Kandel, Matthew Webster Peng Wang
The growth of single crystal CsPbBr3 from pure materials for radiation detecting purposes has been reported; however, synthesis and growth of this material from accessible and low-cost starting materials has yet to be optimized. We report the synthesis, growth, and purification of CsPbBr3 from solution using PbO and CsCO3. Samples of CsPbBr3 were synthesized from solutions of starting material in HBr and H2O, which were filtered and mixed to produce CsPbBr3 powder. Analysis of these powders via Powder X-ray Diffraction showed a purity of phases. This powder was then melted and reduced under a H2 atmosphere, which aided in the removal of oxides and organics. Samples of reduced CsPbBr3 were then purified via a modified zone refining process to form single crystalline CsPbBr3. Boules of CsPbBr3 obtained from this process showed the segregation of impurities to the tip and heel of the crystal. Wafers cut from the crystal were polished with ethanol and fabricated into devices with Ga contacts. Devices fabricated from these samples exhibited high photocurrents with good response to light. This procedure provides a method of producing high quality single crystalline CsPbBr3 at a lower cost than traditional methods.
Donnie McFarlane
Supervisor: Dr. Avena Ross
Heterologous expression of a novel antibiotic discovered in marine bacteria
Donnie McFarlane, Aditi Kanwar, Avena Ross
With the rise of antibiotic-resistant “superbugs”, the need for novel antibiotics cannot be overstated. The main source of novel antibiotics has historically been microorganisms that produce active compounds called natural products. Early evidence indicates that the marine bacteria species Pseudoalteromonas flavipulchra (PFL) produces two natural products with potential antibiotic properties: flavipuricin I and II. Interestingly, the same set of genes seem to produce these compounds, using divergent modifications that result in some rarely seen features. Pending further investigation, these may be some of the first lanthipeptide antibiotics shown to protect against gram negative bacteria. It would also be the first confirmed discovery of lanthipeptides produced by Proteobacteria.
Having garnered these insights, we aimed to confirm our hypothesized chemical structures for flavipuricin I and II. Initially, the major obstacle was poor PFL growth, which led to insufficient amount of product for spectroscopic analysis. To address this challenge, we improved our yield by cloning the four flavipuricin-creating genes found in PFL and inserted them into E. coli. After inducing E. coli to activate those genes, we isolated and purified the extracts from these E. coli cultures. We were unable to detect flavipuricins in the extracts, but we did confirm their presence by direct bacterial sampling. Nevertheless, we did successfully prove that flavipuricins are formed using the genes which we cloned. After optimizing our process, we hope to obtain sufficient yields to study the chemical structure, biosynthetic pathway, and antibiotic activity of these new compounds.
Helen Lord
Supervisor: Dr. Diane Beauchemin
Risk Assessment of Seaweed as an Alternative Protein Source using Inductively Coupled Plasma Mass Spectrometry
Helen Lord, Qiqi Zhang, Andre Castillo, Diane Beauchemin
Heavy toxic metals Pb, Hg, Zn, Cd, As, Se, Ni and Cr have the potential to pollute alternative protein sources like seaweed. Artificial saliva, gastric and intestinal solutions are prepared and heated at 37°C. Using the on-line leaching method coupled to the Inductively Coupled Plasma Mass Spectrometer (ICPMS), the maximum bio-accessible fraction of a heavy toxic metal is measured in a seaweed sample. The ICPMS is equipped with a collision-reaction interface (CRI) with hydrogen as the reaction gas, minimizing carbon- and chlorine-based polyatomic interferences. The on-line continuous leaching method allows for real-time monitoring of potentially toxic elements as they leach out of the seaweed samples. Mass balance was verified at the 99% confidence level. Results obtained from seaweed showed the majority of elements were leached by saliva juice, followed by gastric juice. Leaching of elements in intestinal juices were very small. The on-line leaching method indicated that 18.9 – 97.4% of toxic heavy metals in seaweed are bio-accessible. However, comparison of bio-accessible fractions to tolerable uptake limits suggest that seaweed is safe to eat as a primary alternative protein source for all ages. Extraction of the total bio-accessible concentrations of elements was optimized.
