Towards the development of a novel colourimetric nucleic acid biosensor based on peptide nucleic acid-functionalised polydiacetylene liposomes

Abstract

The aim of the research project described here was to develop a novel colourimetric nucleic acids biosensor based on polydiacetylene liposomes containing lipophilic peptide nucleic acids. Preliminary investigations in this area have shown that PNA-containing PDA liposomes can be constructed and that they are blue in colour as expected. However, their poor water solubility and the resulting precipitation made it necessary to synthesise and evaluate a second generation. In these, the PNA head-group is separated from the lipid tail by an amino diethylene glycol-type spacer molecule. The first hydrophilic spacer synthesised was 8-(tert-butoxycarbonyl)amino-3,6- dioxaoctanoic acid. Three methods based on the O-alkylation of mono-Boc, di-Boc and dibenzyl 5-amino-3-oxapentanol were devised. The Boc strategy afforded the corresponding ether in low yield of 20% while in the case of the dibenzyl approach a suitable methodology to effectively cleave the protecting groups from the amino function could not be found. Moreover, the model reaction between dibenzyl 8-amino-3,6- dioxaoctanoic acid and thyminyl PNA monomer afforded the corresponding conjugates in only 6%. An alternative type of connection to link the spacer to the PNA headgroup was thus sought. A 1,4-disubstituted [1,2,3]-triazole moiety was obtained in 60% yield by reacting spacer, 8-(tert-butoxycarbonyl)amino-3,6-dioxaoctan-1-azide and N-alkynyl PNA monomers bearing thymine, Cbz protected adenine and Cbz protected cytosine according to the conditions of the ‘Click’ reaction. These ‘spacer-PNA monomer’ intermediates were then functionalised at their N-terminus using 10,12-pentacosadiynoyl fluoride, to afford ‘lipid-spacer-PNA monomer’ models. The saturated conjugates were obtained by first preparing the ‘lipid-spacer’ intermediate, N-(8-azido-3,6- dioxaoctanyl)stearamide which was then connected to the N-alkynyl PNA monomers using the ‘Click’ reaction. Following this last strategy, a homothymine PNA dimer, prepared in solution phase, was also functionalised with the saturated lipid chain. This second generation of lipid functionalised-PNA monomer models were incorporated into PDA-liposomes. Upon photo-polymerisation, the liposome solutions became blue. They were found to be stable in solution, at 4 oC over long period of time. Their colour changed to red upon environmental changes (i.e. pH and temperature).