Introduction

Biocatalysis, harnessing enzymes to drive organic transformations, is a powerful approach with broad applications across many fields1. Employing enzymes for precise asymmetric synthesis carries immense significance and has undergone extensive investigation, as biocatalysts facilitate the generation of chiral molecules under environmentally benign conditions while offering not only high efficiency but also exquisite chemo-, regio-, and stereoselectivity. Despite the astonishing diversity of natural enzymatic transformations, biocatalysts frequently fall short in catalyzing a wide array of abiological reactions preferred by synthetic chemists. Since the escalating demand for efficient, selective, and versatile synthetic methods, the expansion of new enzymatic capabilities is both highly sought-after and challenging. In light of the evolutionary catalytic promiscuity of natural enzymes2, researchers are motivated to explore enzymes capable of catalyzing new-to-nature reactions and fine-tune these emerging reactions to achieve desired catalytic performances through the exceptional technique of directed evolution3. Leveraging enzyme active sites for non-natural functions is a valid strategy that has led to significant advances in biocatalysis, resulting in many novel and useful transformations previously unknown in nature4,5,6,7,8,9.

Oxetane, a four-membered heterocyclic compound characterized by the incorporation of an oxygen atom, is notably significant due to its distinctive structural features and reactive nature10. Although the oxetane motif is relatively rare in natural products and metabolites11,12, its presence, when it does occur, frequently imparts vital biological activities, underscoring its importance in the realm of medicinal chemistry. The most representative example is paclitaxel (Fig. 1a), a well-known anticancer drug13. Additionally, oxetane motifs have received enormous interest in drug development as bioisosteric replacements for gem-dimethyl and carbonyl groups, offering favorable physicochemical properties and improved metabolic stability14. A recent example is the development of danuglipron (Fig. 1a), a potent GLP-1R agonist (glucagon-like peptide-1 receptor) designed for the treatment of diabetes15. Furthermore, the intrinsic strain associated with the oxetane ring imparts distinctive reactivity patterns10, which can be harnessed to advance synthetic applications (Fig. 1a). These includes their use as versatile precursors for the synthesis of an array of organic compounds16,17, their role as pivotal intermediates in the total synthesis of natural products18,19, their value as monomers in polymer construction20,21, and their function as potential scaffolds for protein modification22,23. Undoubtedly, the exploration of synthetic methodologies for the synthesis and transformation of oxetane-related compounds has consistently represented a vibrant area of research10,24. The inherent strain of oxetanes imparts them with high reactivity; however, this characteristic also poses challenges in controlling stereoselectivity during their synthesis and transformations, resulting in limited available stereoselective synthetic strategies10. Therefore, the development of practical and efficient methodologies for constructing and manipulating oxetane stereocenters remains a crucial yet formidable endeavor in modern chemical research.

Fig. 1: Design of a biocatalytic platform for enantioselective formation and ring-opening of oxetanes.
figure 1

a The oxetane scaffold is a privileged substructure in natural bioactive products, drug discovery and synthetic chemistry. b Previous studies have exploited the HHDHs’ catalytic repertoire for the enantioselective formation and ring-opening of epoxides. c The architectural features of epoxide and oxetane motifs are compared to highlight the similarities and differences in their chemical structures. d This work expands the HHDHs’ catalytic repertoire for the enantioselective formation and ring-opening of oxetanes.

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Halohydrin dehalogenase (HHDH), first reported by Castro and Bartnicki in 196825, belongs to the superfamily of short-chain dehydrogenases/reductases26. HHDHs exhibit intriguing catalytic promiscuity, mediating not only the dehalogenation of β-haloalcohols to form epoxides with the release of halide ions but also the ring-opening of epoxides into β-substituted alcohols in the presence of several negatively charged nucleophiles (e.g., N3−, CN−, and NO2−)27,28,29. The Janssen and other research groups have conducted extensive investigations into the HHDH’s catalytic mechanisms30,31,32,33, enzyme identification and characterization34,35,36,37, structural features38,39,40,41, enzyme immobilization42,43,44,45, and synthetic applications for the enantioselective formation and ring-opening of epoxides (Fig. 1b)46,47,48,49,50,51,52. Additionally, protein engineering provides a powerful means to improve the catalytic efficiency, enantioselectivity, stability, and substrate specificity of halohydrin dehalogenases (HHDHs), further expanding their utility in synthetic and industrial chemistry53,54,55. For instance, scientists at Codexis have developed a stable and efficient HHDH mutant specifically tailored for the industrial synthesis of ethyl (R)-4-cyano-3-hydroxybutyrate, a key precursor in the manufacturing of atorvastatin56. In recent years, our laboratory has also successfully explored the catalytic repertoire of HHDHs for the stereoselective transformations of epoxides57,58,59,60,61,62.

