David Perrin

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Mar 2019)
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).

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

No abstract available.

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

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

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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.

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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.

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

No abstract available.

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.

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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.

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

No abstract available.

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.

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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.

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Master's Student Supervision (2010-2017)
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.

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Studies on radiotracer precursor components : stabilities of phenylboronic acids and erroneous sources of fluoride (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.

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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.

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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.

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