Synthesis and Optimisation of Red/NIR Fluorophores for Assessment of Antimicrobial Susceptibility

Antimicrobial resistance is a major crisis facing the world today due to the overuse of antibiotic treatments and, therefore, new methods for the diagnosis of bacterial infections are urgently required. Worldwide, around 100 million people each year are diagnosed with a urinary tract infection (UTI), and these infections specifically are responsible for nearly a quarter of all antibiotic prescriptions in the UK. The development of a 30-minute diagnostic toolkit which aim to improve the accuracy and efficacy of prescribed antibiotics in order to reduce the development of resistance is well-underway but requires the development of optimised fluorescent dyes to enable successful identification of the causative bacterial agent. Red/NIR fluorescent dyes are preferred as they avoid the autofluorescence signals of bacteria within the green region of visible light. The current commercially available dyes either do not fluoresce at the desired wavelengths or lack suitable photophysical characteristics, such as quantum efficiency and photostability.
The aims of this project were to synthesise of a variety of improved cyanine and diketopyrrolopyrrole (DPP) based dyes possessing various functional groups, accompanied by their biological evaluation in strains of both gram-negative and gram-positive bacteria. Initially synthesis of some novel DPP cores was conducted, but issues were encountered with solubility and therefore restricted further use of these compounds. Optimisation of an existing DPP dye by introducing functionalisation at the lactam nitrogen of Pigment Red 254, with both charged and non-charged groups, was also probed as a method to improve solubility of these analogues to enable full characterisation and biological evaluation.
Efforts were then directed towards optimisation of another fluorophore core - thiacarbocyanine based fluorescent dyes. DiSC3(5) is a compound of this class that shows good bacterial accumulation, however, also possesses less desirable properties such as poor photostability. Studies were conducted to determine the effects of introducing various aromatic substituents to the benzothiazole core of symmetrical thiacarbocyanines and measure the effect on the photophysical properties of the dyes, as well as its accumulation in bacterial cells. The effects of increasing alkyl chain length at the benzothiazole core were also probed, to see if the upper limit for accumulation in bacterial cells could be determined. Work was also carried out to synthesise some asymmetric pentamethine thiacarbocyanine dyes and optimise the synthetic route for their formation. Flow cytometry and fluorescence microscopy were employed for biological evaluation of all new DPP dyes, alongside the symmetric and asymmetric cyanine dyes; in comparison to the currently available 'gold-standard' red/NIR fluorescent dye, DiSC(3)5, and the monomethine hemicyanine dye SYBR Green.