Pickup, Michael Brennan; (1997) Towards a new methodology for the determination of sequence selectivity in small molecule-DNA binding. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Described in this thesis is work aimed towards the determination of the sequence selectivity of aclacinomycin, an anthracycline, for dsDNA, by an in vitro selection experiment. In particular the work was directed at the development of a technique to distinguish between pieces of dsDNA which were, and were not, being bound by the drug. The first methodology attempted was the attachment to aclacinomycin to a solid support, with the aim of performing an affinity chromatography experiment on a random dsDNA library, which had been synthesised. The successful synthesis of a biotinylated linker, for the immobilisation of aclacinomycin on a streptavidin based support, is described, with an overall yield of 48%, starting from dodecan-1,12-diol, over a five step protocol. Attempts to attach this to aclacinomycin via its methyl ester group, however proved to be futile. A wide range of methods were used, both chemical and enzymatic, but only breakdown products or starting material were observed in the reaction mixtures. In order to study the hydrolysis of the aclacinomycin methyl ester, attempts to synthesise a two-ring model compound of the aglycone portion of the drug were made. Several potential routes were investigated starting from [alpha]- tetralone, however no viable method was found to produce the desired target compound. The most successful methodology was found only to result in a mixture of compounds which were unidentifiable by spectroscopic methods. An alternative means of performing in vitro selection experiments involves the use of gel shift reactions. A method of producing consistent gel shifts for aclacinomycin and dsDNA was developed, and the extraction of the recovered DNA from the gel was optimised. However, it was found that PCR amplification of the resulting DNA was not as facile as had been expected, and although several protocols were used, no reliable method for the amplification of the DNA was found.

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