In Situ Synthesis of Enzyme-Confined Crystalline Porous Frameworks: A Triad of Linkage, Pore, and Interface...

Enzymatic catalysis represents a sustainable and selective approach to chemical synthesis, yet its practical implementation is frequently limited by the instability of enzymes under cell-free conditions. Confinement─a principle fundamental to early biochemical evolution─has emerged as a key strategy for maintaining enzymatic activity in non-native environments. This has motivated the design of robust biocatalysts through the encapsulation of enzymes within synthetic porous scaffolds. Crystalline porous frameworks (CPFs), which exhibit ultrahigh porosity, tunable pore architectures, and programmable compositions, offer an ideal platform for such confinement. In this context, the in situ growth of CPFs using enzymes as nucleation sites (biotemplates) constitutes a cutting-edge strategy to fabricate enzyme-confined CPF (E@CPF) biocatalysts. Nevertheless, this approach has been constrained by two formidable challenges: the incompatibility of conventional CPF crystallization conditions with fragile enzymes and the pervasive stability-activity trade-off in the resulting heterogeneous biocatalysts. This Account outlines our strategies to overcome these barriers through the synergistic integration of molecular linkage design, pore-channel optimization, and host-guest interface engineering. We detail a biocompatible in situ synthetic methodology enabled by moderately energetic linkages─specifically Zn-N coordination and carboxylic acid dimer hydrogen bonds─which facilitate enzyme-templated crystallization of metal-organic and hydrogen-bonded organic frameworks under aqueous ambient conditions. We further illustrate how reticular chemistry can be leveraged to precisely tailor pore channels and interfacial interactions between the enzyme guest and the CPF host. Such control not only facilitates substrate diffusion but also can predispose the enzyme into a catalytically favorable conformation, providing a viable pathway to overcome the classic stability-activity trade-off in heterogeneous biocatalysis. Translating these fundamental insights, we showcase functional E@CPFs systems for biocatalytic sensing, therapeutic nanodrugs, and photoenzyme coupled catalysis for environmental remediation. Finally, we discuss enduring challenges and future directions, advocating for advanced characterization, predictive design, and increased functional complexity to fully harness the potential of CPF-confined enzymes for multidisciplinary applications. This body of work offers both a strategic blueprint for hybrid biocatalyst design and a deeper understanding of enzymatic behavior under nanoconfinement.