The Systematic Study of BODIPY-based Fluorescent Molecular Rotors

Abstract

Small molecular fluorescent probes have been widely used in biology, chemistry and chemical biology due to their advantages of high sensitivity, resolution, selectivity and synthetic simplicity. 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) and its derivatives have been one type of the most famous and well-studied fluorophores since its discovery in 1968. Because of the structural modification diversity, BODIPY has been extensively applied in the bioimaging of the cellular biomolecules and parameters such as polarity, pH, temperature and viscosity. Molecular rotors are a series of compounds of which the viscosity of the environment influences the fluorescence. The BODIPY-based molecular rotor was firstly applied for the measurement of cellular viscosity in 2008. Since then, numerous BODIPY viscosity sensors have been developed to map the intracellular organelle microviscosity. However, the relationship between molecular structures of the rotors and their effect on viscosity sensitivity and temperature response remains largely unaddressed. In this thesis, the mechanisms and origination of the viscosity sensitivities of BODIPY-based molecular rotors are systematically studied to access the practical application for molecular sensing and bioimaging. In chapter II, mechanistic origins of viscosity sensitivity in BODIPY molecular rotors, which can be applied to design new motion-based sensors, were presented. Through a systematical experimental and computational investigation of two series of BODIPY rotors, two rotation modes resulting in planar and butterfly conformations were discovered. Moreover, the transformation energy barrier from the planar conformation to the butterfly conformation displays a direct correlation with the viscosity response of BODIPY rotors. When this barrier decreases, the viscosity sensitivity of BODIPY molecular rotors increases. Based on these findings, two motion-based sensors were developed for protein detection and recognition. This work clearly shows the applicability of the mechanistic study for the design and development of highly effective molecular rotors. In chapter III, a systematic study on the viscosity and temperature responses of BODIPY rotors was described. By investigating twelve BODIPY rotors, a correlation between viscosity sensitivity and temperature-dependency was discovered. For ineffective and effective viscosity sensors, they are insensitive to temperature change and show little temperature dependence. For rotors with a moderate viscosity response, they exhibit a relatively large temperature dependency. Therefore, molecular rotors with excellent viscosity sensitivity may be more suitable as viscosity sensors, while rotors with moderate viscosity sensitivity are probably better thermometers. Moreover, through the systematic study of BODIPY rotors, the most viscosity-sensitive BODIPY rotors so far were found. In chapter IV, a new series of fluorescent thermometers, Thermo Greens (TGs), was developed to visualize the temperature fluctuation in almost all typical organelles. Through fluorescence lifetime-based cell imaging (FLIM), it was proven that TGs allow the organelle-specific monitoring of temperature gradients created by external heating. TGs were further demonstrated in the quantitative imaging of the heat production at different organelles such as mitochondria and lipid droplets in brown adipocytes. The FLIM thermometry showed that each organelle experiences a distinct temperature increment which depends on the distance away from the heat source. To date, TGs are the first palette batch of fluorescent thermometers that can cover almost all typical organelles. These findings can inspire the development of new fluorescent thermometers and enhance the understanding of thermal biology in the future. In chapter V, a robust dual turn-on fluorescent molecular probe, BOS (Bad Oil Sensor), was demonstrated for the straightforward and sensitive detection of bad cooking oil. By an unbiased high-throughput screening of diversity-oriented fluorescence library and structure-activity relationship analysis, BOS was discovered as the most sensitive probe for bad oil so far. BOS can detect cooking oil through the inevitable intrinsic change of viscosity and pH during the cooking process. Therefore, BOS enables the monitoring of cooking extent for any cooking oil regardless of the origins, including adulterated oil. In addition, a portable bad oil sensing system (BOSS) was constructed. With straightforward procedures, BOSS can successfully detect the cooking oil from home cooking and restaurants. As such, BOS provides an incredible tool applicable to the rapid detection of cooking oil in food safety for the public. By careful experiments and computational analysis, with a deeper understanding of the relationship between viscosity sensitivity and temperature dependency, this thesis can potentially inspire more discoveries in the design and development of viscosity sensors and thermometers for molecular sensing and bioimaging with higher accuracy and resolution.