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<p> The ansamycin antibiotics are unique antibacterial agents that inhibit bacterial DNAdependent RNA polymerase II. Clinical use of this class of antibiotics has primarily been focused on the treatment oftuberculosis using the semi-synthetic rifamycin derivative, rifampin. As drug resistance among different classes of antibiotics continues to rise, there is increased interest in new applications ofrifamycins for diseases other than tuberculosis. Clinical resistance to rifampin has largely been the result of point mutations in the target, RpoB, however chromosomal and transposon mediated enzyme-associated resistance is well documented. As rifamycin antibiotic use becomes more widespread, enzymatic resistance will inevitably become more prevalent. Here we describe the characterization of one of the principle enzymes associated with rifampin inactivation, the rifampin ADP-ribosyl transferase enzyme (ARR). Two chromosomally encoded ARR enzymes from MYcobacterium smegmatis, and Streptomyces coelicolor, and the Tn-encoded ARR-2, widely distributed in Gram negative pathogens, were overexpressed and characterized. These enzymes exhibit comparable, substrate specific steady state kinetic features, and substrate-induced conformational changes that suggest ARR enzymes may demonstrate a preferred order of substrate binding. To gain further insight into the interaction between ARR enzymes and rifampin and NAD+, the three-dimensional crystal structure of ARR from M smegmatis was solved in complex with rifampin. Based on the threedimensional structure of ARRm, an SNl type reaction has been predicted for rifampin ADPribosyl transferase enzymes. This is the first detailed examination of these novel antibioticmodifying enzymes, relevant to their increased use in the clinic. </p> / Thesis / Master of Science (MSc)

Ansamycin natural products have historically been a rich source of new drugs for the treatment of bacterial infections and cancer. The C17-benzene ansamycins in particular have shown excellent preclinical results as potential anti-fungal and anti-cancer medicines. However, their thorough clinical evaluation has been hampered by the absence of a concise synthetic strategy. In order to address this issue, recently developed hydrogenative carbon-carbon bond forming methods were applied toward a short total synthesis of C17-benzene ansamycins. This class of natural products provides a challenging testing ground for these methods while facilitating the further development of compounds which may be used as treatments for life threatening diseases. In the first synthetic approach to the C17-benzene ansamycins key bond formations include direct iridium catalyzed carbonyl crotylation from the alcohol oxidation level followed by chelation-controlled dienylation to form the stereotriad, which is attached to the arene via Suzuki cross-coupling. The diene-containing carboxylic acid is prepared using rhodium catalyzed acetylene-aldehyde reductive C-C coupling mediated by gaseous hydrogen. Finally, ring-closing metathesis delivers the cytotrienin core. The second approach toward triene-containing C17-benzene ansamycins resulted in the syntheses of trienomycins A and F, which were prepared in 16 steps (longest linear sequence) and 28 total steps. The C11-C13 stereotriad was generated via enantioselective ruthenium-catalyzed alcohol CH syn crotylation followed by chelation-controlled carbonyl dienylation. Finally, diene-diene ring closing metathesis to form the macrocycle. The present approach is 14 steps shorter (LLS) than the prior syntheses of trienomycins A and F, and eight steps shorter than any prior synthesis of a triene-containing C17-benzene ansamycin. / text