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一大危机，因此迫切需要新的细菌感染诊断方法。在全球范围内，每年约有1亿人被诊断为尿路感染(UTI)，这些感染特别是导致了英国近四分之一的抗生素处方。开发一种30分钟的诊断工具包的工作正在进行中，该工具包旨在提高处方抗生素的准确性和有效性，以减少耐药性的发展，但需要开发优化的荧光染料，以便能够成功识别病原体。红色/近红外荧光染料是首选的，因为它们避免了细菌在可见光绿色区域内的自发荧光信号。目前市场上可用的染料要么不能在所需的波长上发光，要么缺乏合适的光物理特性，如量子效率和光稳定性。本项目的目的是合成各种具有不同官能团的改进的花菁和二酮基吡咯(DPP)染料，并对它们在革兰氏阴性菌和革兰氏阳性菌株中的生物学特性进行评价。初步合成了一些新型的DPP核，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。还探讨了通过在颜料红254的内酰胺氮上引入官能化来优化现有的DPP染料，作为一种改善这些类似物的溶解性的方法，以实现全面的表征和生物学评价。然后，努力优化另一个荧光团核心-硫代碳菁基荧光染料。DiSC3(5)是这类化合物中的一种，具有良好的细菌积累能力，但也具有较差的性能，如较差的光稳定性。研究了在对称硫代碳菁的苯并噻唑核上引入不同芳香族取代基对染料光物理性质的影响，以及染料在细菌细胞中的积累。还探讨了增加苯并噻唑核心烷基链长度的影响，以确定细菌细胞中积累的上限。合成了一些不对称的五甲基硫代碳菁染料，并优化了它们的合成路线。利用流式细胞仪和荧光显微镜对所有新的DPP染料以及对称和不对称的菁染料进行了生物学评价；并与目前可用的‘金标准’红色/近红外荧光染料DISC(3)5和单亚甲基半花青染料SYBR Green进行了比较。

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.