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<p>This thesis is divided into two parts based on a total of 8 papers: Part 1: <i>Synthesis, physicochemical and biochemical studies of chemically modified oligonucleotides and their duplexes and triplexes</i>. Potency of the chromophore conjugated DNA oligonucleotides as antigene and antisense gene repressors was evaluated. The effect of geometry, bulk and ¥ð-electron density of a series of chromophores, tethered at the 5'-end of oligonucleotides, as well as the effect of the linker nature, length and the attachment site of the chromophore to the oligo were explored based on the stability of the duplexes and triplexes. A dramatic improvement in the triplex stability with <i>ara</i>-U linked phenazine oligo (potent antigene) was achieved (¥ÄT_m = 16.5¢ª C). A number of selected phenazine and dipyridophenazine tethered antisense oligos (AONs) and their phosphorothioate analogues were shown to form the AON/RNA hybrid duplexes with enhanced thermal stability. CD experiments revealed that these duplexes have the global structure unaltered from that of the native counterpart. RNase H degradation studies on three RNA targets having different degrees of folded structures showed that tethering of phenazine and dipyridophenazine increases the hydrolysis rates (potent antisense) of the target RNA, and that chemical nature of the chromophore influences the RNase H cleavage pattern. Further investigation at the RNA saturated conditions revealed that 3'-tethered chromophores influence the substrate recognition, and the kinetics of the cleavage by RNase H. Conjugation of different chromophores, charged polyaromatic systems and metal complexes with polyaromatic ligands at different sites of the AON revealed that RNase H is very sensitive to any modifications in the middle region of the AON/RNA duplex. On the contrary, any modification at the 3'-end of the AON regardless of the bulk of the substituent or presence of positive charge can be easily tolerated by the enzyme. Sensitivity of the RNase H towards the local structural changes in the AON/RNA hybrid was probed with a number of AONs containing a single 1-(1',3'-O-anhydro-©¬-<u>D</u>-psicofuranosyl)thymine with locked 3'-<i>endo</i> sugar conformation at different sites of AON. RNase H degradation studies revealed that the local conformational changes brought by the constrained nucleoside, although invisible by CD, span in the hybrid as far as 5 nucleotides toward the 5'-end of the AONs (3'-end of RNA), showing the unique transmission of the structural distortion from a single modification site. The results also showed that the structural requirements for the substrate binding and substrate cleavage by RNase H appear to be different. Part 2: <i>Preparation of biologically important isotope labelled oligo-RNAs for the NMR structure determination in solution</i>. Synthesis of the non-uniformly ¹³C₅ labelled 29mer HIV-1 TAR RNA was achieved by solid-phase synthesis using ¹³C₅ labelled ribonucleosides from ¹³C₆-<u>D</u>-glucose). Two hammerhead forming RNAs (16mer and 25mer) were synthesized according to the Uppsala NMR-window strategy, where the sugar residues of the nucleosides forming stem I, II and the loop of the stem III of the resulting hammerhead complex were deuterated. UV melting and high resolution NMR structural studies showed that the 16mer RNA under quasiphysiological condition folds to a very stable hairpin structure, which prevents formation of a hammerhead RNA with the 25mer, primarily owing to thermodynamic reasons.</p>

