David Perrin


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.

Methodologies for synthesis of transcription inhibiting peptides, amatoxins, and their bioconjugates for targeted cancer therapy (2022)

This thesis describes my contribution to the development of synthetic methods directed at accessing bicyclic toxic peptides, amatoxins, as well as amatoxin-based bioconjugates for targeted treatment of cancer. Alpha-amanitin, the principal toxin of the amatoxin family and a potent transcription inhibitor, is of significant interest for applications in cancer therapy in a form of conjugates targeting cancer-specific receptors. Despite being known for over 70 years, the complex structure of α-amanitin has challenged the development of synthetic methods. In Chapter 2, C-3a-halopyrroloindolines are successfully employed as precursors to a tryptathionine crosslink, a key structural feature shared by amatoxins. Notably, fluoropyrroloindoline (FPI) was utilized to access previously inaccessible, oxidation-sensitive 6'-hydroxy-tryptahtionine found in α amanitin. Furthermore, the distinct reactivity of FPIs under H-bonding conditions was explored whereby hexafluoroisopropanol was used to ionize the C-F bond for nucleophilic attack yielding C-2-thiol-substituted tryptophans and C-3a-functionalized pyrroloindolines.Chapter 3 describes the synthesis and evaluation of (RSO)-5'-hydroxy-6'-deoxy-amanitin, a novel, bioactive analog of α-amanitin for use as a payload in targeted therapies. This synthesis employs naturally abundant 5-OH-tryptophan to incorporate an indole conjugation handle in three steps, in contrast to the synthetically challenging 6-OH-tryptophan. In vitro cell viability and RNA polymerase II transcription inhibition assays complete the biological evaluation of (RSO)-5'-hydroxy-6'-deoxy-amanitin and sulfur-modified analogs thereof.Chapter 4 describes two diastereoselective sulfoxidation methods providing access to amatoxins with the biologically active (R)-sulfoxide, thereby solving the last-standing challenge in the synthesis of amatoxins. The late-stage sulfoxidation approach utilizes Ti(IV)/L-DET-based system and is applied to the bicyclic precursor as the last step of the synthesis yielding the (R)-sulfoxide in large excess ((R:S) 19:1). Additionally, facile and cost-effective early-stage sulfoxidation employing meta-chloroperoxybenzoic acid in dimethylformamide is described. In Chapters 5 and 6, two synthetic amanitin analogs, conjugated at different sites, are used to assemble peptide drug conjugates targeted to cancer-specific receptors. The development of a modular synthetic platform for the construction of cleavable and non-cleavable amanitin-peptide conjugates that can be adapted to various targeting moieties is reported. In vitro cell proliferation studies on antigen-positive cell line revealed target-specific toxicity of conjugates with up to 1000-fold bioactivity enhancement.

View record

Exploiting boron-fluorine bonds for fluorination and synthesis of potential bi-modal imaging agents (2019)

The B-F bond has an expansive and rich history in chemical transformations and the versatility of the B-F bond has also shown immense utility in fields as far reaching as PET and NIRF imaging. The application of B-F bonds for nucleophilic C-F bond formation and the development of novel fluorophores with potential applications in PET/NIRF bi-modal imaging are investigated in this work.Organotrifluoroborates are well known as indispensable synthetic tools for the elaboration of organic molecules. However, the trifluoroborate functionality is generally regarded as an auxiliary group, and its potential as a nucleophilic F- source has remained untested. In this context, the ability of organotrifluoroborates to serve as competent sources of F- was investigated. In an update to the historic Balz-Schiemann reaction, that traditionally uses BF₄- as a source of F-, organotrifluoroborates were shown to mediate the fluorodediazoniation of in situ generated aryl diazonium salts under mild reaction conditions (chapter 2). In reactions with an analogous substrate class, unsymmetrical diaryliodonium salts could also be fluorinated using phenyltrifluoroborate via thermal decomposition of the ion pair (chapter 3). Finally, the ability of the trifluoroborate moiety to mediate intramolecular fluorination reactions was investigated through the synthesis of various trifluoroborate-containing molecular scaffolds (chapter 4).I also investigated novel fluoroborate (B-Fn) complexes of the 2-aryl-benzothiazole scaffold, known for its interaction with Aβ aggregates, as fluorophores. Three complexes were isolated, characterised and their photophysical properties are reported. A 2,4-diaryl-benzothiazole monofluoroborate complex displaying the most promising photophysical properties, was shown to be stable to aqueous conditions. Fluoride abstraction from this complex was also demonstrated, providing promising preliminary results towards the viability of radiolabelling via ¹⁹F-¹⁸F isotope exchange. The monofluoroborate complex also showed interesting structural properties given the asymmetric boron centre. Enantiomers of the complex were successfully chirally resolved from the racemic mixture, characterised by Circular Dichroism and their stability to racemization via inversion investigated (chapter 5).