The biosynthesis of oxetane-containing natural products proves that natural enzymes indeed facilitate synthetic pathways for constructing oxetane motifs11,12. However, the specific enzymes responsible for these biotransformations remain elusive, as they have not yet been identified or are involved in complex reaction processes63,64,65, resulting in the absence of an available biocatalytic platform for oxetane synthesis to date. The oxetane ring harbors an inherent strain energy of approximately 106 kJ/mol and features a larger C-O-C bond angle compared to an epoxide ring, which has a marginally higher strain energy of around 112 kJ/mol (Fig. 1c). The similar structural properties encourage us to envision the possibility of exploiting HHDHs for catalyzing the enantioselective dehalogenation of γ-haloalcohols and also the nucleophile-mediated enantioselective ring-opening of oxetanes (Fig. 1d).

In this work, we present the identification and protein engineering of halohydrin dehalogenase HheD8, which enables the enzymatic enantioselective dehalogenation of γ-haloalcohols and the ring-opening of oxetanes using azide, cyanide, or nitrite as nucleophiles. This biocatalytic methodology facilitates the preparative-scale synthesis of not only chiral oxetanes (up to 49% yield, >99 e.e.) but also various chiral γ-substituted alcohols (up to 50% yield, >99 e.e.)—including γ-haloalcohol, γ-azidoalcohol, γ-cyanoalcohol, γ-nitroalcohol, and γ-diol. Both the enzymatic enantioselective oxetane formation and the ring-opening processes have been demonstrated to be scalable for large-scale transformations (20 mmol) at high substrate concentrations (200 mM). Moreover, the two processes can also be efficiently integrated into a one-pot, one-catalyst cascade system.

Results

Screening of HHDHs

To commence the study, we chose the dehalogenation of racemic 3-chloro-1-phenylpropan-1-ol (1a) and the azide-mediated ring-opening of racemic 2-phenyl-oxetane (1b) as the respective model reactions for dehalogenation and ring-opening processes. Dozens of recombinant E. coli BL21 (DE3) strains harboring HHDH enzymes (Supplementary Table 1), maintained in our laboratory collection57,58,59,60,61, were initially evaluated for their catalytic efficiency in the dehalogenation model reaction (Supplementary Table 2). Background dehalogenation reaction was not observed either in the absence of E. coli cells or with E. coli cells that do not express HHDH (entries 1-2, Fig. 2a). Although the majority of tested enzymes demonstrated negligible or very low catalytic activity, several HHDHs were found to show relatively good catalytic efficiency with the yields of 1b > 10% (entries 3–10, Fig. 2a). Among them, the enzymes HheA5, HheC, and HheD8 exhibited a moderate to good enantioselectivity (E = 13–76). Although non-enzymatic conversion of γ-haloalcohols to oxetanes can be achieved66, finding an enantioselective catalyst for this transformation remains elusive. Subsequently, the active HHDHs underwent further evaluation with the model ring-opening reaction (Supplementary Table 3). No background ring-opening reactions were detected (entries 1–2, Fig. 2b). Unexpectedly, nearly all exhibited very low catalytic efficiency for the oxetane ring-opening, except for the enzymes HheD8 and HheD15. It is noteworthy that HheD8, which originates from strain Thauera aminoaromatica S2, also demonstrated moderate R enantioselectivity (E = 8), resulting in the formation of γ-azidoalcohol (R)-1c with 74% e.e. and 18% yield (entry 7, Fig. 2b). To obtain effective biocatalysts for establishing a biocatalytic platform for the enantioselective formation as well as ring-opening of oxetanes, HheD8 was chosen as the progenitor enzyme for further directed evolution study, as it has demonstrated relatively good catalytic performances in both dehalogenation and ring-opening model reactions.

