Chemoenzymatic synthesis of pentalenolactones via stereoselective Riley oxidation by engineered P450_BM3

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: major_revision

Abstract
Riley oxidation is a powerful method for introducing oxygen functionality but typically relies on toxic reagents and generally lacks enantiocontrol. This limitation has historically restricted the utility of this transformation for the direct asymmetric construction of complex scaffolds. Here, we show that fusion library screening and directed evolution enable the highly enantio- and diastereoselective Riley-type oxidation of a cis-diquinane substrate catalyzed by engineered P450 BM3. This enzymatic desymmetrization strategy provides streamlined access to key chiral cis-diquinane building block on a gram scale, facilitating rapid downstream diversification. Furthermore, by merging this enzymatic desymmetrization with chemical ring formation and late-stage biosynthetic oxidation, we achieve asymmetric total synthesis of pentalenolactone D and neo-pentalenolactone D. This work highlights the power of integrating protein engineering with synthetic chemistry to synthesis of complex natural products.

Introduction
Natural products and drugs featuring a cis-diquinane scaffold typically possess architecturally complex frameworks and display notable biological activities1–5. Natural products containing the diquinane motif have long attracted the interest of synthetic chemists as targets for total synthesis6,7. However, efficient, and scalable preparation of chiral diquinane fragments remains a significant challenge. The C2-symmetric cis-diquinane core represents a meso framework that can, in principle, be efficiently accessed via desymmetrization strategies (Fig. 1A). Among previously reported strategies, desymmetrization by allylic oxidation has emerged as a powerful method for the synthesis of enantioenriched allylic alcohols (Fig. 1B)8–12. However, reported reactions of this type have thus far been limited to relatively simple substrates, and no examples of enantioselective Riley-type hydroxylation have been described to date13–21. Given the previous success of enzyme library screening and directed evolution strategies in synthetic applications22–34, we hypothesized that enzyme-catalyzed oxidation could offer a more effective solution for structurally complex substrates.

In this work, we develop a synthetic approach starting from an accessible, unsubstituted meso cis-diquinane, in which oxidative desymmetrization is employed to construct a chiral cis-diquinane core (Fig. 1C). This approach enables enantioselective Riley-type direct hydroxylation catalyzed by engineered P450 BM3. A readily accessible meso substrate bearing carbonyl and olefin functionalities serves as a versatile intermediate for downstream functionalization. Through subsequent side-chain installation, cyclization, and late-stage oxidation catalyzed by biosynthetic enzymes, this strategy provides access to the tricyclic skeleton and enables the asymmetric total synthesis of pentalenolactone D and neo-pentalenolactone D.

Results
We selected the known compound 135 as a substrate to investigate the enzymatic desymmetrizing allylic oxidation (Fig. 2). By screening oxidative transformations using our oxidase libraries of P450 monooxygenases and α-ketoglutarate-dependent dioxygenases33, we identified that the wild-type P450 BM3 and its F87V mutant afforded the major hydroxylated product 2 with 73% e.e. and 1.6:1 d.r., 88% e.e. and 4.3: 1 d.r., respectively. Guided by the crystal structure of P450 BM3 (PDB: 1JPZ)36, we rationally selected and mutated several residues predicted to play critical roles in shaping the active-site pocket size and modulating substrate–residue interactions. Building upon the F87V mutant, we performed two rounds of saturation mutagenesis at residues 47 and 49. The resulting variant, AAO3, exhibited improved enantioselectivity and diastereoselectivity, affording compound 2 with 98% e.e. and 6.9:1 d.r. Mutations at positions 78 and 82 individually did not lead to further enhancement in selectivity. Inspired by Reetz’s strategy of combinatorial active-site mutagenesis37, we introduced a double mutation at these two positions. The resulting mutant, AAO4, significantly improved the diastereoselectivity (>20:1 d.r.) of the reaction while maintaining high enantioselectivity. We further achieved gram-scale synthesis of compound 2 with excellent diastereoselectivity and enantioselectivity. In addition, the absolute configuration of compound 2 was unambiguously confirmed by X-ray crystal structure of its derivative 4.

