Biocatalytic enantioselective formation and ring-opening of oxetanes

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Nevertheless, the currently narrow spectrum of enzymatic reactions available imposes limitations on synthesizing diverse desired compounds. Consequently, there continues to be a high demand for developing novel biocatalytic processes to access reactions that were previously unattainable. Herein, we report the discovery and subsequent protein engineering of a unique halohydrin dehalogenase to develop a biocatalytic platform for enantioselective formation and ring-opening of oxetanes. This biocatalytic platform, exhibiting high efficiency, excellent enantioselectivity, and broad scopes, facilitates the preparative-scale synthesis of not only both enantiomers of chiral oxetanes (up to 49% yield, >99 e.e.) but also a variety of chiral γ-substituted alcohols (up to 53% yield, >99 e.e.). Additionally, both the enantioselective oxetane formation and ring-opening processes have been proven scalable for large-scale transformations (20 mmol) at high substrate concentrations (200 mM), and can be integrated efficiently in a one-pot, one-catalyst cascade system. Moreover, useful derivatizations highlight the potential synthetic applications of the biocatalytic platform. This work expands the enzymatic toolbox for non-natural reactions and will promote further exploration of the catalytic repertoire of halohydrin dehalogenases in synthetic and pharmaceutical chemistry. Physical sciences/Chemistry/Catalysis/Biocatalysis Biological sciences/Chemical biology/Biocatalysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Biocatalysis, harnessing enzymes to drive organic transformations, is a powerful approach with broad applications across many fields 1 . 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 enzymes 2 , 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 evolution 3 . 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 nature 4 – 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 nature 10 . Although the oxetane motif is relatively rare in natural products and metabolites 11 , 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. 1 a), a well-known anticancer drug 13 . 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 stability 14 . A recent example is the development of danuglipron (Fig. 1 a), a potent GLP-1R agonist (glucagon-like peptide-1 receptor) designed for the treatment of diabetes 15 . Furthermore, the intrinsic strain associated with the oxetane ring imparts distinctive reactivity patterns 10 , which can be harnessed to advance synthetic applications (Fig. 1 a). These includes their use as versatile precursors for the synthesis of an array of organic compounds 16 , 17 , their role as pivotal intermediates in the total synthesis of natural products 18 , 19 , their value as monomers in polymer construction 20 , 21 , and their function as potential scaffolds for protein modification 22 , 23 . Undoubtedly, the exploration of synthetic methodologies for the synthesis and transformation of oxetane-related compounds has consistently represented a vibrant area of research 10 , 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 strategies 10 . Therefore, the development of practical and efficient methodologies for constructing and manipulating oxetane stereocenters remains remains a crucial yet formidable endeavor in modern chemical research. Halohydrin dehalogenase (HHDH), first reported by Castro and Bartnicki in 1968 25 , belongs to the superfamily of short-chain dehydrogenases/reductases 26 . 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., N 3 − , CN − , and NO 2 − ). The Janssen and other research groups have conducted extensive investigations on the catalytic mechanisms, structural features, and synthetic applications of HHDHs 27 – 30 , particularly focusing on the enantioselective formation and ring-opening of epoxides (Fig. 1 b). Moreover, scientists at Codexis have exploited the promiscuous functions of HHDHs for the industrial production of ethyl ( R )-4-cyano-3-hydroxybutyrate, a key precursor to atorvastatin 31 . In recent years, our laboratory has also successfully expanded the catalytic repertoire of HHDHs, enhancing their utility in the stereoselective transformations of epoxides 32 – 36 . The biosynthesis of oxetane-containing natural products proves that natural enzymes indeed facilitate synthetic pathways for constructing oxetane motifs 11 , 12 . However, the specific enzymes responsible for these biotranformations remain elusive, as they have not yet been identified or are involved in complex reaction processes 37 – 39 , 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. 1 c). 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. Pursuit of this objective has the potential to unveil previously unknown enzymatic functionalities, establishing a practical biocatalytic approach for the synthesis of chiral oxetanes and a diverse range of chiral γ-substituted alcohols (Fig. 1 d). Results and discussion 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 collection 32 – 36 , 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. 2 a). 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. 2 a). 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 achieved 40 , 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. 2 b). 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. 2 b). 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. 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. In an attempt to enhance both the catalytic efficiency and enantioselectivity for the formation and ring-opening reactions of oxetane, we strategically targeted eight amino acid residues (F19, A69, Y168, M124, R127, Q160, N161 and R182) lining the active site pocket (Fig. 2 c). Sequential rounds of site-saturation mutagenesis (SSM) were carried out on these selected target residues, with the resulting variants being screened by whole-cell biotransformation for enhanced enantioselectivities and/or substrate conversions. In each round, 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, four rounds of iterative SSM led to the identification of beneficial variants: HheD8-M1 (A69F), HheD8-M2 (A69F/R127G), HheD8-M3 (A69F/M124P/R127G), and HheD8-M4 (A69F/M124P/R127G/R182W). These variants exhibited remarkably improved enantioselectivity and/or catalytic activity compared to the HheD8-WT in the dehalogenation of ( rac )- 1a (Fig. 2 d). 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. 2 e), the mutant HheD8-M1 (A69F) was also identified in the first round of SSM and exhibited exceptional enantioselectivity in stark contrast to the HheD8-WT ( E > 200 vs E = 8). Using the HheD8-M1 variant as the parent enzyme, three additional rounds of iterative SSM 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 (Fig. 2 e). 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 of mutant HheD8-M3 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). 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). Through detailed comparison of these mutated residues (Extended Data Fig. 1 a), 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. 2 c). Mutations M124P and R127G introduce smaller amino acid residues, enlarging the active site pocket, which is thought to enhance catalytic efficiency (Extended Data Fig. 1 b). This speculation is supported by the observed stepwise increase in activity for the mutants HheD8-M2 and HheD8-M3, following the sequential introduction of these mutations (Fig. 2 c). 