Relevant Thesis-Based Degree Programs
Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
This thesis will present the study of a peptide sequence that is favourable for forming a five-helix bundle through the use of template assembled synthetic proteins (TASPs), and applications of our synthetic proteins in rate enhancement of ester hydrolysis and protein-protein interactions. Chapter 1 will introduce proteins, de novo protein design, and TASPs. It will also review past and current research on the applications of the synthetic molecules mimicking biological behavior. Chapter 2 will focus on several modified de novo designed peptide sequences that are intended to be more favourable in folding into a five-helix bundle than a four-helix bundle. We found that our designed sequence narrowed the free energy gap between the five-helix bundle and the four-helix bundle relative to the systems where the peptide sequence was designed to favour a four-helix bundle. Chapter 3 will concentrate on investigating the rate enhancement of ester hydrolysis by histidine-containing TASPs. These TASPs increased the rate of ester hydrolysis, and the position of the histidines was found to be relevant to activity. Chapter 4 will detail the attempts at using a template-assembled synthetic protein to inhibit protein-protein interactions between a Bak peptide and a Bcl-xL protein. Our synthesized protein was found to be a good binding partner towards the Bcl-xL protein and manifested moderately enhanced proteolytic resistance. Several heterocaviteins were also synthesized to study both their inhibitive activities to the Bcl-xL protein, and their proteolytic stability. Chapter 5 will summarize and conclude the work throughout this thesis.
Pyrimidine-based quartets and quadruplexes are unstable and thus are rarely encountered innature. Uracil (U) and thymine (T) quartets in the solution state have only been found as part ofpre-existing G-quadruplex scaffolds and the corresponding quadruplexes have not been reported.Studies on such systems might shed light on their role in nucleic acid topology and stability. Thisthesis describes the assembly and structural characterization of these motifs in vitro as a result ofgrafting the respective nucleosides onto resorcinol-based cavitands. These rigid macrocyclesserve as molecular templates on which these motifs are preorganized. Reduction of entropic lossimproves thermodynamic stability and promotes self-assembly.A convergent synthetic strategy was employed for accessing these cavitand-nucleosideconjugates. Cavitands and nucleosides were prepared separately using established literaturemethods, and the final coupling step of the two components entailed a copper (I)-catalyzedazide-alkyne cycloaddition, or a "click" reaction. NMR spectroscopy was used extensively insignal assignment, structure elucidation and oligomeric state analysis. CD spectroscopy wasemployed in some cases to provide further confirmation of defined structure.Findings indicated the spontaneous self-assembly of a U-quartet in CDCl3 at both 25 ºC and –20ºC. In the presence of a metal cation (Sr²⁺), symmetric homodimerization of two U-quartetsoccurs at 25 ºC. The corresponding U-quadruplex unit was identified in DMSO-d₆ at 25 ºC. TheT-quartet was shown to be nonexistent at 25 ºC, but assembles at a low temperature of –40 ºC.iiiNo evidence for metal cation uptake was found at 25 ºC. Assembly of the T-quadruplex wasconfirmed in DMSO-d₆ at 25 ºC. In all of these systems, stacking of the nucleobase and triazolelinker rings was indicated suggesting π-stacking interactions to be a significant contributor tooverall stability.
This thesis will investigate the sequence to structure relationship of protein folding through the use of template assembled synthetic proteins (TASPs). A cavitand template will be used to bring together four de novo designed peptides to form a four-helix bundled TASP, referred to as a cavitein. The first chapter will detail the rationale behind de novo protein design and use of TASPs to study protein folding. Chapter 1 will also review past and current work relating to the design and structural characterization of synthetic de novo proteins. Chapter 2 will investigate the context dependency of leucine residues within the hydrophobic core of a cavitein. Single leu to ala substitutions resulted in similar destabilization of the cavitein. However, replacing the middle leucine compromised the structure and stability of the cavitein. It was concluded that the middle leucine acted as a linchpin within the peptide sequence. Chapter 3 will examine the first reported crystallization of a TASP. The crystal structure is revealed as a dimer. The cavitein (Q4) was thought to be monomeric. The chapter will compare the solution structures of both the parent and Q4 cavitein. It was found that both parent and Q4 have a weak dimer association at relatively high concentrations.Chapter 4 will discuss glutamine variants used to probe the tolerance of dimer crystallization with respect to sequence variation. The chapter uncovers a key lattice contact for cavitein dimer lattice nucleation and stabilization. Furthermore, in an attempt to control the oligomeric state of caviteins, Chapter 4 will also discuss histidine metal binding caviteins and disulfide caviteins. A cavitein designed to favor a dimer via histidine metal binding was synthesized and crystallized revealing a potential metal binding center. Caviteins designed to favor a monomer conformation were also designed involving either histidine metal binding or disulfide bridges. Both methods proved successful in their monomeric design as shown by solution characterization. Chapter 5 will conclude and summarize the work throughout this thesis. The chapter will also suggest future projects expanding on studies within this thesis as well as suggest other projects pertaining to cavitein research.