Fig. 2: Identification and directed evolution of HheD8.
figure 2

a Enzymes are screened for the dehalogenation of (rac)-1a. See Supplementary Table 2 for details. b Enzymes are screened for the ring-opening of (rac)-1b with azide. See Supplementary Table 3 for details. c Possible docking pose of (R)−1b in the active site of the predicted HheD8-WT/AF model structure is illustrated. Catalytic triad S117-Y130-R134, the hot-spot residues, and (R)−1b are highlighted in sticks or spheres. H-bonds are displayed in dashed lines. d The HheD8 is engineered for enantioselective dehalogenation of (rac)−1a. See Supplementary Table 4 for details. e The HheD8 is engineered for enantioselective ring-opening of (rac)−1b with azide. See Supplementary Table 5 for details. NR= no reaction. ND= not detected. Bar charts: the conversion of substrates, dots: the e.e. values of products. The conversion and e.e. values were calculated from the average of triplicate experiments (n = 3). Data are presented as mean values ± SD.

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Directed evolution of HheD8

Given that the protein structure of the wild-type HheD8 (HheD8-WT) was not resolved at that time, we acquired a predicted structure model of HheD8-WT from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk, AFDB code: AF-N6YXW4-F1). The molecular docking of the oxetane (R)-1b into the active site of the HheD8-WT/AF model was then performed to obtain a probable binding pose for subsequent protein engineering efforts. Based on the docking result (Fig. 2c), eight residues (F19, A69, Y168, M124, R127, Q160, N161, and R182) lining the active site pocket were selected for iterative saturation mutagenesis (ISM)67. These residues can be categorized into three groups: the first group includes A69, M124, R127, and R182, which are believed to interact with the substrate’s phenyl ring; the second group consists of F19 and Y168, which are thought to interact with the substrate’s oxetane ring; and the third group comprises Q160 and N161, which are located in the halide binding site and interact with the nucleophile azide during the ring-opening reaction or with the halogen group of the γ-haloalcohol substrate during the dehalogenation reaction. In each round, the resulting variants were screened through whole-cell biotransformation to evaluate enantioselectivities and/or substrate conversions. Variants exhibiting enhancements underwent further evaluation through separate model reactions: the dehalogenation of (rac)-1a and the ring-opening of (rac)-1b with azide.

In the dehalogenation reaction screening (Fig. 2d), A69, F19, and N161 were selected from each group for the first round of site-saturation mutagenesis study. This led to the identification of the variant HheD8-M1 (A69F) with improved enantioselectivity (E = 123), while no enhanced mutants were obtained at the F19 and N161 sites. Subsequently, sequential rounds of ISM were performed on the HheD8-M1, focusing on the other residues (R127, M124, and R182) of the first group. This process generated beneficial variants HheD8-M2 (A69F/R127G), HheD8-M3 (A69F/M124P/R127G), and HheD8-M4 (A69F/M124P/R127G/R182W) with improved catalytic activity and enantioselectivity. Notably, the quadruple-mutant HheD8-M4 excelled, achieving 49% conversion of 20 mM (rac)-1a and formation of (R)-1b with >99% e.e. after 8 h.

In the case of ring-opening reaction screening (Fig. 2e), the ISM study was also focused on the four residues of the first group. Interestingly, the same single-mutant, HheD8-M1 (A69F), was identified in the first round of ISM, exhibiting exceptional enantioselectivity in stark contrast to HheD8-WT (E > 200 vs E = 9). Using the HheD8-M1 variant as the parent enzyme, three additional rounds of ISM were conducted to create mutants HheD8-M5 (A69F/R127L), HheD8-M6 (A69F/M124P/R127L), and HheD8-M7 (A69F/M124P/R127L/R182W), which displayed progressive improvements in catalytic activity while maintaining excellent enantioselectivity. The exceptionally performant quadruple-mutant HheD8-M7 delivered (R)-1c with >99% e.e. and achieved 46% conversion of 20 mM (rac)-1b after 10 h. In a comparative assessment of the dehalogenation reaction, the mutants HheD8-M5, HheD8-M6, and HheD8-M7 were found to exhibit inferior catalytic performance relative to mutant HheD8-M4 (entries 7-9 vs entry 6, Supplementary Table 4). Concurrently, the mutants HheD8-M2, HheD8-M3, and HheD8-M4 were assessed with the ring-opening reaction. The results revealed that the HheD8-M4 outperformed HheD8-M7 in catalytic efficiency, achieved 50% conversion of 20 mM (rac)-1b and produced (R)-1c with 99% e.e. (entry 9 vs entry 6, Supplementary Table 5).

