Exhaustive Catalytic ortho-Alkoxylation of Azobenzenes: Flexible Access to Functionally Diverse Yellow-Light-Responsive Photoswitches.

We develop the first method for catalytic, exhaustive ortho-alkoxylation of azobenzene photoswitches. Alkoxylation is known to improve the photoswitch properties that control azobenzenes' success in chemical biology or materials sciences, e.g., better completeness of both E → Z and Z → E photoisomerizations and >100 nm red shift of photoresponse. Our method enables straightforward late-stage diversification of photoswitches with interesting functional handles. We showcase four applications: using it to rationally tune lipophilicity, prepare isotopic tracers for metabolism studies, install full water solubility without ionic charges, and efficiently access previously difficult mixed-substituent photoswitches. We also identified a previously unexplored mixed-substituent tetra-ortho family, difluoro-dialkoxy-azobenzenes, whose photoresponse can outperform previous 'gold standard' tetrafluoro-, dichloro-difluoro-, and tetrachloro-azobenzenes in significant ways. We thus expect that both the scaffolds we showcase and the method we develop will impact broadly on photochemistry and photopharmacology.

Photoswitchable Epothilone-Based Microtubule Stabilisers Allow GFP-Imaging-Compatible, Optical Control over the Microtubule Cytoskeleton.

Optical methods to modulate microtubule dynamics show promise for reaching the micron- and millisecond-scale resolution needed to decrypt the roles of the cytoskeleton in biology. However, optical microtubule stabilisers are under-developed. We introduce "STEpos" as GFP-orthogonal, light-responsive epothilone-based microtubule stabilisers. They use a novel styrylthiazole photoswitch in a design to modulate hydrogen-bonding and steric effects that control epothilone potency. STEpos photocontrol microtubule dynamics and cell division with micron- and second-scale spatiotemporal precision. They substantially improve potency, solubility, and ease-of-use compared to previous optical microtubule stabilisers, and the structure-photoswitching-activity relationship insights in this work will guide future optimisations. The STEpo reagents can contribute greatly to high-precision research in cytoskeleton biophysics, cargo transport, cell motility, cell division, development, and neuroscience.

A photo-SAR study of photoswitchable azobenzene tubulin-inhibiting antimitotics identifying a general method for near-quantitative photocontrol.

Azobenzene analogues of the tubulin polymerisation inhibitor combretastatin A4 (PSTs) were previously developed to optically control microtubule dynamics in living systems, with subsecond response time and single-cell spatial precision, by reversible in situ photoswitching of their bioactivity with near-UV/visible light. First-generation PSTs were sufficiently potent and photoswitchable for use in live cells and embryos. However, the link between their seconds-scale and hours-scale bioactivity remained untested. Furthermore, the scope for modifications to tune their photo-structure-activity-relationship or expand their function was unknown. Here, we used large-field-of-view, long-term tandem photoswitching/microscopy to reveal the temporal onset of cytostatic effects. We then synthesised a panel of novel PSTs exploring structural variations that tune photoresponse wavelengths and lipophilicity, identifying promising blue-shifted analogues that are better-compatible with GFP/YFP imaging. Taken together, these results can guide new design and applications for photoswitchable microtubule inhibitors. We also identified tolerated sites for linkers to attach functional cargos; and we tested fluorophores, aiming at RET isomerisation or reporter probes. Instead we found that these antennas greatly enhance long-wavelength single-photon photoisomerisation, by an as-yet un-explored mechanism, that can now drive general progress towards near-quantitative long-wavelength photoswitching of photopharmaceuticals in living systems, with minimal molecular redesign and broad scope.

Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton.

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.

Parallel Approaches for the Functionalization of Thietes: α-Metalation versus C-H Activation.

For the first time, an approach to 3,4-disubstituted thietes was developed through two complementary paths. While the first one relies on α-metalation, the second is based on direct C-H functionalization. A new library of sophisticated sulfur-containing four-membered rings is described, paving the way to new bioactive analogues and small heterocycle incorporation.