No description available.

Synthesizing proteins containing unnatural amino acids inserted at specific positions within the protein sequence has been a longstanding goal of biological chemists. This poses unique challenges, as aminoacyl tRNA synthetases, the enzymes responsible for protein synthesis, are highly specific. To overcome this, a lanthanum-catalyzed, biomimetic tRNA aminoacylation method has been developed(1). However, due to unproductive lanthanum coordination of ethyl phosphate, a reaction byproduct, a full equivalent of lanthanum must be added to each reaction. This may threaten the integrity of tRNA, as lanthanides are known to catalyze the hydrolysis of RNA (2, 3). Using uridine as a simplified tRNA mimic, magnesium, which is known to coordinate strongly with phosphate ions, has been utilized to optimize this reaction and increase the selectivity of lanthanum towards esterification. In the presence of magnesium, ester yield is substantially increased. In addition to this, optimal pH and buffer reaction conditions were determined.

Synthesizing proteins containing unnatural amino acids inserted at specific positions within the protein sequence has been a longstanding goal of biological chemists. This poses unique challenges, as aminoacyl tRNA synthetases, the enzymes responsible for protein synthesis, are highly specific. To overcome this, a lanthanum-catalyzed, biomimetic tRNA aminoacylation method has been developed(1). However, due to unproductive lanthanum coordination of ethyl phosphate, a reaction byproduct, a full equivalent of lanthanum must be added to each reaction. This may threaten the integrity of tRNA, as lanthanides are known to catalyze the hydrolysis of RNA (2, 3). Using uridine as a simplified tRNA mimic, magnesium, which is known to coordinate strongly with phosphate ions, has been utilized to optimize this reaction and increase the selectivity of lanthanum towards esterification. In the presence of magnesium, ester yield is substantially increased. In addition to this, optimal pH and buffer reaction conditions were determined.

No description available.

As part of an ongoing investigation of new bioactive metabolites from rainforest flora, extracts from five different plants were determined to have interesting compounds that were new and/or cytotoxic. These phytochemicals were isolated by various separation techniques and then characterized by common spectroscopic methods. A bark extract of a Tambourissa species yielded a new hydroxybutanolide with moderate cytotoxicity. The long hydrocarbon chain in this molecule is unique, and its structure was determined by various NMR techniques. A fruit extract from Macaranga alnifolia yielded four new prenylated stilbenes, one new geranylated dihydroflavanol, and five known compounds. The stilbenoids are highly cytotoxic, and the National Cancer Institute (NCI) further evaluated one of the new compounds. Bark and leaf extracts from Cerbera manghas yielded a known iridoid and a known cardiac glycoside, both of which showed good bioactivity. The cytotoxicity associated with the iridoid is unprecedented, and it also was further evaluated by the NCI. An extract of a Cordia species yielded two known compounds - a naphthoquinone dimer and a triterpene. Both of these structures are new to the Cordia genus of plants and showed moderate bioactivity. An extract of a Monoporus species yielded a known triterpene saponin. The compound has been previously located in the same plant family, but it is new to this genus and has no prior record of cytotoxicity. / Ph. D.

<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