To showcase the applicability of the chiral cis-diquinane scaffold, we employed it in the total synthesis of pentalenolactone D and neo-pentalenolactone D3. Biological studies of the pentalenolactones have shown that they selectively inhibit glyceraldehyde-3-phosphate dehydrogenase, suggesting their potential as lead compounds for the development of novel antibiotic agents38–42. Accordingly, these pharmacologically important natural products have garnered significant attention from synthetic chemists43–60. Here, the retrosynthetic analysis of pentalenolactone D and neo-pentalenolactone D was undertaken (Fig. 3). Our retrosynthetic logic features a three-stage synthesis to access the target natural products. Initially, late-stage oxidation of biosynthetically recognizable 1-deoxypentalenic acid (7) enable the synthesis of pentalenolactones. In the next stage, 1-deoxypentalenic acid (7) can be obtained from pentalenene (8) via Riley oxidation. In addition, the skeleton of pentalenene (8) can be constructed by 9 through a sequence of Cu-catalyzed 1,4-addition and intramolecular alkylation. Compound 9 can be obtained from compound 2, generated via enzymatic desymmetrizing allylic oxidation, through an enantioretentive elimination. The key feature of this retrosynthetic analysis lies in the integration of engineered P450 BM3-catalyzed oxidation, chemical cyclization, and late-stage biosynthetic enzymatic oxidation.

Our synthetic route is depicted in Fig. 4, which commenced with commercially available compound 10. Although the synthesis of compound 1 had been previously reported via a three-step sequence35, we employed the method reported by H. Lebel and co-workers61 to access compound 1 directly from 10 in a single step, thereby improving step economy. With a substantial quantity of 1 in hand, compound 2 was prepared on a gram scale via our developed enzymatic desymmetrizing allylic oxidation, exhibiting high enantio- and diastereoselectivity. Simmons-Smith reaction of 2 afforded compound 11 which was subsequently subjected to hydrogenation to furnish the diol 12. Attempts to achieve selective oxidation of diol 12 to the compound 13 were undertaken. However, extensive screening of various oxidation conditions (DMP, PCC, SO3·Pyridine, etc.) lacked selectivity. Fortunately, treatment 12 with Ph3C+BF4- successfully afforded the desired compound 13 with excellent selectivity. The inability of the sterically congested carbocation to approach the hydroxyl group adjacent to the geminal dimethyl substituents may account for this observation. Then, we sought to dehydration of the C1-hydroxyl group of 13 to generate an alkene, followed by isomerization to afford enone 9. Presumably due to the presence of the geminal dimethyl group, dehydration reagents such as Burgess reagent and Martin sulfurane failed to yield the desired compound 9. Consequently, compound 13 was converted to the xanthate ester 14 and subsequent pyrolysis afforded 15. Notably, compound 15 underwent smoothly alkene isomerization under the condition to furnish enone 9. It is noteworthy that the elimination proceeded with retention of chirality. Conjugate addition to enone 9 proceeded smoothly, establishing the crucial quaternary carbon center. Subsequent desilylation afforded compound 16. Hydrogenation of 16 using Crabtree’s catalyst proceeded with moderate stereoselectivity, and the intermediate alcohol was tosylated to yield compound 17. Exposure of compound 17 under basic condition induced cyclization, affording the tricyclic product 18. Compound 18 was elaborated over two steps to furnish the natural product pentalenene (8). Pentalenene (8) was transformed to 1-deoxypentalenic acid (7) accessible to recognition by biosynthetic enzymes via allylic oxidation with SeO2, followed by Pinnick oxidation.

With 1-deoxypentalenic acid (7) in hand, we proceeded to investigate late-stage biosynthetic enzyme-catalyzed oxidation based on native enzymes originally reported by the group of D. E. Cane (Fig. 5)62,63. Among them, the α-ketoglutarate-dependent dioxygenase PtlH catalyzed hydroxylation at C11 of 1-deoxypentalenic acid (7), providing this transformation in high yield with exclusive regio- and stereoselectivity. Introduction the oxidation state is essential for achieving the total syntheses of pentalenolactone D (5) and neo-pentalenolactone D (6). Then, the resulting 19 was oxidized under Dess–Martin periodinane condition to provide ketone 20. Due to the observed instability of compound 20 on silica gel, it was carried forward without purification. Chemical Baeyer–Villiger oxidation conditions (m-CPBA, H2O2, etc.) proved unsatisfactory, which might be due to regioselectivity issues and incompatibility with the alkene moiety. Consequently, enzymatic Baeyer–Villiger oxidation was pursued. Screening of our Baeyer–Villiger monooxygenases library confirmed that the coexpression of PntE or PtlE, and the phosphite dehydrogenase Opt13 for NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) regeneration in a single E. coli BL21(DE3) strain allowed remarkable regioselectivity to afford pentalenolactone D (5) and neo-pentalenolactone D (6), respectively. Other Baeyer–Villiger monooxygenases (AndJ, Rhodo, EthA, AusZ, etc), however, displayed no catalytic activity toward this substrate. Thus, through late-stage oxidation mediated by biosynthetic enzymes, we accomplished the asymmetric total syntheses of pentalenolactone D (5) and neo-pentalenolactone D (6).

