H₂O₂-Mediated Covalently Cross-Linked Assemblies with Phosphodiesterase-like Activity for Spectral Sensing

Integrating catalytic chromogenic or fluorogenic reactions into molecular spectroscopic sensing systems represents a robust strategy for achieving high sensitivity. In this work, a thiol-functionalized 1,4,7-triazacyclononane (TACN) derivative, designated TACN-2(C₇-SH), was synthesized and employed to construct a H₂O₂-responsive catalytic assembly. Upon exposure to H₂O₂, in situ oxidation of thiol groups triggers disulfide bond formation to mediate covalent cross-linking that can facilitate spatial proximity among TACN functional sites. Subsequent coordination of Zn²⁺ with the TACN moieties yields multiple copies of TACN·Zn²⁺ units to create multivalent catalytic pockets, composed of two adjacent TACN·Zn²⁺ units, significantly enhancing the catalytic performance toward the hydrolysis of RNA model substrate 2'-hydroxypropyl-4-nitrophenyl phosphate (HPNP). A new fluorescence-based phosphodiester substrate containing a 7-hydroxycoumarin (7HC) moiety, designated 7HC-P-E, was synthesized to develop a fluorescent sensing system for further enhanced sensitivity. By modulating the H₂O₂ concentration during in situ oxidation of TACN-2(C₇-SH) and subsequent addition of an equimolar amount of Zn²⁺ (vs TACN units), remarkable fluorescence responses were observed, allowing quantitative spectral sensing of H₂O₂ with a limit of detection (LOD) of 19.5 μM. Moreover, a cascade spectral sensing platform was developed by coupling glucose oxidase-catalyzed oxidation of glucose to generate H₂O₂ in situ, which subsequently activated TACN-2(C₇-SH) to a covalently cross-linked assembly via a disulfide bond. Following Zn²⁺ addition, efficient catalytic hydrolysis of 7HC-P-E afforded sensitive spectral sensing of glucose with a LOD of 76.4 μM. The in situ formation of target-triggered multivalent covalent assemblies, which demonstrate highly catalytic activity for the hydrolysis of phosphodiester substrates (via chromogenic or fluorogenic reactions), is capable of offering an innovative strategy for highly sensitive molecular spectroscopic sensing of targets.