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. 3 , Supplementary Table 6). 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 HheD-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-disittuted 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 tolerance toward the alkyl-substituted substrate 21a , affoding chiral alkyl oxetane ( R )- 21b (55% yield, > 99% e.e.) and alkyl γ-chloroalcohol ( S )- 21a (48% yield, > 99% e.e.). Furthermore, the biocatalytic system was applied to the enanioselective generation of chiral γ-bromoalcohol ( S )- 22a (50% yield, > 99% e.e) and γ-iodoalcohol ( S )- 23a (48% yield, > 99% e.e). Scope for enantioselective ring-opening of oxetanes We next turned our attention to the substrate generality for the enantioselective ring-opening of oxetanes (Fig. 4 , Supplementary Table 7). 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 contaning 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 . Furthermore, we challenged the ring-opening reaction with other anionic nucleophiles. The reaction accepted the nucleophilic cyanide, generated in situ from mandelonitrile 36 , 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 8). Further efforts in enzyme screening and protein engineering are required to facilitate oxetane ring-opening reactions utilizing these nucleophilic agents. 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 (Extended Data Fig. 2 , Supplementary Table 9). 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. 5 a). For the enantioselective ring-opening of 40–200 mM oxetane ( rac )- 1b , reactions consistently reached 50% conversions within 12–36 h (Extended Data Fig. 3 , Supplementary Table 10). A large-scale reaction of 200 mM ( rac )- 1b (20 mmol, 27 g/L) was successfully conducted to completion after 39 h (Fig. 5 a). 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 while maintaining catalytic efficiency and enantioselectivity, thereby emphasizing its synthetic potential for industrial applications. Biocatalytic cascade reactions The favorable compatibility of enzymatic reactions facilitates their application in biocatalytic cascade processes, thus omitting the need for intermediate purification and isolation steps 41 . 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 unisolated oxetane intermediates. As depicted in Fig. 5 b, 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.). The successful integration of the biocatalytic cascade reactions further highlighted the operational flexibility of the biocatalytic platform. Transfomations 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. 5 c). The γ-chloroalcohol ( S )- 1a can be transformed into the chiral ( R )-3-phenylisoxazolidine ( 1aa ), an important heterocyclic motif found in anticancer agents 42 . Smooth conversion of the chiral ( S )- 1a to furnish ( S )- 1ab was also realized, providing a key precursor for the synthesis of ( R )-dapoxetine 43 . Additionally, the chiral oxetane ( R )- 1b was successfully converted into ( R )-2-phenyltetrahydrofuran ( 1ba ) through a straightforward ring-expansion reaction 44 . We also performed the conversion of ( R )- 1b into the chiral ( R )- 1bb , a crucial precursor in the synthesis of ( R )-tomoxetine 45 . Moreover, the presence of an azide group enables copper-catalyzed click chemistry 46 , 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 aslo performed to deliver medium-sized ring lactam ( R )- 1cb , a commonly important intermediate in the synthesis of nitrogen-containing compounds 47 . The absolute stereochemistry of ( R )- 1cb was ascertained by single-crystal X-ray diffraction analysis (Supplementary Table 11), 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 )-norfluoxetine 48 . For the nitrite-mediated ring-opening products, the γ-nitroalcohol ( R )- 1e can be readily reduced to generate the chiral γ-aminoalcohol ( R )- 1ea 49 . On the other hand, esterification of chiral γ-diol ( R )- 1f yielded a key intermediate, ( R )- 1fa , for the systhesis of natural piperidine alkaloids 50 . Taken together, these representative transformations resulting in the generation of useful derivatives showcased the synthetic scalability of the biocatalytic platform. Conclusion In summary, our findings constitute the inaugural 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 both ( R )- and ( S )-enantiomers of oxetanes, in addition to a variety 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. Overall, this work endows HHDH with new 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, drug discovery, and synthetic biology. Declarations Data availability All data are available within the paper and its Supplementary Information (detailed methods, NMR spectra, chiral HPLC/GC traces, Supplementary Figures and Tables). Crystallographic data of compound ( R )- 1cb are available free of charge from the Cambridge Crystallographic Data Centre (CCDC; https://www.ccdc.cam.ac.uk/structures/) reference number 2350047. The coordinate files and structure factors have been deposited in the PDB with accession number 8XXB. Data collection statistics are provided in Extended Data Table 1. Acknowledgments We appreciate the financial support from the National Natural Science Foundation of China (22167023 to W. N.W.), the Program for Technology Elite of Zunyi Medical University (ZYSE-2022-03 to W.N.W.), and the Science and Technology Department of Zunyi (No. ZSKRPT-2020-5 to Y.Z.C.). We also thank NovoPro Bioscience Inc. (Shanghai, China) for technical assistance in crystallization. Author contributions W.N.W. devised and supervised the project. X.H. and Y.F.W. performed most of the experiments. X.J., H.Y.Y and H.H.W. assisted in enzyme screening, synthetic experiments, crystallographic and docking studies. W.N.W., H.H.W. and Y.Z.C. wrote the manuscript and generated the figures. Competing interests The authors declare no competing interests. References Buller, R. et al. From nature to industry: harnessing enzymes for biocatalysis. Science 382 , eadh8615 (2023). Leveson-Gower, R. B., Mayer, C. & Roelfes, G. 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B., Majerić-Elenkov, M., Hasnaoui, G., Hauer, B. & Spelberg, J. H. L. Enantioselective formation and ring-opening of epoxides catalysed by halohydrin dehalogenases. Biochem. Soc. T. 34 , 291-295 (2006). You, Z.-Y., Liu, Z.-Q. & Zheng, Y.-G. Properties and biotechnological applications of halohydrin dehalogenases: current state and future perspectives. Appl. Microbiol. Biot. 97 , 9-21 (2013). Schallmey, A. & Schallmey, M. Recent advances on halohydrin dehalogenases—from enzyme identification to novel biocatalytic applications. Appl. Microbiol. Biot. 100 , 7827-7839 (2016). Findrik Blažević, Z., Milčić, N., Sudar, M. & Majerić Elenkov, M. Halohydrin dehalogenases and their potential in industrial application – a viewpoint of enzyme reaction engineering. Adv. Synth. Catal. 363 , 388-410 (2021). Fox, R. J. et al. Improving catalytic function by ProSAR-driven enzyme evolution. Nat. Biotechnol. 25 , 338-344 (2007). Zhang, F.-R. et al. Enzymatic kinetic resolution of bulky spiro-epoxyoxindoles via halohydrin dehalogenase-catalyzed enantio- and regioselective azidolysis. ACS Catal. 11 , 9066-9072 (2021). Xu, Q. et al. Stereodivergent synthesis of epoxides and oxazolidinones via the halohydrin dehalogenase-catalyzed desymmetrization strategy. ACS Catal. 12 , 6285-6293 (2022). Wang, H.