Methods
Generation of pET22b(+)-P450 BM3 and pRSF-Opt13 Co-expression Vector
The respective gene fragment of P450 BM3 was synthesized and assembled directly into pET22b(+) between NdeI and XhoI by Universe Gene Technology (Tianjin) Co. Ltd to yield vector pET22b(+)-P450 BM3. Opt-13 gene were inserted between NcoI and XhoI restriction sites of pRSF-1b to yield vector pRSF-Opt13. The resulting vectors pET22b(+)-P450 BM3 and pRSF-Opt13 were used to co-transform competent E. coli strain BL21(DE3).

Gram-Scale Synthesis of Compound 2
An overnight culture of E. coli BL21(DE3) cells harboring pET-22b(+)-based vector for expressing P450 BM3 (AAO4) and pRSF-Opt13 plasmid was used to inoculate 500 mL TB media (in 2L Erlenmeyer flask) containing 50 µg/mL kanamycin, 50 µg/mL ampicillin. The cultures were shaken at 250 rpm at 37 ˚C until an optical density of OD600=0.7–1.0 was reached. The cultures were cooled to 20°C (20 min) and then induced with 5-aminolevulinic, IPTG and FeSO4·7H2O to final concentrations of 1.0 mM, 0.5 mM and 0.05 mM, respectively. The cultures were shaken at 250 rpm at 20°C for a further 20 h. Cells were harvested by centrifugation (4°C, 15 min, 2100 × g), resuspended in 50 mM KPi (pH=8.00) to an OD600=10 into a 2L Erlenmeyer flask. Cells were disrupted by sonication (20 min, cycle= 1s on/2s off, 50% amplitude) and pelleted by centrifugation (4°C, 15 min, 2100 × g). The supernatant was used directly for subsequent reactions. Reactions were carried out in parallel across twelve 2L Erlenmeyer flasks, each charged with the following amounts of substrate and reagents. A pre-dissolved solution of 1 (300 mg, 2.0 mmol, 4.0 mM final concentration) in 25 mL DMSO was added to the lysate, followed by Na2NADP (250 mg, 0.32 mmol, 0.16 equiv.) and Na2HPO4∙5H2O (6.25 g, 28.9 mmol, 14.5 equiv.). The flask was shaken at 150 rpm at 23°C under air for 20 h. Then, 25 g NaCl was added to flask and the mixture was extracted with EtOAc (400 mL × 3). The combined organic extracts of twelve 2L Erlenmeyer flasks from the twelve parallel reactions were washed with sat. aq. NaHCO3 (400 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The crude material was purified by flash column chromatography to yield compound 2 of 1.60 g (44%) as yellow oil.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Li and coworkers report an excellent strategy to constructed enantio- and diastereoselectivity chiral cis-diquinane via Riley-type oxidation by engineered P450BM3. This work achieved the total syntheses of the natural products pentalenolactone D and neo-pentalenolactone D by combining chemical synthesis with enzyme-catalyzed synthesis.\nHere I have a few questions for the author:\n1. The description of protein engineering is relatively brief and lacks the rationale for selecting mutation sites regarding the section on mutation engineering of enzymes. For instance, the experimental design in which saturation mutagenesis was performed on residues 47 and 49 using the F87V mutant as a template (lines 58-61 in manuscript) lacks a clear justification. It is unclear what evidence or reasoning guided the decision-making to (i) employ the F87V mutant specifically, and (ii) focus the saturation efforts on residues 47 and 49. Providing this information is crucial for evaluating the soundness of the experimental strategy. In the section \"Library screening of P450BM3 mutants\" within SI, the 35 P450BM3 mutants are mentioned. The description lacks the precise criteria used for their selection. Please clarify the screening methodology and ensure that the corresponding experimental results for these mutants are fully presented in SI.\n2.The manuscript describes the first asymmetric total syntheses of pentalenolactone D and neo-pentalenolactone D, which indeed represents a highly innovative achievement. To better contextualize the significance of this work, we inquire whether there have been any prior synthetic efforts toward these or closely related compounds. Furthermore, given the multi-step nature of the synthetic route, we seek clarification on the overall yield. Finally, we are interested in the authors' perspective on the potential of this strategy to serve as a general method for preparing this class of natural products.\n3.The manuscript mentions that pentanolactones can specifically inhibit glyceraldehyde-3-phosphate dehydrogenase and have potential antibacterial effects. The manuscript cites three references related to the study of such compounds, but all of which were conducted 30 years ago. May I ask if there are any recent research results to support the antibiotic activity of pentanolactones. Is there any relevant antibacterial activity test in this work to confirm the antibacterial activity of the compound？Finally, a forward-looking discussion on the therapeutic potential of this compound class as novel antibiotics, particularly within the current landscape of antimicrobial resistance, would greatly enhance the impact and relevance of the study.