Bioorthogonal Modification of the Major Sheath Protein of Bacteriophage M13: Extending the Versatility of Bionanomaterial Scaffolds

This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Biconjugate Chemistry, copyright © American Chemical Society after peer review. To access the final edited and published work see http://dx.doi.org/10.1021/acs.bioconjchem.6b00460With a mass of ~1.6 x 107 Daltons and com- posed of approximately 2700 proteins, bacteriophage M13 has been employed as a molecular scaffold in bionanomaterials fabrication. In order to extend the versatility of M13 in this area, residue-specific unnatural amino acid incorporation was employed to successfully display azide functionalities on specific solvent-exposed positions of the pVIII major sheath protein of this bacteriophage. Employing a combination of engineered mutants of the gene coding for the pVIII protein, the methionine (Met) analog, L-azidohomoalanine (Aha), and a suitable Escherichia coli Met auxotroph for phage pro- duction, conditions were developed to produce M13 bacteri- ophage labeled with over 350 active azides (estimated by fluorescent dye labeling utilizing a strain-promoted azide- alkyne cycloaddition) and capable of azide-selective attach- ment to 5 nm gold nanoparticles as visualized by transmis- sion electron microscopy. The capability of this system to undergo dual labeling utilizing both chemical acylation and bioorthogonal cycloaddition reactions was also verified. The above stratagem should prove particularly advantageous in the preparation of assemblies of larger and more complex molecular architectures based on the M13 building block.Natural Sciences and Engineering Research Council University of Waterloo NSERC Canadian Graduate Scholarship (CGS-M) Ontario Graduate Scholarship Waterloo Institute for Nanotechnology Nanofellowshi

A Unified Synthetic Approach Toward the Kalihinanes

This dissertation describes our efforts toward developing a unified synthesis of the kalininane family of antimalarial marine isocyanoterpenes. Chapter 1 focuses on the isolation, structure determination, subclass specification, biological activity, and proposed biogenesis of the kalihinanes. Additionally, methods for the synthesis of isonitriles and previous syntheses of kalihinanes and related isocyanoterpenes are described.Chapter 2 describes our divergent synthetic plan, featuring an oxa Michael/Robinson annulation sequence and a Piers-type annulation for the rapid synthesis of the decalin framework of the kalihinanes. This strategy was validated with a formal synthesis of 10 isocyano 4 cadinene, and was subsequently applied toward the synthesis of kalihinanes bearing a pendant tetrahydropyran (kalihinol A) or tetrahydrofuran (kalihinol B). While the synthesis of the tetrahydropyran-containing kalihinanes has yet to be accomplished, our efforts toward tetrahydrofuran-containing kalihinanes culminated in the first synthesis of kalihinol B. An unexpected hydride shift has thwarted efforts to extend the synthesis to other tetrahydrofuran containing kalihinanes.Chapter 3 focuses on the application of our strategy to the synthesis of unnatural kalihinane analogues. Several analogues have been prepared and subjected to an antiplasmodial assay. All of the synthetic isocyanoterpenes exhibited antiplasmodial activity against drug sensitive and drug-resistant strains of Plasmodium falciparum (IC50 < 1.2 µM). Furthermore, kalihinane-based small-molecule probes have been prepared, and will be used for protein profiling in P. falciparum

A Unified Synthetic Approach Toward the Kalihinanes

This dissertation describes our efforts toward developing a unified synthesis of the kalininane family of antimalarial marine isocyanoterpenes. Chapter 1 focuses on the isolation, structure determination, subclass specification, biological activity, and proposed biogenesis of the kalihinanes. Additionally, methods for the synthesis of isonitriles and previous syntheses of kalihinanes and related isocyanoterpenes are described.Chapter 2 describes our divergent synthetic plan, featuring an oxa Michael/Robinson annulation sequence and a Piers-type annulation for the rapid synthesis of the decalin framework of the kalihinanes. This strategy was validated with a formal synthesis of 10 isocyano 4 cadinene, and was subsequently applied toward the synthesis of kalihinanes bearing a pendant tetrahydropyran (kalihinol A) or tetrahydrofuran (kalihinol B). While the synthesis of the tetrahydropyran-containing kalihinanes has yet to be accomplished, our efforts toward tetrahydrofuran-containing kalihinanes culminated in the first synthesis of kalihinol B. An unexpected hydride shift has thwarted efforts to extend the synthesis to other tetrahydrofuran containing kalihinanes.Chapter 3 focuses on the application of our strategy to the synthesis of unnatural kalihinane analogues. Several analogues have been prepared and subjected to an antiplasmodial assay. All of the synthetic isocyanoterpenes exhibited antiplasmodial activity against drug sensitive and drug-resistant strains of Plasmodium falciparum (IC50 < 1.2 µM). Furthermore, kalihinane-based small-molecule probes have been prepared, and will be used for protein profiling in P. falciparum

Synthesis and Potent Antimalarial Activity of Kalihinol B

Of the 50+ kalihinane diterpenoids reported to date, only five had been tested for antimalarial activity, in spite of the fact that kalihinol A is the most potent among the members of the larger family of antimalarial isocyanoterpenes. We have validated a strategy designed to access many of the kalihinanes with a 12-step enantioselective synthesis of kalihinol B, the tetrahydrofuran isomer of kalihinol A (a tetrahydropyran). Kalihinol B shows similarly high potency against chloroquine-resistant Plasmodium falciparum