AbstractComplex biological systems, such as cells and their organelles, are composed of regions of varying viscosity, temperature and polarity. Fluorescent molecular rotors (FMRs) based on the popular dye boron-dipyrromethene (BODIPY) have been shown to be very effective sensors of these different conditions. BODIPY-based FMRs have fluorescence lifetimes and quantum yields which have large ranges and are dependent on their local environment. The probes also fluoresce in the visible region and can be adapted to target specific organelles. However, although they are known to be excellent bio-sensors, their working mechanisms are not yet fully understood. This makes adapting them, and improving their capabilities for research in the life sciences, challenging.
My thesis aims to gain a deeper understanding of the working mechanism of BODIPYbased FMRs in sensing viscosity, temperature and polarity by using a range of computational techniques, assisted with results from experimental collaborations. My studies range in scale from single molecule quantum chemical investigations of BODIPY’s fundamental photophysics, to large scale simulations of full organelles. This means I use a range of tools of differing theoretical levels, from multireference wavefunction methods through to large-scale, long timeframe classical molecular dynamics.
After outlining the theory of the investigatory tools I use, I begin my thesis with an examination of the fundamental photophysics of a prototypical BODIPY FMR, phenyl-BODIPY. Not only does this serve as an opportunity to explore its basic properties, such as the effect of the rotation of its phenyl ring on fluorescence, but it also allows me to benchmark many of the computational methods involved in future chapters. Following this, I study the transition dipole moment (TDM) vector of its first excited singlet state, whose direction was unclear. I show that the TDM is aligned along the BODIPY core but that the core can become misaligned with the molecular principal axes of inertia, causing a tilted TDM. My calculations of the molecular structure also aid the interpretation of results from the Toptygin method, an experimental technique for calculating the TDM orientation in fluorophores.
My second results chapter examines the viscosity-sensing mechanism of phenyl-BODIPY. I begin with ab initio molecular dynamics simulations of the probe in vacuo, to act as a control, before comparing these results to ab initio quantum mechanical/molecular mechanical (QM/MM) dynamics simulations in two solvents of different viscosities: methanol and glycerol. I consider the dynamics of the first excited singlet state in these solvents; in particular, I show that two aspects of the molecular motion, rotation of the phenyl ring and bending of the core dye with respect to the ring, are sensitive to viscosity, and, therefore, likely to underpin the mechanism for sensing local viscosity based on free-volume. In the second half of the chapter, I present the results of a joint theoretical-experimental collaboration examining the viscosity-sensing capabilities of BODIPY-C10 in lipid droplets. We show that when the probe’s time-resolved fluorescence anisotropy and its fluorescence lifetime are simultaneously measured in these environments, they are able to sense four different micro-viscosity components. By comparing the experimental measurements to classical molecular dynamics simulations, we match these components to different configurations of the dye within the organelle.
In my third results chapter, I explore the temperature-sensing mechanism of red BODIPYCyclopropyl FMRs. Experimental colleagues synthesised two red BODIPY-FMRs which were small enough to internalise into cells. One probe sensed both viscosity and environmental temperature, while the second was only sensitive to temperature, and the reason behind these differences were not understood. I use time-dependent density functional theory (TDDFT), compared to experimental results and a control FMR, BODIPY-C10, to show that the closer proximity of the cyclopropyl substituents in one probe causes an energy barrier between its radiative and non-radiative decay states which is unlikely to be surmountable, even in high temperatures. In the other probe, however, the barrier is smaller and more likely to be overcome at lower temperatures. In the second half of the chapter, I examine the FMR’s structural properties in greater depth, including locating their ground-first excited state minimum energy conical intersections. Finally, I perform some initial tests in order to assess the best computational tools for dynamically exploring the temperature-sensing mechanism in future works.
In my final results chapter, I bring together what I have learnt, thus far, in my thesis to explain the polarity-sensing mechanism in OCH3-based phenyl-BODIPY FMRs. These probes have long been known to be sensitive to polarity. In low viscosities, the non-radiative decay rates have consistently been shown to be higher in higher polarity solvents. On the other hand, the red BODIPY-Cyclopropyl FMRs examined in the previous chapter show increased temperature sensitivities in low polarities. Despite computational investigations into the polaritysensing mechanism, however, it had not yet been explained. In this chapter, I demonstrate that, rather than being a result of the energy barriers between the non-radiative and radiative decay structural regions, the mechanism is governed by coupling between the ground and excited states at the barrier between the different decay types. I use TDDFT to show that in lower polarity solvents, greater deformation of the molecular structure is possible, which changes the excited state electronic density distribution and lowers the strength of the transition dipole moment. This, in turn, reduces the non-radiative decay rate.
|Date of Award
|1 Dec 2022
|Carla Molteni (Supervisor), Klaus Suhling (Supervisor) & Chris Lorenz (Supervisor)