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Bleomycins (BLMs) are a class of water soluble, glycopeptide-derived antitumor antibiotics consisting of a structurally complicated unnatural hexapeptide and a disaccharide, clinically used as an anticancer chemotherapeutic agent at an exceptionally low therapeutic dose. The efficiency of BLM is likely achieved both by selective localization within tumor cells and selective binding to DNA followed by efficient double-strand cleavage. The disaccharide moiety is responsible for the tumor cell targeting properties of BLM. A recent study showed that both BLM and its disaccharide, conjugated to the cyanine dye Cy5**, bound selectively to cancer cells. Thus, the disaccharide moiety alone recapitulates the tumor cell targeting properties of BLM. Work presented here describes the synthesis of the fluorescent carbohydrate conjugates. A number of dye-labeled modified disaccharides and monosaccharides were synthesized to study the nature of the participation of the carbamoyl moiety in the mechanism of tumor cell recognition and uptake by BLM saccharides. It was demonstrated that the carbamoylmannose moiety of BLM is the smallest structural entity capable for the cellular targeting and internalization, and the carbamoyl functionality is indispensible for tumor cell targeting. It was also confirmed that BLM is a modular molecule, composed of a tumor cell targeting moiety (the saccharide) attached to a cytotoxic DNA cleaving domain (the BLM aglycone). These finding encouraged us to further synthesize carbohydrate probes for PET imaging and to conjugate the saccharide moiety with cytotoxins for targeted delivery to tumor cells.
The misacylated suppressor tRNA technique has enabled the site-specific incorporation of noncanonical amino acids into proteins. The focus of the present work was the synthesis of unnatural lysine analogues with nucleophilic properties for incorporation at position 72 of the lyase domain of human DNA polymerase beta, a multifunctional enzyme with dRP lyase and polymerase activity.
Mitochondrial dysfunction has been proposed in the etiology of various pathologies, including cardiovascular and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, ischemia-reperfusion (IR) injury, diabetes and aging. To treat these disorders, it is imperative to target mitochondria, especially the electron transport chain. One of the methodologies currently used for the treatment of mitochondrial and neurodegenerative diseases where endogenous antioxidant defenses are inadequate for protecting against ROS involves the administration of exogenous antioxidants.
As part of our pursuit of effective neuroprotective drugs, a series of pyridinol and pyrimidinol analogues have been rationally designed and synthesized. All the analogues were evaluated for their ability to quench lipid peroxidation and reactive oxygen species (ROS), and preserve mitochondrial membrane potential (Δm) and support ATP synthesis. These studies are summarized in Chapter 2.
Drug discovery and lead identification can be reinforced by assessing the metabolic fate of orally administered drugs using simple microsomal incubation experiments. Accordingly, in vitro microsomal studies were designed and carried out using bovine liver microsomes to screen available pyridinol and pyrimidinol analogues for their metabolic lability. The data obtained was utilized for an initial assessment of potential bioavailability of the compounds screened and is summarized fully in Chapter 3.
The objective of this study is to create a spectrophotometric assay that can measure quinone reduction in the HbRC. The key techniques used in the project consisted of a PCR, a pseudo golden gate, a transformation into E. coli, a conjugation into Heliomicrobium modesticaldum, a growth study, a HbRC prep, and absorbance spectroscopy. PCR was crucial for amplifying the Cyt c553-PshX gene for the pseudo golden gate. The pseudo golden gate ligated Cyt c553-PshX into the plasmid pMTL86251 in order to transform the plasmid with the desired gene into the E. coli strain S17-1. This E. coli strain allows for conjugation into H. modesticaldum. H. modesticaldum cannot uptake DNA by itself, so the E. coli creates a pilus to transfer the desired plasmid to H. modesticaldum. The growth study was crucial for determining if H. modesitcaldum could be induced using xylose without killing the cells or inhibiting the growth in such a way that the project could not be continued. The HbRC prep was used to isolate and purify the Cyt c553-PshX protein. Absorbance spectroscopy and JTS kinetic assay was used to characterize and confirm that the protein eluted from the affinity column was Cyt c553-PshX. The results of the absorbance spectra and JTS kinetic assay confirmed that Cyt c553-PshX was not made. The study is currently being continued using a new system that utilizes SpyCatcher SpyTag covalent linkages in order to attach cytochrome to reduce P800 to the HbRC.