-H. et al. Identification and structure analysis of an unusual halohydrin dehalogenase for highly chemo-, regio- and enantioselective bio-nitration of epoxides. Angew. Chem. Int. Ed. 61 , e202205790 (2022). Ma, R. et al. Biocatalytic thionation of epoxides for enantioselective synthesis of thiiranes. Angew. Chem. Int. Ed. 61 , e202212589 (2022). Guan, X.-E. et al. Biocatalytic enantioselective synthesis of chiral β-hydroxy nitriles using cyanohydrins as cyano sources. ACS Catal. 13 , 13597-13606 (2023). Meng, S., Tang, G.-L. & Pan, H.-X. Enzymatic formation of oxygen-containing heterocycles in natural product biosynthesis. ChemBioChem 19 , 2002-2022 (2018). Zhao, Y. et al. Oxetane ring formation in taxol biosynthesis is catalyzed by a bifunctional cytochrome P450 Enzyme. J. Am. Chem. Soc. 146 , 801-810 (2023). Jiang, B. et al. Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III. Science 386 , 622-629 (2024). Coppi, D. I., Salomone, A., Perna, F. M. & Capriati, V. 2-Lithiated-2-phenyloxetane: a new attractive synthon for the preparation of oxetane derivatives. Chem. Commun. 47 (2011). Benítez-Mateos, A. I., Roura Padrosa, D. & Paradisi, F. Multistep enzyme cascades as a route towards green and sustainable pharmaceutical syntheses. Nat. Chem. 14 , 489-499 (2022). Lee, Y. H. et al. Preparation of heteroaryl derivative as anticancer. WO2022245085A1 (2022). Bai, J., Wu, J., Liu, S., Li, Y. & Zhang, W. Preparation method of 3-(1-naphthyloxy)-1-phenylpropanol. CN108083991 (2018). Butova, E. D. et al. Stereospecific consecutive epoxide ring expansion with dimethylsulfoxonium methylide. J. Org. Chem. 75 , 6229-6235. Bertolini, F., Crotti, S., Bussolo, V. D., Macchia, F. & Pineschi, M. Regio- and stereoselective ring opening of enantiomerically enriched 2-aryl oxetanes and 2-aryl azetidines with aryl borates. J. Org. Chem. 73 , 8998-9007 (2008). Hein, J. E. & Fokin, V. V. Copper-catalyzed azide–alkyne cycloaddition (CuAAC) and beyond: new reactivity of copper(i) acetylides. Chem. Soc. Rev. 39 , 1302-1315 (2010). Ribelin, T. P. & Aubé, J. Synthesis of enantiomerically enriched ( R )-5-tert-butylazepan-2-one using a hydroxyalkyl azide mediated ring-expansion reaction. Nat. Protoc. 3 , 137-143 (2008). Hilborn, J. W. et al. A practical asymmetric synthesis of ( R )-fluoxetine and its major metabolite ( R )-norfluoxetine. Tetrahedron Lett. 42 , 8919-8921 (2001). Otevrel, J. & Bobal, P. Diamine-tethered bis(thiourea) organocatalyst for asymmetric henry reaction. J. Org. Chem. 82 , 8342-8358 (2017). Yadav, J. S., Reddy, M. S., Rao, P. P. & Prasad, A. R. Enantioselective synthesis of (+)-sedamine and (-)-allosedamine. Synthesis 2006 , 4005-4012 (2006). Additional Declarations There is NO Competing Interest. Supplementary Files checkcifforR1cb.pdf checkcif for compound (R)-1cb R1cb.cif cif file for compound (R)-1cb SupplementaryInformation.pdf Supplementary Information ExtendedDataFigsandTable.docx Cite Share Download PDF Status: Published Journal Publication published 30 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4316588","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Physical Sciences - Article","associatedPublications":[],"authors":[{"id":296645700,"identity":"77004b40-b64f-4486-aa8d-cc2d91be9037","order_by":0,"name":"Nan-Wei Wan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYNACAyBmbwAzGRuI18JzgCQtICCRQKQWc/azxyQ+FByWN5d8/nQzD4ON7IYDzM8e4NNi2ZOXJjnD4LDhztkJabd5GNKMNxxgMzfAp8XgQI7ZbR6D24wbbiccA2o5nLjhAA+bBF4t59+Y3f5jcNt+w82DbUAt/4nQcgNoC4PB7cQNN5jZgFoOEKPljfnPHoP/yRvOpLHdnGOQbDzzMJsZAYflGBv8+JNmu+H48Wc33lTYyfYdb36GVwu6CUDMTIL6UTAKRsEoGAXYAQCki05c4QI/EAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3561-8380","institution":"Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Nan-Wei","middleName":"","lastName":"Wan","suffix":""},{"id":296645701,"identity":"416ea965-40de-4e30-b610-e22374cdb2d9","order_by":1,"name":"Xia Hua","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Hua","suffix":""},{"id":296645702,"identity":"1b4bfb3a-8634-416c-a81d-c12bd93e7cf7","order_by":2,"name":"Yuan-Fei Wang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuan-Fei","middleName":"","lastName":"Wang","suffix":""},{"id":296645703,"identity":"6c8ddaf6-0bf3-4665-9b8f-9ae882d280e9","order_by":3,"name":"Xiao Jin","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Jin","suffix":""},{"id":296645704,"identity":"7c745d55-a171-41b9-a2a1-76b560bc2eed","order_by":4,"name":"Hong-Yin Yu","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hong-Yin","middleName":"","lastName":"Yu","suffix":""},{"id":296645705,"identity":"22a9f2c1-b389-4e97-9009-197277317f29","order_by":5,"name":"Hui-Hui Wang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hui-Hui","middleName":"","lastName":"Wang","suffix":""},{"id":296645706,"identity":"5bbdaa65-68b4-47e4-9fd5-3a566e92387a","order_by":6,"name":"Yong-Zheng Chen","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yong-Zheng","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-04-24 08:25:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4316588/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4316588/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-56463-z","type":"published","date":"2025-01-30T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56591088,"identity":"32ef79eb-3d75-4ff9-93f0-cff84cb42195","added_by":"auto","created_at":"2024-05-16 09:03:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":428508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of a biocatalytic platform for enantioselective formation and ring-opening of oxetanes.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e The oxetane scaffold is a privileged substructure in natural bioactive products, drug discovery and synthetic chemistry.\u003cstrong\u003e b,\u003c/strong\u003e Previous studies have exploited the HHDHs’ catalytic repertoire for the enantioselective formation and ring-opening of epoxides.\u003cstrong\u003e c,\u003c/strong\u003e The architectural features of epoxide and oxetane motifs are compared to highlight the similarities and differences in their chemical structures.\u003cstrong\u003e d,\u003c/strong\u003e This work expands the HHDHs’ catalytic repertoire for the enantioselective formation and ring-opening of oxetanes.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/fa20d8928192c2b296bff14a.jpg"},{"id":56590509,"identity":"a10c8d72-3e13-407d-8da9-bdd5d406d602","added_by":"auto","created_at":"2024-05-16 08:55:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":725799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification and directed evolution of HheD8.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Enzymes are screened for the dehalogenation of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e. See Supplementary Table 2 for details. \u003cstrong\u003eb,\u003c/strong\u003e Enzymes are screened for the ring-opening of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e with azide. See Supplementary Table 3 for details. \u003cstrong\u003ec,\u003c/strong\u003e The possible docking pose of (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1b \u003c/strong\u003ein the active site of the predicted HheD8-WT/AF model structure is illustrated. Catalytic triad S117-Y130-R134, the hot-spot residues, and (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e are highlighted in sticks or spheres.H-bonds are displayed in dashed lines. \u003cstrong\u003ed, \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003eHheD8 is engineered for enantioselective dehalogenation of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e. See Supplementary Table 4 for details. \u003cstrong\u003ee,\u003c/strong\u003e The\u003cstrong\u003e \u003c/strong\u003eHheD8 is engineered for enantioselective ring-opening of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e with azide. See Supplementary Table 5 for details. NR= no reaction. ND= not detected.