Jack Babulic
Supervisor: Dr. Chantelle Capicciotti
Precision photo-cross-linking of glycan—protein interactions through cell-surface glycan engineering
Jonathan L. Babulic and Chantelle J. Capicciotti
Glycans mediate vital biological processes such as cell growth, differentiation, and immune recognition through selective interaction with glycan-binding proteins. Dysregulation of glycan-mediated signaling is implicated in disease states including cancer, inflammation, and pathogen infection. While the importance of glycan-mediated interactions is becoming increasingly recognized, detecting native interactions is challenging with current methods. Monovalent glycan-protein interactions are typically short-lived and have low affinities, making it difficult to capture and isolate interacting partners. A multivalent display is thus necessary to study relevant glycan-protein binding. Recognition of glycan ligands is also dependent on interactions with terminal sugar epitopes, glycan subclass-specific presentation, and conjugation to specific protein or lipid anchors. The complexity of glycan-protein interactions thus makes it challenging to identify the specific glycoconjugates involved in critical biological processes and consequently there is a need for novel tools to discover and probe these interactions.
Introduction of photo-cross-linking groups into cellular glycans provides an attractive strategy to capture, isolate, and analyze native binding partners involved in glycan-protein interaction complexes. Herein, we present a precision labeling and photo-cross-linking approach through cell-surface glyco-engineering using a photo-cross-linking CMP-Neu5Ac (CMP-sialic acid) derivative by exogenous enzymatic transfer. Our toolset harnesses the inherent specificity of various sialyltransferases to install sugar probes with high linkage and class specificity onto native glycans on live cells. These cross-linking probes are then used to interrogate sialic acid-based interactions between glycoconjugates and important receptors such as Siglecs, which are involved in modulation of the immune system. This approach thus represents an exciting tool to improve our fundamental understanding how these interactions play a role in health and disease.
Jennifer McLeod
Supervisor: Dr. Zhe She
LABEL-FREE PATHOGEN DETECTION USING BROAD SPECTRU BIORECOGNITION ELEMENT
McLeod, J. F., Park, C., Cunningham, A., She, Z., Kelly, F.
The 2021 UN World Water Development Report highlights the undervaluation of water, impacting water accessibility and climate action. Despite Canada’s relative wealth, there is a serious water crisis amongst many First Nations communities, where several short- and long-term drinking water advisories are in effect. The SARS-CoV-2 pandemic highlighted the necessity for access to clean water to guarantee appropriate sanitation. Ensuring the potability of water for basic human needs requires time consuming and costly analytical methods, highly qualified personnel, and equipment. Point-of-care (POC) devices eliminate the need for highly qualified persons and equipment, allowing tests to be performed on site.
The demand for rapid detection with high sensitivity and low cost has increased attention toward electrochemical biosensors. A biosensor contains three key parts: a biorecognition element (BRE), a sensor chip, and a linker molecule which connects the two. Using a gold sensor chip, whole cell pathogen detection by toll-like receptor BREs is demonstrated.1,2 The result demonstrates the versatility in the biosensor scaffold, with implications for multi-analyte detection.
[1] McLeod, J. et al. Analyst 2020, 145 (18), 6024-6031
[2] Singh, I. et al. Chem Commun 2021, 57 (68), 8421-8424
Jess Deng
Supervisor: Dr. Richard Oleschuk
LMJ-SSP For Rapid Analysis of Prophylatic Lubricant, Spermicides and Residues
Jessie Deng, Haidy Metwally, Rebecca Richardson, Richard Oleschuk
Jennifer Kolwich
Rapid natural product screening and dereplication using on-plate mass spectrometry imaging
Jennifer L. Kolwich, Jian Yu, Haidy Metwally, Hailey A. Tomm, Richard D. Oleschuk, and Avena C. Ross.
