Highly Efficient Chiral Separation Based on Alkali‐proof Protein Immobilization by Covalent Organic Frameworks

Abstract Chiral separation plays a pivotal role in both practical applications and industrial productions. However, traditional chiral stationary phases (CSPs) exhibit inherent instability in alkaline environments, presenting a significant challenge despite their importance. Herein, basophilic alcalase is creatively developed to fabricate ultrastable protein‐based CSPs that can efficiently work under alkaline conditions. An in‐depth theoretical simulation is conducted to unveil the unique three‐dimensional conformation of alcalase, showing selective affinity towards various enantiomers of chiral amino acids and drugs, especially acidic substrates. Subsequently, an in situ assembly strategy is used to immobilize alcalase within a hydrazone‐linked covalent organic framework (COF) platform. The generated protein‐based CSPs enable successful baseline separation (R s ≥1.50) for various value‐added compounds (e.g., non‐steroidal drug, RS‐flurbiprofen; nucleotide analog, RS‐tenofovir) via high‐performance liquid chromatography, surpassing the commercial chiral column. Furthermore, a systematic study reveals that increasing hydrophilicity and pore sizes of COFs can enhance the separation performance. Remarkably, the obtained CSPs demonstrated exceptional durability, maintaining performance for >2,400 runs. This study provides a new member to the protein library for CSPs, and represents an innovative and effective platform for CSPs with immense potential for the enantioseparation of acidic drugs.