Design and Optimisation of Red/NIR Fluorescent Dyes for the Assessment of Antimicrobial Susceptibility

由于抗生素治疗的过度使用，抗生素耐药性是当今世界面临的主要危机，因此迫切需要用于诊断细菌感染的新方法。在全球范围内，每年约有1亿人被诊断患有尿路感染（UTI），这些感染占英国所有抗生素处方的近四分之一。旨在提高处方抗生素的准确性和有效性以减少耐药性发展的30分钟诊断工具包的开发正在顺利进行，但需要开发优化的荧光染料，以成功识别致病细菌。红色/NIR荧光染料是优选的，因为它们避免了细菌在可见光的绿色区域内的自发荧光信号。目前可商购的染料在所需波长下不发荧光或缺乏合适的光物理特性，例如量子效率和光稳定性。本项目的目的是合成各种改进的花青和二酮吡咯并吡咯（DPP）为基础的染料具有不同的功能基团，伴随着他们的生物学评价在革兰氏阴性和革兰氏阳性细菌菌株。最初进行了一些新型DPP核的合成，但遇到了溶解性问题，因此限制了这些化合物的进一步使用。通过在颜料红254的内酰胺氮处引入具有带电和不带电基团的官能化来优化现有DPP染料，也被探索为改善这些类似物的溶解度的方法，以实现全面表征和生物学评价。然后努力针对另一荧光团核心-基于硫碳菁的荧光染料的优化。DiSC 3（5）是这类化合物，其显示出良好的细菌积累，然而，也具有不太理想的性质，例如差的光稳定性。进行了研究，以确定引入各种芳香族取代基的对称thiacarbocyanines的苯并噻唑核心的影响，并测量对染料的物理性质的影响，以及其在细菌细胞中的积累。还探讨了苯并噻唑核心处烷基链长度增加的影响，以观察是否可以确定细菌细胞中累积的上限。本论文还合成了一些不对称五甲川硫碳菁染料，并优化了合成路线。采用流式细胞术和荧光显微镜对所有新DPP染料以及对称和不对称花青染料进行生物学评价;与目前可用的“金标准”红色/NIR荧光染料DiSC（3）5和单次甲基半花青染料SYBR绿色进行比较。

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.