Detection of Biomolecules: Syntheses and Studies of Minor Groove Binding Unsymmetrical Cyanine Dyes and Chromogenic Enzyme Substrates

Doctoral thesis, 2003

Visualisation or detection of biomolecules is usually achieved by the use of fluorescent or highly coloured chromophores. The work presented in this thesis deals with the development of new chromogenic substrates for the detection of enzyme activity, and new DNA-binding fluorescent dyes for staining of DNA. Paper I reports on the syntheses and studies of new indolyl acetates for chromogenic detection of esterase activity. The main advantages of these new compounds are that they can detect activity at longer wavelengths and with larger shifts in absorption upon enzymatic action compared to the commonly used p-nitrophenyl acetate. In Papers II-VI, syntheses and DNA-binding studies of new crescent-shaped unsymmetrical cyanine dyes are described. Some of these dyes were shown by optical spectroscopy to exhibit a large preference for binding in the minor groove of natural DNA. Different facile synthetic routes have been developed to obtain these dyes, involving either palladium-catalysed cross-couplings or various cyclisation reactions. Similarly to the widely used intercalating unsymmetrical cyanine dyes, the minor groove binding dyes demonstrate a large enhancement in fluorescence intensity upon binding to DNA. It is shown that the extent of minor groove binding to mixed sequence DNA varies significantly between the dyes, depending on the nature of the heteroaromatic groups. The results suggest that hydrophobicity and size are the crucial parameters for recognition of the minor groove of this type of dyes. In Paper IV it is shown that the groove-bound dyes dissociate more slowly and exhibit a reduced sensitivity to ionic strength compared to corresponding intercalating dyes. Finally, one of the dyes, BEBO, has been compared to SYBR Green I as a reporter dye in real time PCR and was shown to behave similarly in all important aspects.