Synthesis and quantitative characterization of coumarin-caged D-luciferin
Synthesis and quantitative characterization of coumarin-caged D-luciferin
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香豆素笼D-荧光素的合成及定量表征
DOI：
10.1016/j.jphotobiol.2018.10.002
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Akiyama Hidehumi
中科院分区：
文献类型：
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作者：
Kurata Maki;Hiyama Miyabi;Narimatsu Takuma;Hazama Yuji;Ito Takashi;Hayamizu Yuhei;Qiu Xingping;Winnik Francoise M.;Akiyama Hidehumi
萤火虫荧光蛋白的笼状荧光蛋白化合物最近引起了人们的广泛关注，因为萤火虫生物发光，其中D-荧光蛋白作为底物，被广泛用于非侵入性基因表达成像，体内细胞运输的研究，以及酶活性的检测。本研究的目的是开发新的笼状蛋白和定量测定其光分解的物理参数。我们合成了7-（二乙基氨基香豆素）-4-（基）甲基笼状D-胡萝卜素（DEACM-笼状D-胡萝卜素），并利用手性HPLC色谱对其吸收光谱、生物发光和光产物进行了定量表征。我们观察到，4分钟的UV照射产生最大的D-D浓度，其对应于原始DEACM笼-D-D浓度的16.2%。此外，我们不仅评估了从DEACM笼化的D-麦考因到麦考因的光裂解速率（0.20/min），而且评估了在暴露于70 mW/cm 2高压汞灯（254-600 nm）照射后不产生麦考因的笼化麦考因降解速率（0.28/min）和麦考因分解速率（0.20/min）。在光裂解过程中，DEACM笼状D-半乳糖苷形成L-半乳糖苷的速率比D-半乳糖苷的速率小1/10。这些定量测量和光裂解，降解和分解的同时评价是本研究中提出的最重要和原始的方法。
Caged luciferin compounds of firefly luciferins have recently drawn much attention since firefly bioluminescence, in which D-luciferin acts as a substrate, is widely used in noninvasive gene-expression imaging, studies of in vivo cell trafficking, and the detection of enzyme activity. The objectives of this study are the development of new caged luciferins and the quantitative determination of the photophysical parameters of their photo-decomposition. We synthesized 7-(diethylaminocoumarin)-4-(yl)methyl caged D-luciferin (DEACM-caged D-luciferin) and quantitatively characterized its absorption spectrum, bioluminescence, and photoproducts using chiral HPLC chromatography, as a function of light-irradiation time. We observed that 4 min of UV irradiation generated maximum D-luciferin concentrations, which corresponds to 16.2% of the original DEACM-caged-D-luciferin concentration. Moreover, we evaluated not only the rate of photocleavage (0.20/min) from DEACM-caged D-luciferin to luciferin but also the rate of caged-luciferin degradation that did not produce luciferin (0.28/min) and the rate of luciferin decomposition (0.20/min) after exposure to irradiation with a 70 mW/cm2high-pressure mercury lamp (254–600 nm). The formation rate of L-luciferin via DEACM-caged–D-luciferin photocleavage was smaller by a factor of 1/10 compared with that of D-luciferin. These quantitative measurements and simultaneous evaluations of photocleavage, degradation, and decomposition are the most important and original methodology presented in this study.