Supervisor: Dr. Avena Ross
Jian Yu
Supervisor: Dr. Richard Oleschuk
Hyperspectral visualization based real-time microbial mass spectrometry imaging
Jian Yu, Haidy Metwally, Jennifer Kolwich, Hailey Tomm, Avena Ross, Richard Oleschuk
Mass spectrometry imaging (MSI) has been widely adopted for its exceptional performance in combining spatial and molecular information. However, dynamic monitoring of bacteria growth has been out of reach as sample preparation usually alters the sample. We have been examining the use of liquid micro-junction surface sampling probe (LMJ-SSP) to directly sample and image Pseudoalteromonas colonies without sample preparation. Real-time sampling on different strains is performed, while hyperspectral visualization method is employed to give a straightforward interpretation of their dynamic interaction based on principal component analysis (PCA). RGB color-coding translated from PCA score is used to find corresponding m/z values. One can witness the appearance/expression of prodiginine and tambjamine compounds in parallel with the growth of P. rubra and P.tunicata. Different growth rates of different strains, observed through the expansion of colony boundaries, are easily observed in hyperspectral images. The m/z values of metabolites other than prodiginine and tambjamine can be spotted in spectra at later stages of the life circle. Although not all the novel m/z have yet been identified, different spectra typically signify a dynamic metabolism status. Here, hyperspectral visualization-based in-situ MSI is demonstrated to be valid for the dynamic monitoring of bacteria growth.
Joshua Kofsky
Supervisor: Dr. Chantelle Capicciotti
Efficient synthesis of azido sugars using fluorosulfuryl azide diazotransfer reagent
Joshua M. Kofsky, Gour C. Daskhan, Matthew S. Macauley, Chantelle J. Capicciotti
Azide-containing carbohydrates are important and versatile tools in synthetic chemistry and chemical biology. They are widely used in the synthesis of amino sugars, 1,2-cis glycosides, and for biorthogonal labelling strategies to study cellular glycans. Typical strategies to install a non- participating C2-azido functionality use harsh and hazardous conditions and require long reaction times. These strategies involve azidonitration methods that can suffer from poor yields and stereoselectivity, or diazotransfer protocols that use particularly hazardous (i.e. explosive and toxic) reagents or intermediates.
We report in this presentation the synthesis of azido sugars using fluorosulfuryl azide as a safe and efficient diazotransfer reagent. Diazotransfer on various amino sugars was demonstrated with varying reaction times, quantities of diazotransfer reagent, and with the addition of several metal catalysts. Common hexosamine substrates were converted to 2-azido-2-deoxy sugars in less than 5 minutes in quantitative yield using a Cu(II) catalyst, which can also acts as a colour indicator for diazotransfer reaction completion. The diazotransfer protocol was applied to other amino sugar derivatives and the protocol was shown to be orthogonal to O-sulfation. This diazotransfer method will expand access to important non-participating C2-azido protecting groups and other azido sugar derivatives. We also demonstrate an application of this methodology for the synthesis of C2-azido glycosyl donors with orthogonal protecting groups from their amino sugar analogues with good overall yield and a single column purification. This optimized synthetic strategy has been applied for the preparation of 2-azido-2-deoxygalactosides, allowing faster access to useful intermediates for the chemoenzymatic synthesis of O-glycan derivatives.
Jordan Rensing
Supervisor: Dr. Steven Brown
Assessment of fecal indicators to correct analysis of fecal components of wastewater samples
Jordan Rensing, Abdul Alashraf, Nathan Mullins, and Stephen Brown
To monitor community cases, wastewater based epidemiological tracking of SARS CoV 2 is a valuable asset, as it allows monitoring without requiring individual testing. Normalization of SARS CoV 2 signal based on biomarkers from fecal matter is therefore important, to get a more accurate measurement. In this study, multiple fecal markers are tested as normalization factors for SARS-CoV-2 signal in 3 sites across Ontario. PMMoV and pellet weight show good correlation with each other (r=0.789, 0.749), and E. coli and total coliform show strong correlation with each other (r=0.889,0.984), but neither pair show good correlation with the other pair (r=0.293,0.599,0.375,0.272). In addition, the normalizations seem to not match up well to the case data, though there may be some predictive element involved.