View record

Bicyclic octapeptide alpha-Amanitin, the death cap mushroom toxin: the total synthesis and derivatives of the hydroxyproline residue (2018)

This thesis presents the first total synthesis of the death cap mushroom toxin α-amanitin and the synthesis of its derivatives containing analogues of the hydroxyproline residue. In Chapter 2, an enantioselective route to the synthesis of (2S,3R,4R)-dihydroxyisoleucine, an unnatural oxidized amino acid found in α-amanitin, is presented. This includes the synthetic challenges that needed to be overcome, previous non-enantioselective syntheses of this amino acid, my failed attempts, and eventually the route to successfully obtain the desired enantiomer of this residue.Chapter 3 describes an unprecedented method to synthesize the unique, oxidant-sensitive 6-hydroxy-L-tryptathionine linkage. First, C-6 borylation of a suitably protected L-tryptophan was performed according to recent literature. Then, fluorocyclization of 6-boronate-L-tryptophan yielded a fluoropyrrolo indoline (Fpi) moiety that was shown to engage in the Savige-Fontana reaction with trifluoroacetic acid to furnish the 6-boronate-tryptathionine crosslink. In this synthesis, a boronate was used as a latent hydroxy group that could be revealed on the fully elaborated toxin following an oxidative deborylation reaction.In Chapter 4, the first total synthesis of α-amanitin is concluded. First, incorporation of 6-boronate-Fpi yielded a 6-hydroxy-tryptathionine crosslink. Then, the synthetic (2S,3R,4R)-dihydroxyisoleucine was introduced to the peptide sequence of α-amanitin. Following a macrolactamization step and a diastereoselective sulfoxidation of the tryptathionine thioether to the corresponding (R)-sulfoxide found in the natural product, the synthetic α-amanitin was afforded. Juxtaposition of the synthetic and authentic α-amanitins and extensive comparison of their physical, chemical and biological properties validated the synthetic analogue.The analogues of trans-hydroxyproline and the method for their incorporation into α-amanitin derivatives are disclosed in Chapter 5. The hydroxyproline residue of α-amanitin has been shown to be critical for the toxicity of this toxin. However, surprisingly, there is little traction in the literature regarding the structure-activity relationships (SAR) of the hydroxyproline space and how it could affect the binding of the toxin to RNA polymerase II. Hence, a series of hydroxyproline analogues, including a photocleavable hydroxyproline derivative, were synthesized and aimed to be incorporated into amanitin via an improved solid-phase strategy.

View record

Synthesis on the solid phase of a bioactive tryptathionine octreotate (2017)

Development of somatostatin analogues (SSAs) plays a central role in the improvement of diagnostic and therapeutic tools in the treatment of neuroendocrine neoplasms (NENs). These analogues target the somatostatin receptors (sst) 1-5 which are often overexpressed on the outer membrane surface of many tumor cells. Poor selective modulation of a single sst receptor subtype by SSAs leads to a wide range of side effects, limiting their clinical impact. The high affinity of somatostatin (SST) and its analogues that bind to sst receptors results from a short subsequence (5-9 amino acids), usually within a rigid beta-turn motif. Tryptathionine bridges (Ttn) represent a privilege scaffold for the beta turn pharmacophore and contribute to high affinity, selectivity and significant metabolic stability of peptidic toxins. In the present thesis a trimmed somatostatin analogue, in which the disulfide bond found in octreotate was replaced by a tryptathionine bridge ((Ttn)-TATE), was made on solid phase. The resulting soluble (Ttn)-TATE displayed a high affinity in vitro for membrane solubilized sst2 receptor and for sst2 receptor expressed on whole Ar42J cells. Similar results were obtained from (Ttn)-TATE anchored on TentaGel microbeads. This work lays down the foundation for a one-bead-one-compound (OBOC) combinatorial tryptathionine peptidic library to isolate selective and avid binders of a desired sst receptor subtype and cellular phenotype outcome.

View record

A pursuit of sequence specific RNase A mimicking DNAzymes (2016)

