DNA photosensitizers are compounds that are capable of binding in to DNA strands through groove binding, intercalation, or electrostatic interactions. Excitation of these agents by light generates reactive oxygen species which causes extensive photo-oxidative damage to genomic DNA. Physiological concentrations of NaCl and KCl are ~ 150 mM and 260 mM within the cell nucleus where DNA is contained. Unfortunately, the ability of most photosensitizers to bind to double-helical DNA is reduced and photocleavage yields are diminished as concentrations of salt increase. The aim of this project is to observe the photocleavage of pUC19 plasmid DNA induced by N1,N1-bis(9-anthrymethyl)triethylenetetraamine tetrahydrochloride (AL-VIII 23) 1 or N,N-dimethyl-N’-(9-methylanthracenyl)ethylenediamine (NMEA) 2 in presence of salt. Spectroscopic titrations and DNA melting assays were used to study binding modes and affinities of both dyes to the helix upon the addition of salt.

This thesis represents results of experiments involved in ultra-high resolution x-ray crystal structures of DNA. Detailed analysis of DNA coordinates of increased accuracy, refinement of individual anisotropic B-factors, visualization of disorder, counterions and hydration are presumably facilitated by the increased information of ultra-high resolution structures. However, recent survey of available DNA crystal structures with the data that extends beyond 1Ã... has brought a few areas of considerations; local conformational flexibility of DNA and counterions and how they have been interpreted in previous studies. While the results given here demonstrate both structural and surrounding details of DNA that may be undetected at lower resolution, some of the technical aspects of interpreting ultra-high resolution data is also addressed.

The first section of this work describes the synthesis of a library of novel rhein analogues that are potential anticancer agents. The design of these compounds takes advantage of the ability for rhein to intercalate into DNA and as the incorporation of an alkylating agent, which serves to covalently modify DNA. In three cell lines, these compounds showed potent cytotoxicity with IC50 in the low to mid-μM range. The second project was focused on the development of an efficient synthesis of 6H-Isoindolo[2,1-α]indol-6-one (24), a core structure for a number of biologically active compounds. The approach is metal-free and uses a Beckmann rearrangement followed by an intramolecular cyclization.

Our laboratory has used the 1,4,5,8 Naphthalenetetracarboxylic diimine (NDI) unit to develop threading polyintercalators that bind DNA with the NDI units intercalated in between GpG steps and two different peptide linkers, which connect the NDI units, situated in either the major or minor grooves. The first generation bisintercalators, G₃K and [beta]Ala₃K, were shown to bind two different sequences of DNA, where the peptide linkers reside in the major and minor grooves respectively. These binding modules were then combined to generate threading polyintercalators that bound different DNA sequences with simultaneous occupation of both grooves. In particular, a cyclic bisintercalator was designed and DNAse I footprinting revealed a strong preference for the sequence 5'-GGTACC-3'. NMR structural studies of the complex with d(CGGTACCG)₂ verified a pseudocatenane structure in which the NDI units reside four base pairs apart, with one linker located in the minor groove and the other in the major groove. This was the first structurally well-characterized pseudocatenane complex between a sequence-specific cyclic bisintercalator and its preferred binding sequence. The ability to simultaneously occupy both groves of the same sequence is interesting for several reasons. Most significantly, it raises questions about a complex DNA intercalator's ability to locate its preferred sequence within a long strand of DNA. In order to directly assess this, the intercalator was modified (CBI-Cys) to incorporate a gold nanoparticle probe to allow for the direct visualization of the intercalator locating its preferred sequence within a long DNA strand. The appropriate protocols to visualize DNA using electron and atomic force microscopy were unsuccessful; however, the foundation has been set for future work to develop the appropriate method to determine the mechanism by which the cyclic bisintercalator locates its preferred sequence. Additionally, the bisintercalators developed in our laboratory offered a unique opportunity to exploit their sequence specificity for controlled nanoparticle assembly. Over the past decade, nanoparticles and DNA have been used to develop novel nanoparticle assembly systems with the goal of developing electronic devices and nanomaterials. The G₃K bisintercalator was synthetically modified to incorporate a gold nanoparticle probe. This intercalator-nanoparticle conjugate, BisKC·Au, maintained its binding specificity (5'-GGTACC-3') to a modified DNA fragment containing multiple G₃K binding sites. The atomic force microscope has become the most promising tool in visualizing individual DNA molecules. A modified procedure utilized APS to allow for the direct visualizing of plasmid DNA. The framework is now in place to confirm the controlled assembly of the gold nanoparticles. This protocol can then be used for the [beta]Ala₃K bisintercalator to lead to the development of a nanoparticle assembly system that can precisely control the organization of multiple types of nanoparticles. / text