Kailey Browne
Supervisor: Dr. Zhe She
Ultrasensitive Electrochemical Phosphate Detection by Pyridine-zinc(II) Complex
Kailey Browne, Yu Pei, Ishwar Singh, Sarah Jane Payne, Zhe She
The presence of phosphate within water systems is a driving force in the prevalence of harmful algal blooms or cyanobacteria blooms. Accordingly, phosphates are important environmental health parameters that are monitored using analytical techniques. Currently, phosphates are monitored using chromatographic and colorimetric techniques. Major disadvantages of these techniques include their poor selectivity towards specific anions, as well as the high cost of analysis. This work explores a new, electrochemical detection method, which can provide a cheaper yet sensitive technique. By implementing self-assembled monolayers through the modification of a gold working electrode with pyridine-zinc(II) complex, we were able to improve the selectivity of the sensor towards phosphate anions. This modification allows for an indirect and ultrasensitive method of detection of the H2PO4- species, ranging in concentrations between 0.0 and 1.2 fM phosphate. Electrochemical techniques such as cyclic voltammetry (CV) and square-wave voltammetry (SWV) were used to explore phosphate detection abilities. These techniques allowed for detection limits of 4.0 x 10-16 and 4.0 x 10-17 M H2PO4- to be obtained with CV and SWV, respectively. Selectivity of this sensor was also successfully explored for phosphate detection in the presence of potential interfering agents such as sulfate, carbonate and chlorine. Trace levels of phosphate within a tap water sample were also successfully detected with the use of this ultrasensitive sensor. We anticipate that this sensitive and reproducible technique will have applications for low-level phosphate detection in environment and treated waters.
Kristin Partanen
Supervisor: Dr. Zhe She
Fabricating Electrodes using Conductive 3-D Printing Materials and Electrochemical Gold Deposition
Kristin Partanen, Yu Pei, Phillip Hillen, Malek Hassan, Kevin McEleney, Sarah Jane Payne, Richard Oleschuk, Zhe She*
Bacterial and viral detections are important in environmental monitoring and clinical diagnostics. Current methods, such as bacteria culture tests and polymerase chain reactions, are successful when detecting bacteria and viruses, but are costly, require trained personnel, can take up to 5 days to receive results, and are inaccessible in resource poor settings.1 Therefore, it is important to move towards point-of-care testing (POCT) to decrease the detection time of bacterial species. There is a growing interest towards electrochemical (EC) sensors, such as the glucose meter, as they are based on compact potentiostats which offer portable and low-cost solutions for POCT, further decreasing the need of trained personnel.2 For an effective EC sensor, an electrode chip needs to be fabricated for sensing. Techniques, such as electron beam lithography and photolithography, have been used to design features on chips but they are costly, can be time-consuming, and may not be accessible.3 Therefore, there is a need to investigate new methods of fabricating chips that are capable for sensing applications. By using 3-Dimensional (3-D) printing technology, this can reduce the costs of chip fabrication due to the low-cost of the printer and allow for rapid prototyping capability. In this study, we explore enhancing the conductivity of 3-D printed electrodes along with EC gold deposition using two commercial conductive filament brands. These electrodes were then able to demonstrate a practical application by detecting Cu2+ using anodic stripping voltammetry at drinking water concentration thresholds.
Leila Pujal Gómez
Supervisor: Dr. Farnaz Heidar-Zadeh
Malek Hassan
Supervisor: Dr. Richard Oleschuk
SINGLE SPOT ANALYSIS OF PARAFFIN-EMBEDDED TISSUE SECTIONS USING LIQUID MICROJUNCTION SAMPLING PROBE (LMJ-SSP) MASS SPECTROMETRY
Haidy Metwally, Malek Hassan, Jessie Deng, and Richard Oleschuk
Freezing fresh tissues is the optimum method for conserving nucleic acids and proteins. However, fresh frozen (FF) tissues require strict storage conditions such as -80 temperature for up to one year. In addition, FF tissues suffer from the formation of ice crystals, and they became fragile with time. Formalin fixation and paraffin embedding (FFPE) of tissues improve their processing and extend their shelf life. FFPE tissues are an important source of archived biological data. FFPE tissues cannot be used directly with mass spectrometry imaging as paraffin needs to be removed. The protocol used to remove the paraffin from the tissues usually starts with exposing the FFPE tissue to xylene, followed by washing with serial dilutions of ethanol. This study aims to decrease the amount of time spent on tissue cleaning and processing as well as preserve the tissue for further storage. Using the LMJ-SSP mass spectrometry; we were able to dewax certain spots on the FFPE chicken liver and detect some bile acid signals.
