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.
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.
Photoresponsive materials feature properties that can be adjusted by light near-instantaneously, reversibly, and with high spatiotemporal precision. There is considerable interest in maximising the degree of photoswitching, and in measuring this degree during illumination in complex environments. We study the switching of photoresponsive lipid membranes that allow for precise and reversible manipulation of membrane shape, permeability, and fluidity. Though these macroscopic responses are clear, it is unclear how large the changes of trans/cis ratio are, and whether they can be improved. Here, we used small-angle X-ray scattering to measure the thickness of photoswitchable lipid membranes, and we correlate lipid bilayer thickness to trans/cis ratios. This reveals an unexpected dependency of photoswitching ratio upon aqueous phase composition. In buffer with ionic strength, we observe thickness variations twice as large as previously observed. Furthermore, soft X-rays can quantitatively isomerise photolipid membranes to the all-trans state; enabling X-ray-based membrane control. High energy X-rays do not influence the state of the photoswitches, presumably because they deposit less dose in the sample.
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.
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.
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