DNAzymes are single-stranded DNA molecules capable of catalysis, and they are the DNA counterparts of ribozymes and protein enzymes. M²⁺-independent RNA cleaving DNAzymes are intriguing due to their robust activity that is not compromised by low Mg²⁺ that is found in cells. Hence, they hold great promise for RNA regulation in vivo. To mimic the metal independent protein endonuclease RNase A, three chemically modified nucleotides dAimTP, dCaaTP, and dUgaTP, that are adorned with respective side-chain functionalities of histidine, lysine, and arginine, have been simultaneously introduced in in vitro selections by our group, and led to the development of families of M²⁺-independent DNAzymes targeting chimeric DNA/RNA substrates, attaining kobs as high as ~0.6 min-¹ at pH 7.4, 37°C. In order to further select such DNAzymes capable of highly efficient all-RNA cleavage and multiple-turnover, a novel unimolecular selection scheme containing an all-RNA substrate derived from the HIV-1 LTR-promoter allows a direct selection of all-RNA cleavers meanwhile fostering the subsequent conversion of the cis-cleaving species into a trans-acting catalyst was constructed. An optimized in vitro selection cycle combining selection, re-selection, and evolution that permitted greater sequence space sampling and pursuit of catalytically improved sequences through generation-specific mutagenesis was designed. The application of the novel construct in the optimized in vitro selection cycle gave rise to two families of desired DNAzyme candidates. Under simulated physiological conditions (pH 7.45, 150 mM K⁺, 0.5 mM Mg²⁺, 37°C), the best representative, Dz7-38-32t, attained kcat and KM values of ~0.24 min-¹ and 2.72 µM, respectively, corresponding to a catalytic efficiency of ~10⁵ M-¹min-¹. Dz7-38-32 can be spontaneously taken up by HeLa cells after 45 h incubation at 0.9 µM due to its similarity to cell penetrating peptides (CPPs) regarding the appended functionalities. To understand fundamental aspects by which these three modified bases function in in vitro selection, investigations of biophysical and enzymatic properties of them in the context of discretely modified oligonucleotides were performed. These studies identified certain shortcomings in the use of modified nucleosides while providing clear evidence of negligible mutagenicity in terms of both primer extension and enzymatic read-through.

View record

Selecting and improving the functionality of DNAzymes (2015)

DNAzymes are strands of catalytic DNA first discovered in 1994. These species are isolated through in vitro selection and are capable of catalyzing many different types of reactions. RNA-cleaving DNAzymes are one subset that have many biological implications; however, more work needs to be done to make them suitable for therapeutic in vivo applications. Modifying DNAzymes with protein functionalities represents a promising strategy to evolve efficient cleavage in vivo.Chapter 2 described the syntheses of five modified 2'-deoxyuridine triphosphates and the enzymatic incorporation of these modified dUTPs. The modifications were introduced at the 5-position, and consist of a carboxylate group, indole group and napthyl group. The enzymatic incorporation of these modified nucleotide triphosphates evaluated their suitability for use in an in vitro selection. It was found that Vent (exo-) DNA polymerase was able to incorporate all the modified dUTPs successfully. In Chapter 3 and Chapter 4, two all-RNA-cleaving DNAzyme selections were described. DNAzyme clone 25 was selected in Chapter 3 against an HIV RNA target, which had a self-cleavage rate constant of 3.3 min-¹. However, when the DNAzyme was tested for intermolecular cleavage activity, the result was unsatisfactory. It was found that the maximum rate constant had not been reached under 2 µM substrate, indicating a low substrate binding affinity. With this disappointing result, DNAzyme clone 25 was not considered for in vivo studies.Conversely, DNAzyme clone 11 was selected in Chapter 4 and displayed a robust trans-cleavage activity and a high binding affinity towards a c-Myc oncogene target sequence. DNAzyme clone 11 was obtained from this process, which had a self-cleavage rate constant of 0.84 min-¹. The intermolecular cleavage study showed that it had a cleavage kmax of 4.3 min-¹ and Km of 297 nM. The DNAzyme was then shown to be highly sequence-specific. Solid-phase synthesis of the modified DNAzyme was attempted, and the crude oligonucleotide mixture obtained showed trans-cleavage activity. Lastly, Chapter 5 described several failed DNAzyme selections in which no promising active strands were obtained.

View record

Developing a Broadly Applicable and Facile 18F-Labeling Method for PET Imaging (2014)

Positron emission tomography (PET) is on the forefront of cancer diagnosis, clinical drug evaluation, and patient management. Among the numbers of β+-emitting nuclides, ¹⁸F (t₁/₂=109.8 min) is a mainstay isotope for PET imaging owing to its excellent nuclear properties and on-demand production at Curie levels. Despite the success of PET and increasing interests of ¹⁸F-radiochemistry, a facile ¹⁸F-labeling method that can be broadly applied to biomolecules has been a long-standing challenge. Most known ¹⁸F-labeling methods are relatively onerous and lengthy processes, which is a particularly serious problem given the short half-life of ¹⁸F. This thesis describes the design, synthesis and in vivo evaluation of novel ¹⁸F-radioprosthetics based on B-¹⁸F formation, and aims at developing a facile and broadly applicable ¹⁸F-labeling method for PET imaging. Previously, the Perrin group has established ¹⁸F-aryltrifluoroborates as a promising radiosynthon to radiolabel bioligands. In light of this success, this thesis has dedicated to expand B-¹⁸F labeling method from a scientific design to a generic clinical-friendly tool for developing new PET tracers. The first highlight of this thesis is to create new labeling methods (Chapter 4 and Chapter 5) to increase the specific activity of ¹⁸F-radiotracers to 15 Ci/μmol, which is about a magnitude higher than normal maximum. The second highlight is to discover a heretofore-unknown linear correlation between the solvolytic stability for a given organotrifluoroborate and the pKa of the corresponding carboxylic acids (Chapter 3). This discovery has fundamental interests for Suzuki-Miyaura coupling and also leads me to find a novel B-¹⁸F radiosynthon that combines high in vivo stability and “kit-like” ¹⁸F-labelling technology, which is the third highlight and also the core of this body of work (Chapter 6). Along with this, numbers of bioligands have been biologically evaluated, and some of them demonstrate excellent in vivo performance. Particularly, TATE-AMBF₃, which is an octreotate derivative, showing the best performance of any ligands for imaging somatostatin receptors in several decades (Chapter 7). In addition, for seamless bench-to-bed translation, a dual-modal strategy of synthesizing fluorescent PET tracer is presented (Chapter 8).

