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We synthesize polyamide compounds containing bithiophene functional group and lexitropsin analogs and study their biological activity.

Natural and synthetic heterocyclic cations that bind to the DNA minor groove have demonstrated effectiveness as therapeutic agents for cancer, parasitic and viral diseases, as well as powerful probes for use to extend our fundamental understanding of DNA molecular recognition. Crystal and NMR structures with a variety of minor groove binding compounds have shed light on the structural varieties of these systems, the important solvent molecules in the complexes, and the induced fit effects for binding of both DNA and the bound small molecule. Topics of specific importance in DNA recognition are the development of a greater variety of cell-permeable minor groove agents that have increased DNA binding sequence selectivity. In this dissertation, the structural and energetic basis of the interaction between DNA and minor groove binders has been systematically investigated. A set of powerful and complementary biophysical methods have been used: gel electrophoresis with ligation ladder assay, circular dichroism, mass spectrometry, surface plasmon resonance and isothermal titration calorimetry have been applied to determine the binding stoichiometry, binding affinity, kinetics and thermodynamics, and also the structural influence that minor groove binders can have on DNA. The results of several minor groove complexes clearly show that based on DNA sequences, minor groove binders can have multiple binding modes and consequently affect the geometry of DNA minor groove and the overall DNA curvature in distinct manners. In addition, the binding enthalpy of a minor groove binder is essentially salt concentration and binding mode independent. Besides the investigation of DNA-minor groove binder complex, the binding and inhibition of transcription factor PU.1 has also been studied. The highly positive charged PU.1 targets DNA by inserting an α-helix in the major groove of the 5’-GGAA-3’ site, and displays a strong salt concentration dependency. A set of minor groove binders have been rationally designed based on the high-affinity DNA sequence for PU.1 to target the flanking sequences of the 5’-GGAA-3’ site. They display a structure-related PU.1 inhibition efficacy. This work demonstrates that minor groove binders are capable of modulating PU.1 by targeting the opposite groove and supports future efforts to develop agents for other transcription factors.

Thesis advisor: Larry W. McLaughlin / Nucleic acids are fundamental biomolecules responsible for all activities of a living cell. DNA serves as an instruction manual to the cell, containing blueprints and directions for all cellular processes, while RNA serves to carry out the messages held within DNA. Research into the structure, stability, and function of nucleic acids has revealed much about the origin and evolution of life. The ultimate goal of this work is to understand how molecules bind and associate within the minor groove of double stranded, helical DNA. A series of 2'-deoxyadenosine analogues are modified at the three position by replacing the N3-nitrogen with carbon. Substitution at this position is designed to emulate the effects of removing hydrogen bond acceptors, introducing steric bulk, and tethering functional groups of interest into the minor groove. These functional groups mimic small molecules that have been shown to bind within the minor groove of A-T rich sequences as well as serve as a platform for further substitution by fluorescent tags. The synthetic effort needed to obtain purine nucleosides containing each of these modifications was non-trivial. New methodologies unveiled directing and protecting strategies towards the desired isomer of these modified nucleosides in higher yields than those previously deemed acceptable. Application of these modified nucleosides into duplex DNA reveals thermodynamic parameters for how small molecules bind to the minor groove and the effects of introducing biomarkers into an unprecedented region of DNA. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Diamidines are small molecules that generally possess antiparasitic properties and bind preferentially to the minor groove of AT rich DNA. With the goal of getting a better understanding of the thermodynamic driving forces and binding affinities, a series of pentamidine analogs were investigated with various AT rich DNA by ITC, UV-Vis and fluorescence spectroscopic methods. Findings suggest that the substitution of the linker oxygen of pentamidine to a nitrogen slightly improves the binding affinity. All of the investigated compounds are entropically driven at 25 oC with non-alternating AT DNA. Additionally, the increased fluorescence of the nitrogen and sulfur linked analogs will enable future work to be done with fluorescence microscopy to help determine if and where these compounds accumulate in the target organism.

It has been shown that heterocyclic diamidines, a class of minor groove binders, are promising antimicrobial agents. These compounds bind none covalently to the minor groove of A/T rich regions of the kinetoplast DNA and kill the parasite. The mechanism of action of these compounds is not well understood, yet many hypotheses have been proposed. One of the methods that improve the specificity is cooperative binding. Since there are many binding sites available in k-DNA thus the cooperativity in adjacent binding sites is desirable. A library of compounds has been scanned and few of those compounds identified that are able to bind to two adjacent A/T binding sites separated by a single G. Many biophysical methods such as isothermal titration calorimetry, surface Plasmon resonance, circular dichroism and thermal melting have been used to explore the thermodynamic profiles and binding mode of these compounds. The pulsed field gradient NMR was used to investigate the structural changes to the DNA sequence upon binding of the minor groove binders and find a correlation between their biological difference and structural changes. The molecular dynamics was applied to look at the interaction of some of the heterocyclic diamidines to the DNA with more details and predict the unknown structures.

Certain natural products, Ditamycin and Netropsin are considered as models of designing new DNA binding agents. A variety of DNA binding ligands were synthesized and accordingly characterized by different bioassays. In the series of azo-polyamide, it showed slight DNA binding affinity but has the properties of DNA photo-cleavage and recognition of mixed sequence. The carbohydrate-azo-polyamide series show the properties of DNA photo-cleavage and more effective in vitro experiment. In the quinoline series proves the attenuation effect for G-quadruplex stabilization which provides a novel strategy for development of G-quadruplex binding ligands.

A series of combilexin-like monoamidines has been synthesized by linking an intercalative unit with the DNA minor groove binder DB 818 via “Click chemistry.” DB 818 is a dicationic minor groove binder that has shown strong binding affinity to AT sequences. The aim was to synthesize novel classes of DNA minor groove binders that are combilexin-like – minor groove binder / intercalator hybrid – as potential unique DNA binding agents and therapeutics against African Sleeping Sickness. Additionally, a series of novel benzo[d]1,3,2-diazaboroles DAPI derivatives were also synthesized and investigated. These boron compounds have the potential to be strong DNA minor groove binders because of their lower pKa and act as potential chromophores for Boron Neutron Capture Therapy.

A series of combilexin-like monoamidines has been synthesized by linking an intercalative unit with the DNA minor groove binder DB 818 via “Click chemistry.” DB 818 is a dicationic minor groove binder that has shown strong binding affinity to AT sequences. The aim was to synthesize novel classes of DNA minor groove binders that are combilexin-like – minor groove binder / intercalator hybrid – as potential unique DNA binding agents and therapeutics against African Sleeping Sickness. Additionally, a series of novel benzo[d]1,3,2-diazaboroles DAPI derivatives were also synthesized and investigated. These boron compounds ave the potential to be strong DNA minor groove binders because of their lower pKa and act as potential chromophores for Boron Neutron Capture Therapy.

The objective of this thesis was to study whether heterocyclic dicationic compounds that are minor groove binders have the ability to inhibit the digestive properties of type II restriction enzymes which bind to the major groove of the DNA. If these compounds do possess the ability to inhibit restriction enzymes, then what factors influence their ability to inhibit the restriction enzymes? The methods used to study the interactions of DNA, compounds, and enzymes are gel electrophoresis, DNA thermal melting, and circular dichroism. The results from this project reveal that the minor grove binding compounds are able to inhibition type II restriction enzymes. The inhibition is heavily influenced by compound structure and the DNA binding sequence of the enzyme.