Crovetto Luis, Orte Angel, Paredes Jose M, Resa Sandra, Valverde Javier, Castello Fabio, Miguel Delia, Cuerva Juan M, Talavera Eva M, Alvarez-Pez Jose M
Department of Physical Chemistry, Faculty of Pharmacy, University of Granada , Cartuja Campus, 18071 Granada, Spain.
Department of Organic Chemistry, Faculty of Sciences, University of Granada , C. U. Fuentenueva s/n, 18071 Granada, Spain.
J Phys Chem A. 2015 Nov 5;119(44):10854-62. doi: 10.1021/acs.jpca.5b07898. Epub 2015 Oct 23.
Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophysical behavior of a new silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity constant of the dye, pKN-A, was determined by absorbance and steady-state fluorescence at very low buffer concentrations. Next, we determined whether the addition of phosphate buffer promoted the excited-state proton-transfer (ESPT) reaction among the neutral and anion form of 2-Me TM in aqueous solutions at near-neutral pH. For this analysis, both the steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were employed. The TRES experiments demonstrated a remarkably favored conversion of the neutral form to the anion form. Then, the values of the excited-state rate constants were determined by global analysis of the fluorescence decay traces recorded as a function of pH, and buffer concentration. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate constant for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity constant pK*N-A and therefore an enhanced photoacid behavior of the neutral form. Finally, we determined whether 2-Me TM could be used as a sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells.
具有近红外发射的染料因其在活细胞中用作探针具有毋庸置疑的优势而备受关注。深入了解其光物理性质对于此类染料的应用至关重要。在本文中，通过吸收光谱、稳态荧光光谱和时间分辨荧光光谱研究了一种新型硅取代呫吨染料7-羟基-5,5-二甲基-10-(邻甲苯基)二苯并[b,e]硅杂蒽-3(5H)-酮(2-Me TM)的光物理行为。首先，在极低缓冲液浓度下，通过吸光度和稳态荧光测定了染料在近中性pH下的基态酸度常数pKN-A。接下来，我们研究了在近中性pH的水溶液中，添加磷酸盐缓冲液是否会促进2-Me TM的中性和阴离子形式之间的激发态质子转移(ESPT)反应。为此分析，采用了稳态荧光法和时间分辨发射光谱法(TRES)。TRES实验表明中性形式向阴离子形式的转化非常有利。然后，通过对作为pH和缓冲液浓度函数记录的荧光衰减曲线进行全局分析，确定了激发态速率常数的值。所揭示的动力学参数与TRES结果一致，去质子化速率常数高于质子化速率常数，这意味着激发态酸度常数pK*N-A的值异常低，因此中性形式的光酸行为增强。最后，通过测量HeLa 229细胞不同细胞区室中探针的强度分布，我们确定了2-Me TM是否可以用作活细胞内的传感器。