Marie Boddington
Supervisor: Dr. Chantelle Capicciotti
Combining chemoenzymatic synthesis and cell-surface glycan engineering to probe glycan-protein interactions
Marie E. Boddington, Chantelle J. Capicicotti
Glycans are complex and structurally diverse carbohydrates, found on the surface of all cells, that are critical for a wide range of biological functions and are directly involved in every major disease. The biological roles of oligosaccharides range from trivial to essential functions in development, growth, and survival of the organism. More specifically, complex type N-glycans are involved in signal transduction, fertilization, cell-cell recognition and adhesion, immune regulation, and cancer. Due to the importance of N-glycans, there is an interest in understanding the role that defined structures play in biological functions. However, understanding which complex structures are involved in crucial protein-binding events and biological function is challenging. N-Glycans are branched, highly diverse complex structures and their biosynthesis is not template driven. The branches of complex type N-glycans consist of Type II LacNAcs and/or Type I LacNAcs that can be decorated with fucose and terminal sialic acids, forming important glyco-epitopes such as Lewis.
Novel chemical biology approaches are needed to be able to synthesize these structures and study how variations in structures impact interactions with glycan-binding proteins. This poster presentation will describe a two-pronged approach to study glycan-binding protein interactions with defined glycans and glyco-epitopes on cell surfaces. We combine chemoenzymatic synthesis with cell-surface glyco-engineering to selectively install defined small glyco-epitopes on cell surfaces. The glyco-engineered cells are then probed for glycan-binding protein interactions to understand how the epitopes modulate binding interactions. Type II LacNAc epitopes will be synthesized initially, and then extended into poly-LacNAcs and elaborated with sialic acid and fucose to produce sLex and poly-sLex--Lex epitopes. These structures will be used to investigate their binding with glycan-binding proteins such as E-selectin and various Siglecs. The information gained from glyco-engineering with small LacNAc-based epitopes will then be used to guide the chemoenzymatic synthesis of complex N-glycan derivatives with multivalent, extended poly-LacNAc branches bearing sialyl-Lewis X (sLeX) epitopes. Poly-LacNAc epitopes are enzymatically installed on each branch of the N-glycan core to explore the branch-specific presentation of poly-LacNAcs and sLeX epitopes. This will allow us to probe how these glycans interact with glycan-binding proteins and to develop an on-cell glycan array platform as a tool to probe protein binding along with biological function of these glycan structures.
Niranji Thilini Ekanayake C T M Ralahamilage
Supervisor: Dr. Nick Mosey
Michael Trolio
Supervisor: Dr. Diane Beauchemin
COMPARISON OF LOW VOLUME INJECTION TO MORE CONVENTIONAL SAMPLE INTRODUCTION IN INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY FOR ITS EFFECT ON OXIDE FORMATION AND MATRIX EFFECTS
Michael G.A. Trolio, Graham Shearing, and Diane Beauchemin
Oxide interferences and matrix effects may compromise the accuracy of inductively coupled plasma mass spectrometry (ICPMS) measurements. A method consistently reducing the impact of these interferences on empirical results would be valuable. A new sample introduction method was explored, involving mono-segmented (MS) flow injection analysis (FIA) with 1 µL of sample. This technique was compared to both continuous delivery using a peristaltic pump and 50 µL FIA sampling methods. Oxide formation was examined through measuring CeO+ /Ce+ ratios while injecting a solution of 10 mg/L Ce. A positive correlation between volume and interference was observed and the 1 µL MS-FIA offered up to a 68.8% reduction in CeO+ /Ce+ . Matrix effects were examined across 12 elemental solutions of 10 mg/L, with and without 400 mg/L of Na. The average changes in signal with the addition of Na for peristaltic, 50 µL FIA, and 1 µL MS-FIA were -4.7%, -1.11%, and 0.18% respectively. Finally, CRM analysis was performed using calibrations without internal standard, for all three techniques. A negative correlation between sample volume and accuracy was observed and the 1 µL MS-FIA technique was the only one to yield concentrations within the range of inclusion for all successfully observed elements. Hence, both oxide interferences and matrix effects can be reduced using low volume MS-FIA, thereby increasing measurement accuracy.