\n4.The manuscript would benefit from a careful proofread to correct a number of minor linguistic errors. kPi-KPi, TCL-TLC. Several variations were noted in the expression of rotational speed values (e.g., \"4200 rpm\" vs. \"4,200 rpm\"). To maintain rigorous scientific presentation and adhere to journal style, please select one formatting of numbers (e.g., use of commas).", "reviewer_2": "The authors report a chemoenzymatic synthesis of pentalenolactone D and its regioisomer neo-pentalenolactone D. First, the authors utilize a p450BM3 enzyme and through directed evolution achieve highly selective desymmetrization of diquinanes by enzymatic Riley oxidation. Second, chemical synthesis is used for a concise synthesis of triquinanes pentalenene and 1-deoxypentalenic acid. Finally, using known biosynthetic enzymes in the biosynthesis of pentalenolactone, the chemoenzymatic synthesis was achieved. These enzymes include an alpha-KG dependent hydroxylase, PtlH, and the regiodivergent Baeyer-Villiger monooxygenases (PntE and PtlE).\nBy combining synthetic chemistry with enzymatic reactions, the authors have developed a unique chemoenzymatic synthesis of triquinane sesquiterpenes. The enzymatic reactions would be difficult to replicate with synthetic methods, particularly the enantioselective Riley oxidation catalyzed by p450BM3 and the undirected methylene C-H hydroxylation catalyzed by PtlH. Thus, the use of enzymatic reactions in the chemoenzymatic synthesis is justifiable. Moreover, this is the first chemoenzymatic synthesis of this class of natural products, so the retrosynthetic strategy is a novel aspect of this work.\nDespite being able to achieve enantioselective allylic C-H hydroxylation, the application to the synthesis of pentalenene is indirect. The hydroxyl group installed by enzymatic hydroxylation is removed by elimination to form the enone which renders the optimized dr of the hydroxylation inconsequential as the axial alcohol could also eliminate through an E2 reaction. Essentially, the primary purpose of the initial enzymatic step is to achieve a desymmetrization. The authors also compare their p450 hydroxylation method to synthetic reactions and mention that theirs is the first \"direct\" method for enantioselective allylic oxidation. While this reviewer agrees that this method allows for the direct installation of alcohols (OH) as opposed to other methods that incorporate acetate (OAc) or benzoyl (OBz) group, in the context of the synthesis, the acetate or the benzoyl group could be preferrable given that they are better leaving groups as the OH is eliminated to form an alkene. In addition, one of the benefits of asymmetric catalysis is that the enantiomer can also be synthesized by using the enantiomer of the ligand or catalyst.\nThe chemical steps include an unselective reduction of the cyclopropane to the gem dimethyl group which also resulted in the undesired reduction of the ketone to an alcohol. A selective oxidation of that alcohol back to the ketone was developed using tritylium tetrafluoroborate.\nThe PtlH C-H hydroxylation is a known reaction in the biosynthesis of pentalenolactone (Ref 59). This should be mentioned explicitly in the text. The PntE and PtlE regiodivergent Baeyer-Villiger (BV) oxidation have also been previously reported (Ref 60). This should also be mentioned explicitly in the text rather than attributing the identification of these enzymes to screening libraries for BV monooxygenases.\nGiven the potential applications of combining enzymatic reactions with chemical synthesis, this work is a significant example of biocatalysis enabled natural product synthesis. Moreover, this work utilizes both engineered and wild type biosynthetic enzymes at both early and late-stages of the synthesis. This reviewer believes this work could merit publication in Nature Communications if the authors expand on the enzymatic Riley oxidation. This reviewer recommends the following major revisions.\n1. Can the authors through directed evolution of p450 enzymes access the enantiomer of 2 by selective oxidation of Hb2? If so, then this reviewer would agree that this would be consider an advance to the field of enantioselective allylic oxidation. This example would broaden the scope of this work and potentially allow for a chemoenzymatic synthesis pentalenic acid.\n2. Can the authors through directed evolution of p450 enzymes access the diastereomer of 2 by selective oxidation of Ha1? The WT enzyme has 1.6:1 dr, so it should be possible to evolve the enzyme to achieve selectivity for the axial hydrogen. This alcohol could be more susceptible to elimination through E2 mechanism.\nSome minor corrections and suggestions:\n3. The yield of the p450 Riley oxidation should be provided in the Figures and main text rather than just in the methods section.\n4. The authors should show a figure of the active site of p450BM3 and where the mutations in AAO4 are located. Are the mutations close to the heme co-factor?\n5. Compound 14 is not a xanthate. It is a O-thiocarbamate.\n6. In the methods sections, lysaste should be \"lysate\"."