Synthesis of Sulfonated Carbofluoresceins for Voltage Imaging
Synthesis of Sulfonated Carbofluoresceins for Voltage Imaging
复制标题
DOI：
10.1021/jacs.9b01261
复制
发表时间：
2019-04-24
影响因子：
15
通讯作者：
Miller, Evan W.
中科院分区：
文献类型：
--
作者：
Ortiz, Gloria;Liu, Pei;Miller, Evan W.
我们提出了一类新的电压敏感的荧光指示剂的设计，合成和应用建立在一个修改的碳荧光素支架。碳荧光素是响应性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，同时具有与荧光素相比红移近50 nm的激发和发射谱。然而，碳荧光素染料的高pK（a），加上它们环化为非荧光构型的倾向，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pK（a）降低2个单位至5.2和磺化以防止环化为非吸收形式来克服碳荧光素的局限性。为了实现这一目标，我们设计了一种合成路线卤代磺化碳荧光素从容易获得的，廉价的起始材料。新的氯化砜碳荧光素具有低pK（a）值（5.2），并且可以被并入到亚苯基亚乙烯基分子线支架中以产生碳电压敏感的荧光团（碳VF染料）。最好的新carboVF染料carboVF 2.1（OMe）中心点Cl具有>560 nm的激发和发射曲线，显示出高电压灵敏度（>30% Δ F/F/100 mV），并且可以在其他蓝色激发的荧光团如绿色荧光蛋白的存在下使用。由于carbovF2.1（OMe）中心点Cl含有酚氧，它可以被纳入荧光标记策略。烷基化与空间上庞大的环丙基甲基衍生的乙酰氧基甲基醚呈现carboVF弱荧光，我们表明，荧光可以恢复猪肝酯酶在体外和活细胞和神经元的表面上的作用。总之，这些结果表明，氯化砜碳荧光素可以是有前途的候选人混合化学遗传电压成像在波长超过典型的荧光素激发和发射。
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 pK(a) 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 pK(a) 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 pK(a) 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)center dot Cl, possesses excitation and emission profiles of >560 nm, displays high voltage sensitivity (>30% Delta 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)center dot 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.