J Am Chem Soc. 2019 Apr 24;141(16):6631-6638. doi: 10.1021/jacs.9b01261. Epub 2019 Apr 12.
We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high p K of carbofluorescein dyes, coupled with their tendency to cyclize to nonfluorescent configurations, precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the p K by 2 units to 5.2 and sulfonation to prevent cyclization to the nonabsorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low p K values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles of >560 nm, displays high voltage sensitivity (>30% Δ F/ F per 100 mV), and can be used in the presence of other blue-excited fluorophores such as green fluorescent protein. Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase both in vitro and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.
我们提出了一类基于改良碳荧光素骨架的新型电压敏感荧光指示剂的设计、合成和应用。碳荧光素是响应探针的有吸引力的靶标，因为它们在 3'和 6'位置保持氧取代模式，类似于荧光素，同时具有比荧光素红移近 50nm 的激发和发射谱。然而，碳荧光素染料的高 pK 值，加上它们倾向于环化形成非荧光构型，排除了它们在电压成像应用中的使用。在这里，我们通过氯化将其 pK 值降低 2 个单位至 5.2，并通过磺化来防止环化形成非吸收形式，从而克服了碳荧光素的局限性。为此，我们设计了一种从易得、廉价的起始原料合成卤化磺化碳荧光素的路线。新型的氯磺化碳荧光素具有低 pK 值（5.2），可以掺入苯并二乙烯分子线支架中，以创建碳电压敏感荧光团（carboVF 染料）。新型 carboVF 染料中最好的 carboVF2.1（OMe）.Cl，具有>560nm 的激发和发射谱，显示出高电压灵敏度（每 100mV 超过 30%ΔF/F），并且可以在存在其他蓝色激发荧光团（如绿色荧光蛋白）的情况下使用。由于 carboVF2.1（OMe）.Cl 含有酚氧，因此可以纳入荧光标记策略。用空间位阻较大的环丙甲基衍生的乙酰氧基甲基醚烷基化使 carboVF 具有弱荧光；我们表明，在体外和活细胞和神经元表面，荧光可以通过猪肝酯酶的作用恢复。总的来说，这些结果表明，氯化磺化碳荧光素可以成为在典型荧光素激发和发射波长之外进行杂交化学遗传电压成像的有前途的候选物。