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/9cf3eafbfc5971577d120378.jpg"},{"id":56591437,"identity":"0c17fda2-fb6a-4178-93be-4cedf637c203","added_by":"auto","created_at":"2024-05-16 09:11:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":799518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScope of the biocatalytic enantioselective dehalogenation of γ-haloalcohols.\u003c/strong\u003e General reaction conditions: 100 mL PB buffer (50 mM, pH 8.5), 10 g dcw/L \u003cem\u003eE.coli\u003c/em\u003e (HheD8-M4) cells, 2 mmol (20 mM)\u003cstrong\u003e \u003c/strong\u003esubstrates \u003cstrong\u003ea\u003c/strong\u003e, 30 °C. The isolated yields were obtained by silica gel chromatography, and the e.e. values were determined by chiral HPLC or GC. Enantioselectivity (\u003cem\u003eE\u003c/em\u003e) values were calculated using the e.e. values of the γ-haloalcohol\u003cstrong\u003e \u003c/strong\u003esubstrates and the oxetane products. See section 5 in the Supplementary Materials for details.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/175bcac51e2616b882110852.jpg"},{"id":56590512,"identity":"e80371d4-22f7-46a4-87a3-9de10331a110","added_by":"auto","created_at":"2024-05-16 08:55:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":786369,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScope of the biocatalytic enantioselective ring-opening of oxetanes.\u003c/strong\u003e General reaction conditions: 100 mL PB buffer (50 mM, pH 7.5), 10 g dcw/L \u003cem\u003eE.coli\u003c/em\u003e (HheD8-M4) cells, 2 mmol (20 mM) substrates\u003cstrong\u003e b\u003c/strong\u003e, 2 mmol (20 mM) NaN\u003csub\u003e3\u003c/sub\u003e, 30 °C. The isolated yields were obtained by silica gel chromatography, and the e.e. values were determined by chiral HPLC or GC. Enantioselectivity (\u003cem\u003eE\u003c/em\u003e) 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.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/1b36c6eb683ae8fe1aa5ee06.jpg"},{"id":56590513,"identity":"3804670a-35da-4817-b360-d2b19b673816","added_by":"auto","created_at":"2024-05-16 08:55:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":621527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthetic applicability, flexibility, and scalability of the biocatalytic platform.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Biocatalytic enantioselective dehalogenation of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e and ring-opening of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e are scaled up to gram-scale reactions. \u003cstrong\u003eb\u003c/strong\u003e, Biocatalytic sequential dehalogenation and ring-opening reactions are conducted in a one-pot cascade manner. \u003cstrong\u003ec\u003c/strong\u003e, Synthetic applications of chiral products are exemplified by representative transformations. See section 7-9 in the Supplementary Materials for detailed rection conditions.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/915412d8d2e8a92edc121acd.jpg"},{"id":75148587,"identity":"25311192-8f56-4e84-8982-e607773bd763","added_by":"auto","created_at":"2025-01-31 08:05:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4423559,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/0f635e68-fcd2-4195-8de7-6e333f14a312.pdf"},{"id":56590507,"identity":"77c01cdd-51c1-4235-8fe5-165da8ec65e4","added_by":"auto","created_at":"2024-05-16 08:55:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":74778,"visible":true,"origin":"","legend":"checkcif for compound (R)-1cb","description":"","filename":"checkcifforR1cb.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/f22e2f400f8b9918d9107ae1.pdf"},{"id":56591087,"identity":"ef79b409-7a80-4cef-a796-a1b702add759","added_by":"auto","created_at":"2024-05-16 09:03:38","extension":"cif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":294510,"visible":true,"origin":"","legend":"cif file for compound (R)-1cb","description":"","filename":"R1cb.cif","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/e3df795183637275929e4de3.cif"},{"id":56590515,"identity":"6413f2b4-909e-4ece-9b64-e16e037482a4","added_by":"auto","created_at":"2024-05-16 08:55:39","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21435184,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/6fab56a8ffa5bc955906c50f.pdf"},{"id":56590514,"identity":"4f5f03c7-4d79-4d8e-bc0e-d5e99e4cc46a","added_by":"auto","created_at":"2024-05-16 08:55:39","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12895635,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigsandTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-4316588/v1/e92d8717c388e01bca52073f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Biocatalytic enantioselective formation and ring-opening of oxetanes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiocatalysis, harnessing enzymes to drive organic transformations, is a powerful approach with broad applications across many fields\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. 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 enzymes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, 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 evolution\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. 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 nature\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOxetane, a four-membered heterocyclic compound characterized by the incorporation of an oxygen atom, is notably significant due to its distinctive structural features and reactive nature\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Although the oxetane motif is relatively rare in natural products and metabolites\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea), a well-known anticancer drug\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. 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 stability\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. A recent example is the development of danuglipron (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea), a potent GLP-1R agonist (glucagon-like peptide-1 receptor) designed for the treatment of diabetes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, the intrinsic strain associated with the oxetane ring imparts distinctive reactivity patterns\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, which can be harnessed to advance synthetic applications (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). These includes their use as versatile precursors for the synthesis of an array of organic compounds\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, their role as pivotal intermediates in the total synthesis of natural products\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, their value as monomers in polymer construction\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and their function as potential scaffolds for protein modification\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Undoubtedly, the exploration of synthetic methodologies for the synthesis and transformation of oxetane-related compounds has consistently represented a vibrant area of research\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. 