This work deals with carborane and metallacarborane clusters, in terms of their fundamental chemistry and complexation with cyclodextrins, and in the context of emerging pharmacophores applicable in medicinal chemistry. Arguably, the most important part of this work is the preparation of cobalt bis(dicarbollide) sulfamide derivatives. The sulfamido group is attached to the metallacarborane carbon vertex by an alkyl chain that may be modified in its length. This was accomplished by, firstly, the abstraction of the acidic hydrogen, located on the {CH}-vertex from the metallacarborane, by reaction with lithium base, followed by, secondly, reaction with electrophilic agents (PFA, oxirane and oxetane), which leads to a cascade of reactions to give the desired sulfamide derivatives. These compounds were then tested by collaborators in other institutes for in vitro and in vivo activity towards Carbonic Anhydrase IX (CA IX), which is an enzyme associated with tumour growth. In vivo tests on mice have shown that these types of substances are able to effectively reduce tumour size by 30%. The synthetic research continued with the preparation of sulfonamide compounds of the isomers of the carborane series. The reactions began exclusively with propylhydroxy carborane starting materials, which provide optimum...

This work deals with carborane and metallacarborane clusters, in terms of their fundamental chemistry and complexation with cyclodextrins, and in the context of emerging pharmacophores applicable in medicinal chemistry. Arguably, the most important part of this work is the preparation of cobalt bis(dicarbollide) sulfamide derivatives. The sulfamido group is attached to the metallacarborane carbon vertex by an alkyl chain that may be modified in its length. This was accomplished by, firstly, the abstraction of the acidic hydrogen, located on the {CH}-vertex from the metallacarborane, by reaction with lithium base, followed by, secondly, reaction with electrophilic agents (PFA, oxirane and oxetane), which leads to a cascade of reactions to give the desired sulfamide derivatives. These compounds were then tested by collaborators in other institutes for in vitro and in vivo activity towards Carbonic Anhydrase IX (CA IX), which is an enzyme associated with tumour growth. In vivo tests on mice have shown that these types of substances are able to effectively reduce tumour size by 30%. The synthetic research continued with the preparation of sulfonamide compounds of the isomers of the carborane series. The reactions began exclusively with propylhydroxy carborane starting materials, which provide optimum...

<p>Synthetic and physicochemical studies on appropriately functionalized ODN-conjugates have been performed to evaluate their abilities to act as antisense agents against RNA or as intramolecular DNA cross-linking agents. Intercalating aromatic systems [phenazine (Pnz), dipyridophenazine (DPPZ)] and metallointercalators such as Ru²⁺(phen)₂(DPPZ) and Ru²⁺(tpy)(DPPZ)<b>L</b> [where <b>L</b> = chemically or photochemically labile ligand, phen = phenanthroline, tpy = terpyridine], which are covalently tethered to the oligo-deoxynucleotides (ODNs), have been chosen for this purpose. The ODN-conjugates were typically prepared by automated solid phase synthesis using phosphoramidite building blocks, or on solid supports, both functionalized with the chromophore groups. The photosensitive metal complex, Ru²⁺(tpy)(DPPZ)(CH₃CN), has been incorporated by post-synthetic coupling to the amino-linker modified ODNs <i>via</i> an amide bond. The intercalating ability of the tethered chromophores gave enhanced stability of the duplexes and triplexes formed with ODN-conjugates and their complementary targets: DNA, RNA, or double-stranded DNA. The conjugation of DPPZ chromophore to ODN (at 3', 5' or at the middle) led us to incorporate Ru²⁺(phen)₂(DPPZ) through the DPPZ ligand, for the first time. The corresponding (Ru²⁺-ODN)•DNA duplexes showed dramatic stabilization (ΔT_m = 19.4 – 22.0ºC). The CD and DNase I footprinting experiments suggest that the stabilization is owing to metallointercalation by threading of the Ru²⁺(phen)₂ moiety through the ODN•DNA duplex core, thus "stapling" the two helical strands from the minor to major groove. On the other hand, Ru²⁺(tpy)(DPPZ)(CH₃CN)-ODN conjugates represent a new class of oligonucleotides containing the photoactivatible Ru²⁺ complexes, which can successfully crosslink to the complementary strand. The mechanism of cross-linking upon photoirradiation of [Ru²⁺(tpy)(DPPZ)(CH₃CN)-ODN]•DNA involves <i>in situ</i> conversion to the reactive [Ru²⁺(tpy)(DPPZ)(H₂O)-ODN]•DNA which are subsequently cross-linked through the G residue of the complementary DNA strand. All starting materials and products have been purified by HPLC and/or by PAGE and subsequently characterized by MALDI-TOF as well as ESI mass spectroscopy. Terminal conjugation of the planar Pnz and DPPZ groups through the flexible linkers were also shown to improve thermal stability of the ODN•RNA hybrid duplexes without alteration of the initial AB-type global helical structure as revealed from CD experiments. As a result, RNase H mediated cleavage of the RNA strand in the intercalator-tethered ODN•RNA duplexes was more efficient compared to the natural counterpart. The RNase H cleavage pattern was also found to be dependent on the chemical nature of the chromophore. It appeared that introduction of a tether at the 3'-end of the ODN can be most easily tolerated by the enzyme regardless of the nature of the appending chromophore. The tethered DPPZ group has also been shown to chelate Cu²⁺ and Fe³⁺, like phenanthroline group, followed by the formation of redox-active metal complex which cleaves the complementary DNA strand in a sequence-specific manner. This shows that the choice of appropriate ligand is useful to (i) attain improved intercalation giving Tm enhancement, and (ii) sequence-specifically inactivate target RNA or DNA molecules using multiple modes of chemistry (RNase H mediated cleavage, free-radical, oxidative pathways or photocross-linkage).</p>

