Design, synthesis and application of long wavelength fluorophores for bioimaging

项目摘要 神经元和心肌细胞利用膜电位的快速变化进行细胞信号传导。了解如何 膜电位的变化影响整个神经元或心肌细胞群体的细胞行为是一个重要的 生物学的挑战广泛使用的直接或间接测量膜电位的技术包括贴片法， 钳位电生理学和钙离子显像剂。膜片钳电生理技术在膜测量中的应用 电位导致在动作电位的时间尺度上精确测量膜电位。但这些 实验仅限于单细胞膜，这排除了使用膜片钳电生理学来研究更大的细胞 人口。钙离子显像剂由于具有较高的通量和较膜片钳更小的创伤性而被广泛应用 电生理学通过测量钙离子浓度的暂时变化，我们能够间接测量 膜电位的变化。不幸的是，该技术的主要缺点是钙离子的长持续时间 在神经元动作电位期间发现的摄取（10-100 ms），其显著长于实际动作电位 （1-2毫秒）。这是我们理解细胞行为的能力，以及它与这些快速信号事件的关系。 为了应对这一挑战，我们的团队开发了几种不同的荧光探针，称为VoltageFluor染料， 测量这些膜电位的快速变化。VoltageFluor染料由一个连接在一起的荧光氧杂蒽核心组成 疏水分子线。分子线定位在细胞膜中，而氧杂蒽核心自身定向 在细胞外空间。使用这个系统，我们可以测量大群体的膜电位的快速变化， 与动作电位本身的时间尺度相同。 虽然我们以前的VoltageFluors按预期工作，但我们希望开发新的VoltageFluors， 红外线红移的发射光谱将允许这些VoltageFluor染料在体内使用以及结合 与其它在<600 nm处发光的指示剂一起使用。为了实现这一目标，我们将开发新的VoltageFluor染料， 在咕吨骨架中含有吸电子基团。根据杜瓦规则， 撤回基团将导致吸收和发射光谱红移。这一假设得到了 我们进行的计算研究。这些咕吨核的计划合成也将更加稳健， 比我们以前合成的二甲基碳和二甲基硅更耐受各种官能团 染料。 与我们先前合成的利用二甲基碳和二甲基硅的VoltageFluor染料相比， 在氧杂蒽核心的骨架中，在所提出的VoltageFluors中发现的吸电子基团提供了新的功能性 可以轻松实现多样化的手柄。这扩大了这些染料可用于的应用范围，包括 将三重态猝灭剂拴系到咕吨核心，并使用遗传靶向技术定位这些VoltageFluors 在特定的细胞中。我们还预计，氧杂蒽核的容易多样化将意味着这些染料将更多地用于染料。 广泛适用。通过在没有分子导线的情况下合成这些染料，我们还预计我们将能够使用 这项技术在钙离子成像研究中的应用。

Project Summary Neurons and cardiomyocytes utilize rapid changes in membrane potential for cellular signaling. Understanding how changes in membrane potential affect cell behavior across a population of neurons or cardiomyocytes is an important challenge in biology. Widely used techniques to measure membrane potential, either directly or indirectly, consist of patch clamp electrophysiology and calcium ion imaging agents. Using patch clamp electrophysiology to measure membrane potential results in accurate measurements of membrane potential on the timescale of the action potentials. However, these experiments are limited to a single cell membrane, which precludes using patch clamp electrophysiology to study larger cell populations. Calcium ion imaging agents are widely used, as it has a higher throughput and is less invasive than patch clamp electrophysiology. By measuring temporary changes in calcium ion concentrations, we are able to indirectly measure changes in membrane potential. Unfortunately, a major drawback in this technique is the long durations of calcium ion uptake (10-100 ms) found during a neuronal action potential, which is significantly longer than the actual action potential itself (1-2 ms). This our ability to understand cell behavior and how it relates to these rapid signaling events. To address this challenge, our group has developed several different fluorescent probes called VoltageFluor dyes to measure these rapid changes in membrane potential. The VoltageFluor dyes consist of a fluorescent xanthene core tethered to a hydrophobic molecular wire. The molecular wire localizes in the cell membrane while the xanthene core orients itself in the extracellular space. Using this system, we can measure rapid changes in membrane potential in large populations of cells on the same time scale as the action potentials themselves. While our previous VoltageFluors work as intended, we wish to develop new VoltageFluors that emit light in the near infrared. A red-shifted emission spectrum would allow these VoltageFluor dyes to be used in vivo as well as in conjunction with other indicators that emit light at <600 nm. To accomplish this goal, we will develop new VoltageFluor dyes that contain electron withdrawing groups in the xanthene backbone. In accordance with Dewar’s rule, the inclusion of electron withdrawing groups will result in a red-shifted absorption and emission spectra. This hypothesis is supported by computational studies we have undertaken. The planned syntheses of these xanthene cores will also be more robust and tolerant of various functional groups than the syntheses of our previous dimethylcarbon and dimethylsilicon VoltageFluor dyes. In comparison to our previously synthesized VoltageFluor dyes that utilize a dimethylcarbon and dimethylsilicon backbone in the xanthene core, the electron withdrawing groups found in the proposed VoltageFluors offer new functional handles that can easily be diversified. This expands the range of applications that these dyes can be utilized for, including tethering triplet state quenchers to the xanthene core and using genetic targeting techniques to localize these VoltageFluors in specific cells. We also anticipate that the facile diversification of the xanthene core will mean that these dyes will be more broadly applicable. By synthesizing these dyes without the molecular wire, we also anticipate that we will be able to use this technology in calcium ion imaging studies.