View record

Applying aryltrifluoroborates as PET imaging agents (2012)

This dissertation is focused on applying aryltrifluoroborates (ArBF₃s) as PETimaging agents. Several aspects of this new ¹⁸F-labeling technique are addressed.These include the hydrolytic stability of heteroaryltrifluoroborates (HetArBF₃s), thefluoridation of arylboronic acids/esters and the radiosyntheses of several ¹⁸F-ArBF₃labeled biomolecules for potential PET imaging applications. The solvolysis ofseveral N-HetArBF₃s under physiological conditions was studied with ¹⁹F NMRspectroscopy in Chapter 2. All the N-HetArBF₃s tested therein displayed excellentsolvolytic stability under physiological conditions. It is expected that theseHetArBF₃s can be further applied as ¹⁸F-labeled PET imaging agents.In Chapter 3, a rapid fluoridation was carried out under conditions with low fluorideconcentrations in a short reaction time (~ one hour). Via TLC-fluorescentdensitometry, ¹⁹F NMR spectroscopy, and radio-HPLC, the fluoridation of differentarylboronic acids/esters was investigated. It was found that the fluoridation occursrelatively rapidly in the presence of 3 to 5 equivalents of fluoride in acidic aqueousCH₃CN at room temperature. Under such conditions, radiochemical yields of 20-30%can be achieved. It was also noticed that arylboronates with acid-sensitive protectinggroups could undergo fluoridations rapidly comparable to the arylboronic acids.In Chapter 4, marimastat, an MMP inhibitor, was labeled with an ¹⁸F-ArBF₃ toimage breast cancer in mice. An unoptimized isolated radiochemical yield of ~ 1.5%and specific activities of 0.179 and 0.396 Ci/µmol were obtained within two hoursincluding packaging. The blocking experiment suggested that the tumor uptake ofMar-¹⁸F-ArBF₃ was MMP specific. This one-step aqueous fluoridation was alsoapplied to label a urea-based PSMA inhibitor (Chapter 5) and RGD-containingcyclopeptides (Chapter 8). Radiochemical yields ranging from 10% to 25% wereobtained within one hour and good HPLC separation was achieved. In addition, aone-pot two-step labeling strategy was developed in Chapter 6 to label acid-sensitivebiomolecules with ¹⁸F-ArBF₃s. The copper(I) catalyzed 1,3-dipolar cycloaddition wassuccessfully used to conjugate ¹⁸F-ArBF₃s with biomolecules includingoligonucleotides (Chapter 6), folate (Chapter 7), and a cyclic RGD-peptide (Chapter 8)with radiochemical yields of 20-30% over two steps in one hour.

View record

Improving DNAzyme catalysis through synthetically modified DNAzymes and probing DNA polymerase function to improve selection methodology (2011)

DNA enzymes (DNAzymes) are part of a growing field of nucleic acid-based catalysts. Unlike ribozymes, DNAzymes have no apparent precedence in nature to date and can only be discovered through in vitro selection. These selections can accommodate both natural and chemically-functionalized nucleotides. Functionalities provide DNAzymes with enhanced novel function or catalytic rates normally not attainable using natural nucleotides. The overall purpose of this thesis is to evaluate and improve the utility of modified nucleotides as components in a DNAzyme selection. This dissertation discusses an updated synthesis of the functionalized 8-(2-(4-imadazolyl)aminoethyl)-2'-deoxyadenosine triphosphate, dAimeTP, that was based on the synthesis used to produce the phosphoramidite analog. dAimeTP bears the imidazole which was crucial in the discovery of several divalent metal cation-independent DNAzymes. A DNAzyme selection using a derivative nucleoside triphosphate, 8-(4-imidazolyl)aminomethyl-2'-deoxyadenosine triphosphate, investigated shortening the linker of the appended imidazole group. The most efficient clone, Dz20-49, was determined to have a catalytic rate of 3.5 ± 0.4 • 10-³ min-¹. In the context of eight different DNAzyme selections, the replication of modified DNA is examined, and it was found that DNA templates modified with 8-(4-imidazolyl)aminomethyl-2'-deoxyadenosine are poorly amplified compared to an oligonucleotide template of the same sequence modified with 8-(2-(4-imidazolyl)aminoethyl)-2'-deoxyadenosine. Also examined was the use of 5-modified dUTP derivatives as substrates in PCR. The resulting doubly-modified dsDNA was used for restriction enzyme digestions and cloning. Restriction sites containing doubly-modified dsDNA were found to be resistant to restriction enzyme digestion. The doubly-modified amplicons were ligated into a vector and transfected into cells. Plasmids copied from the modified dsDNA were sequenced. Fidelity appeared to be maintained through PCR and cell-mediated replication. Due to the limitations of incorporation and read-through of modified nucleotides, steps were taken towards the directed evolution of Thermus aquaticus DNA polymerase I, commonly referred to as Taq, for improved incorporation and read-through of modified nucleotides. Short patch compartmentalized self-replication (spCSR) was chosen for the directed evolution. Three unnatural nucleoside triphosphates targeted for use in the polymerase evolution included 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)thymine 5'-triphosphate, 5-aminoallyl-2'-deoxycytidine triphosphate and 8-(4-imidazolyl)aminomethyl-2'-deoxyadenosine triphosphate.

