Exploring naphthalimide derivatives as sensitisers for genosensors

This thesis is divided into five chapters. The first chapter describes the structure of DNA and discusses the ways that small molecules can bind with DNA and the driving forces for each mode of binding. Furthermore, the chapter describes how these small molecules that bind with DNA can be applied as sensitizers to detect DNA. Lastly, I briefly describe several techniques that can be used to detect ligand-DNA binding, including electrochemical techniques that can be applied in biosensors and other biophysical techniques such as UV-visible titration, isothermal titration calorimetry (ITC), electron paramagnetic resonance (EPR). In chapter 2, a series of ferrocene-naphthalimide conjugates 2.12a-f and 2.13a,c,e were successfully synthesized and characterized using NMR spectroscopy and mass spectrometry. In these compounds, the naphthalimide scaffold is linked with a ferrocene moiety via a terminal diamine linker. Several solubilizing groups were introduced on C4 in the naphthalimide scaffold to produce compounds 2.12a-f and 2.13a,c,f. These solubilizing groups (1°- amine, 2°-amine and thiol group) have different effects on the solubility of the obtained ferrocene-naphthalimide conjugates. Moreover, the solubilizing groups affect the DNA-binding affinities for these compounds which have been studied using UV-visible titrations. Although most synthesised ferrocene-naphthalimide conjugates can be considered weak binders, 2.12f and 2.13c bind strongly with DNA with binding affinities of 3.8x105 and 1.01x105 M-1, respectively. Generally, using linkers with different length to connect the naphthalimide scaffold and ferrocene moieties did not play a significant role in terms of binding with DNA except for one case when morpholine was used as a solubilizing group. In this case changing the length of the linker between the ferrocene and the naphthalimide has a remarkable effect on improving the binding affinity where the compound with a short linker 2.13c exhibits stronger binding affinity than the compound with the longer ether-based linker. In chapter 3, a series of bisnaphthalimide derivatives has been synthesized where two naphthalimide units were connected via several different linkers, including aliphatic, aromatic, and ether-containing linkers. Several solubilizing groups, viz. 1°-amine, 2°-amine and thiol groups, were introduced to both C4 positions in the resulting bisnaphthalimide scaffolds. The solubility for these compounds has been tested in MOPS buffer with solubilities typically in the range of 10-6 M –1 ~10-5 M –1 except in case of using thioglycolic acid as a solubilizing group where the solubility is higher and in the range of ~ x10-4 M. DNA-binding affinities were investigated using UV-visible titrations. According to the DNA-binding affinities, most of the tested bisnaphthalimides such as 3.9e and 3.12d can be considered good DNA binders with binding affinities of 6.13x104 and 1.24x104 M-1, respectively. On the other hand, compound 3.10b is the most promising compound among this series as it binds relatively strongly with DNA with binding affinity of 3.95 x105 M-1. Unsurprisingly, bisnaphthalimides connected with thioglycolic acid do not bind with DNA due to the negatively charged carboxylates when the carboxylic acids get deprotonated in solution and electrostatically prevent bisnaphthalimide from binding with negatively charged DNA. In chapter 4, a series of fused-ring-extended 1,8-naphthalimide derivatives were successfully synthesized. The synthesis involves synthesizing and separating two isomers of a building block for potential intercalators. Several solubilizing groups were introduced in C4 of the naphthalene ring. The DNA-binding properties for these compounds were studied using UV-visible titrations. It is found that this class of compounds binds strongly with DNA, however the titration curves show unusual behavior where we had to analyse the data in a slightly different way to obtain apparent DNA-binding affinities Kapp. In general, there is no difference between these isomers in terms of binding with DNA. The binding properties for two isomers has remarkably improved when we used different groups (1°-amin, 2°-amine, thiol group) compared to bromo-substituted isomers. Compound 4.16a, with ethanolamine as the solubilizing group, is the most promising compound among this series with an apparent DNAbinding affinity Kapp of 8.45´104 M-1. Using 2-mercaptoethanol 4.16d and 4.17d on the other hand show the lowest DNA-binding affinities (~103 M-1). We note, however, that these binding affinities are apparent binding affinities and therefore likely underestimate the actual affinities of these compounds for DNA. In chapter 5, a series of mono- and bisnaphthalimide-TEMPO conjugates has been successfully synthesized. The TEMPO moiety was connected to the naphthalimide scaffold using an amide-coupling reaction between 4-amino TEMPO and a carboxylic acid group connected to the naphthalimide unit. The solubility of the synthesized monoand bisnaphthalimide TEMPO conjugates has been tested in MOPS buffer. Understandably, bisnaphthalimide derivatives were not sufficiently soluble but the mononaphthalimide-TEMPO conjugates are partially soluble. DNA-binding properties for the synthesized mononaphthalimide derivatives were studied using UV-visible titrations. Based on the binding affinities, this class of compound binds moderately with DNA. Among this series of compound, 5.12e which contains two TEMPO moieties, binds with DNA better than the other compounds while 5.12b that involves morpholine as solubilizing group binds with DNA with lower affinity