Natural Product Biosynthesis: Friend or Foe? From Anti-tumor Agent to Disease Causation

Biosynthetic natural products are invaluable resources that have been gleaned from the environment for generations, and they play an essential role in drug development. Natural product biosynthesis also possesses the latent ability to affect biological systems adversely. This work implements recent advances in genomic, proteomic and microbiological technologies to understand further biosynthetic molecules that may influence progression of human disease. Azinomycin A and B are antitumor metabolites isolated from the terrestrial bacterium Streptomyces sahachiroi. The azinomycins possess an unusual aziridine [1,2-a] pyrrolidine ring that reacts in concert with an epoxide moiety to produce DNA interstrand cross-links. Genomic sequencing of S. sahachiroi revealed a putative azinomycin resistance protein (AziR). Overexpression of AziR in heterologous hosts demonstrated the protein increases cell viability and decreases DNA damage response in the presence of azinomycin. Fluorescence titration indicated AziR functions as an azinomycin binding protein. An understanding of azinomycin resistance is important for future engineering and drug delivery strategies. Additionally, the S. sahachiroi draft genome obtained via 454 pyrosequencing and Illumina sequencing revealed several silent secondary metabolic pathways that may provide new natural products with biomedical application. β-lactoglobulin (BLG), the most abundant whey protein in bovine milk, has been observed to promote the self-condensation of retinal and similar α,β-unsaturated aldehydes. BLG is a possible non-genetic instigator of cycloretinal and A2E accumulation in the macula, a condition associated with age-related macular degeneration. BLG-mediated terpenal condensation has been optimized for in vitro study with the retinal mimic citral. In rabbits fed retinal and BLG or skim milk, cycloretinal formation was detected in the blood by 1H-NMR, and SDS-PAGE analysis indicated BLG was present in blood serum, suggesting the protein survives ingestion and retains catalytic activity. Mass spectrometry and site-directed mutagenesis provided mechanistic insight toward this unusual moonlighting behavior. The experiments described in this dissertation serve to further natural product biosynthesis discovery and elucidation as they relate to consequences for human health. Efforts to solve azinomycin biosynthesis via enzymatic reconstitution, characterize compounds produced by orphan gene clusters within S. sahachiroi, and obtain a clear mechanism for BLG-promoted cycloterpenal formation are immediate goals within the respective projects

EXPLORING SUBSTRATE SPECIFICITY OF FRUCTOSE TRANSPORTERS EN ROUTE TO GLUT SPECIFIC PROBES FOR BIOCHEMICAL AND BIOMEDICAL APPLICATIONS

Carbohydrate Transporters (GLUTs) are responsible for the transportation of sugars into the cell and have been of great interest in research for decades. Alterations or mutations that result in overexpression of GLUTs have been linked to a great number of diseases including, obesity, diabetes, and cancer. Differentiation between transporters has been shown to be incredibly difficult due to the highly conserved nature of the transporter structure, and thus specific targeting of transporters has proven a difficult challenge. Additionally, the GLUTs have been shown to high flexibility in their conformations, so it is difficult to determine what can or cannot pass through the transporter, which has led to many failed attempts at targeting these transporters. So in an attempt to gain a better understanding of one transporter specifically, GLUT 5, a transporter known to be responsible for fructose transporter, new probes were created by conjugating 1-amino-2,5-anhydro-D-mannitol and various fluorescent coumarins. The probes were tested in both normal and cancerous cell lines in order to determine their uptake kinetics and transport specificity. To establish transport specificity the probes were tested in the presence of various competitive and noncompetitive inhibitors. The probe transport was analyzed through various meanings including microplate setting, immunostaining, and confocal microscopy. The combined analysis of the probes has shown to be GLUT5 specific which allows for their use as for biochemical and biomedical imaging an analysis of GLUT5 transporter in cells

Development of Methodologies to Prepare Interstrand Cross-Links in Oligonucleotides