View record

Increasing the chemical functionality of DNA enzymes (2011)

Deoxyribonucleic acid (DNA) has long been known as the storage of genetic material. DNA is chemically stable and predominantly found as a double helix of antiparallel complementary strands. Due to the development of in vitro selection techniques, artificial single-stranded DNA molecules have been discovered that can catalyze a range of reactions including the sequence-specific cleavage of ribonucleic acids (RNA). Such DNA enzymes (DNAzymes) are being studied for the in vivo cleavage of mRNA. Compared to protein enzymes, DNAzymes have far fewer functional groups to employ for catalysis. In order to increase the chemical functionality of DNAzymes, eight modified nucleoside triphosphates were synthesized: three dUTP’s modified at the 5-position with either a phenol, phenylboronic acid or guanidinium and five dATP’s modified at the 8-position with imidazoles that are attached with linkers of various composition, length and flexibility. After the synthesis of the modified nucleotides, the incorporation of seven of them into oligonucleotides by DNA polymerases was studied. Under primer extension conditions, the guanidinium- and phenol-modified dUTP’s were found to be good substrates for several DNA polymerases including Pfu (exo–) and Vent (exo–). Under PCR conditions, both of the modified nucleotides gave rise to properly sized products as well. The modified dATP’s, however, were found to be very poor substrates. Only two of them were incorporated by Sequenase V2.0. After establishing conditions under which the modified nucleotides could be incorporated, the phosphoramidite of the guanidinium-modified nucleotide was used for the solid phase synthesis of oligonucleotides, and the phenol-modified nucleotide was used in an in vitro selection. Oligonucleotides containing the guanidinium groups were found to exhibit enhanced duplex stability. The guanidinium-modified nucleoside phosphoramidite was also used to synthesize variants of the divalent metal cation-dependent DNAzyme, 10-23. Variants containing guanidinium groups in the substrate binding regions were found to display reduced rates of RNA cleavage. Using the phenol-modified dUTP, an in vitro selection process gave rise to DNAzyme STA17, isolated from the eleventh generation, that could catalyze self-cleavage in the presence of divalent metal cations such as Mg⁺², Zn⁺² and Mn⁺². This DNAzyme was found to be inhibited by Hg⁺².

View record

Synthesis and evaluation of probes of DNA polymorase II: Adaptation of the bicyclic scaffold of the octapeptide amanitin (2010)

This thesis covers the synthesis and evaluation of bicyclic octapeptides as intracellular probes of RNAP polymerase II. In Chapter 2, the synthetic methodology used to achieve the rigid bicyclic octapeptide and the introduction of modifications to gain spatio-temporal control and visualization is presented. This includes the synthesis of a nitrorveratryl protected derivative of hydroxyproline, and a diethylaminocoumarin labeled asparagine residue. These amino acids were incorporated into amatoxins through solid-phase peptide synthesis. The synthesis and properties of these amatoxins, including their photolability and fluorescent properties is discussed. Chapter 3 describes the initial evaluation of the amatoxin probes in various eukaryotic cell lines. The cytotoxicity of α-amanitin in a variety of cell lines was investigated, where Chinese hamster ovary cells proved to be most sensitive to the toxin. These cytotoxic effects were observed at ~1 μM, which is much higher than the reported binding constant (~ 3 nM). The cytotoxicity was slow, requiring 60-72 hours to achieve 100% cell death. This diminished activity was attributed to cell uptake. Cell permeabilizing agents digitonin and saponin were applied to improve α-amanitin uptake and toxicity. All synthetic amatoxins were shown to have minimal cytotoxic effects. Confocal microscopy demonstrated no cell uptake of a fluorescent amatoxin unless the cells were treated with detergent, pointing to critical issues of cell permeability. The optimization of amatoxin synthesis is presented in Chapter 4. It is shown that up to 20% water can inhibit epimerization during macrolactamization of amatoxins. A convergent synthetic approach to the bicyclic octapeptide was also developed. This allowed for the incorporation of a variety of amino acids at position three. This new synthetic approach was applied to the synthesis of tryptathionine-containing analogs of the opioid receptor agonist enkephalin. The attempted synthesis of the unnatural amino acid (2S),(3R),(4R)-dihydroxyisoleucine is described in Chapter 5. This was achieved through the use of a diastereoselective [3,3] sigmatropic rearrangement followed by Sharpless asymmetric dihydroxylation (AD). Surprising selectivity was noted during the AD reaction using phthalazine-based ligands, but this selectivity was reversed using pyrimidine-based ligands.