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 strategies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, the development of practical and efficient methodologies for constructing and manipulating oxetane stereocenters remains remains a crucial yet formidable endeavor in modern chemical research.\u003c/p\u003e\n\u003cp\u003eHalohydrin dehalogenase (HHDH), first reported by Castro and Bartnicki in 1968\u003csup\u003e25\u003c/sup\u003e, belongs to the superfamily of short-chain dehydrogenases/reductases\u003csup\u003e26\u003c/sup\u003e. HHDHs exhibit intriguing catalytic promiscuity, mediating not only the dehalogenation of \u0026beta;- haloalcohols to form epoxides with the release of halide ions but also the ring-opening of epoxides into \u0026beta;-substituted alcohols in the presence of several negatively charged nucleophiles (e.g., N\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, CN\u003csup\u003e\u0026minus;\u003c/sup\u003e, and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e). The Janssen and other research groups have conducted extensive investigations on the catalytic mechanisms, structural features, and synthetic applications of HHDHs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, particularly focusing on the enantioselective formation and ring-opening of epoxides (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Moreover, scientists at Codexis have exploited the promiscuous functions of HHDHs for the industrial production of ethyl (\u003cem\u003eR\u003c/em\u003e)-4-cyano-3-hydroxybutyrate, a key precursor to atorvastatin\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In recent years, our laboratory has also successfully expanded the catalytic repertoire of HHDHs, enhancing their utility in the stereoselective transformations of epoxides\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe biosynthesis of oxetane-containing natural products proves that natural enzymes indeed facilitate synthetic pathways for constructing oxetane motifs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, the specific enzymes responsible for these biotranformations remain elusive, as they have not yet been identified or are involved in complex reaction processes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). The similar structural properties encourage us to envision the possibility of exploiting HHDHs for catalyzing the enantioselective dehalogenation of \u0026gamma;-haloalcohols and also the nucleophile-mediated enantioselective ring-opening of oxetanes. Pursuit of this objective has the potential to unveil previously unknown enzymatic functionalities, establishing a practical biocatalytic approach for the synthesis of chiral oxetanes and a diverse range of chiral \u0026gamma;-substituted alcohols (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\n\u003ch2\u003eScreening of HHDHs\u003c/h2\u003e\n\u003cp\u003eTo commence the study, we chose the dehalogenation of racemic 3-chloro-1-phenylpropan-1-ol (\u003cstrong\u003e1a\u003c/strong\u003e) and the azide-mediated ring-opening of racemic 2-phenyl-oxetane (\u003cstrong\u003e1b\u003c/strong\u003e) as the respective model reactions for dehalogenation and ring-opening processes. Dozens of recombinant \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) strains harboring HHDH enzymes (Supplementary Table\u0026nbsp;1), maintained in our laboratory collection\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, were initially evaluated for their catalytic efficiency in the dehalogenation model reaction (Supplementary Table\u0026nbsp;2). Background dehalogenation reaction was not observed either in the absence of \u003cem\u003eE. coli cells\u003c/em\u003e or with \u003cem\u003eE. coli\u003c/em\u003e cells that do not express HHDH (entries 1\u0026ndash;2, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). 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 \u003cstrong\u003e1b\u003c/strong\u003e\u0026thinsp;\u0026gt;\u0026thinsp;10% (entries 3\u0026ndash;10, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). Among them, the enzymes HheA5, HheC, and HheD8 exhibited a moderate to good enantioselectivity (\u003cem\u003eE\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13\u0026ndash;76). Although non-enzymatic conversion of \u0026gamma;-haloalcohols to oxetanes can be achieved\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, finding an enantioselective catalyst for this transformation remains elusive. Subsequently, the active HHDHs underwent further evaluation with the model ring-opening reaction (Supplementary Table\u0026nbsp;3). No background ring-opening reactions were detected (entries 1\u0026ndash;2, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). 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 \u003cem\u003eThauera aminoaromatica\u003c/em\u003e S2, also demonstrated moderate \u003cem\u003eR\u003c/em\u003e enantioselectivity (\u003cem\u003eE\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), resulting in the formation of \u0026gamma;-azidoalcohol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1c\u003c/strong\u003e with 74% e.e and 18% yield (entry 7, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eDirected evolution of HheD8\u003c/h2\u003e\n\u003cp\u003eGiven 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 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk\u003c/span\u003e\u003c/span\u003e, AFDB code: AF-N6YXW4-F1). The molecular docking of the oxetane (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e into the active site of the HheD8-WT/AF model was then performed to obtain a probable binding pose for subsequent protein engineering efforts. In an attempt to enhance both the catalytic efficiency and enantioselectivity for the formation and ring-opening reactions of oxetane, we strategically targeted eight amino acid residues (F19, A69, Y168, M124, R127, Q160, N161 and R182) lining the active site pocket (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). Sequential rounds of site-saturation mutagenesis (SSM) were carried out on these selected target residues, with the resulting variants being screened by whole-cell biotransformation for enhanced enantioselectivities and/or substrate conversions. In each round, variants exhibiting enhancements underwent further evaluation through separate model reactions: the dehalogenation of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e and the ring-opening of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e with azide.\u003c/p\u003e\n\u003cp\u003eIn the dehalogenation reaction screening, four rounds of iterative SSM led to the identification of beneficial variants: HheD8-M1 (A69F), HheD8-M2 (A69F/R127G), HheD8-M3 (A69F/M124P/R127G), and HheD8-M4 (A69F/M124P/R127G/R182W). These variants exhibited remarkably improved enantioselectivity and/or catalytic activity compared to the HheD8-WT in the dehalogenation of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). Notably, the quadruple-mutant HheD8-M4 excelled, achieving 49% conversion of 20 mM (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e and formation of (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e with 99% e.e. after 8 h. In the case of ring-opening reaction screening (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee), the mutant HheD8-M1 (A69F) was also identified in the first round of SSM and exhibited exceptional enantioselectivity in stark contrast to the HheD8-WT (\u003cem\u003eE\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;200 vs \u003cem\u003eE\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8). Using the HheD8-M1 variant as the parent enzyme, three additional rounds of iterative SSM 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 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). The exceptionally performant quadruple-mutant HheD8-M7 delivered (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1c\u003c/strong\u003e with \u0026gt;\u0026thinsp;99% e.e. and achieved 46% conversion of 20 mM (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e 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\u0026ndash;9 vs entry 6, Supplementary Table\u0026nbsp;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 (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e and produced (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1c\u003c/strong\u003e with 99% e.e. (entry 9 vs entry 6, Supplementary Table\u0026nbsp;5).