Fluorescent Membrane Tension Probes for Super-Resolution Microscopy: Combining Mechanosensitive Cascade Switching with Dynamic-Covalent Ketone Chemistry

Abstract

We report design, synthesis, and evaluation of fluorescent flipper probes for single-molecule super-resolution imaging of membrane tension in living cells. Reversible switching from bright-state ketones to dark-state hydrates, hemiacetals, and hemithioacetals is demonstrated for twisted and planarized mechanophores in solution and membranes. Broadband femtosecond fluorescence up-conversion spectroscopy evinces ultrafast chalcogen-bonding cascade switching in the excited state in solution. According to fluorescence lifetime imaging microscopy, the new flippers image membrane tension in live cells with record red shifts and photostability. Single-molecule localization microscopy with the new tension probes resolves membranes well below the diffraction limit. The imaging of physical forces in living systems is a central challenge in current biology. 1 To contribute solutions, we have introduced small-molecule chemistry tools to image membrane tension in living cells. 2Based on the concept of planarizable push-pull probes, 3,4 the so-called flipper probes allowed us to image the change of plasma 5 and organellar membrane tension in living cells. 6 Flipper probes are twisted dithienothiophene (DTT) push-pull dimers (Figure 1a). Their mechanical planarization in the ground state

The primary dipole of flipper probes

“…These secondary push-pull systems have been varied extensively. 14,20 However, the role of the primary push-pull system was never verified, Fig. 1 (a) The primary push-pull dipole from sulfide to sulfone bridges, supported by exocyclic donors D and acceptors A, is designed to turn on upon mechanical planarization of fluorescent flipper probes, shift absorption to the red and increase fluorescence lifetimes.…”

“…2). 20 Exocyclic triazole donors and ketone acceptors operate as chalcogen-bonding cascade switches triggered by mechanical planarization. 14 Reversible dynamic covalent conversion of the trifluoromethyl ketone 1 acceptor into the non-fluorescent hydrate 1d affords the blinking needed for super-resolution microscopy (Fig.…”

“…2). 20 Flippers 2 and 3 were the obvious choices to evaluate the importance of the primary dipole in push-pull flipper 1 (Fig. 2).…”

“…This complementary removal of the primary dipole already affected flipper synthesis significantly. Both DTTs 4 and 5, prepared from tetrabromothiophene as described, 20 were readily oxidized into the DTTO2s 6 and 7 (Scheme 1). Stille coupling of the electron-rich DTTs 4 and 5 gave flipper 8 in 25% yield.…”

“…Scheme 1 (a) Five steps from tetrabromothiophene, 20-40%; 20 (b) mCPBA, CHCl 3 , rt, 4 h, 56%; (c) 2 steps, 70%; 20 (d) mCPBA, CH 2 Cl 2 , rt, 8 h, 75%; (e) (1) nBuLi, THF, À78 1C, 15 min; (2) Bu 3 SnCl, À78 1C to rt, 30 min; (3) 5 or 7, Pd(PPh 3 ) 4 , toluene/DMF, 80 1C, 48 h, 4 + 5 -8, 25%; 4 + 7 -9, B8% (max: 23%); 6 + 7 -10, 0%; (f) mCPBA, CH 2 Cl 2 , rt, 12 h, 40%; (g) NIS, AcOH, CHCl 3 , 0 1C, 0.5 h, then rt, 0.5 h (8 -11), 1 h (9 -12), or 3 h (10 -13), quantitative; (h) 3 steps, see ESI. † Fig.…”