Synthesis of a molecularly tethered dual function germinant and antimicrobial agent

Abstract

Clostridium difficile (C. difficile) bacteria are the leading cause of nosocomial diarrhoea in the UK. The problem with the removal of C. difficile from hospitals is that it can sporulate and therefore be difficult to remove/kill using conventional methods. The spores enter the body via the faecal-oral route and in the presence of germinants (taurocholate), germinate into vegetative cells in the intestine, cause infection and produce symptoms via the release of two main toxins. The project’s aim was to produce polymeric steroid-based antimicrobial materials which will be able to germinate spores and then destroy the resulting vegetative cells. Deoxycholic acid, lithocholic acid and cholic acid were chemically manipulated to do this. Various methods were tried to attach di-amines with varying tertiary amine-based groups to the parent bile acids, with success found using ethyl chloroformate to activate the carboxylic acid group via an anhydride group, with yields up to 90 %. Once synthesised, the bile amides were screened for germinatory activity. The variables included the chain length and the nature of the groups on the tertiary amine. Once germination had been achieved the tertiary amine group was quaternized using various alkyl halides to introduce potential antimicrobial functionality. From the manipulation of the tertiary amine, several compounds were found to be germinants. Several quaternized materials also displayed antimicrobial activity. Work was undertaken to attach acryloyl groups to the 3-OH group of chemically modified lithocholic acid, with success of then polymerising the monomer. Co-germinants are amino acids, such as glycine and alanine which assist taurocholate in the germination of C. difficile spores. It was therefore attempted to produce a polymerizable glycine analogue which could be incorporated into the steroidal polymer to produce a germinatory surface. Boc-Lys-OH was converted to its acrylamide derivative with a view to the incorporation of a tethered glycine equivalent into a steroid polymer.