Intracellular Responsive Lanthanide Probes

Abstract

The use of lanthanide complexes for the detection of biologically relevant species such as anions, pH and metal ions has grown significantly over the past decade. Such probes offer significant advantages over conventional probes; sharp narrow emission lines encode detailed spectral information and allow ratiometric analysis, and their luminescence is long-lived allowing selective spectral acquisition. Many lanthanide-based probes operate in aqueous media, but few have been applied to intracellular measurements.

The introductory chapter considers the design of lanthanide based probes for cellular applications. The fundamentals of lanthanide emission are discussed, and how the ligand structure needs to be carefully constructed to maximise emission efficiency. Reported bicarbonate- and pH-responsive probes, both lanthanide based and non-lanthanide based, are reviewed, leading to a set of proposed novel probe structures.

The synthesis of these probes with further reasoning behind their design is described in chapter two. The chapter concludes with an overview of the complexes in terms of their emission spectral form, hydration number, HPLC and mass spectrometry properties.

Chapter three presents work on bicarbonate-responsive probes. Through a series of spectral titrations, affinity constants for a range of anions were assessed. A high selectivity for bicarbonate was observed over other oxy-anions, and in the presence of protein. The complexes exhibited a mitochondrial localisation profile, and the europium luminescence intensity was modulated reversibly in response to pCO2. The terbium analogues showed negligible change under such conditions, and thus a europium to terbium emission intensity ratio could be used to calibrate pCO2. This principle was also applied to the analysis of bicarbonate levels in simulated biological fluid, and in a serum sample.

Several pH-responsive complexes are described in chapter four. Key aspects of pH-probe design were highlighted in early examples, leading to the final set of probes based on reversible sulphonamide ligation. Spectral titrations were performed to assess pKa values, and the use of europium to terbium emission intensity ratios was again found to offer a convenient method for assessing pH. Unprecedented changes in CPL were found in response to pH in these systems, and gem values could be used to report upon pH. The complexes were observed to exhibit a lysosomal distribution.

Finally, chapter five contains experimental procedures for each compound synthesised, as well as general experimental procedures.

Both sets of complexes described in this thesis show great promise for use as well-defined intracellular probes of bicarbonate or pH.