View record

DNA-inspired Janus AT and GC heterocycles : synthesis, structural analysis and self-organization (2009)

Inspired by the significance of hydrogen bond driven self-organization, especially fromthe base-pairing interactions of double helical DNA, this dissertation discusses the synthesis andcharacterization of a number of DNA-inspired self-complementary heterocycles and thesupramolecular ensembles derived from them. Specifically, two projects have been completed.Each of these projects addresses the high yielding syntheses of heterocycles with definedhydrogen bond accepting and donating capabilities designed to self-assemble under the generalpurview of base pairing.The first chapter provides an introduction to general concepts such as base-pairing aswell as an outline of the diverse synthetic supramolecular ensembles that have been prepared byutilizing such interactions. Chapter 2 focuses on the syntheses and solid-state structures of threeself-complementary DNA-inspired heterocycles which contain ADA-DAD hydrogen bondacceptor-donor patterns (Janus AT 1-3). These novel heterocycles represent diaminopurinethymine hybrids that, in two of the three cases, relate to previously reported heterocyclic hybridsof guanine and cytosine. All three heterocycles crystallized and afforded the first X-ray crystalstructures of such heterocycles and revealed their extended H-bonded arrays. This chapter alsointroduces the synthetic development to build Janus AT deoxynucleosides, capable of beingoligomerized, as the current trend of this project. The potential use of Janus AT heterocycles inDNA and RNA recognition is briefly discussed as well. Chapter 3 will disclose the synthesis andcharacterization of a DNA-inspired self-complementary heterocycle capable of AAD-DAAhydrogen bond pairing, which self-organizes to a tetrameric rosette, that unlike a G-quartet,needs no metal binding or peripheral component for pre-organization (Janus GC 1). Notably,ESI-MS, variable temperature ¹H-NMR, 2D-NOESY and DOSY ¹H-NMR have been exploitedto validate the tetrameric stoichiometry in this non-covalent rosette comprising twelve H-bonds.At the end of each of these chapters, a section pertaining to ongoing efforts and proposed futureresearch is included.

View record

Mechanistic studies of natural ribozymes and a synthetic DNAzyme (2009)

This thesis reports the results of experiments designed to help elucidate the catalytic mechanisms of three RNA cleaving nucleic acid catalysts (the 9₂₅-11 DNAzyme, and the hammerhead and hairpin ribozymes). A number of enzymological and chemical probing experiments were employed in this regard, which were inspired by antecedent studies of RNA cleaving protein enzymes. First, a novel affinity labeling technique for nucleic acid catalysts was developed to probe general base catalysis. The affinity labeling substrate analogues bear a 2'-bromoacetamide modification at their cleavage sites. The second mechanistic probe employed was 5'-bridging phosphorothioate (S-link) substrate analogues, in which sulphur replaces the native oxygen leaving group. Investigation of S-link substrate cleavage in the context of active site mutations provided insight into general acid catalysis. A novel, simplified method for synthesizing S-link substrates is also presented. Several other experiments provided further mechanistic insight including pH-rate profiling, pKa perturbation, and the use of nonbridging phosphorothioate substrates. Using many of the aforementioned experiments, the catalytic mechanism 9₂₅-11 DNAzyme (a synthetically modified DNAzyme that contains unnatural protein-like functional groups) was investigated in detail. The data suggest that 9₂₅-11 uses its synthetic functional groups to mimic the active site mechanism of the protein enzyme RNaseA. Affinity labeling, pKa perturbation, and S-link cleavage experiments were also applied to the hammerhead ribozyme. The affinity labeling data suggest that the deprotonated N1 position of G12 acts as a general base catalyst. The results of pKa perturbation and S-link cleavage experiments provide strong evidence for a unique general acid mechanism in the hammerhead ribozyme. Therein, metal coordination is used to acidify a 2'-hydroxyl and thereby improve its ability to act as general acid. Finally, affinity labeling was used to characterize the role of hairpin ribozyme G8 residue, which occupies a similar structural position to the hammerhead G12 residue. Affinity labeling indeed identified G8 as a potential general base in the hairpin ribozyme. The properties of the hairpin and hammerhead affinity labeling reactions are compared and the mechanistic implications are discussed.

View record

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.