}, "author_response": "Responses to the Reviewers' comments\nWe thank the reviewers for their professional and helpful suggestions, which were useful in improvement of the quality of the manuscript. The point-by-point responses to the reviewer’s comments are detailed as follows.\nReviewer # 1\nComments:\nThe manuscript by Li and coworkers report an excellent strategy to constructed enantio- and diastereoselectivity chiral cis-diquinane via Riley-type oxidation by engineered P450 . This BM3 work achieved the total syntheses of the natural products pentalenolactone D and neo-pentalenolactone D by combining chemical synthesis with enzyme-catalyzed synthesis.\nResponse: We thank the reviewer for the positive and constructive comments.\n1. The description of protein engineering is relatively brief and lacks the rationale for selecting mutation sites regarding the section on mutation engineering of enzymes. For instance, the experimental design in which saturation mutagenesis was performed on residues 47 and 49 using the F87V mutant as a template (lines 58-61 in manuscript) lacks a clear justification. It is unclear what evidence or reasoning guided the decision-making to (i) employ the F87V mutant specifically, and (ii) focus the saturation efforts on residues 47 and 49. Providing this information is crucial for evaluating the soundness of the experimental strategy. In the section \"Library screening of P450 mutants\" within SI, the BM3 35 P450 mutants are mentioned. The description lacks the precise criteria used for their BM3 selection. Please clarify the screening methodology and ensure that the corresponding experimental results for these mutants are fully presented in SI.\nResponse:\nWe sincerely thank the reviewer for professional and insightful comments. Here, we will provide a more detailed description regarding the rationale for selecting the mutation sites used in our directed evolution strategy.\nFirst, the wild-type P450 yielded only low amounts of product, approximately 10 mg/L. BM3 By analyzing the crystal structure of P450 and its heme domain, we identified position BM3 87 as a key determinant of the active-site pocket size. Because our substrate possesses a bowl-shaped, rigid scaffold, we anticipated that an enlarged active-site pocket would be required for efficient catalysis. The bulky phenylalanine at position 87 in the wild-type enzyme likely imposes significant steric hindrance. In fact, during our enzyme library screening, we had already observed that the F87V mutant led to an improvement in both the dr value and the ee value. Interestingly, we observed a positive correlation between product yield and both the enantioselectivity and diastereoselectivity of the reaction.\nBased on the F87V mutation, we subsequently carried out further rounds of directed evolution.\nTo further increase the probability of substrate entry into the active-site pocket, we focused on residues located at the pocket entrance and performed saturation mutagenesis at these positions. Inspired by Reetz’s work (ref. 37), we found that residues 47 and 49 are particularly important for substrate access to the active-site pocket. Saturation mutagenesis at these two sites led to the identification of an improved oxidative biocatalyst, AAO3, which exhibited significantly enhanced selectivity.\nSubsequently, we performed site-directed mutagenesis on residues near the active-site pocket. Mutations at positions 78 and 82 individually did not lead to further enhancement in selectivity. Inspired by Reetz’s strategy of combinatorial active-site mutagenesis, we introduced a double mutation at these two positions. To our delight, saturation mutagenesis at positions 78 and 82 yielded the enzyme AAO4, which exhibited greatly enhanced diastereoselectivity. The product yield reached up to 200 mg/L. We further achieved gram-scale synthesis of compound 2 with excellent diastereoselectivity and enantioselectivity.\nIn addition, we sincerely thank the reviewer for helpful suggestion about library screening of P450 mutants. Here, we present the screening methodology and results of the BM3 P450BM3 enzyme library.\nMethod: The 35 P450 mutants were picked from library and cultured in a sterilized 96- BM3 well culture plate (each well contains 200 μL of LB medium with 50 μg/mL kanamycin and 100 μg/mL ampicillin). The cultures were grown at 37 °C, 900 rpm for 12–15 hours. 400 μL of TB medium containing 50 μg/mL kanamycin, 100 μg/mL ampicillin, 1 mM 5-ALA, 0.05 mM FeSO ·7H O and 0.5 mM IPTG (final concentrations) were added into each well. 4 2 The plate was expressed at 23 °C and 900 rpm for 24 hours. The cells were pelleted (4 °C, 15 min, 4,200 rpm), and 200 μL of KPi buffer (pH 8.0, 50 mM) containing 1 mg/mL lysozyme were added to each well. After fully resuspended by shaking at 900 rpm, the suspension was quick frozen by liquid nitrogen and restored to room temperature by a water bath. Then, each well was filled with another 200 μL of KPi buffer containing 20 μL pre-dissolved solution of compound 1 (10 mg/mL in DMSO), 5 mg of Na HPO ·5H O and 2 3 2 200 μg of Na NADP per well. The plate was shaken at 900 rpm at room temperature 2 overnight. The mixture was extracted with EtOAc (200 μL/well). The resulting suspension in the wells were mixed by shaken at 1100 rpm for 15 minutes. The organic extracts were separated by centrifuged (4 °C, 15 min, 4,200 rpm) and monitored by TLC (PE: EA 1:1). Once the spot on the TLC showed higher conversion or stereoselectivity. These ‘hits’ were recultured, the ratios of different stereo-isomers and the enantiomeric excess of the compound 2 were revalidated in analytical scale reactions by 1H-NMR spectrum and HPLC traces respectively.