Structural analysis and molecular dynamics (MD) simulations

For possible understanding the impact of these mutagenesis on catalytic efficiency and enantioselectivity, we sought to determine the crystallographic structure of the HheD8-M4. However, this mutant tended to precipitate during the purification process. The mutant HheD8-M3 (A69F/M124P/R127G), which lacks the R182W mutation, showed improved solubility that favored the crystallization process. We successfully resolved the X-ray crystal structure of the HheD8-M3 complex with chloride as a ligand in the halide-binding site to a resolution of 2.40 Å (PDB code: 8XXB, Supplementary Fig. 1 and Supplementary Table 6). Our further attempts to obtain the crystals of the HheD8-M3 complexed with oxetane or azide were not successful.

Overlap analysis revealed that the mutant HheD8-M3 adopts an overall architecture similar to the HheD8-WT/AF (Supplementary Fig. 2), with a highly matched catalytic triad composed of residues (S117-Y130-R134). We further conducted site-directed mutagenesis of the mutant HheD8-M3 focusing on the residues of the catalytic triad S117-Y130-R134. The results showed that all ten generated mutants (S117-C/T/A, Y130-F/T/A, and R134-K/D/E/A) completely lost catalytic activity in both the dehalogenation of γ-haloalcohol and the azide-mediated oxetane ring-opening reactions (Supplementary Fig. 3). These findings provide additional evidence that S117-Y130-R134 serves as a key catalytic triad, facilitating the formation and ring-opening of oxetanes through intramolecular and intermolecular SN2 reaction mechanisms, respectively. This is similar to the mechanism for the formation and ring-opening of epoxides catalyzed by halohydrin dehalogenases30.

Through detailed comparison of the three mutated residues (A69F, M124P and R127G) in HheD8-M3 (Fig. 3a), we speculate that the A69F mutation, which replaces the nonpolar amino acid alanine (A) with the larger aromatic amino acid phenylalanine (F), leads to enhanced catalytic enantioselectivity through aromatic π-π stacking interactions, while it slightly decreases catalytic efficiency by reducing the space size of the active site pocket. Our inference is consistent with the experimental results comparing the catalytic performances of HheD8-WT and HheD8-M1 (Fig. 2d, e). Mutations M124P and R127G introduce smaller amino acid residues, enlarging the active site pocket, which is thought to enhance catalytic efficiency (Fig. 3b). This speculation is reinforced by the striking increase in activity observed in the mutant HheD8-M3 compared to HheD8-M1 (entry 2 vs entry 4 in Supplementary Table 4, and entry 2 vs entry 8 in Supplementary Table 5).

Fig. 3: Comparison of mutated residues of the mutant HheD8-M3 with the wild type HheD8-WT/AF.
figure 3

a Close-up view of the A69F mutation in the active site. b Close-up view of the M124P and R127G mutations in the active site. HheD8-M3 (PDB code: 8XXB) is shown as blue cartoon. HheD8-WT (AFDB code: AF-N6YXW4-F1) is shown as gray cartoon. Catalytic triad S117-Y130-R134, the mutated residues (A69F, M124P, and R127G), and (R)−1b are highlighted in sticks. The ligand Cl− is highlighted in green sphere.