Fabrication of functional supramolecular structures requires a certain degree of controlwhich may not be achieved by relying solely on noncovalent interactions. The current studyaims to investigate the effect of a rigid cavitand template on morphology, function and stabilityof lipophilic G-quadruplexes. The first Chapter of this thesis introduces different aspects of Gquadruplex chemistry and explains how these structures are particularly suited for the creation ofsupramolecular architectures.The second Chapter of this thesis presents the synthesis and self-assembly of a new classof supramolecular architectures composed of four guanosines attached to a rigid cavitandtemplate. These structures, named template-assembled synthetic G-quartets (TASQs), weresynthesized via the “click” reaction and manifest an ordered topology dictated by the template.The lipophilic TASQs were found to self-associate spontaneously to form a singular basket-likestructure in chloroform. Moreover, it was found that TASQs form cation-free G-quartets whichexhibit remarkable stability under this condition.The third Chapter of this thesis describes the preparation, characterization and solutionstudy of the cation-bound complexes TASQNa⁺, TASQK⁺, TASQCs⁺, and TASQSr²⁺.Cations play a major role in controlling the morphology and stability of G-quadruplexes. Theanalysis of the cation-specific structures of TASQs reveals the formation of a monomeric Gquartet for Na⁺ and Sr²⁺,a dimeric system for Cs⁺ and a mixture of monomers and dimers for K⁺.The factors governing the formation of these structures were evaluated, the selectivities ofTASQs for cations were determined, and the cation-dependent structural transformations werestudied.The fourth Chapter describes the efforts towards synthesizing a hydrophilic TASQ viathe “click” reaction. The following steps have been taken: 1) a water-soluble cavitand has beensuccessfully synthesized and characterized, which can potentially serve as a hydrophilictemplate, and 2) two oligonucleotides have been appropriately functionalized and preliminarycoupling reactions were attempted. The next phases of this research along with potential futuredirections are discussed in Chapter five.
Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
DNA is a classic target for small-molecule ligands. In order to reduce significant toxicities of anticancer drugs resulting from unspecific interactions with DNA duplexes, it aroused great interest to investigate the specific interactions of ligands with a secondary DNA structure, G-quadruplex, formed by a guanine-rich DNA sequence. Induction and stabilization of G-quadruplex structures by ligands have been shown to inhibit telomerase activities and regulate the transcription and expression levels of oncogenes in cancer cells; therefore, the design of synthetic G-quartets under physiological conditions as minimal models of G-quadruplexes or artificial receptors of anticancer drugs has become an important and promising approach to clarify binding mechanisms, as well as to develop practical high-performance anticancer drugs. This thesis explores the recognition behavior of the second generation of hydrophilic template-assembled synthetic G-quartets (TASQs) using fluorescence spectroscopy and CD spectroscopy with PIPER, TMPyP4, AZATRUX, BSU 1051 and BRACO-19. The results show that PIPER, TMPyP4, AZATRUX can stack on top of a G-tetrad plane via π-π stacking with stoichiometries of 1:1 and high binding affinities (KPIPER=1.65×10⁷ M⁻¹, KTMPyP4= 8.5×10⁵ M⁻¹, KAZATRUX=2.55×10⁶ M⁻¹); however, BSU 1051 and BRACO-19 have no such behavior with TASQs. All the spectra and binding mechanisms are similar to known mechanisms or computer-aided molecular simulation models, suggesting that the second generation of hydrophilic TASQs can imitate the natural terminal G-tetrad planes of G-quadruplexes. Moreover, this artificial receptor has selectivity over different ligands with an ability to contribute to the screening of small-molecule ligands, as well as the investigations of binding mechanisms of new anticancer ligands. The main works in this thesis are shown as follows:1. Introduction to DNA duplexes, G-quadruplexes, template-assembled synthetic G-quartets, anticancer drugs (i.e. PIPER, TMPyP4, AZATRUX, BSU-1051, BRACO 19, telomestatin), and characteristic methods.2. Synthesis of water-soluble template-assembled G-quartets (TASQ 13), PIPER, and AZATRUX. The binding properties of these ligands with TASQ 13 were characterized by spectroscopic methods so as to show the binding abilities and binding modes of different drugs to TASQ 13. 3. Conclusions and future work.