\nThe corresponding experimental results are shown here.\nEntry Name Mutation Sites (compared with WT) e.e.(major conv. d.r. product)\n2 1E47TH T49F/V78I/A82M/F87A 56% 41% 5.1 : 1\n3 1E49TH R47Y/V78I/A82M/F87A 57% 70% 6.3 : 1\n4 1E78TH R47Y/T49F/A82M/F87A 68% 74% 7.2 : 1\n5 1E82TH R47Y/T49F/V78I/F87A 47% 8.00% 4.3 : 1\n6 1E87TH R47Y/T49F/V78I/A82M - <5% -\n8 1GLG23HR R47W/S72W/F77Y/V78L/F81I/A82L/F87G/T88S/ - <5% - M177T/L181F/M185Q/L188Q/I209T/A328G/A330W\n2. The manuscript describes the first asymmetric total syntheses of pentalenolactone D and neo-pentalenolactone D, which indeed represents a highly innovative achievement. To better contextualize the significance of this work, we inquire whether there have been any prior synthetic efforts toward these or closely related compounds. Furthermore, given the multi-step nature of the synthetic route, we seek clarification on the overall yield. Finally, we are interested in the authors' perspective on the potential of this strategy to serve as a general method for preparing this class of natural products.\nResponse: We thank the reviewer for their constructive suggestions. Biological studies of the pentalenolactones have shown that they selectively inhibit glyceraldehyde-3-phosphate dehydrogenase, suggesting their potential as lead compounds for the development of novel antibiotic agents. Accordingly, the these pharmacologically important natural products have garnered significant attention from synthetic chemists. Here, we provide a summary of progress in their total synthesis. (ref. 41-60). At present, most reported syntheses are racemic. This work establishes a strategy for enantioselective synthesis.\nStarting from compound 1, the overall yield of our total synthesis is 0.23%. The novelty of our study lies in two major aspects. First, we establish that an enzyme-catalyzed desymmetrization provides a concise and efficient approach to access enantioenriched cis-quinane. Second, we put forward a new retrosynthetic logic for the synthesis of complex natural products. A biosynthetically relevant intermediate is rapidly assembled through the complementary use of artificial enzymes and chemical methods, after which biosynthetic enzymes relay enables key transformations that are otherwise difficult or inaccessible using conventional chemical approaches. As additional late-stage biosynthetic enzymes are identified, this strategy has the potential to access most members of this class of natural products.\nIn addition, by integrating the advantages of engineered enzymes, synthetic chemistry, and biosynthetic enzymes, we believe that this strategy has the potential to facilitate more efficient access to structurally complex and biologically active natural products.\n3. The manuscript mentions that pentanolactones can specifically inhibit glyceraldehyde-3-phosphate dehydrogenase and have potential antibacterial effects. The manuscript cites three references related to the study of such compounds, but all of which were conducted 30 years ago. May I ask if there are any recent research results to support the antibiotic activity of pentanolactones. Is there any relevant antibacterial activity test in this work to confirm the antibacterial activity of the compound？Finally, a forward-looking discussion on the therapeutic potential of this compound class as novel antibiotics, particularly within the current landscape of antimicrobial resistance, would greatly enhance the impact and relevance of the study.\nResponse: We sincerely appreciate the reviewer for constructive comments concerning the antibacterial activity. As you correctly pointed out, investigating the antibacterial activity of natural products is particularly important in the current landscape of antimicrobial resistance. Two references Int. J. Mol. Sci. 2024, 25, 13328; Molecules 2021, 26, 7377）relevant that were previously overlooked have now been added to the manuscript.\nFollowing the reviewer’s helpful suggestion, we have now evaluated antifungal and antibacterial activities of the tricyclic products 5, 6, 7, 8, 19. The corresponding experimental results are shown here. We found that only compound 8 exhibited weak antifungal activity against certain fungal strains. Compared with previously reported antibacterial pentalenolactones, we hypothesize that the epoxide moiety in this class of compounds may be important for their biological activity. These results provide helpful insights into the relationship between structure and biological activity of this class of natural products.