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Molecular docking of (R)-1b into the X-ray structure of HheD8-M3 was then carried out to obtain a possible HheD8-M3/(R)-1b binding pose (Supplementary Fig. 4). Further MD simulations of both complexes, HheD8-WT/(R)-1b and HheD8-M3/(R)-1b, were conducted for 150 ns in duplicate. The trajectories were analyzed to determine the distances between the oxetane-O and both S117-O and Y130-O during the simulations (Fig. 4, and Supplementary Fig. 5). Comparing the fluctuation of these distances revealed that (R)-1b is more stable in the active site of the mutant HheD8-M3 than in that of HheD8-WT. Moreover, the expression levels of HheD8-WT and mutants (M1-M7) were analyzed, and enzyme HheD8-WT and mutants (M1, M3, M5, and M6) were purified for the characterization of the kinetic parameters in the ring-opening reactions of oxetane 1b (Supplementary Fig. 6). By comparing the kinetic parameters of HheD8-M3 (Km: 11.93 mM; kcat: 83.77 min−1) and HheD8-WT (Km: 18.85 mM; kcat: 11.30 min−1), it was found that HheD8-M3 exhibited higher substrate affinity, which is consistent with the results from MD simulations. We therefore speculate that the increased substrate affinity might be one of the factors contributing to the enhanced activity and enantioselectivity. Additional thermostability studies demonstrated that the thermal resistance of mutant HheD8-M3 is lower than that of HheD8-WT (Supplementary Fig. 7). This result would suggest that the enhancement in catalytic activity and enantioselectivity of the mutant HheD8-M3 is achieved at the expense of enzyme stability to a certain extent. This also indicates that the easily precipitated mutant HheD8-M4 is best used as a whole-cell catalyst to ensure its stability.

Fig. 4: Molecular dynamics simulation of HheD8-WT/(R)−1b and HheD8-M3/(R)−1b.
figure 4

a The fluctuation of the distances of the 1b-O between S117-O (d1) and Y130-O (d2) during the MD simulations of HheD8-WT/(R)−1b. b The fluctuation of the distance of the 1b-O between S117-O (d1) and Y130-O (d2) during the MD simulations of HheD8-M3/(R)−1b. Catalytic triad S117-Y130-R134, and (R)−1b are highlighted in sticks. Distances are displayed in dashed lines.

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Scope for enantioselective formation of oxetanes

Subsequently, the substrate scope of the biocatalytic platform for the enantioselective dehalogenation of γ-haloalcohols was explored on a preparative-scale (Fig. 5, Supplementary Table 7). Various aryl γ-chloroalcohols bearing electron-donating or electron-withdrawing substituents on the phenyl ring (1-15a) were accepted to furnish both chiral (R)-oxetanes 1-15b (32-46% yield, 93- > 99% e.e.) and (S)-γ-chloroalcohols 1-15a (47-53% yield, 86- > 99% e.e.) with good yields and high optical purities. It is worth noting that the presence of steric hindrance at the ortho position of the aromatic ring (2-5a) was found to be compatible with the biotransformation. Substitutions were well tolerated at both the para and meta positions (6-14a). Smooth conversion of substrate 14a, featuring a strong electron-withdrawing trifluoromethyl group, to chiral oxetane (R)-14b was achieved, albeit with a slight decrease in enantioselectivity. Interestingly, introducing two fluoro substituents on the phenyl ring (15a) did not hamper the reaction; rather, it successfully delivered both chiral (R)-15b and (S)-15a with excellent enantioselectivity (E > 200). The substrate containing a bulky naphthalene moiety (16a) underwent effective and enantioselective dehalogenation using the mutant HheD8-M3, yielding chiral oxetane (R)-16b and γ-chloroalcohol (S)-16a with 37% yield, >99% e.e. and 50% yield, 96% e.e., respectively. Additionally, enantioselective dehalogenation of α-,α-disubstituted γ-chloroalcohol 17a also proceeded smoothly to furnish the (R)-17b (36% yield, >99% e.e) and (S)-17a (49% yield, >99% e.e). Remarkably, the reaction also tolerated structural perturbations, accommodating the substitution of the aryl ring with several heterocyclic moieties such as pyrimidine (18a), quinoline (19a), and thiophene (20a). Chiral heterocyclic γ-chloroalcohols (18-20a, 48-50% yields and 89- > 99% e.e.) and oxetanes (18-19b, 42-44% yields and 96-97% e.e.) were successfully isolated with the exception of the unstable oxetane 20b. Moreover, the reaction exhibited good tolerance toward the alkyl-substituted substrates, affording chiral alkyl γ-chloroalcohols 21-22a (41-48% yields and 93-98% e.e.) and 2-cyclohexyloxetane (R)-21b (35% yield, >99% e.e.), with the exception of the unstable 2-propyloxetane 22b. Furthermore, the biocatalytic system was applied to the enantioselective generation of chiral γ-bromoalcohol (S)-23a (50% yield, >99% e.e.) and γ-iodoalcohol (S)-24a (48% yield, >99% e.e.). Unfortunately, additional attempts to synthesize 3-substituted and 2,3- or 2,4-disubstituted oxetanes from the corresponding γ-haloalcohols were not successful due to the undetectable catalytic efficiency of all the HheD8 enzymes (Supplementary Fig. 8).