A pursuit of self-cleaving RNase-A mimicking DNAzymes without an imidazole functionality and their further characterization (2022)

DNAzymes are catalytic DNA molecules that resemble ribozymes and, to a lesser extent, protein-based enzymes. Many of the known DNAzymes have been selected in the presence of metal ions (M²⁺) that are present at non-physiological levels. As a result, there has been much less interest in developing a candidate DNAzyme for therapeutic use. M²⁺-independent DNAzymes are intriguing as they can function even under the low magnesium (Mg²⁺) concentrations usually found in an intracellular environment. Previous works in our lab relied on the use of DNAzymes containing three or two modified nucleosides such as an amine (lysine side chain), a guanidine (arginine side chain) and an imidazole (histidine side chain) with an emphasis on the imidazole group to select metal-independent DNAzymes resembling the RNase A mechanism. Using the functional group modifications they have been able to select several fast cleaving DNAzymes. However, to date there has not been a systematic study on the necessity of all three modifications (in particular the imidazole) as the value of each nucleoside have been contextualized at the beginning of the selection. I hypothesized that two of the positively charged dNTP analogs, excluding the imidazole, i.e. 5-aminoallyl-2'-deoxycytidine triphosphate (dCªªTP) and 5-guanidinoallyl-2'-deoxyuridine triphosphate (dUᵍªTP), are sufficient to select in-vitro self-cleaving DNAzymes in the absence of Mg²⁺ ions in a pH-independent fashion. I was able to identify two DNAzyme candidates that performed the best (Dz11-23 and Dz11-33). The DNAzymes appeared to show a monophasic rate constant (~0.03 min⁻¹) on-par with previous selections without the imidazole; when the DNAzyme species were inquired using a biphasic kinetics model two phases were identified with one of the phases showing a rate constant that was similar to the fast phase in the imidazole containing DNAzymes (kcat= 0.7-0.9 min⁻¹). When studied over different pH values and Mg²⁺ concentration the DNAzymes also appeared to show a self-cleavage activity that was independent of pH and Mg²⁺ with an optimal temperature around 25 °C and sufficiently high cleavage observed even at 37 °C. The results in the thesis support my hypothesis that the imidazole moiety may be redundant in selecting fast cleaving metal-independent DNAzymes.

View record

Leveraging boron and fluorine: synthesis of organoboronates for radiolabeling and suzuki coupling of acylboronate MIDA esters (2022)

This thesis presents a new synthesis of acylboronate esters from vinylboronates. These acylboronate esters are synthesized from vinylboronate MIDA (N-methyliminodiacetyl) esters that are either bought or hydroborylated from alkynyl-derivatives. In Chapter 2, a two-step oxidative process involving Upjohn dihydroxylation and Lemieux-Johnson conditions to make the desired acylboronate MIDA ester is disclosed. This includes the work by myself including the synthesis of some key substrates that would be used for the synthesis of bi-modal sensors for acyl trifluoroborates. Chapter 3 describes the application of the previously synthesized acylboronate MIDA esters for Suzuki-Miyaura cross coupling (SMCC). There are no prior studies on the cross-coupling of this organoboron species, therefore, I designed and optimized a scheme for the cross-coupling between phenacylboronate MIDA ester and aryl bromides. I successfully synthesized benzophenone derivatives from the previously mentioned materials at moderate yields. Further research is done on non-aroyl starting material derivatives, but did not yield cross-coupling and possibly led to ß-hydride elimination or rapid acylboronate decomposition.

View record

Generation of Modified DNA Aptamers Toward a Complex Whole Cell Target and Investigations into Improving Selection Methodology with Modified DNA (2015)

Since the discovery was made that DNA is capable of functions beyond genetic storage and propagation, much work has been done to explore the non-canonical abilities of this molecule. Two functionalities of DNA are of particular interest. The first is the ability of DNA to act as a catalyst, and the second is the ability of the molecule to bind with high affinity to various biological and non-biological surfaces. Although numerous DNA catalysts and binders (termed “aptamers”) have been identified using the five nucleoside triphosphates found in nature, the functionality of the molecule can be expanded with the addition of protein-like functional groups to the base moiety of the nucleotide. Several modified nucleotides have been developed previously and used to discover novel catalysts and aptamers. However, the successful development of modified-DNA aptamers has thus far been limited, particularly with respect to biological targets that might be of use in a clinical or commercial context. Moreover, technical challenges in the field exist that have not yet been adequately addressed. Most significantly, the employment of the modified molecules in the discovery process can be problematic as they are not ideal substrates for many basic molecular biology procedures, notably polymerase chain reaction. New techniques to overcome these difficulties are needed and few have been developed. This thesis will focus on two aspects of modified DNA research. The first is the application of modified DNA in aptamer discovery. Starting from the hypothesis that additional functional groups will confer chemical advantages in traditionally challenging aptamer selections, the objective was to identify a modified DNA aptamer for whole bacterial cells in vitro. Several promising phenol-dUTP-modified aptamer sequences were identified using a modified SELEX procedure. The specificity and affinity of these sequences for the target bacterial strain were investigated. The second aim of this work is to address technical problems encountered when working with modified DNA in order to develop catalysts or aptamers. Specifically, a novel selection scheme was designed that eliminated the need to amplify modified DNA. Research was conducted to develop and validate this selection system as a viable route to discovery of DNA catalysts.

