Lim Mi Hee, Wong Brian A, Pitcock William H, Mokshagundam Deepa, Baik Mu-Hyun, Lippard Stephen J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Am Chem Soc. 2006 Nov 8;128(44):14364-73. doi: 10.1021/ja064955e.
A series of FL(n) (n = 1-5) ligands, where FL(n) is a fluorescein modified with a functionalized 8-aminoquinoline group as a copper-binding moiety, were synthesized, and the chemical and photophysical properties of the free ligands and their copper complexes were investigated. UV-visible spectroscopy revealed a 1:1 binding stoichiometry for the Cu(II) complexes of FL(1), FL(3), and FL(5) in pH 7.0 buffered aqueous solutions. The reactions of FL(2) or FL(4) with CuCl(2), however, appear to produce a mixture of 1:1 and 1:2 complexes, as suggested by Job's plots. These binding modes were modeled by the synthesis and X-ray crystal structure determination of Cu(II) complexes of 2-[(quinolin-8-ylamino)methyl]phenol (modL), employed as a surrogate of the FL(n) ligand family. Two kinds of crystals, Cu(modL)(2)(2) and Cu(2)(modL')(2)(CH(3)OH)(2) (modL' = 2-[(quinolin-8-ylamino)methyl]phenolate), were obtained. The structures suggest that one oxygen and two nitrogen atoms of the FL(n) ligands most likely bind to Cu(II). Introduction of nitric oxide (NO) to pH 7.0 buffered aqueous solutions of Cu(FL(n)) (1 microM CuCl(2) and 1 microM FL(n)) at 37 degrees C induces an increase in fluorescence. The fluorescence response of Cu(FL(n)) to NO is direct and specific, which is a significant improvement over commercially available small molecule-based probes that are capable of detecting NO only indirectly. The NO-triggered fluorescence increase of Cu(FL(5)) occurs by reduction of Cu(II) to Cu(I) with concomitant dissociation of the N-nitrosated fluorophore ligand from copper. Spectroscopic and product analyses of the reaction of the FL(5) copper complex with NO indicated that the N-nitrosated fluorescein ligand (FL(5)-NO) is the species responsible for fluorescence turn-on. Density functional theory (DFT) calculations of FL(5) versus FL(5)-NO reveal how N-nitrosation of the fluorophore ligand brings about the fluorescence increase. The copper-based probes described in the present work form the basis for real-time detection of nitric oxide production in living cells.
合成了一系列FL(n)（n = 1 - 5）配体，其中FL(n)是一种用功能化的8 - 氨基喹啉基团修饰的荧光素作为铜结合部分，并研究了游离配体及其铜配合物的化学和光物理性质。紫外 - 可见光谱显示，在pH 7.0缓冲水溶液中，FL(1)、FL(3)和FL(5)的铜（II）配合物的结合化学计量比为1:1。然而，Job曲线表明，FL(2)或FL(4)与CuCl₂的反应似乎产生了1:1和1:2配合物的混合物。通过合成和X射线晶体结构测定2 - [(喹啉 - 8 - 基氨基)甲基]苯酚（modL）的铜（II）配合物（用作FL(n)配体家族的替代物）对这些结合模式进行了建模。得到了两种晶体，Cu(modL)(2)(2)和Cu₂(modL')(2)(CH₃OH)(2)（modL' = 2 - [(喹啉 - 8 - 基氨基)甲基]苯酚盐）。结构表明，FL(n)配体的一个氧原子和两个氮原子最有可能与铜（II）结合。在37℃下，将一氧化氮（NO）引入到Cu(FL(n))（1 microM CuCl₂和1 microM FL(n)）的pH 7.0缓冲水溶液中会导致荧光增强。Cu(FL(n))对NO的荧光响应是直接且特异的，这比仅能间接检测NO的市售小分子探针有显著改进。Cu(FL(5))由NO引发的荧光增强是通过将铜（II）还原为铜（I），同时N - 亚硝化荧光团配体从铜上解离而发生的。FL(5)铜配合物与NO反应的光谱和产物分析表明，N - 亚硝化荧光素配体（FL(5)-NO）是导致荧光开启的物质。FL(5)与FL(5)-NO的密度泛函理论（DFT）计算揭示了荧光团配体的N - 亚硝化如何导致荧光增强。本工作中描述的基于铜的探针构成了实时检测活细胞中一氧化氮产生的基础。