Mitchell Jeffs
Supervisor: Dr. Christopher Lohans
Development of a Whole-Cell Biosensor for Beta-Lactamase Inhibitor Discovery
Mitchell Jeffs, Jennifer Kolwich, Avena Ross, Christopher Lohans
The clinical utility of β-lactam antibiotics is endangered by the production of β-lactamases by multi-drug resistant bacterial pathogens, a global health threat. Although extensive efforts have been made to develop inhibitors for these enzymes, inhibitor-resistant variants emerge rapidly. In addition, there are no clinically available inhibitors for many currently observed β -lactamases. To facilitate inhibitor discovery efforts, new assays are required to assess inhibitor efficacy in a cellular context. We describe the development of a whole-cell biosensor which can be applied to the identification of β-lactamase inhibitors, prioritizing hits that are effective against bacterial cells. Upon administration of an effective inhibitor, the co-administered β-lactam antibiotic is rescued from β-lactamase-catalyzed degradation, causing the biosensor to produce a luminescent signal. This platform was validated using a panel of clinically relevant β-lactamases and was applied to quantitatively measure the potency of currently used β-lactamase inhibitors. This assay is cost-effective and does not require the use of potentially expensive substrates. It also overcomes limitations associated with purified protein assays, accounting for inhibitor permeability and efflux, and has a significantly shorter time-to-result than the growth-based methods traditionally used for evaluating β-lactamase inhibitors. We are currently using the biosensor platform to screen Pseudoalteromonas natural product libraries for novel beta-lactamase inhibitors.
Max van Zyl
Supervisor: Dr. Heidar-Zadeh
Parimah Aminfar
Supervisor: Dr. Kevin Stamplecoskie
Photochemical Synthesis of Novel Fluorescent Peptide-Protected Gold Nanoclusters for Biomedical Applications
Parimah Aminfar and Kevin Stamplecoskie
Accelerated Size-Focusing Photochemical Synthesis of Novel Fluorescent Peptide-Protected Gold Nanoclusters for Biomedical Applications
The synthesis and isolation of atomically precise gold and silver metal clusters have recently attracted enormous research interest. This is especially true for aqueous soluble clusters that typically display more challenges in achieving pure, atomically precise samples and in crystallization for characterization. Among them, peptide-protected gold nanoclusters (AuNCs) have been of particular interest for their good biocompatibility, photo-stability, along with the retained biological activity of the template.1 Owing to their tunable optical, electronic, and physiochemical properties thiolate-protected gold clusters have been highly attractive for biomedical applications compared to traditional gold nanoparticles. In particular, AuNCs emitting in NIR window have been studied for in vivo therapeutic and diagnostic (theranostic) applications. Before clinical use, it is important to ascertain that these clusters are pure and single size to be able to track them in cells and understand their function in the body. However, the typical synthesis of these clusters often lead to a distribution of sizes with various number of metal atoms and ligands. Most of these studies investigate the properties of a mixture of clusters without exploring the size-dependent properties.2
In this work, we propose light activated synthesis of Au clusters using a sequence of peptides as a ligand. Using Norrish type 1 photochemistry3, a unique control over the most challenging aspects of metal cluster synthesis is provided. The obtained Clusters were found to be optically pure using fluorescence excitation-emission matrix (EEM) spectroscopy and PARAFAC analysis. These novel clusters were then characterized by high resolution Electrospray Ionization (ESI) and Time-of-Flight (TOF) Mass Spectrometry and were prepared to be tested in a cell environment.
Reference:
1. Yuan, Q.; Wang, Y.; Zhao, L.; Liu, R.; Gao, F.; Gao, L.; Gao, X., Peptide protected gold clusters: chemical synthesis and biomedical applications. Nanoscale 2016, 8 (24), 12095-12104.
2. Ramsay, H. S.; Silverman, M. M.; Simon, D.; Oleschuk, R. D.; Stamplecoskie, K. G., Light activated synthesis of the atomically precise fluorescent silver cluster Ag18(Capt)14. Nanoscale 2019, 11 (43), 20522-20526.
