Squalamine, chemically known as 3β-[[3-((4-aminobutyl)amino)propyl]amino]-5α-cholestan-7α,24R-diol-24-sulfonate, was initially identified as a broad-spectrum aminosterol antibiotic with potent bactericidal activity [1]. Subsequent studies had revealed its additional pharmacological properties, including inhibition of retinal neovascularization for treating retinopathy [2], suppression of mitosis-induced endothelial cell proliferation and migration for cancer therapy [3], and interference with the interaction between α-synuclein oligomers and lipid membranes to eliminate their toxicity in human neuroblastoma cells for neurodegenerative disease treatment [4]. These multifaceted biological activities highlighted its significant medicinal value.

Currently, the chemical synthesis of squalamine typically employed steroidal compounds such as hyodeoxycholic acid, soy sterol, or bisnoralcohol as raw materials ‌ [5]. However, the synthetic route faced considerable challenges, particularly in constructing key intermediate (7α, 24R)-dihydroxy-5α-cholestan-3-one (24R-OH) [6], [7]. The introduction of the 24R-hydroxylation often required protective groups and chiral reagents, involving harsh reaction conditions and tedious steps, which limited the efficient, green, and large-scale production of squalamine [8], [9].

Biocatalysis, especially ketoreductase (KRED)-mediated conversion, had emerged as an environmentally friendly alternative compared to traditional metal and organic catalysts for chiral alcohol synthesis due to its high stereoselectivity and mild reaction conditions [10], [11], [12]. KREDs had been successfully applied in the preparation of various chiral pharmaceutical intermediates, such as miconazole analogs, statins, and γ-butyrolactones [13], [14], [15]. Strategies including directed evolution, semi-rational design, and rational design enabled the engineered KRED mutants with enhanced catalytic activity, regio- and stereo-selectivity, and stability, offering a feasible approach for the chiral modification of steroids [16], [17].

To construct the 24R-OH intermediate in squalamine synthesis, our laboratory previously had employed an engineered KRED mutant from Novosphingobium aromaticivorans to achieve biocatalytic synthesis of this chiral center [18]. However, this biocatalyst exhibited critical limitations with poor tolerance to high substrate loading, and in the subsequent chemical oxidation of the enzymatic product (b1) to the key intermediate 24R-OH (B1), insufficient oxidative selectivity led to the formation of multiple impurities, including A1, resulting in difficult purification and low yield (Fig. 1, Route I). Together, these shortcomings underscored the need for a more reliable and efficient biocatalyst to enable practical synthesis.

To address this issue, a zinc-dependent medium-chain KRED from Rhodococcus sp. (RhKRED). was screened out from a KRED library [19]. This enzyme could catalyze the regio- and stereo-selective reduction of the 24‑carbonyl group in substrate A1 to produce the 24R-OH product B1 with 38.9% conversion at 6 mg/mL, coupled with glucose dehydrogenase (GDH)-NADH regeneration system (Fig. 1, Route II). However, the wild-type RhKRED exhibited poor tolerance to high substrate concentrations and organic solvents, with nearly zero conversion for A1 at 10% (v/v) isopropanol.

To improve the catalytic activity and enhance the industrial applicability, a mechanism-guided computational design strategy was employed to engineer RhKRED through regional modifications, including the substrate channel, flexible regions, and structural stabilization zones. This strategy yielded the quadruple mutant D42A/P89G/P158S/A327P (M4) with markedly improved tolerance to elevated substrate and organic solvent concentrations. Under optimized conditions at 10 mg/mL substrate and 23% (v/v) isopropanol at 40 °C for 8 h, the mutant achieved 99.1% conversion and d.e. > 99.9%. This strategy effectively overcame the limitations of synthesis, such as low optical purity, harsh reaction conditions, showcasing promising prospects for industrial application.