Cellular DNA is susceptible to damage by chemical agents that causes modifications which includes interstrand cross-links (ICL). ICLs covalently attach the complementary DNA strands, and interfere with replication and transcription by preventing strand separation. The deliberate formation of ICL in DNA by bi-functional alkylating chemotherapeutic agents leads to the death of cancer cells. Development of tumors resistant to these agents is a factor in the lack of response in some patients, with removal of ICL believed to play a role in resistance. In mammalian cells, the precise role of excision repair in eliminating ICL is not completely understood. For better understanding of the repair pathways involved in removing ICL damage, ICL DNA duplexes containing well-defined modified moieties are required to mimic the lesions induced by chemotherapeutic agents. This thesis describes two major approaches that have been investigated to synthesize ICL DNA. The first describes a method to prepare DNA duplexes containing cross-linked N3-butylene-N3 thymidines that enables the preparation of asymmetric nucleotide sequences around cross-linked sites. Protective groups for the 3’- and 5’-hydroxyl moieties were screened for compatibility of subsequent extensions from a cross-linked thymidine dimer incorporated in a support bound oligonucleotide by automated DNA synthesis. Two cross-linked dimer phosphoramidites were prepared, one with dimethoxytrityl (DMT) and allyloxycarbonyl (Alloc) protective groups at 5’-O positions and a 3’-O-t-butyldimethylsilyl (TBS) group which enabled the production of completely asymmetric ICL DNA duplexes in good yields. After coupling of the cross-linked phosphoramidite to a linear strand assembled on the solid support, the DMT group was cleaved on the synthesizer to allow for the synthesis of the second arm of the duplex. The Alloc group was then removed via an off-column strategy to expose the 5'-hydroxyl group to complete assembly of one strand of the duplex to form a "Y-shaped" intermediate. Final removal of a 3'-O-TBS group off column followed by coupling with deoxynucleoside 5'-phosphoramidites yielded ICL DNA duplexes containing completely asymmetric nucleotide composition around the cross-link site. The identity and composition of the ICL duplexes were confirmed by mass spectrometry (ESI-TOF) and enzymatic digestion. The synthesized ICL duplexes displayed characteristic features of a B-form duplex and had stabilities that were higher than those of the unmodified controls assessed by circular dichroism (CD) spectroscopy and UV thermal denaturation experiments. The second major project describes approaches to prepare a 7-deaza-2’-deoxyguanosine cross-linked dimer where the C7 atoms are attached by an alkylene linker. The chemical instability of alkylated N7 2’-deoxyguanosine (dG) represents a major challenge for preparing ICL DNA containing an alkylene linkage between the N7 atoms. The incorporation of a C7-alkylene cross-linked dimer of 7-deaza-2’-deoxyguanosine in DNA would allow for the preparation of a chemically stable ICL which mimic lesions formed by bifunctional alkylating agents (i.e. mechlorethamine and hepsulfam). Two synthetic methods were explored to prepare 7-deazaguanine (and other 7-deazapurines). These involved two cyclization strategies to prepare these molecules starting from a pyrimidine or a pyrrole to produce the purine. In both synthetic methods it was challenging to purify some of the intermediates. All intermediates in the synthetic method starting from the pyrimidine precursors to produce the 7-deazapurines were more stable while the production of the 7-deazapuines from the pyrroles resulted in higher yields. An attempt to produce a C7 cross-linked dimer of 7-deaza-2’-deoxyguanosine containing a heptamethylene linker is described. Starting from 7-deazaguanine, 7-iodo-7-deaza-2’-deoxyguanosine was prepared in good yield. This nucleoside was converted to 5’-O-DMT-7-iodo-7-deaza-2’-deoxyguanosine and the Sonogashira reaction used with 1,6-heptadiyne to introduce the heptamethylene linker. Unfortunately, multiple challenges were encountered with the dimerization and hydrogenation reactions which did not allow for the synthesis of the desired dimer for solid-phase synthesis of the ICL DNA