3. Yousefalizadeh, G.; Stamplecoskie, K. G., Norrish type I photochemistry as a powerful tool in the isolation of thiol protected Au25SR18 clusters. Journal of Photochemistry and Photobiology A: Chemistry 2018, 353, 251-254.
Qiqi Zhang
Supervisor: Dr. Diane Beauchemin
Raymond Lai
Supervisor: Dr. Guojun Liu
Facile Preparation of a Transparent and Rollable Omniphobic Coating with Exceptional Hardness and Wear Resistance
Raymond (Ziruo) Lai, Guojun Liu
The encapsulating film for the touchscreen of a foldable smartphone consists of a flexible polymer layer covered by a hard coating and an antismudge coating, and the two coating layers are currently deposited via separate steps. This study reports the preparation of a bilayer bifunctional coating via the deposition of a single polymer mixture. The base material for the coating is a ladder-like polysilsesquioxane (LASQ) that is derived from the sol−gel chemistry of 2-(3,4-epoxycyclohexyl)- ethyltrimethoxysilane. Reacting a limiting amount of the liquid antismudge agent FP-COOH, which is a perfluorinated poly(propylene oxide) bearing a terminal carboxyl group, with LASQ yields m-LASQ-FP, a mixture of unreacted LASQ, and a graft copolymer LASQ-FP. m-LASQ-FP at a fluorine mass fraction of 6.0% is photocured to yield a coating with a surface energy of 12.3 ± 1.5 mJ/m2. At a thickness of 40 μm, the coating has at 500 nm a transmittance of >99% measured against its glass substrate, a remarkable nanoindentation hardness H value of 1.4 GPa, and a pencil hardness of > 9H. After being abraded for 300 strokes under a pressure of 26 kPa with steel wool, the coating exhibits no noticeable degradation in its ink contraction properties. At a thickness of 10 μm on a poly(ethylene terephthalate) film, the coating can undergo inward (on the inner surface of the bend) and outward bending to radii <1 and <2 mm, respectively, without cracking. Aside from being a superb candidate as a protective antismudge coating for foldable smartphones, this marvelous material should also have many other applications.
Samantha Hollands
Supervisor: Dr. David Zechel
Exploring uncharted territory in RiPP biosynthesis: studying the biosynthesis of curacozole from Streptomyces curacoi
Samantha Hollands*, Dr. Manisha Patel, Dr. David Simon, Isobel Barber, and Dr. David Zechel
The majority of antibiotics, antiviral medications, and cancer drugs used in modern medicine are natural products and derivatives.1 The cyanobactins are a class of ribosomally synthesized and post-translationally modified peptides produced by cyanobacteria that have notable anticancer, antiviral, and antibacterial properties. In recent years, cyanobactin-like compounds have been discovered in Streptomyces species of the actinobacteria phylum (the YM-216391 family). Structurally, molecules from the cyanobactin and YM-216391 families are small, cyclic peptides that are heavily modified after translation, for example by side chain cyclization, epimerization, and oxidation.2 This project aims to characterize the biosynthetic pathway of curacozole, a YM-216391 family compound produced by the soil bacterium Streptomyces curacoi (S. curacoi). Curacozole has potent anticancer activity, and many of the reactions required to form curacozole (such as epimerization, cyclodehydration, and peptide macrocyclization) are novel and difficult to perform synthetically. A cluster of 10 genes within the genome of S. curacoi has been identified for curacozole biosynthesis, but the functions of the encoded enzymes are unknown. In vitro assays with the precursor peptide (CzlA and variants) were undertaken to biochemically define the functions of these enzymes. By elucidating the curacozole biosynthesis pathway, we aim to discover useful enzymes for industrial biocatalysis, and to set the stage for engineering the pathway to create new cyanobactins with useful bioactivities.
1. Calixto, J. B. The Role of Natural Products in Modern Drug Discovery. An Acad Bras Cienc 2019, 91 Suppl 3, e20190105.
2. Sivonen, K.; Leikoski, N.; Fewer, D. P.; Jokela, J. Cyanobactins—Ribosomal Cyclic Peptides Produced by Cyanobacteria. Appl Microbiol Biotechnol 2010, 86 (5), 1213–1225.