\u003c/p\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eStructural analysis of mutant HheD8-M3\u003c/h2\u003e\n\u003cp\u003eFor 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 \u0026Aring; (PDB code: 8XXB, Supplementary Fig.\u0026nbsp;1). 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.\u0026nbsp;2), with a highly matched catalytic triad composed of residues (S117-Y130-R134). Through detailed comparison of these mutated residues (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea), 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 \u0026pi;-\u0026pi; 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). Mutations M124P and R127G introduce smaller amino acid residues, enlarging the active site pocket, which is thought to enhance catalytic efficiency (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). This speculation is supported by the observed stepwise increase in activity for the mutants HheD8-M2 and HheD8-M3, following the sequential introduction of these mutations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eScope for enantioselective formation of oxetanes\u003c/h2\u003e\n\u003cp\u003eSubsequently, the substrate scope of the biocatalytic platform for the enantioselective dehalogenation of \u0026gamma;-haloalcohols was explored on a preparative-scale (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table\u0026nbsp;6). Various aryl \u0026gamma;-chloroalcohols bearing electron-donating or electron-withdrawing substituents on the phenyl ring (\u003cstrong\u003e1-15a\u003c/strong\u003e) were accepted to furnish both chiral (\u003cem\u003eR\u003c/em\u003e)-oxetanes \u003cstrong\u003e1-15b\u003c/strong\u003e (32\u0026ndash;46% yield, 93-\u0026gt;99% e.e.) and (\u003cem\u003eS\u003c/em\u003e)-\u0026gamma;-chloroalcohols \u003cstrong\u003e1-15a\u003c/strong\u003e (47\u0026ndash;53% yield, 86-\u0026gt;99% e.e.) with good yields and high optical purities. It is worth noting that the presence of steric hindrance at the \u003cem\u003eortho\u003c/em\u003e position of the aromatic ring (\u003cstrong\u003e2-5a\u003c/strong\u003e) was found to be compatible with the biotransformation. Substitutions were well tolerated at both the \u003cem\u003epara\u003c/em\u003e and \u003cem\u003emeta\u003c/em\u003e positions (\u003cstrong\u003e6-14a\u003c/strong\u003e). Smooth conversion of substrate \u003cstrong\u003e14a\u003c/strong\u003e, featuring a strong electron-withdrawing trifluoromethyl group, to chiral oxetane (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e14b\u003c/strong\u003e was achieved, albeit with a slight decrease in enantioselectivity. Interestingly, introducing two fluoro substituents on the phenyl ring (\u003cstrong\u003e15a\u003c/strong\u003e) did not hamper the reaction; rather, it successfully delivered both chiral (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e15b\u003c/strong\u003e and (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e15a\u003c/strong\u003e with excellent enantioselectivity (\u003cem\u003eE\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;200). The substrate containing a bulky naphthalene moiety (\u003cstrong\u003e16a\u003c/strong\u003e) underwent effective and enantioselective dehalogenation using the mutant HheD-M3, yielding chiral oxetane (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e16b\u003c/strong\u003e and \u0026gamma;-chloroalcohol (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e16a\u003c/strong\u003e with 37% yield, \u0026gt;\u0026thinsp;99% e.e. and 50% yield, 96% e.e., respectively. Additionally, enantioselective dehalogenation of \u0026alpha;-,\u0026alpha;-disittuted \u0026gamma;-chloroalcohol \u003cstrong\u003e17a\u003c/strong\u003e also proceeded smoothly to furnish the (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e17b\u003c/strong\u003e (36% yield, \u0026gt;\u0026thinsp;99% e.e) and (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e17a\u003c/strong\u003e (49% yield, \u0026gt;\u0026thinsp;99% e.e). Remarkably, the reaction also tolerated structural perturbations, accommodating the substitution of the aryl ring with several heterocyclic moieties such as pyrimidine (\u003cstrong\u003e18a\u003c/strong\u003e), quinoline (\u003cstrong\u003e19a\u003c/strong\u003e), and thiophene (\u003cstrong\u003e20a\u003c/strong\u003e). Chiral heterocyclic \u0026gamma;-chloroalcohols (\u003cstrong\u003e18-20a\u003c/strong\u003e, 48\u0026ndash;50% yields and 89-\u0026gt;99% e.e.) and oxetanes (\u003cstrong\u003e18-19b\u003c/strong\u003e, 42\u0026ndash;44% yields and 96\u0026ndash;97% e.e.) were successfully isolated with the exception of the unstable oxetane \u003cstrong\u003e20b\u003c/strong\u003e. Moreover, the reaction exhibited tolerance toward the alkyl-substituted substrate \u003cstrong\u003e21a\u003c/strong\u003e, affoding chiral alkyl oxetane (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e21b\u003c/strong\u003e (55% yield, \u0026gt;\u0026thinsp;99% e.e.) and alkyl \u0026gamma;-chloroalcohol (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e21a\u003c/strong\u003e (48% yield, \u0026gt;\u0026thinsp;99% e.e.). Furthermore, the biocatalytic system was applied to the enanioselective generation of chiral \u0026gamma;-bromoalcohol (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e22a\u003c/strong\u003e (50% yield, \u0026gt;\u0026thinsp;99% e.e) and \u0026gamma;-iodoalcohol (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e23a\u003c/strong\u003e (48% yield, \u0026gt;\u0026thinsp;99% e.e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eScope for enantioselective ring-opening of oxetanes\u003c/h2\u003e\n\u003cp\u003eWe next turned our attention to the substrate generality for the enantioselective ring-opening of oxetanes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Table\u0026nbsp;7). As expected, aromatic substituted oxetanes (\u003cstrong\u003e1-14b\u003c/strong\u003e) with mono-substituent at the \u003cem\u003epara\u003c/em\u003e-, \u003cem\u003emeta\u003c/em\u003e-, and \u003cem\u003eortho\u003c/em\u003e positions were well tolerated, affording the corresponding chiral (\u003cem\u003eR\u003c/em\u003e)-\u0026gamma;-azidoalcohols (41\u0026ndash;50% yield, 91-\u0026gt;99% e.e) and (\u003cem\u003eS\u003c/em\u003e)-oxetanes (30\u0026ndash;49% yield, 86-\u0026gt;99% e.e) with good to excellent enantioselectivities (\u003cem\u003eE\u003c/em\u003e\u0026thinsp;=\u0026thinsp;72-\u0026gt;200). The electronic characteristics of the substituents show a modest effect on both the efficiency and enantioselectivity of the ring-opening reaction. Oxetane \u003cstrong\u003e15b\u003c/strong\u003e, bearing two fluoro \u003cem\u003emeta\u003c/em\u003e-substituents, was