Dual-strategy guided bioinspired catalysts for efficient synthesis of pyrazole derivatives via hydrazide–ketone condensation

Abstract

Pyrazole derivatives find widespread applications in fields such as pharmaceuticals. Their synthesis typically proceeds via a condensation-driven C–N bond coupling followed by a dehydration step. The condensation reaction mediated by natural enzymes offers a sustainable gateway to this process, circumventing the requirement for acidic activation (protic or metallic) of the carbonyl group and the high-temperature necessary to drive irreversible water removal demanded. Nevertheless, the practical deployment of natural enzymes is thwarted by a fragile, hierarchically structured protein matrix that imposes intrinsic limits on turnover frequency, thermal tolerance and substrate scope. Here we present a dual-strategy guiding principle for the rational assembly of minimalist enzyme mimics. By surgically eliminating non-essential structural motifs while conserving the catalytic core, the resulting hybrids replicate—and often surpass—the capacity of their mimic objects. The condensation reaction between benzohydrazide and acetylacetone to form (3,5-dimethyl-1H-pyrazol-1-yl)(phenyl)methanone was selected as the model reaction. The mimics outperform cognate enzymes across the board: HM1 (39%) vs. PURO (0%), HM2 (44%) vs. CAL-B (21%) and HM3 (35%) vs. SGP (4%). Iterative spatial reconstruction—precise addition, deletion and repositioning of catalytic residues—delivers the optimized variant HM9, which furnishes the product in 91% yield and exhibits a 646-fold enhanced catalytic efficiency (k_cat/k_m) relative to CAL-B. The simplified yet robust scaffold broadens the operational window from 40–60 °C to 0–80 °C and accommodates disparate substrates with varying electronic and steric properties, enabling efficient condensation of substrates that are recalcitrant to natural enzymes. The enzyme mimics synthesized via this novel design strategy retain the inherently green catalytic conditions of natural enzymes while exhibiting superior catalytic properties, thereby establishing a versatile and extensible platform for different reaction types.