Synergistic Carbocation Quenching and Bioprotective Shielding for Uncondensed Lignin and In Situ Saccharification

Abstract The “lignin-first” biorefinery using deep eutectic solvents (DESs) offers a promising route to valorize lignocellulose, yet conventional acidic DESs suffer from severe lignin condensation and cellulase incompatibility, necessitating energy-intensive washing steps. Herein, we develop a multifunctional DES comprising choline prolinate ([Ch][Pro]) and gallic acid (GA) that enables one-pot biomass fractionation and in situ saccharification without any intermediate washing. Conductor-like screening model for real solvents (COSMO-RS) calculations guided the selection of [Ch][Pro] for its high lignin affinity. The [Ch][Pro]/GA DES operates through a synergistic three-layer protection mechanism. The nucleophilic –NH– group of prolinate quenches reactive Cα+ carbocations, preserving 91.0% uncondensed lignin with 82.3% β–O–4 linkages and only 8.4% condensation. GA's multiple phenolic hydroxyls cooperatively stabilize lignin via hydrogen bonding and π–π stacking, as evidenced by COSMO-RS thermodynamics (HE from –4.587 to –6.623 kJ·mol–1, lnγ from –2.586 to –4.974). Simultaneously, GA forms a dense hydrogen-bonding shield on cellulase, retaining 98.2% of the native α-helical conformation and 92.9% relative activity. Moreover, GA protects cellulose via a hydrogen-bond network, achieving >95% cellulose preservation. Consequently, a total reducing sugar yield of 90.2% is obtained after 24 h in situ enzymolysis at 50 °C, significantly outperforming conventional DES processes. The DES shows excellent recyclability, with optimized nanofiltration (150 Da + diafiltration) restoring delignification to 93.5% and enzyme compatibility to 91.8% after five cycles through effective removal of accumulated lignin fragments (94.8% removal), furfural/HMF (91.2% removal), and GA dimers (85.6% removal). This work establishes a rational multifunctional DES platform for lignin-first biorefining, combining high performance with economic and environmental sustainability, and provides a preliminary optimization framework for process scale-up.