also successfully accommodated to yield the chiral (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e15c\u003c/strong\u003e and (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e15b\u003c/strong\u003e with \u0026gt;\u0026thinsp;99% e.e. and high yields. Additionally, efficient ring-opening of sterically hindered oxetanes \u003cstrong\u003e16b\u003c/strong\u003e and \u003cstrong\u003e17b\u003c/strong\u003e was achieved using the mutants HheD8-M3 and HheD8-M7, respectively. Moreover, heterocyclic oxetanes contaning pyrimidine (\u003cstrong\u003e18b\u003c/strong\u003e) or quinoline (\u003cstrong\u003e19b\u003c/strong\u003e) served as competent substrates for the mutant HheD8-M4. Alkyl oxetane \u003cstrong\u003e21b\u003c/strong\u003e was also smoothly converted to deliver the corresponding chiral alkyl \u0026gamma;-azidoalcohol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e21c\u003c/strong\u003e and oxetane (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e21b\u003c/strong\u003e. Furthermore, we challenged the ring-opening reaction with other anionic nucleophiles. The reaction accepted the nucleophilic cyanide, generated in situ from mandelonitrile\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, yielding the chiral \u0026gamma;-cyanoalcohol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1d\u003c/strong\u003e with 46% yield and \u0026gt;\u0026thinsp;99% e.e.. The nitrite demonstrated as an ambident nucleophile, leading to the formation of chiral \u0026gamma;-nitroalcohol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1e\u003c/strong\u003e and \u0026gamma;-diol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1f\u003c/strong\u003e 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\u0026nbsp;8). Further efforts in enzyme screening and protein engineering are required to facilitate oxetane ring-opening reactions utilizing these nucleophilic agents.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eLarge-scale reactions\u003c/h2\u003e\n\u003cp\u003eSubstrate 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: \u003cem\u003en\u003c/em\u003e-hexane\u0026thinsp;=\u0026thinsp;5:1), employing the variant \u003cem\u003eE. coli\u003c/em\u003e (HheD8-M4) whole cells as biocatalysts. The enantioselective dehalogenation reactions of 40\u0026ndash;140 mM (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e proceeded smoothly to completion with 47\u0026ndash;50% conversions over a period of 3\u0026ndash;48 h (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table\u0026nbsp;9). 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\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 through the addition of aqueous sodium hydroxide solution, a large-scale reaction to convert 200 mM of (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e (20 mmol, 34 g/L) was performed and completed smoothly within 33 h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). For the enantioselective ring-opening of 40\u0026ndash;200 mM oxetane (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e, reactions consistently reached 50% conversions within 12\u0026ndash;36 h (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table\u0026nbsp;10). A large-scale reaction of 200 mM (\u003cem\u003erac\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e (20 mmol, 27 g/L) was successfully conducted to completion after 39 h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Notably, in both the large-scale dehalogenation and ring-opening reactions, all chiral compounds were obtained in gram quantities, achieving high isolated yields (39\u0026ndash;49%) and excellent enantiopurities (97-\u0026gt;99% e.e.). These results substantiate that the biocatalytic platform accommodates large-scale synthesis at high substrate concentrations while maintaining catalytic efficiency and enantioselectivity, thereby emphasizing its synthetic potential for industrial applications.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eBiocatalytic cascade reactions\u003c/h2\u003e\n\u003cp\u003eThe favorable compatibility of enzymatic reactions facilitates their application in biocatalytic cascade processes, thus omitting the need for intermediate purification and isolation steps\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. 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 \u0026gamma;-haloalcohols into \u0026gamma;-azidoalcohols via the unisolated oxetane intermediates. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb, three \u0026gamma;-haloalcohols (\u003cstrong\u003e1a\u003c/strong\u003e, \u003cstrong\u003e6-7a\u003c/strong\u003e) underwent evaluation using \u003cem\u003eE. coli\u003c/em\u003e (HheD8-M4) cells. The results indicated that all reactions proceeded efficiently, resulting in the formation of both chiral \u0026gamma;-azidoalcohols and \u0026gamma;-haloalcohols with excellent isolated yields (47\u0026ndash;50%) and as single enantiomers (\u0026gt;\u0026thinsp;99% e.e.). The successful integration of the biocatalytic cascade reactions further highlighted the operational flexibility of the biocatalytic platform.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eTransfomations of chiral products\u003c/h2\u003e\n\u003cp\u003eThe developed biocatalytic platform enables the synthesis of both (\u003cem\u003eR\u003c/em\u003e)- and (\u003cem\u003eS\u003c/em\u003e)-enantiomers of oxetanes as well as various chiral \u0026gamma;-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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). The \u0026gamma;-chloroalcohol (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e can be transformed into the chiral (\u003cem\u003eR\u003c/em\u003e)-3-phenylisoxazolidine (\u003cstrong\u003e1aa\u003c/strong\u003e), an important heterocyclic motif found in anticancer agents\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Smooth conversion of the chiral (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e1a\u003c/strong\u003e to furnish (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e1ab\u003c/strong\u003e was also realized, providing a key precursor for the synthesis of (\u003cem\u003eR\u003c/em\u003e)-dapoxetine\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Additionally, the chiral oxetane (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e was successfully converted into (\u003cem\u003eR\u003c/em\u003e)-2-phenyltetrahydrofuran (\u003cstrong\u003e1ba\u003c/strong\u003e) through a straightforward ring-expansion reaction\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. We also performed the conversion of (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1b\u003c/strong\u003e into the chiral (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1bb\u003c/strong\u003e, a crucial precursor in the synthesis of (\u003cem\u003eR\u003c/em\u003e)-tomoxetine\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Moreover, the presence of an azide group enables copper-catalyzed click chemistry\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, allowing for the modification of \u0026gamma;-azidoalcohol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1c\u003c/strong\u003e with a simple alkyne to afford the chiral \u0026gamma;-hydroxytriazole (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1ca\u003c/strong\u003e. The reaction of (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1c\u003c/strong\u003e with a symmetrical ketone was aslo performed to deliver medium-sized ring lactam (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1cb\u003c/strong\u003e, a