View record

Studies on radiotracer precursor components: Stabilities of phenylboronic acids and erroneous sources of flouride (2015)

The use of aryltrifluoroborate radiotracers previously developed in the Perrin lab at the University of British Columbia has been prolific in showcasing boron as an ¹⁸F-fluoride capturing agent. Their in-vivo use for PET-Imaging has been widely discussed and supported with radioimages, publications, and applications in PET including advantages for higher specific activity than other tracers currently in use. However the final synthesis of these ArBF₃-radiotracers include synthetic challenges, peptide-arylboronic acids were observed to deboronate giving rise to low radiochemical yields along with low effective specific activity for the final tracer. The interest and study in determining conditions, where deboronation of arylboronic acids may occur, and where increased ¹⁸F production or conversely limitations on ¹⁹F contaminants, would potentially increase the value of aryltrifluoroborates use in PET-imaging with fundamental improvements in transition from lab bench to clinical settings.The stabilities of ayrlboronic acids under alkaline conditions have not been previously explored in literature. Previous reports indicate that electron rich arylboronic acids decomposing under acidic media but does not address alkalinity. Chapter 2 details the kinetic data concerning a set of electronically withdrawing arylboronates that are evaluated in basic aqueous-organic conditions. It was discovered that only subsets of electron deficient diortho substituted arylboronates undergo deboronation under base-typical solutions. These findings were then related to insights towards reactions concerning C-C bond formation through the stabilities and their uses as aryltrifluoroborates in cancer imaging.Apart from the use of ArBF₃s as radioprosthetics, specific activity is of primary concern. Cyclotrons around the world use ¹⁸O-H₂O, for the nuclear production of ¹⁸F. It has widely been acknowledged that ¹⁹F pervades within the system. Chapter 3 identifies some of the potential sources of ¹⁹F-fluoride in order to enhance radiofluoridation yields of ¹⁸F-fluoride with the aryltrifluoroborates. This fundamental analysis has led to the discovery of increased ¹⁹F-fluoride following irradiation, as well as possible leaching from the tubing and channels used for primary transfer. This work re-opens the discussion of the basics of the scientific research where acceptable standards of low specific activity may yet be in question and improved upon.

View record

Synthesis of a peptide nucleic acid oligomer of a Janus heterocycle (2014)

This thesis describes the oligomerization of the Janus AT heterocycle on a peptide nucleic acid scaffold, a molecule that is capable of concurrently hydrogen bonding with adenine and thymine. An oligonucleotide incorporating this moiety should form sequence specific Watson-Crick based triplexes with DNA by strand invasion. Initially, the synthesis of a diamino Janus AT peptide nucleic acid monomer was attempted, but the monomer was too insoluble for solid phase synthesis. To curtail this issue, attempts were made to synthesize a more hydrophobic surrogate, wherein the exocyclic amines were masked. The synthesis of an undecamer was then undertaken, wherein the Janus monomer was alternated with a 6-methyluracil spacer. Lastly, a dodecamer of 6MU was synthesized to examine its biophysical properties in the context of base pairing with DNA.

View record

Towards the synthesis of Janus-AT phosphoramidites (2013)

A Janus heterocycle is one kind of heterocycle that possesses hydrogen bonding donors or acceptors at both sides compared to regular nuclear bases. This provides an interesting synthetic target as well as a tool for testing an intriguing biological hypothesis related to DNA recognition, namely can both strands in the DNA duplex be recognized simultaneously by a single oligomeric strand of Janus heterocycles? In order to test this hypothesis, oligonucleotides containing at least one Janus heterocycle must be synthesized. One challenge involved in the synthesis of oligonucleotides with a Janus heterocycle is the extremely low solubility of Janus heterocycle phosphoramidites in organic solvents, namely MeCN. To solve the solubility issue, two sulfone-masked Janus-AT phosphoramidites were designed and constructed in this thesis. In addition to questions of solubility, the other obstacle related with Janus heterocycle oligonucleotide construction is the potential for deglycosylation, analogous to depurination, which might occur under the acidic conditions used during solid phase synthesis of oligonucleotides. The carbocyclic analog of deoxyribose, in lieu of 2-deoxyribose was employed to construct Janus heterocycle phosphoramidites in order to obviate the potential for depurination-like elimination of the nucleobase from the sugar.

View record

Synthesis of 18F-Radiolabeled LLP2A for use in PET imaging via aryltrifluoroborate formation (2012)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

View record


Membership Status

Member of G+PS
View explanation of statuses



If this is your researcher profile you can log in to the Faculty & Staff portal to update your details and provide recruitment preferences.


Discover the amazing research that is being conducted at UBC!