Experimental and computational studies of spectroscopic properties of 2-aminopurine and pyrrolocytosine

Ghazal, Davide (2014) Experimental and computational studies of spectroscopic properties of 2-aminopurine and pyrrolocytosine. PhD thesis, Birkbeck, University of London.

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Pyrrolocytosine (Pc) and 2-aminopurine (2-Ap) are modified fluorescent nucleobases that can respectively replace the non-fluorescent natural nucleobases cytosine (C) and adenine (A) and therefore, can be used to probe the structure and dynamics of nucleic acids. Changes in fluorescence emission intensity of these modified bases as a function of polarity (general solvent effect) were measured. Lippert plots were generated and the difference between the excitation and emission maxima (Stokes shift) wavelengths of Pc and 2-Ap in water and acetonitrile mixtures indicate that the measured Stokes shifts are mainly due to hydrogen bonding (specific solvent effect) rather than polarity. The data of the quantum yield measurements show that the fluorescence intensity of 2-Ap decreases in organic solvents as opposed to that of Pc. The big difference in dipole moment of the bases in the excited and ground state experimentally found may be due to intramolecular charge transfer. Computational studies performed at DFT (Density Functional Theory) level by using the B3LYP functional revealed that the most stable 2-Ap tautomer is the N9H amino tautomer in both gas and liquid phase. In the case of Pc, our results show that the most stable tautomer in the gas phase is the N9H enol tautomer whereas the N1H N9H keto one is the most stable tautomer in solution. The theoretical absorption and emission maxima are in excellent agreement with the experimental data. The optimized geometry of 2-Ap A in the ground state was found to be non-planar, i.e., with an out of plane pyramidal amino group with respect to the purine ring whereas the optimized molecule appears to be planar in the first excited state. When 2-Ap A is explicitly bound to water molecules, the energy gap between the dark state n-π* and the bright state π-π* is bigger than the electronic energy gap between the two states previously predicted in implicit solvent. The quantum yield of 2-Ap in phosphate buffers is lower than the one measured in water. This decrease in fluorescence emission is almost certainly due to dynamic quenching. The fluorescence emission of both 2-Ap and Pc is drastically reduced when the bases are incorporated into single and double-stranded oligonucleotides but, the degree of fluorescence depression is more marked in water than in buffer solutions. Oligonucleotides may form secondary structures in buffers because of the presence of salts; in particular, G-quadruplexes. The spectroscopic data acquired by using UV, fluorescence, CD (circular dichroism), and anisotropy steady-state techniques seem to rule out the presence of G-quadruplex secondary structure in selected fluorescent nucleobase containing oligonucleotides.

Item Type: Thesis (PhD)
Copyright Holders: The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted.
School/Department: School of Science > Biological Sciences
Depositing User: ORBIT Editor
Date Deposited: 25 Sep 2014 08:59
Last Modified: 02 Dec 2016 12:47
URI: http://bbktheses.da.ulcc.ac.uk/id/eprint/89

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