commonly important intermediate in the synthesis of nitrogen-containing compounds\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The absolute stereochemistry of (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1cb\u003c/strong\u003e was ascertained by single-crystal X-ray diffraction analysis (Supplementary Table\u0026nbsp;11), which also demonstrated \u003cem\u003eR\u003c/em\u003e-enantioselectivity during the enzymatic dehalogenation and ring-opening reactions. Furthermore, the transformations of chiral \u0026gamma;-cyanoalcohol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1d\u003c/strong\u003e yielded the chiral lactone (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1da\u003c/strong\u003e and the \u0026gamma;-hydroxyamide (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1db\u003c/strong\u003e, both serving as building blocks in the synthesis of (\u003cem\u003eR\u003c/em\u003e)-fluoxetine and (\u003cem\u003eR\u003c/em\u003e)-norfluoxetine\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. For the nitrite-mediated ring-opening products, the \u0026gamma;-nitroalcohol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1e\u003c/strong\u003e can be readily reduced to generate the chiral \u0026gamma;-aminoalcohol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1ea\u003c/strong\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. On the other hand, esterification of chiral \u0026gamma;-diol (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1f\u003c/strong\u003e yielded a key intermediate, (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1fa\u003c/strong\u003e, for the systhesis of natural piperidine alkaloids\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Taken together, these representative transformations resulting in the generation of useful derivatives showcased the synthetic scalability of the biocatalytic platform.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our findings constitute the inaugural 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 both (\u003cem\u003eR\u003c/em\u003e)- and (\u003cem\u003eS\u003c/em\u003e)-enantiomers of oxetanes, in addition to a variety 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\u0026rsquo;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. Overall, this work endows HHDH with new 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, drug discovery, and synthetic biology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available within the paper and its Supplementary Information (detailed methods, NMR spectra, chiral HPLC/GC traces, Supplementary Figures and Tables). Crystallographic data of compound (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e1cb\u003c/strong\u003e are available free of charge from the Cambridge Crystallographic Data Centre (CCDC; https://www.ccdc.cam.ac.uk/structures/) reference number 2350047. The coordinate files and structure factors have been deposited in the PDB with accession number 8XXB. Data collection statistics are provided in Extended Data Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the financial support from the National Natural Science Foundation of China (22167023 to W. N.W.), the Program for Technology Elite of Zunyi Medical University (ZYSE-2022-03 to W.N.W.), and the Science and Technology Department of Zunyi (No. ZSKRPT-2020-5 to Y.Z.C.). We also thank NovoPro Bioscience Inc. (Shanghai, China) for technical assistance in crystallization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.N.W. devised and supervised the project. X.H. and Y.F.W. performed most of the experiments. X.J., H.Y.Y and H.H.W. assisted in enzyme screening, synthetic experiments, crystallographic and docking studies. W.N.W., H.H.W. and Y.Z.C. wrote the manuscript and generated the figures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBuller, R.\u003cem\u003e et al.\u003c/em\u003e From nature to industry: harnessing enzymes for biocatalysis. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e382\u003c/strong\u003e, eadh8615 (2023).\u003c/li\u003e\n\u003cli\u003eLeveson-Gower, R. B., Mayer, C. \u0026amp; Roelfes, G. The importance of catalytic promiscuity for enzyme design and evolution. \u003cem\u003eNat. Rev. Chem.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 687-705 (2019).\u003c/li\u003e\n\u003cli\u003eChen, K. \u0026amp; Arnold, F. H. Engineering new catalytic activities in enzymes. \u003cem\u003eNat. 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Enantioselective synthesis of (+)-sedamine and (-)-allosedamine. \u003cem\u003eSynthesis\u003c/em\u003e \u003cstrong\u003e2006\u003c/strong\u003e, 4005-4012 (2006).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4316588/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4316588/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The remarkable selectivity, sustainability, and efficiency afforded by biocatalytic strategies position them as complements or alternatives to traditional synthetic methods. Nevertheless, the currently narrow spectrum of enzymatic reactions available imposes limitations on synthesizing diverse desired compounds. Consequently, there continues to be a high demand for developing novel biocatalytic processes to access reactions that were previously unattainable. Herein, we report the discovery and subsequent protein engineering of a unique halohydrin dehalogenase to develop a biocatalytic platform for enantioselective formation and ring-opening of oxetanes. This biocatalytic platform, exhibiting high efficiency, excellent enantioselectivity, and broad scopes, facilitates the preparative-scale synthesis of not only both enantiomers of chiral oxetanes (up to 49% yield, \u003e99 e.e.) but also a variety of chiral γ-substituted alcohols (up to 53% yield, \u003e99 e.e.). Additionally, both the enantioselective oxetane formation and ring-opening processes have been proven scalable for large-scale transformations (20 mmol) at high substrate concentrations (200 mM), and can be integrated efficiently in a one-pot, one-catalyst cascade system. Moreover, useful derivatizations highlight the potential synthetic applications of the biocatalytic platform. This work expands the enzymatic toolbox for non-natural reactions and will promote further exploration of the catalytic repertoire of halohydrin dehalogenases in synthetic and pharmaceutical chemistry.","manuscriptTitle":"Biocatalytic enantioselective formation and ring-opening of oxetanes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-16 08:55:34","doi":"10.21203/rs.3.rs-4316588/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c87d5c04-f3a3-450f-971e-0f09735c1f4f","owner":[],"postedDate":"May 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31285449,"name":"Physical sciences/Chemistry/Catalysis/Biocatalysis"},{"id":31285450,"name":"Biological sciences/Chemical biology/Biocatalysis"}],"tags":[],"updatedAt":"2025-01-31T08:05:38+00:00","versionOfRecord":{"articleIdentity":"rs-4316588","link":"https://doi.org/10.1038/s41467-025-56463-z","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-30 05:00:00","publishedOnDateReadable":"January 30th, 2025"},"versionCreatedAt":"2024-05-16 08:55:34","video":"","vorDoi":"10.1038/s41467-025-56463-z","vorDoiUrl":"https://doi.org/10.1038/s41467-025-56463-z","workflowStages":[]},"version":"v1","identity":"rs-4316588","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4316588","identity":"rs-4316588","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}