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Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton
Results . Design and synthesis . We chose the azobenzene photoswitch for installing photoswitchable potency onto the taxane core. This photoswitch offers a substantial geometric change upon isomerisation, which we hoped would differentiate the isomers’ binding constants, and it allows reliable, high-quantum-yield, near-UV/visible-light-mediated, highly robust E?Z photoisomerisability, which enables repeated photoswitching in situ in living cells. Taxanes feature a number of chemically modifiable positions; we chose to focus on sites where substituents can be tolerated, but where their geometric changes might impact binding potency through steric interactions or by modulating the orientation of key interacting groups nearby. Potent taxanes feature a side-chain 3′-amine acylatedsubstituted with mid-size hydrophobic groups (e.g., Boc group in docetaxel and Bz in paclitaxel) 8 , 34 which abut the tubulin protein surface yet are projected away from the protein interior (Fig.? 1a , highlighted in pink); the other side-chain positions (e.g., the 3′-phenyl or 2′-hydroxyl) offer less tolerance for substitution as they project into the protein 8 . The 3′-amine also tolerates the attachment of somewhat polar cargos such as the large silarhodamine fluorophore, as long as they are attached via a long spacer, with only moderate potency loss 35 , making it desirable for photopharmaceutical tuning as it might tolerate azobenzenes with a range of structural characteristics. However, we anticipated that attenuating the high potency of paclitaxel itself (low nM range) might be required, in order that the relatively small structural change of a E / Z isomerisation at the molecular periphery could substantially modify the overall potency. Fig. 1: Design and synthesis of AzTax . a Paclitaxel:tubulin structure (PDB: 3J6G 36 ) with the benzamide indicated in pink. b Synthesis of AzTax from docetaxel. c Panel of AzTax examined in this study. Full size image We accordingly designed a panel of 3′-azobenzamide-taxanes ( AzTax ) for biological testing. As taxanes have famously poor aqueous solubility (still worsened by attaching an azobenzene), we initially determined to focus on compounds displaying satisfactory potency at concentrations substantially below their solubility limit. This avoids the case that the compounds’ apparent potencies would be dictated by solubility effects, and so should enable robust use as reagents across a variety of systems and settings. Theorising that the sterics around the azobenzene phenyl ring proximal to the taxane core would be the greatest potency-affecting factor, we first focussed on testing which orientations of photoswitch would be best tolerated. We therefore scanned orientations of the diazene in ortho , meta and para relative to the amide ( AzTax2 / 3 / 4 compound sets, Fig.? 1b, c ), and when early cellular testing showed that the AzTax2 set had the lowest potency, we abandoned it at this stage. Next, examination of the published tubulin:paclitaxel cryo-EM structures (Fig.? 1a ) 36 , 37 indicated that the azobenzene’s distal ring can project freely away from the protein. Therefore, we hypothesised that steric variation to the distal ring would not greatly impact binding potency of either isomer, but could be used orthogonally to tune their photochemical properties, by substitutions in para to the diazene that chiefly mesomerically affect the photochemistry of the N=N double bond. We accordingly synthesised unsubstituted (“H”), para- methoxy (“MP”) and para- dimethylamino (“DMA”) derivatives of the AzTax3 / 4 sets. These were chosen to vary the photochemical properties of most relevance to photopharmacology: the completeness of the E→Z and the Z→E photoisomerisations at fixed wavelengths, which dictate the dynamic range of isomer photoswitchability, and τ (the halflife of the spontaneous unidirectional Z→E relaxation). Lastly, when the AzTax3 set proved promising in early studies, we also examined installing an electron-donating 3,4,5-trimethoxy motif on the distal ring ( AzTax3TM ) as well as an additional R 3 methoxy group to reduce the rotatability of the proximal ring in case this could amplify the difference between isomer potencies ( AzTax3MTM ), and we controlled for solubility effects by exchanging the dimethylamino substituent for a more soluble diethanolamino (“DEA”) group ( AzTax3DEA ). The target AzTax were synthesised by degradation of commercial docetaxel followed by amide couplings to various azobenzenecarboxylic acids in moderate yields (Fig.? 1b, c and Supplementary Note? 1 ). Photochemical characterisation . The AzTax all displayed robust and repeatable E?Z photoswitching under near-UV/visible illuminations, as expected from the literature 19 (Supplementary Fig.? 1 ). Since their protein target is located in the cytosol, we first wished to evaluate their photoswitching in physiological aqueous media. Since taxanes are too poorly water-soluble to perform reliable photoswitching studies easily by UV–Vis spectroscopy, we synthesised fully water-soluble diethanolamides of all the azobenzenecarboxylic acids and used them in aqueous photoswitching tests (see Supplementary Note? 2 ). The photochemical properties within each substituent set were similar. The unsubstituted (H) compounds displayed a 3-fold dynamic range of Z- isomer photoswitchability between the photostationary states (PSSs) at 375?nm (80% Z ) and 410?nm (26% Z ), and had substantially slower relaxation than biological timescales ( τ ca. 50?days). The methoxylated compounds (MP, TM and MTM) had been chosen to improve the dynamic range of isomer photoswitching by relative shifting of the isomers’ absorption bands 16 (Supplementary Fig.? 2 ). Indeed, they delivered a ca. 9-fold dynamic range of Z- isomer photoswitchability (375?nm: 96% Z ; 530?nm: 11% Z ), and their relaxation remained substantially slower than biological timescales ( τ ca. 24?h). Advantageously for practical work, the metastable Z -isomers of all AzTaxs could be quantitatively relaxed to E by warming DMSO stocks to 60?°C overnight (which increases the practical ease-of-use of these reagents as compared to irreversibly photouncaged reagents). The para -amino (DMA and DEA) compounds featured τ values too small to observe bulk photoswitching in aqueous media under biologically applicable conditions. Yet, since less water-solvated environments such as lipid vesicles, membranes, or protein-adsorbed states are likely intracellular localisations for hydrophobic taxane conjugates, we then determined their photochemistry in moderately polar aprotic media (EtOAc). Here, they were easily bulk-switchable ( τ ca. 11?min), giving a 4-fold dynamic range of Z- isomer photoswitchability (410?nm: 91% Z ; 530?nm: 21% Z ) (further detail in Supplementary Note? 2 ). As the AzTax reagents were intended for use with microscopy, we also examined photoswitching of all compounds over a broader range of wavelengths, to determine what dynamic range of isomer photoswitchability would be accessible in practice, with standard (405, 488 and 514?nm) or more exotic (380, 440 and 532?nm) microscopy laser wavelengths (Fig.? 2a , Supplementary Fig.? 2 and Supplementary Table? 1 ). Fig. 2: Photoswitchable performance of AzTax . a Photostationary state UV–Vis absorption spectra of AzTax3MP under a range of cell-compatible wavelengths similar to microscopy laser lines. b, c Resazurin antiproliferation assays of AzTax highlight their structure- and light-dependent cell cytotoxicity. HeLa cells, 40?h incubation in dark conditions (all- E ) or under pulsed illuminations with low-power LEDs (75?ms per 15?s near-UV at
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