Conditional probes and fluorogenic reagents for manipulating and imaging biology

This thesis focuses on the development of chemical molecular tools to visualise and manipulate biological systems, aiming to study the role of proteins and biochemical analytes in health and disease. The first part develops fluorogenic xanthene probes for imaging membrane damage (Paper 1) and enzyme activity (Paper 2). The second part (Paper 3) develops a cageable HaloTag ligand that selectively ligates to the HaloTag protein only after activation by light or a biochemical stimulus. Fluorogenic Probes Paper 1: Damage to cell membranes plays a key role in diverse pathologies, including bacterial infections and multiple sclerosis. DNA-intercalating fluorogens are commonly used to detect such damage, but they can be toxic and are restricted to identifying permeabilisation in cell volumes that contain a nucleus. To overcome these limitations, this work develops disulfonated fluorogenic probes that selectively label the entire cytosol of damaged cells, with near zero background fluorescence for sensitive detection. These probes reliably reveal membrane damage induced by biological, biochemical, or physical means and are compatible with multicolour microscopy. Their advantages over DNA fluorogens are demonstrated by imaging neuronal axon damage in vitro and discriminating membrane damage at single-cell resolution in Drosophila wound models in vivo. Paper 2: Fluorogenic bioactivity probes are powerful tools for research and diagnostics, widely used to study enzyme activity in disease contexts or for fluorescence assisted surgery. While these probes must be membrane permeable to efficiently enter cells, often their fluorescent products also leak out, leading to signal loss, poor cell-by-cell resolution, and low sensitivity for low-turnover processes. Prior cell-retention strategies are typically inefficient or disrupt native biology through non-specific alkylation or precipitation. To overcome this, Paper 2 screened charge- and polarity-based designs and identified a rhodol scaffold that can switch from cell-permeable to cell-retained states. This modular scaffold is compatible with sensing diverse species (e.g. glutathione, thioredoxin reductase, hydrogen peroxide), and releases a bright, soluble, cell-retained fluorophore enabling sensitive, cell-resolved activity imaging. Conditional HaloTag Ligand Paper 3: The HaloTag self-labelling protein system enables the covalent attachment of diverse chemical reagents to fusion proteins of interest (POIs). It is widely used for fluorogenic imaging, analyte sensing, or for tethering to increase local reagent concentrations. However, its utility has been limited by the lack of control: existing ligands only link chemical and biological components unconditionally. This absence of ligands that can respond to a (bio-)chemical stimulus, is a major design and performance gap in the HaloTag toolbox. Paper 3 (and the Patent) introduce a conditional HaloTag ligand that ligates only after uncaging e.g. by light or enzymatic activity. This new ligand expands the HaloTag system with new capabilities: (1) photo-triggered fluorogenic reagents enable spatiotemporally precise protein labelling, (2) photo-triggered heterodimerisers allow light-controlled protein recruitment forcing two POIs into proximity, and (3) enzyme-triggered fluorogens permit durable recording and ratiometric quantification of enzyme activities (e.g. peptidases, oxidoreductases) in ways that should prove easily multiplexable. Overall, this thesis develops molecular tools for probing and manipulating biological systems with unprecedented precision. The fluorogenic probes enhance the sensitivity for studying membrane integrity and enzyme activity, while the conditional HaloTag ligand brings programmable control to one of the most widely used protein labelling systems. This sets the stage for dissecting cellular processes with spatial and temporal precision, advancing basic research and translational applications.