This thesis is divided into two parts based on a total of 8 papers: Part 1: Synthesis, physicochemical and biochemical studies of chemically modified oligonucleotides and their duplexes and triplexes. Potency of the chromophore conjugated DNA oligonucleotides as antigene and antisense gene repressors was evaluated. The effect of geometry, bulk and ¥ð-electron density of a series of chromophores, tethered at the 5'-end of oligonucleotides, as well as the effect of the linker nature, length and the attachment site of the chromophore to the oligo were explored based on the stability of the duplexes and triplexes. A dramatic improvement in the triplex stability with ara-U linked phenazine oligo (potent antigene) was achieved (¥ÄTm = 16.5¢ª C). A number of selected phenazine and dipyridophenazine tethered antisense oligos (AONs) and their phosphorothioate analogues were shown to form the AON/RNA hybrid duplexes with enhanced thermal stability. CD experiments revealed that these duplexes have the global structure unaltered from that of the native counterpart. RNase H degradation studies on three RNA targets having different degrees of folded structures showed that tethering of phenazine and dipyridophenazine increases the hydrolysis rates (potent antisense) of the target RNA, and that chemical nature of the chromophore influences the RNase H cleavage pattern. Further investigation at the RNA saturated conditions revealed that 3'-tethered chromophores influence the substrate recognition, and the kinetics of the cleavage by RNase H. Conjugation of different chromophores, charged polyaromatic systems and metal complexes with polyaromatic ligands at different sites of the AON revealed that RNase H is very sensitive to any modifications in the middle region of the AON/RNA duplex. On the contrary, any modification at the 3'-end of the AON regardless of the bulk of the substituent or presence of positive charge can be easily tolerated by the enzyme. Sensitivity of the RNase H towards the local structural changes in the AON/RNA hybrid was probed with a number of AONs containing a single 1-(1',3'-O-anhydro-©¬-<u>D</u>-psicofuranosyl)thymine with locked 3'-endo sugar conformation at different sites of AON. RNase H degradation studies revealed that the local conformational changes brought by the constrained nucleoside, although invisible by CD, span in the hybrid as far as 5 nucleotides toward the 5'-end of the AONs (3'-end of RNA), showing the unique transmission of the structural distortion from a single modification site. The results also showed that the structural requirements for the substrate binding and substrate cleavage by RNase H appear to be different. Part 2: Preparation of biologically important isotope labelled oligo-RNAs for the NMR structure determination in solution. Synthesis of the non-uniformly 13C5 labelled 29mer HIV-1 TAR RNA was achieved by solid-phase synthesis using 13C5 labelled ribonucleosides from 13C6-<u>D</u>-glucose). Two hammerhead forming RNAs (16mer and 25mer) were synthesized according to the Uppsala NMR-window strategy, where the sugar residues of the nucleosides forming stem I, II and the loop of the stem III of the resulting hammerhead complex were deuterated. UV melting and high resolution NMR structural studies showed that the 16mer RNA under quasiphysiological condition folds to a very stable hairpin structure, which prevents formation of a hammerhead RNA with the 25mer, primarily owing to thermodynamic reasons.

ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor. Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis. UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.

ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor. Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis. UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.

ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor. Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis. UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities. / Science, Faculty of / Chemistry, Department of / Graduate

Since the isolation of azinomycins A and B in 1954 from the soil bacterium, Streptomyces sahachiroi, these natural products have been synthetic targets. Both compounds exhibit in vitro cytotoxic activity at submicromolar levels and demonstrate anti-tumor activities comparable to that of mitomycin C in vivo. Unique to this class of natural products is the presence of an aziridine [1,2-a] pyrrolidine ring system. Coupled with an epoxide moiety, these structural functionalities impart the ability to form interstrand cross-links with DNA via the electrophilic C10 and C21 carbons of azinomycin and the N7 positions of suitably disposed purine bases. This dissertation investigates the global impact of azinomycin B treatment in a yeast model with special emphasis on DNA damage response, the resulting cell cycle effects, and cellular localization of the compound. The results provide the first demonstration of the in vivo actions of azinomycin B and are consistent with the proposed role of the drug as a DNA crosslinking agent. Biosynthesis of azinomycin B was investigated and appears to have polyketide, non-ribosomal peptide synthetase and alkaloid origins. In pursuit of elucidating the biosynthetic origin we developed both a cell culturing system and a cell-free extract procedure capable of supporting azinomycin synthesis; we used these. These were employed with labeled metabolites to probe the biosynthetic origins of the molecule. Investigations with this enzyme preparation imparted important information regarding the substrate and cofactor requirements of the pathway. These results supported the premise of a mixed origin for the biosynthesis of the molecule and paved the way for expansive stable isotope labeling studies, which largely revealed the biosynthetic precursors and probable construction of the azinomycins. Some of these studies corroborate while other results conflict with initial proposed biosynthetic routes based upon the azinomycin biosynthetic gene cluster sequence. Future azinomycin biosynthetic gene cluster enzyme characterization, mechanistic investigations, and genetic modifications will ultimately provide definitive proof for the intermediacy of proposed biosynthetic precursors and the involvement of specific cofactors. Better understanding of how nature constructs unique molecule may provide insight into eventual chemoenzymatic/gene thearapy based approaches toward cancer therapy.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Bruce Banks; submitted to the Dept. of Chemistry. Title from PDF t.p. (viewed Jul. 31, 2009). Includes bibliographical references (p. 95-102).

