Badugu Ramachandram, Lakowicz Joseph R, Geddes Chris D
Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, Medical Biotechnology Center, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
Institute of Fluorescence and Center for Fluorescence Spectroscopy, Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA.
Anal Chim Acta. 2004 Sep 20;522(1):9-17. doi: 10.1016/j.aca.2004.06.040. Epub 2004 Jul 29.
We characterize six new fluorescent probes that show both intensity and lifetime changes in the presence of free uncomplexed aqueous cyanide, allowing for fluorescence based cyanide sensing up to physiological safeguard levels, i.e. <30 µM. One of the probes, -BMQBA, shows a ≈15-fold reduction in intensity and a ≈10% change in mean lifetime at this level. The response of the new probes is based on their ability to bind the cyanide anion through a boronic acid functional group, changing from the neutral form of the boronic acid group R-B(OH) to the anionic R-B(CN) form, a new cyanide binding mechanism which we have recently reported. The presence of an electron deficient quaternary heterocyclic nitrogen nucleus, and the electron rich cyanide bound form, provides for the intensity changes observed. We have determined the disassociation constants of the probes to be in the range ≈ 15-84 µM. In addition we have synthesized control compounds which do not contain the boronic acid moiety, allowing for a rationale of the cyanide responses between the probe isomers to be made. The lifetime of the cyanide bound probes are significantly shorter than the free R-B(OH) probe forms, providing for the opportunity of lifetime based cyanide sensing up to physiologically lethal levels. Finally, while fluorescent probes containing the boronic acid moiety have earned a well-deserved reputation for monosaccharide sensing, we show that strong bases such as CN and OH preferentially bind as compared to glucose, enabling the potential use of these probes for cyanide safeguard and determination in physiological fluids, especially given that physiologies do not experience any notable changes in pH.
我们对六种新型荧光探针进行了表征，这些探针在存在游离未络合的氰化氢水溶液时会表现出强度和寿命变化，能够基于荧光检测氰化物，检测上限可达生理安全水平，即<30 μM。其中一种探针，-BMQBA，在此水平下强度降低约15倍，平均寿命变化约10%。这些新型探针的响应基于它们通过硼酸官能团结合氰根阴离子的能力，从硼酸基团的中性形式R-B(OH)转变为阴离子形式R-B(CN)，这是我们最近报道的一种新的氰化物结合机制。缺电子的季杂环氮核的存在以及富电子的氰化物结合形式导致了观察到的强度变化。我们已确定这些探针的解离常数在≈15 - 84 μM范围内。此外，我们合成了不含硼酸部分的对照化合物，以便对探针异构体之间的氰化物响应做出合理说明。与游离的R-B(OH)探针形式相比，结合氰化物的探针的寿命明显更短，这为基于寿命检测氰化物直至生理致死水平提供了机会。最后，虽然含硼酸部分的荧光探针在单糖检测方面赢得了当之无愧的声誉，但我们表明，与葡萄糖相比，CN和OH等强碱更优先结合，这使得这些探针有潜力用于生理流体中的氰化物防护和测定，特别是考虑到生理过程中pH值没有明显变化。