Fig. 5: Scope of the biocatalytic enantioselective dehalogenation of γ-haloalcohols.
figure 5

General reaction conditions: 100 mL PB buffer (50 mM, pH 8.5), 10 g dcw/L E. coli (HheD8-M4) cells, 2 mmol (20 mM) substrates a 30 °C. The isolated yields were obtained by silica gel chromatography, and the e.e. values were determined by chiral HPLC or GC. Enantioselectivity (E) values were calculated using the e.e. values of the γ-haloalcohol substrates and the oxetane products. See section 5 in the Supplementary Materials for details.

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Scope for enantioselective ring-opening of oxetanes

We next turned our attention to the substrate generality for the enantioselective ring-opening of oxetanes (Fig. 6, Supplementary Table 8). As expected, aromatic substituted oxetanes (1-14b) with mono-substituent at the para-, meta-, and ortho positions were well tolerated, affording the corresponding chiral (R)-γ-azidoalcohols (41-50% yield, 91- > 99% e.e.) and (S)-oxetanes (30-49% yield, 86- > 99% e.e.) with good to excellent enantioselectivities (E = 72- > 200). The electronic characteristics of the substituents show a modest effect on both the efficiency and enantioselectivity of the ring-opening reaction. Oxetane 15b, bearing two fluoro meta-substituents, was also successfully accommodated to yield the chiral (R)-15c and (S)-15b with >99% e.e. and high yields. Additionally, efficient ring-opening of sterically hindered oxetanes 16b and 17b was achieved using the mutants HheD8-M3 and HheD8-M7, respectively. Moreover, heterocyclic oxetanes containing pyrimidine (18b) or quinoline (19b) served as competent substrates for the mutant HheD8-M4. Alkyl oxetane 21b was also smoothly converted to deliver the corresponding chiral alkyl γ-azidoalcohol (R)-21c and oxetane (S)-21b. As expected, further attempts at the ring-opening reactions of 3-substituted and 2,3-disubstituted oxetanes were not successful (Supplementary Fig. 9). Furthermore, we challenged the ring-opening reaction with other anionic nucleophiles. The reaction accepted the nucleophilic cyanide, generated in situ from mandelonitrile61, yielding the chiral γ-cyanoalcohol (R)-1d with 46% yield and >99% e.e. The nitrite demonstrated as an ambident nucleophile, leading to the formation of chiral γ-nitroalcohol (R)-1e and γ-diol (R)-1f through attack by its nitrogen and oxygen atoms, respectively. Cyanate and thiocyanate were also examined but exhibited very low reactivity and enantioselectivity in the ring-opening reactions (Supplementary Table 9). Further efforts in enzyme screening and protein engineering are required to facilitate oxetane ring-opening reactions utilizing these nucleophilic agents.

Fig. 6: Scope of the biocatalytic enantioselective ring-opening of oxetanes.
figure 6

General reaction conditions: 100 mL PB buffer (50 mM, pH 7.5), 10 g dcw/L E. coli (HheD8-M4) cells, 2 mmol (20 mM) substrates b, 2 mmol (20 mM) NaN3, 30 °C. The isolated yields were obtained by silica gel chromatography, and the e.e. values were determined by chiral HPLC or GC. Enantioselectivity (E) values were calculated using the e.e. values of the oxetane substrates and the γ-substituted alcohol products. See section 6 in the Supplementary Materials for details.