<p>This thesis is based on ten publications (Papers I-X). The phosphodiester backbone makes DNA or RNA to behave as polyelectrolyte, the pentose sugar gives the flexibility, and the aglycones promote the self-assembly or the ligand-binding process. The hydrogen bonding, stacking, stereoelectronics and hydration are few of the important non-covalent forces dictating the self-assembly of DNA/RNA. The pH-dependent thermodynamics clearly show (Papers I and II) that a change of the electronic character of aglycone modulates the conformation of the sugar moiety by the tunable interplay of stereoelectronic anomeric and <i>gauche</i> effects, which are further transmitted to steer the sugar-phosphate backbone conformation in a cooperative manner. 3'<i>-</i>anthraniloyl<b> </b>adenosine<b> </b>(a mimic of 3'-teminal CC<u>A</u>_OH of the aminoacyl-tRNA^Phe) binds to EF-Tu*GTP in preference over 2'<i>-</i>anthraniloyl<b> </b>adenosine<b>, </b>thereby showing (Paper III) that the 2’-<i>endo</i> sugar conformation is a more suitable mimic of the transition state geometry than the 3’<i>-endo</i> conformation in discriminating between correctly and incorrectly charged aminoacyl-tRNA^Phe by EF-Tu during protein synthesis. The presence of 2'-OH in RNA distinguishesit from DNA both functionallyas well as structurally. This work (Paper IV) provides straightforward NMR evidence to show that the 2'-OH is intramolecularly hydrogen bonded with the vicinal 3'-oxygen, and the exposure of the 3'<i>-</i>phosphate of the ribonucleotides to the bulk water determines the availability of the bound water around the vicinal 2'-OH, which then can play various functional role through inter- or intramolecular interactions. The pH-dependent ¹H NMR study with nicotinamide derivatives demonstrates (Paper V) that the cascade of intramolecular cation (pyridinium)-π(phenyl)-CH(methyl) interaction in edge-to-face geometry is responsible for perturbing the p<i>K</i>_a of the pyridine-nitrogen as well as for the modulation of the aromatic character of the neighboring phenyl moiety, which is also supported by the T₁ relaxation studies and <i>ab initio</i> calculations. It has been found (Papers VI-IX) that the variable intramolecular electrostatic interaction between electronically coupled nearest neighbor nucleobases (steered by their respective microenvironments) can modulate their respective pseudoaromatic characters. The net result of this pseudoaromatic cross-modulation is the creation of a unique set of aglycones in an oligo or polynucleotide, whose physico-chemical properties are completely dependent upon the propensity and geometry of the nearest neighbor interactions (extended genetic code). The propagation of the interplay of these electrostatic interactions across the hexameric ssDNA chain is considerably less favoured (effectively up to the fourth nucleobase) compared to that of the isosequential ssRNA (up to the sixth nucleobase). The dissection of the relative strength of basepairing and stacking in a duplex shows that stability of DNA-DNA duplex weakens over the corresponding RNA-RNA duplexes with the increasing content of A-T/U base pairs, while the strength of stacking of A-T rich DNA-DNA duplex increases in comparison with A-U rich sequence in RNA-RNA duplexes (Paper X).</p>