\nMICs (μM) of tricyclic products against bacteria and common pathogenic fungi.\nMICs (μM)\nStrains\n8 5 6 7 19 Gentamicin Fluconazole\nS. aureus >100 >100 >100 >100 >100 0.18 -\nE. coli >100 >100 >100 >100 >100 0.18 -\nC. albicans SC5314 >100 >100 >100 >100 >100 - 0.82\nC. tropicalis 112936 >100 >100 >100 >100 >100 - 1.63\nATCC\nC. glabrata >100 >100 >100 >100 >100 - 3.27\nC. krusei 463 100 >100 >100 >100 >100 - 26.12\nC. auris Au1 100 >100 >100 >100 >100 - 6.53\nC. neoformans H99 100 >100 >100 >100 >100 - 13.06\nC. krusei, C. glabrata, C. auris, and C. neoformans were cultured in RPMI 1640 medium for 48 h; all other strains were cultured for 24 h.\n4. The manuscript would benefit from a careful proofread to correct a number of minor linguistic errors. kPi-KPi, TCL-TLC. Several variations were noted in the expression of rotational speed values (e.g., \"4200 rpm\" vs. \"4,200 rpm\"). To maintain rigorous scientific presentation and adhere to journal style, please select one formatting of numbers (e.g., use of commas).\nResponse: Thank you for pointing out these errors. Consistency in presentation is indeed very important, and we have made the corresponding corrections in both the main manuscript and the Supporting Information. We thank the reviewer once again for the helpful and constructive comments.\nReviewer # 2\nComments:\nThe authors report a chemoenzymatic synthesis of pentalenolactone D and its regioisomer neo-pentalenolactone D. First, the authors utilize a p450BM3 enzyme and through directed evolution achieve highly selective desymmetrization of diquinanes by enzymatic Riley oxidation. Second, chemical synthesis is used for a concise synthesis of triquinanes pentalenene and 1-deoxypentalenic acid. Finally, using known biosynthetic enzymes in the biosynthesis of pentalenolactone, the chemoenzymatic synthesis was achieved. These enzymes include an alpha-KG dependent hydroxylase, PtlH, and the regiodivergent Baeyer-Villiger monooxygenases (PntE and PtlE).\nBy combining synthetic chemistry with enzymatic reactions, the authors have developed a unique chemoenzymatic synthesis of triquinane sesquiterpenes. The enzymatic reactions would be difficult to replicate with synthetic methods, particularly the enantioselective Riley oxidation catalyzed by p450BM3 and the undirected methylene C-H hydroxylation catalyzed by PtlH. Thus, the use of enzymatic reactions in the chemoenzymatic synthesis is justifiable. Moreover, this is the first chemoenzymatic synthesis of this class of natural products, so the retrosynthetic strategy is a novel aspect of this work.\nDespite being able to achieve enantioselective allylic C-H hydroxylation, the application to the synthesis of pentalenene is indirect. The hydroxyl group installed by enzymatic hydroxylation is removed by elimination to form the enone which renders the optimized dr of the hydroxylation inconsequential as the axial alcohol could also eliminate through an E2 reaction. Essentially, the primary purpose of the initial enzymatic step is to achieve a desymmetrization. The authors also compare their p450 hydroxylation method to synthetic reactions and mention that theirs is the first \"direct\" method for enantioselective allylic oxidation. While this reviewer agrees that this method allows for the direct installation of alcohols (OH) as opposed to other methods that incorporate acetate (OAc) or benzoyl (OBz) group, in the context of the synthesis, the acetate or the benzoyl group could be preferrable given that they are better leaving groups as the OH is eliminated to form an alkene. In addition, one of the benefits of asymmetric catalysis is that the enantiomer can also be synthesized by using the enantiomer of the ligand or catalyst.\nThe chemical steps include an unselective reduction of the cyclopropane to the gem dimethyl group which also resulted in the undesired reduction of the ketone to an alcohol. A selective oxidation of that alcohol back to the ketone was developed using tritylium tetrafluoroborate.\nThe PtlH C-H hydroxylation is a known reaction in the biosynthesis of pentalenolactone (Ref 59). This should be mentioned explicitly in the text. The PntE and PtlE regiodivergent Baeyer-Villiger (BV) oxidation have also been previously reported (Ref 60). This should also be mentioned explicitly in the text rather than attributing the identification of these enzymes to screening libraries for BV monooxygenases.\nGiven the potential applications of combining enzymatic reactions with chemical synthesis, this work is a significant example of biocatalysis enabled natural product synthesis. Moreover, this work utilizes both engineered and wild type biosynthetic enzymes at both early and late-stages of the synthesis. This reviewer believes this work could merit publication in Nature Communications if the authors expand on the enzymatic Riley oxidation. This reviewer recommends the following major revisions.