3. Kaweewan, I.; Komaki, H.; Hemmi, H.; Hoshino, K.; Hosaka, T.; Isokawa, G.; Oyoshi, T.; Kodani, S. Isolation and Structure Determination of a New Cytotoxic Peptide, Curacozole, from Streptomyces curacoi Based on Genome Mining. J Antibiot 2019, 72 (1), 1–7.
Valerii Chuiko
Supervisor: Dr. Paul W. Ayers, McMaster University
HOW TO MAKE A MOLECULE SING…AND REVEAL ITS PROPERTIES
Valerii Chuiko, Alireza Tehrani, Fanwang Meng, Farnaz Heidar-Zadeh and Paul W. Ayers
To design a drug, one must identify a small molecule that is complementary in shape and charge to the biomolecular target with which it interacts. Tremendous effort has been expended in the search for small molecules that bind to a biomolecular targets, using both experimental and computational approaches. Many recent computational strategies utilize machine-learning algorithms. The main problem face by machine-learning approaches is that they need to be intelligent and flexible enough to accommodate the inherent uncertainty of in vivo molecular data. We are proposing a completely new approach in which important features of molecules are represented as a sound. Specifically, inspired by Mark Kac’s classic paper, “Can One Hear the Shape of a Drum”[1], we compute the acoustic spectrum of a molecule by treating the molecule’s surface as a three-dimensional drum, with the density of the drum’s membrane determined by key molecular properties/features. Using this idea, together with strategies based on graph-theory, hyperparameter optimization, and spectral theory, allows key molecular features to be represented as sounds. These sounds are shown to be consistent with well-known chemical concepts related to functional groups and molecular similarity. Finally, we compute molecular sounds for a database of molecules and use these as input features for a machine-learning method. Even outside the context of machine learning and drug design, molecular sounds provide a new, effective, and efficient descriptor for computational chemists.
References
[1] Mark Kac, Can One Hear the Shape of a Drum?, The American Mathematical Monthly, Vol. 73, No. 4, Part 2: Papers in Analysis (Apr., 1966), pp. 1-23
Youjin Kim
Supervisor: Dr. Chantelle Capicciotti
Developing Antibody-drug Conjugates Through Site-specific Glyco-engineering
Youjin Kim, and Chantelle Capicciotti
Antibody-drug conjugates (ADCs) are complex therapeutic molecules that are a powerful target-specific drug delivery system for various diseases. The design of ADCs involves unique challenges including the control of homogeneity, drug-to-antibody ratio (DAR), and drug load distribution without disturbing the integrity of antibodies. Recently, antibody glyco-engineering has shown potential for drug conjugation to improve various aspects of ADCs. Immunoglobulin G1 (IgG1) has a single N-glycosylation site on its heavy chain, thus enabling site-specific modifications. However, the glycoform within the site is highly heterogeneous, and current approaches only employ N-glycans with small functional groups, which can lead to heterogeneity in the products as it requires an additional drug conjugation step. Therefore, we aim to produce improved glycan-handle ADCs by overcoming some of these barriers in creating IgGs with a defined glycoform.
This poster will describe the chemoenzymatic synthesis of defined N-glycan-cargo conjugates and enzymatic transfer to IgGs through a single step remodeling strategy. The synthesized glycans are capped with sialic acids functionalized with azides, and these azido-glycans are then conjugated to alkyne-functionalized drug or reporter molecules via copper-catalyzed azide-alkyne cycloaddition. The defined glycan-cargo structure will then be enzymatically transferred to IgGs using mutant endo-β-N-acetylglucosaminidases (ENGases) with a single step after the removal of pre-existing IgG glycans catalyzed by wild-type ENGases. Our strategy shows promise to achieve a highly controlled process for producing homogeneous ADCs and to increase DARs by utilizing multivalency of N-glycans. Once validated, it can be easily translated to create various target-specific therapeutics, theranostics, imaging agents, and more.
Zhenyu Hu
Supervisor: Dr. Graeme Howe
