以萘酰亚胺为基础的荧光探针PX-1检测ONOO⁻的机制研究

Mechanism Study of Naphthalimide-Based Fluorescent Probe PX-1 for Detecting Peroxynitrite

摘要: 过氧亚硝基阴离子(ONOO⁻)是生物体系内高活性活性氮自由基，其在肝细胞过氧化物酶体内异常累积是四氯化碳诱导急性肝损伤早期标志性病理事件。靶向过氧化物酶体、可实现活体原位成像的双光子荧光探针是解析肝脏氧化应激通路的关键工具。唐波课题组前期已完成新型“关–开”型双光子荧光探针PX-1的合成、体外光谱性能、细胞成像与小鼠急性肝损伤活体成像完整实验表征，证实PX-1可特异性识别过氧化物酶体内ONOO⁻并实现组织可视化。本文以量子化学计算为核心手段，从分子轨道、电子激发、反应位点、激发态跃迁四个维度系统解析PX-1传感响应内在机理。借助Gaussian16与Multiwfn软件开展几何优化、态密度(DOS)、平均局域离子化能(ALIE)、S₀→S₁空穴–电子分布、原子贡献热图等系列理论计算。轨道能级分析表明，未反应PX-1中1,4-苯二酚识别单元的HOMO轨道能级介于萘酰亚胺荧光团HOMO与LUMO之间，满足光诱导电子转移(PeT)发生条件，探针本底荧光被强效淬灭；当PX-1与ONOO⁻发生氧化裂解反应生成PX-OH后，富电子苯二酚片段脱除，PeT电子转移通路彻底阻断，分子内电荷转移(ICT)效应成为激发态主导跃迁模式，荧光信号大幅恢复。ALIE计算精准定位氧化反应发生于苯二酚羟基取代碳位点，与实验氧化裂解规律完全匹配；空穴–电子可视化与原子贡献定量分析直观区分PX-1与PX-OH两种分子截然不同的激发态电子转移特征，完整阐明PeT淬灭与ICT增强协同调控荧光开关的双重响应机制。通过多肽截断模型对比计算证实，短肽片段对荧光团与识别单元轨道能级、激发态跃迁特征干扰可忽略，现有简化模型具备可靠代表性；多组取代苯二酚衍生物定量计算建立识别基团HOMO能级差值-PeT淬灭效率线性关联指标，可直接用于同类探针分子预筛选与性能预测。本理论研究从微观电子层面解释PX-1优异传感性能的分子本质，同时建立普适性量化设计标准，为后续萘酰亚胺类细胞器靶向ONOO⁻荧光探针分子结构优化、识别基团改造提供可量化、可预测的理论指导。

Abstract: Peroxynitrite (ONOO⁻) is a highly reactive nitrogen species in biological systems. Its abnormal accumulation in hepatic peroxisomes acts as an early pathological marker of carbon tetrachloride-induced acute liver injury. Two-photon fluorescent probes capable of peroxisome-targeted in vivo in situ imaging serve as crucial tools for deciphering hepatic oxidative stress pathways. The Tang Bo group has previously accomplished comprehensive experimental characterizations of a novel “turn-on” two-photon fluorescent probe PX-1, including synthetic preparation, in vitro spectroscopic performance, cellular imaging and in vivo imaging of acute liver injury in mice. Their results verified that PX-1 can specifically recognize peroxisomal ONOO⁻ and realize tissue visualization. In this work, quantum chemical calculations were adopted as the core method to systematically analyze the intrinsic sensing mechanism of PX-1 from four perspectives: molecular orbital, electronic excitation, reactive site and excited-state transition. A series of theoretical calculations, such as geometric optimization, density of states (DOS), average local ionization energy (ALIE), S₀→S₁ hole-electron distribution and atomic contribution heatmap, were performed via Gaussian16 and Multiwfn software. Orbital energy level analysis revealed that the HOMO energy level of the 1,4-benzenediol recognition unit in intact PX-1 lies between the HOMO and LUMO of the naphthalimide fluorophore, satisfying the energy matching requirement for photoinduced electron transfer (PeT) and leading to strong intrinsic fluorescence quenching of the probe. After oxidative cleavage between PX-1 and ONOO⁻ generates PX-OH, the electron-rich benzenediol moiety is eliminated, the PeT electron transfer channel is completely blocked, and intramolecular charge transfer (ICT) dominates excited-state transitions, resulting in drastic fluorescence recovery. ALIE calculations precisely locate the oxidation reaction at the hydroxyl-substituted carbon sites of benzenediol, which is fully consistent with the experimental oxidative cleavage rule. Visualized hole-electron distributions and quantitative atomic contribution analyses distinctly differentiate the disparate excited-state electron transfer behaviors of PX-1 and PX-OH, comprehensively elaborating the dual response mechanism synergistically regulated by PeT quenching and enhanced ICT. Comparative calculations of truncated peptide models verify that short peptide fragments exert negligible interference on the orbital energy levels of fluorophores and recognition units as well as excited-state transition characteristics, demonstrating the reliable representativeness of the simplified model adopted herein. Quantitative calculations on multiple substituted benzenediol derivatives establish a linear correlation index between the HOMO energy level difference of recognition groups and PeT quenching efficiency, which can be directly applied to pre-screening and performance prediction of analogous probe molecules. This theoretical study elucidates the molecular origin underlying the outstanding sensing performance of PX-1 at the microscopic electronic level, and meanwhile establishes universal quantitative design criteria. It provides quantifiable and predictable theoretical guidance for subsequent structural optimization and recognition group modification of naphthalimide-based organelle-targeted fluorescent probes toward ONOO⁻.