Since the development of antibacterial drugs, the emergence of drug-resistant bacteria have posed a huge challenge for treating bacterial infection, especially in case of multidrug-resistant bacteria. There has been a significant efforts from the scientific community to develop novel antibiotics that can counteract these. Complementary to these efforts, the National Vaccines Advisory Committee has called for developing vaccines against drug resistant bacteria. During my graduate studies, I aim to develop platforms to develop live attenuated vaccine candidates. I envision doing this by engineering a biorthogonal system to utilize an unnatural analog of essential cofactor found in central metabolism pathways such as glycolysis, TCA cycle, among others, in virulent bacterial strains. As a result of this, the virulent bacteria will only be able to survive in the presence of a defined unnatural cofactor; consequently, this will control the bacterial growth in vivo which will leading to virulence attenuation after a defined period of time, creating a live attenuate vaccine candidate. Particularly, I am targeting essential Nicotinamide adenine dinucleotide (NAD) dependent enzymes. I have designed synthetic strategies to substitute the adenine moiety with noncanonical hydrophobic bases that disrupt hydrogen bonding interactions in the enzyme’s active site and create a biorthogonal enzyme that utilizes defined unnatural cofactor analog. This will impede scavenging, thus causing the bacterial strain to have deterred growth in the absence of the noncanonical cofactor, leading to in vivo attenuation.