Facile fabrication of tailored Pepsin@COF nanobiohybrid platform for high-performance chiral separation

Enzymes, with their broad chiral recognition capabilities, are promising but limited by stability issues. Covalent organic frameworks (COFs), with high surface area, porous structure, and biocompatibility, offer an ideal platform for enzyme immobilization. However, the non-matching between the large size of enzyme molecules and the pore size of COFs, along with conformational disturbances during the immobilization process, as well as the complex preparation, low efficiency, and poor stability of traditional enzyme-based chiral stationary phases (CSPs), hinders the application of highly chiral selectivity of enzyme in enatioseparation.

A green synthesis strategy is introduced to overcome the limitations of conventional enzyme@COF synthesis, enabling the facile anchoring of pepsin onto TpPa-1 with small pores. The in-situ encapsulation approach facilitates high enzyme loading and excellent stability, which lead to the remarkable potential of Pepsin@TpPa-1 for enhanced chiral selectivity. Inspired by the in-situ concept, an innovative chromatographic column preparation technique is developed by growing Pepsin@TpPa-1 directly within capillaries, creating an capillary electrochromatography open-tube column (OT-CEC) with high-density chiral recognition sites, achieving efficient separation of diverse chiral drugs, including β-blockers, esterase inhibitors, and other types. The complex chiral recognition mechanism of pepsin is revealed at the molecular level, and the positive correlation between Pepsin@TpPa-1 activity and chiral selectivity is explored for offering theoretical support for designing high-performance chiral recognition platforms.

This study represents the first application of enzyme-COF composites in CEC CSPs, overcoming challenges like low enzyme loading and instability. This enzyme-based CSP facilitated the extensive separation of chiral drugs. A novel insight is provided into the recognition mechanism, as well as the relationship between pepsin's activity and its chiral selectivity. Furthermore, the highly stable chiral nanobiohybrid separation platform introduces a pioneering research paradigm for CSP development in OT-CEC.