Synthetic and physicochemical studies on appropriately functionalized ODN-conjugates have been performed to evaluate their abilities to act as antisense agents against RNA or as intramolecular DNA cross-linking agents. Intercalating aromatic systems [phenazine (Pnz), dipyridophenazine (DPPZ)] and metallointercalators such as Ru2+(phen)2(DPPZ) and Ru2+(tpy)(DPPZ)<b>L</b> [where <b>L</b> = chemically or photochemically labile ligand, phen = phenanthroline, tpy = terpyridine], which are covalently tethered to the oligo-deoxynucleotides (ODNs), have been chosen for this purpose. The ODN-conjugates were typically prepared by automated solid phase synthesis using phosphoramidite building blocks, or on solid supports, both functionalized with the chromophore groups. The photosensitive metal complex, Ru2+(tpy)(DPPZ)(CH3CN), has been incorporated by post-synthetic coupling to the amino-linker modified ODNs via an amide bond. The intercalating ability of the tethered chromophores gave enhanced stability of the duplexes and triplexes formed with ODN-conjugates and their complementary targets: DNA, RNA, or double-stranded DNA. The conjugation of DPPZ chromophore to ODN (at 3', 5' or at the middle) led us to incorporate Ru2+(phen)2(DPPZ) through the DPPZ ligand, for the first time. The corresponding (Ru2+-ODN)•DNA duplexes showed dramatic stabilization (ΔTm = 19.4 – 22.0ºC). The CD and DNase I footprinting experiments suggest that the stabilization is owing to metallointercalation by threading of the Ru2+(phen)2 moiety through the ODN•DNA duplex core, thus "stapling" the two helical strands from the minor to major groove. On the other hand, Ru2+(tpy)(DPPZ)(CH3CN)-ODN conjugates represent a new class of oligonucleotides containing the photoactivatible Ru2+ complexes, which can successfully crosslink to the complementary strand. The mechanism of cross-linking upon photoirradiation of [Ru2+(tpy)(DPPZ)(CH3CN)-ODN]•DNA involves in situ conversion to the reactive [Ru2+(tpy)(DPPZ)(H2O)-ODN]•DNA which are subsequently cross-linked through the G residue of the complementary DNA strand. All starting materials and products have been purified by HPLC and/or by PAGE and subsequently characterized by MALDI-TOF as well as ESI mass spectroscopy. Terminal conjugation of the planar Pnz and DPPZ groups through the flexible linkers were also shown to improve thermal stability of the ODN•RNA hybrid duplexes without alteration of the initial AB-type global helical structure as revealed from CD experiments. As a result, RNase H mediated cleavage of the RNA strand in the intercalator-tethered ODN•RNA duplexes was more efficient compared to the natural counterpart. The RNase H cleavage pattern was also found to be dependent on the chemical nature of the chromophore. It appeared that introduction of a tether at the 3'-end of the ODN can be most easily tolerated by the enzyme regardless of the nature of the appending chromophore. The tethered DPPZ group has also been shown to chelate Cu2+ and Fe3+, like phenanthroline group, followed by the formation of redox-active metal complex which cleaves the complementary DNA strand in a sequence-specific manner. This shows that the choice of appropriate ligand is useful to (i) attain improved intercalation giving Tm enhancement, and (ii) sequence-specifically inactivate target RNA or DNA molecules using multiple modes of chemistry (RNase H mediated cleavage, free-radical, oxidative pathways or photocross-linkage).