Enzymatic Assembly of Diverse Lactone Structures: An Intramolecular C-H Functionalization Strategy

Abstract

Lactones are cyclic esters with extensive applications in materials science, medicinal chemistry, and the food and perfume industries. Nature's strategy for the synthesis of many lactones found in natural products always relies on a single type of retrosynthetic strategy, a C-O bond disconnection. Here, we describe a set of laboratory-engineered enzymes that use a new-to-nature C-C bond-forming strategy to assemble diverse lactone structures. These engineered "carbene transferases" catalyze intramolecular carbene insertions into benzylic or allylic C-H bonds, which allow for the synthesis of lactones with different ring sizes and ring scaffolds from simple starting materials. Starting from a serine-ligated cytochrome P450 variant previously engineered for other carbene-transfer activities, directed evolution generated a variant P411-LAS-5247, which exhibits a high activity for constructing a five-membered ε-lactone, lactam, and cyclic ketone products (up to 5600 total turnovers (TTN) and >99% enantiomeric excess (ee)). Further engineering led to variants P411-LAS-5249 and P411-LAS-5264, which deliver six-membered δ-lactones and seven-membered ε-lactones, respectively, overcoming the thermodynamically unfavorable ring strain associated with these products compared to the γ-lactones. This new carbene-transfer activity was further extended to the synthesis of complex lactone scaffolds based on fused, bridged, and spiro rings. The enzymatic platform developed here complements natural biosynthetic strategies for lactone assembly and expands the structural diversity of lactones accessible through C-H functionalization.

Figure 1.

(A) Small-molecule transition-metal-catalyzed intramolecular C–H insertion, which typically produces five-membered lactones. (B) Nature employs C‒O disconnections for the construction of lactones. (C) This work: a biocatalytic platform enables access to diverse lactones via intramolecular carbene transfer reactions. R, organic groups; TM, transition metal.

Figure 2.. Directed evolution and substrate-scope study for γ-lactone synthesis.

Reaction conditions: 2.5 mM 1, E. coli whole cells harboring P411 variants (OD₆₀₀ = 2.5) in M9-N aqueous buffer (pH 7.4), 1.25% v/v acetonitrile (co-solvent), room temperature, anaerobic conditions, 24 h. (A) Directed evolution culminating in P411-LAS-5247. Reactions were performed in triplicate (n = 3). Yields reported are based on calibrated HPLC traces and are the means of three independent experiments. (B) Evolutionary trajectory of P411-LAS-5247 from P411-C10 and locations of beneficial mutations in protein structure based on a close variant E10 (PDB ID: 5UCW). (C) Scope of γ-lactones using P411-LAS-5247. (D) Synthesis of a 5-membered cyclic ketone and a γ-lactam using P411-LAS-5247.

Figure 3.. Directed evolution and substrate-scope study for δ- and ε-lactone synthesis.

Reaction conditions: 2.5 mM 3 or 5, E. coli whole cells harboring P411 variants (OD₆₀₀ = 2.5) in M9-N aqueous buffer (pH 7.4), 1.25% v/v acetonitrile (co-solvent), room temperature, anaerobic conditions, 24 h. Reactions were performed in triplicate (n = 3). Yields reported are based on calibrated HPLC traces and are the means of three independent experiments. (A) Evolutionary trajectories of lactone synthases P411-LAS-5247, P411-LAS-5249, and P411-LAS-5264 from P411-C10. (B) Locations of beneficial mutations in the protein structure based on a close variant E10 (PDB ID: 5UCW); (C) Scope of δ-lactones using P411-LAS-5249. (D) Scope of ε-lactones using P411-LAS-5264. ^aP411-LAS-5244 was used. ^bP411-LAS-5249 was used.

Figure 4.. Enzymatic synthesis of complex lactones.

Reaction condition: 2.5 mM 3, E. coli whole cells harboring P411 variants (OD₆₀₀ = 2.5) in M9-N aqueous buffer (pH 7.4), 1.25% v/v acetonitrile (co-solvent), room temperature, anaerobic conditions, 48 h. Yields reported are based on calibrated HPLC traces and are the means of three independent experiments. Enzymatic synthesis of (A and B) fused lactones, (C and D) spiro lactones, (E) fused- and bridged-lactones. (F) Parallel kinetic resolution toward 8f and 8f’catalyzed by P411-LAS-0003 from racemic diazo 7f. (G) Specific synthesis of spiro lactone 8f catalyzed by P411-LAS-0011 from racemic diazo 7f. (H) Specific synthesis of bridged-lactone 8f catalyzed by P411-LAS-0012 from racemic diazo 7f. ^a76% isolated yield. ^b8% yield, 530 TTN for product 8f’. ^c8f was not detected.