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Large-scale reactions

Substrate tolerance is a critical metric for assessing the effectiveness of biocatalytic approaches. To demonstrate its practical applicability, the biocatalytic platform was then evaluated at higher substrate concentrations within a biphasic system (PB buffer: n-hexane=5:1), employing the variant E. coli (HheD8-M4) whole cells as biocatalysts. The enantioselective dehalogenation reactions of 40-140 mM (rac)-1a proceeded smoothly to completion with 47-50% conversions over a period of 3-48 h (Supplementary Table 10). The decrease in reaction pH, caused by proton release at higher substrate concentrations, may diminish the reaction efficiency. By maintaining the reaction pH at 8.5 ± 0.1 through the addition of aqueous sodium hydroxide solution, a large-scale reaction to convert 200 mM of (rac)-1a (20 mmol, 34 g/L) was performed and completed smoothly within 33 h (Fig. 7a). For the enantioselective ring-opening of 40-200 mM oxetane (rac)-1b, reactions consistently reached 50% conversions within 12-36 h (Supplementary Table 11). A large-scale reaction of 200 mM (rac)-1b (20 mmol, 27 g/L) was successfully conducted to completion after 39 h (Fig. 7a). Notably, in both the large-scale dehalogenation and ring-opening reactions, all chiral compounds were obtained in gram quantities, achieving high isolated yields (39-49%) and excellent enantiopurities (97- > 99% e.e.). These results substantiate that the biocatalytic platform accommodates large-scale synthesis at high substrate concentrations, thereby emphasizing its synthetic potential for industrial applications.

Fig. 7: Synthetic applicability, flexibility, and scalability of the biocatalytic platform.
figure 7

a Biocatalytic enantioselective dehalogenation of (rac)−1a and ring-opening of (rac)−1b are scaled up to gram-scale reactions. b Biocatalytic sequential dehalogenation and ring-opening reactions are conducted in a one-pot cascade manner. c Synthetic applications of chiral products are exemplified by representative transformations. See section 7-9 in the Supplementary Materials for detailed rection conditions.

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Biocatalytic cascades and enantiocomplementary synthesis

The favorable compatibility of enzymatic reactions facilitates their application in biocatalytic cascade processes, thus omitting the need for intermediate purification and isolation steps68. We subsequently explored the feasibility of integrating and executing both the formation and ring-opening reactions of oxetanes in a one-pot, one-catalyst cascade system. This setup was envisaged to enable the enantioselective transformation of γ-haloalcohols into γ-azidoalcohols via the un-isolated oxetane intermediates. As depicted in Fig. 7b, three γ-haloalcohols (1a, 6-7a) underwent evaluation using E. coli (HheD8-M4) cells. The results indicated that all reactions proceeded efficiently, resulting in the formation of both chiral γ-azidoalcohols and γ-haloalcohols with excellent isolated yields (47-50%) and as single enantiomers ( > 99% e.e.). In addition, we attempted to invert the enantioselectivity of HheD8-M4 through further rounds of ISM on six residues, chosen based on the docking pose of HheD8-M3/(R)-1a (Supplementary Fig. 10). This effort led to the creation of an S-enantioselective mutant, HheD8-M12 (A69N/S71P/A119T/M124W/R127T/R182W), which generated (S)-1b with 92% e.e. (Supplementary Table 12). Further substrate scope studies indicated that HheD8-M12 exhibited low to excellent enantioselectivity in both the formation and ring-opening of oxetanes (E = 10- > 200), although its catalytic efficiency remained lower compared to HheD8-M4 (Supplementary Fig. 11). The achievement of biocatalytic cascades and inversion of the enzyme’s enantioselectivity further highlighted the operational flexibility of the biocatalytic platform.

Transformations of chiral products

The developed biocatalytic platform enables the synthesis of both (R)- and (S)-enantiomers of oxetanes as well as various chiral γ-substituted alcohols. These provide a versatile platform for synthesizing many valuable compounds, particularly serving as key intermediates and functional groups in bioactive molecules. Representative transformations were subsequently carried out using the enzymatically synthesized chiral products (Fig. 7c). The γ-chloroalcohol (S)-1a can be transformed into the chiral (R)-3-phenylisoxazolidine (1aa), an important heterocyclic motif found in anticancer agents69. Smooth conversion of the chiral (S)-1a to furnish (S)-1ab was also realized, providing a key precursor for the synthesis of (R)-dapoxetine70. Additionally, the chiral oxetane (R)-1b was successfully converted into (R)-2-phenyltetrahydrofuran (1ba) through a straightforward ring-expansion reaction71. We also performed the conversion of (R)-1b into the chiral (R)-1bb, a crucial precursor in the synthesis of (R)-tomoxetine72. Moreover, the presence of an azide group enables copper-catalyzed click chemistry73, allowing for the modification of γ-azidoalcohol (R)-1c with a simple alkyne to afford the chiral γ-hydroxytriazole (R)-1ca. The reaction of (R)-1c with a symmetrical ketone was also performed to deliver medium-sized ring lactam (R)-1cb, a commonly important intermediate in the synthesis of nitrogen-containing compounds74. The absolute stereochemistry of (R)-1cb was ascertained by single-crystal X-ray diffraction analysis (Supplementary Table 13), which also demonstrated R-enantioselectivity during the enzymatic dehalogenation and ring-opening reactions. Furthermore, the transformations of chiral γ-cyanoalcohol (R)-1d yielded the chiral lactone (R)-1da and the γ-hydroxyamide (R)-1db, both serving as building blocks in the synthesis of (R)-fluoxetine and (R)-norfluoxetine75. For the nitrite-mediated ring-opening products, the γ-nitroalcohol (R)-1e can be readily reduced to generate the chiral γ-aminoalcohol (R)-1ea76. On the other hand, esterification of chiral γ-diol (R)-1f yielded a key intermediate, (R)-1fa, for the synthesis of natural piperidine alkaloids77. Taken together, these representative transformations resulting in the generation of useful derivatives showcased the synthetic scalability of the biocatalytic platform.

