Chiral Alcohols from Alkenes and Water: Directed Evolution of a Styrene Hydratase

Abstract

Enantioselective synthesis of chiral alcohols through asymmetric addition of water across an unactivated alkene is a highly sought-after transformation and a big challenge in catalysis. Herein we report the identification and directed evolution of a fatty acid hydratase from Marinitoga hydrogenitolerans for the highly enantioselective hydration of styrenes to yield chiral 1-arylethanols. While directed evolution for styrene hydration was performed in the presence of heptanoic acid to mimic fatty acid binding, the engineered enzyme displayed remarkable asymmetric styrene hydration activity in the absence of the small molecule activator. The evolved styrene hydratase provided access to chiral alcohols from simple alkenes and water with high enantioselectivity (>99 : 1 e.r.) and could be applied on a preparative scale.

Keywords: Alkene Hydration; Biocatalysis; Directed Evolution; Hydratase; Stereoselective Catalysis.

Figure 1

Synthesis of chiral alcohols from alkenes. a) A typical approach to access chiral secondary alcohols is the enantioselective reduction of the corresponding ketones using chiral chemocatalysts[ ³ , ⁴ ] or enzymes. The ketones can be generated from alkenes by the palladium‐catalyzed Wacker oxidation and this oxidation reduction sequence can also be performed in a one‐pot process. In contrast, the direct enantioselective addition of water across alkenes can generate chiral alcohols with high atom economy. In this desired reaction, water is used as the sole reactant and stoichiometric amounts of reducing agents are avoided.[ ¹ , ² , ⁶ , ⁷ ] b) The proposed mechanism of fatty acid hydratases involves cooperative Brønsted acid‐base catalysis to activate the alkene as well as water for asymmetric alkene hydration.

Figure 2

Identification and directed evolution of a promiscuous FAH for asymmetric hydration of styrenes. a) Sequence similarity network (SSN) of the oleate hydratase family (IPR010354). A sequence similarity network is useful to visualize a relationship among a large number of protein sequences. Each node is a protein sequence and edges between nodes are only shown if protein sequences share at least a defined level of similarity. Here, an alignment score of 160 has been used that corresponds to 48 % sequence identity. The SSN was generated using the Enzyme Function Initiative‐Enzyme Similarity Tool (EFI‐EST). The network connectivity displays the number of edges from one node to other nodes. Consequently, clusters with high network connectivity (red) represent a much higher number of protein sequences than clusters with low network connectivity (light blue). The SSN shows that the more promiscuous enzymes (MhyFAH, CteFAH and SspFAH) and less promiscuous enzymes (e.g. LplFAH and RerFAH) occupy different clusters in sequence space. b) Promiscuous activity of the chosen enzyme panel was studied using styrene and 4‐bromostyrene as substrate. Reactions were performed in the presence of hexanoic acid (C₆ acid, 1 equiv) to mimic fatty acid binding. Product formation was measured as peak area using GC/MS in SIM mode (styrene) as well as HPLC/DAD (4‐bromostyrene). c) Model of MhyFAH with the active site represented as a grey surface. Amino acids that have been mutated during directed evolution are shown as red spheres. d) Directed evolution experiment shown as activity of freshly prepared cell‐free extract (lysate conc. 300 mg wcw mL⁻¹, 5 mM 4‐bromostyrene, 2 mM heptanoic acid, 24 h, room temperature). Although activities in cell‐free extract are lower than in whole cells, these reactions are highly reproducible and this setup has been used to rescreen beneficial mutants. KO represents MhyFAH‐E78A‐Y196F, a variant with two mutations in the catalytically relevant cooperative acid‐base machinery. e) Enantioselectivity of the styrene hydratase (MhyFAH S388M‐C497L‐T500S)‐catalyzed reaction.

Figure 3

Substrate scope of the asymmetric styrene hydration reactions. Reaction conditions: E. coli whole cells (100 mg wcw mL⁻¹), 2 mM alkene and 0.2 equiv heptanoic acid (C₇ acid), 24 h, room temperature. The yields represent mean values from reactions of biological and technical triplicates (n=9). The coefficient of variation (ratio of standard deviation to the mean) in yield determination was within 8 %. [a] Reactions were also performed on a preparative 100 mg scale. [b] Reactions were performed with MhyFAH‐S388M‐T500S. [c] The reaction was performed with MhyFAH. [d] The absolute configuration of the stereocenter has not been determined.