Design and Synthesis of a Fluorescent Probe for Zn²⁺, 5,7-Bis(<i>N</i>,<i>N</i>-dimethylaminosulfonyl)-8-hydroxyquinoline-Pendant 1,4,7,10-Tetraazacyclododecane and Zn²⁺-Dependent Hydrolytic and Zn²⁺-Independent Photochemical Reactivation of Its Benzenesulfonyl-Cage

We previously reported on a 8-quinolinol-pendant cyclen (L5) as a Zn2+ fluorophore (cyclen = 1,4,7,10-tetraazacyclododecane) and its caged derivative, 8-(benzenesulfonyloxy)-5-(N,N-dimethylaminosulfonyl)quinolin-2-ylmethyl-pendant cyclen (BS-caged-L5), which can be reactivated by hydrolysis of benzenesulfonyl group upon complexation with Zn2+ at neutral pH to give a 1:1 Zn2+−L5 complex (Zn(H−1L5)). We report herein on the synthesis of 5,7-bis(N,N-dimethylaminosulfonyl)-8-hydroxyquinolin-2-ylmethyl-pendant cyclen (L6) and its caged derivative (BS-caged-L6) for more sensitive and more efficient cell-membrane permeability than those of L5 and BS-caged-L5. By potentiometric pH, 1H NMR, and UV−vis spectroscopic titrations, the deprotonation constants pKa1−pKa6 of H5L6 were determined to be <2, <2, <2, 2.5 ± 0.1 (for the 8-OH group of the quinoline moiety), 9.7 ± 0.1, and 10.8 ± 0.1 at 25 °C with I = 0.1 (NaNO3). The results of 1H NMR, potentiometric pH, UV−vis, and fluorescent titrations showed that L6 rapidly forms a 1:1 complex with Zn2+ (Zn(H−1L6)), the dissociation constant of which is 50 fM at pH 7.4. The fluorescent emission of Zn(H−1L6) at 478 nm is 32 times as large as that of L6 (excitation at 370 nm), and the fluorescent quantum yield of Zn(H−1L6) (ΦF = 0.41) is much greater than that of Zn(H−1L5) (ΦF = 0.044). The BS-caged-L6 was reactivated by hydrolysis of the benzenesulfonyl moiety more rapidly (completes in 30 min at pH 7.4 at 37 °C) than BS-caged-L5, presumably enabling the practical detection of Zn2+ in sample solutions and living cells. The photochemical deprotection of BS-caged-L6 and the cell membrane permeability of L6 and BS-caged-L6 are also described.本团队此前曾报道过一款以8-羟基喹啉悬挂式环仑（cyclen，即1,4,7,10-四氮杂环十二烷）为骨架的Zn²+荧光探针（L5）及其笼状衍生物8-(苯磺酰氧基)-5-(N,N-二甲基氨磺酰基)喹啉-2-基甲基悬挂式环仑（BS-caged-L5）：该衍生物可在中性pH条件下与Zn²+络合时，通过苯磺酰基的水解反应被活化，得到1:1型Zn²+−L5络合物（Zn(H−1L5)）。 本文报道了5,7-二(N,N-二甲基氨磺酰基)-8-羟基喹啉-2-基甲基悬挂式环仑（L6）及其笼状衍生物（BS-caged-L6）的合成，相较于L5与BS-caged-L5，该系列化合物具备更优异的检测灵敏度与更高效的细胞膜穿透能力。 通过电位pH滴定、1H核磁共振（1H NMR）波谱与紫外-可见（UV−vis）光谱滴定实验，我们在25℃、离子强度I=0.1（以NaNO3为支持电解质）的条件下，测定了H5L6的去质子化常数pKa1~pKa6，其数值分别为<2、<2、<2、2.5±0.1（对应喹啉结构单元的8-OH基团）、9.7±0.1与10.8±0.1。 1H核磁共振谱、电位pH滴定、紫外-可见光谱与荧光滴定实验结果表明，L6可快速与Zn²+形成1:1型络合物（Zn(H−1L6)），该络合物在pH 7.4条件下的解离常数为50飞摩尔（fM）。 在370 nm激发波长下，Zn(H−1L6)在478 nm处的荧光发射强度是L6的32倍，且Zn(H−1L6)的荧光量子产率（ΦF = 0.41）远高于Zn(H−1L5)（ΦF = 0.044）。 相较于BS-caged-L5，BS-caged-L6可通过苯磺酰基结构单元的水解反应更快地被活化（在37 ℃、pH 7.4条件下30分钟内即可完成活化），这有望实现样品溶液与活细胞内Zn²+的实际检测。 本文同时还阐述了BS-caged-L6的光化学脱保护过程，以及L6与BS-caged-L6的细胞膜穿透性能。