Discussion

In summary, we developed a biocatalytic platform capable of the enantioselective formation as well as ring-opening of oxetanes. This biocatalytic method exhibited high catalytic efficiency, excellent enantioselectivity, and broad substrate scopes, thereby enabling the preparative-scale synthesis of chiral oxetanes and a wide range of chiral γ-substituted alcohols, spanning γ-haloalcohol, γ-azidoalcohol, γ-cyanoalcohol, γ-nitroalcohol, and γ-diol classes. Additionally, successful transformations of these chiral products into various valuable chiral compounds further showcase the platform’s synthetic scalability. Moreover, both the enzymatic enantioselective formation and ring-opening reactions of oxetanes have been scaled up to large-scale synthesis at high substrate concentrations, emphasizing the practical applicability of the platform. Furthermore, the integration of the two enzymatic processes within a one-pot, one-catalyst cascade system illustrates the operational flexibility of the biocatalytic approach. Further structural analysis and MD simulations provided potential insights into the origins of the enzyme’s activity and enantioselectivity. Overall, this work endows HHDH with additional non-natural functionalities, significantly expanding their catalytic repertoire in the synthesis of synthetically useful chiral molecules. We anticipate that this abiological system will find potential applications in pharmaceutical manufacturing and drug discovery.

Methods

Biocatalytic enantioselective formation of oxetanes

General procedure: In a 200 mL round-bottom flask, a resting cell suspension of E. coli (HheD8-M4) at a concentration of 10 g dcw/L was prepared in 100 mL of PB buffer (50 mM, pH 8.5). To this suspension, 2 mmol of γ-haloalcohol (rac)-a was added to a final concentration of 20 mM. The reaction mixture was then stirred at 30 °C. Upon completion of the enzymatic reaction, the mixture was subjected to extraction using ethyl acetate (3 × 70 mL). The organic phases were separated by centrifugation (8800 × g, 3 min), combined, dried over anhydrous Na2SO4, and evaporated at reduced pressure. The resulting mixture was purified by flash chromatography (petroleum ether: ethyl acetate = 50:1 ~ 20:1; dichloromethaneethyl: ethyl acetate = 6:1 ~ 3:1) on silica gel to afford the desired chiral oxetane (R)-b and γ-haloalcohol (S)-a.

Biocatalytic enantioselective ring-opening of oxetanes

General procedure: In a 200 mL round-bottom flask, a resting cell suspension of E. coli (HheD8-M4) at a concentration of 10 g dcw/L was prepared in 100 mL of PB buffer (50 mM, pH 7.5). To this suspension, 2 mmol of oxetane (rac)-b and 2 mmol of NaN3 were added to a final concentration of 20 mM. The reaction mixture was then stirred at 30 °C. Upon completion of the enzymatic reaction, the mixture was subjected to extraction using ethyl acetate (3 × 70 mL). The organic phases were separated by centrifugation (8800 × g, 3 min), combined, dried over anhydrous Na2SO4, and evaporated at reduced pressure. The resulting mixture was purified by flash chromatography (petroleum ether: ethyl acetate = 50:1 ~ 1:1) on silica gel to afford the desired chiral oxetane (S)-b and γ-azidoalcohol (R)-c.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.