A 1H NMR assay for measuring the photostationary States of photoswitchable ligands.
A 1H NMR assay for measuring the photostationary States of photoswitchable ligands.
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DOI：
10.1007/978-1-62703-345-9_8
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发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Trauner, Dirk
中科院分区：
文献类型：
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作者：
Banghart, Matthew R;Trauner, Dirk
在小分子配体中掺入可光异构化的发色团代表了一种用光可逆控制蛋白质功能的一般方法。不同波长的光照产生由不同比例的光异构体组成的光稳定态(PSS)。因此，光开关配体的最佳实现需要了解它们的波长敏感性。以偶氮苯为基础的离子通道阻滞剂为例，该协议描述了一种(1)H核磁共振分析方法，该方法可以用于精确地确定光静止状态(PSS)的异构体含量作为光照波长的函数。光开关配体的样品被溶解在重水中，并通过UV/Vis光谱分析来确定产生PSS的照明波长范围。通过单色仪耦合的光缆，在连续照射下，使用(1)氢核磁共振波谱对这些波长产生的PSS进行了量化。由于偶氮苯的反式和顺式异构体的芳香族质子具有明显不同的化学位移，因此它们在每个PSS处的相对丰度可以很容易地通过峰积分来确定。光谱采集过程中的恒定光照对于准确地从在几分钟或更快的时间尺度上热松弛的分子中确定PSS是必不可少的。该通用方案可以容易地应用于在每个光同分异构体状态下显示不同(1)H核磁共振信号的任何光开关。
Incorporation of photoisomerizable chromophores into small molecule ligands represents a general approach for reversibly controlling protein function with light. Illumination at different wavelengths produces photostationary states (PSSs) consisting of different ratios of photoisomers. Thus optimal implementation of photoswitchable ligands requires knowledge of their wavelength sensitivity. Using an azobenzene-based ion channel blocker as an example, this protocol describes a (1)H NMR assay that can be used to precisely determine the isomeric content of photostationary states (PSSs) as a function of illumination wavelength. Samples of the photoswitchable ligand are dissolved in deuterated water and analyzed by UV/VIS spectroscopy to identify the range of illumination wavelengths that produce PSSs. The PSSs produced by these wavelengths are quantified using (1)H NMR spectroscopy under continuous irradiation through a monochromator-coupled fiber-optic cable. Because aromatic protons of azobenzene trans and cis isomers exhibit sufficiently different chemical shifts, their relative abundances at each PSS can be readily determined by peak integration. Constant illumination during spectrum acquisition is essential to accurately determine PSSs from molecules that thermally relax on the timescale of minutes or faster. This general protocol can be readily applied to any photoswitch that exhibits distinct (1)H NMR signals in each photoisomeric state.