\nResponse: We sincerely thank the reviewer for the highly constructive comments, particularly regarding the enzymatic Riley oxidation. First, metal-catalyzed asymmetric allylic oxidation can indeed achieve the desymmetrization of simple alkenes, and both enantiomers can be accessed through appropriate choice of ligands and catalysts. The advantage of enzymes, on the other hand, lies in their ability to perform asymmetric allylic oxidation on structurally complex substrates, thereby generating chiral allylic alcohols under mild and highly selective conditions. Thus, the two approaches are complementary and synergistic.\nIn addition, we thank the reviewer for this helpful reminder. We have now included the relevant reports on the biosynthetic enzymes in the main text to avoid any potential misunderstanding for readers: With 1-deoxypentalenic acid (7) in hand, we proceeded to investigate late-stage biosynthetic enzyme-catalyzed oxidations based on native enzymes originally reported by the group of D. E. Cane (Figure 5).\n1. Can the authors through directed evolution of p450 enzymes access the enantiomer of 2 by selective oxidation of Hb2? If so, then this reviewer would agree that this would be consider an advance to the field of enantioselective allylic oxidation. This example would broaden the scope of this work and potentially allow for a chemoenzymatic synthesis pentalenic acid.\nResponse: We are grateful to the reviewer for the constructive and insightful comments on enzyme-catalyzed allylic oxidation, which inspired new considerations in our reaction.\nRegarding the reviewer’s question concerning the oxidation of H 2, we believe that this is an b issue of enantioselective oxidation a or b site.\nIn this work, we reported a directed evolution study on allylic oxidation at the a site, enabling highly enantioselective synthesis at this position.\nAt first, we did not focus extensively on or elaborate the oxidation at the b site, because we believe that the selective oxidation at the a site via directed evolution to afford product 2 provides the greatest benefit compared to other sites. This intermediate 2 may be readily elaborated, through subsequent chemical transformations and biosynthetically inspired oxidations, toward pentalenolactons.\nWe greatly appreciate the reviewer’s insightful perspective on our enzyme-catalyzed reaction, which prompted us to recognize the importance of enantiodivergent synthesis. In fact, in the wild-type enzyme, the minor product arises from oxidation at the b site, exhibiting an ee of 90% (see below). These results were not included in the original manuscript, as we did not initially realize that this reaction represents a case of enantiodivergent synthesis. We have benefited greatly from the reviewer’s insightful reminder.\nThe optical compound S1 were analyzed by HPLC (CHIRALPAK AD-H column, hexane: i-PrOH 80:20, 1.0 mL/min) to determine enantiomeric excess.\nHPLC traces of S1. (minor product of WT)\nHPLC traces of S1. (product of AAO4)\nIn addition, we are also grateful to the reviewer for bringing to our attention the synthesis of pentalenic acid. Haruo Ikeda and co-workers have reported that the cytochrome P450 enzyme CYP105D7 is capable of converting 1-deoxypentalenic acid (7) into pentalenic acid. In principle, according to the literature (J. Antibiot. 2011, 64, 65–71) on the discovery of the native enzyme, our existing intermediate 7 could be converted to the natural product pentalenic acid. We attempted to follow the reported enzymatic protocol. Accordingly, the P450 oxidase was heterologously expressed in E. coli BL21, and its successful expression was verified by SDS–PAGE analysis.\ntarget protein\net as\nyl\nn\noit c\narf\nc\nel o h\nw\ne\nsll\nHowever, no detectable product formation was observed under our experimental conditions. It is possible that minor experimental details not reported in the original paper contributed to the challenges we observed in replication.\nFinally, we would like to sincerely thank the reviewer once again for their constructive and insightful comments, which have greatly improved our manuscript.\n2. Can the authors through directed evolution of p450 enzymes access the diastereomer of 2 by selective oxidation of Ha1? The WT enzyme has 1.6:1 dr, so it should be possible to evolve the enzyme to achieve selectivity for the axial hydrogen. This alcohol could be more susceptible to elimination through E2 mechanism.\nResponse: We sincerely appreciate the reviewer’s constructive comment regarding the E2 elimination. As pointed out by the reviewer, hydroxyl groups with an upright orientation are likely more prone to elimination. The minor product of WT arises from selective oxidation at the b site with an ee of 90%. However, the minor product has opposite axial chirality. While the minor product is more readily eliminated, it serves as the precursor for chemoenzymatic transformations that afford the enantiomers of the natural products pentalenolactone D and neo-pentalenolactone D.\nSome minor corrections and suggestions:\n3. The yield of the p450 Riley oxidation should be provided in the Figures and main text rather than just in the methods section.\nResponse: We thank the reviewer for the constructive suggestion; the relevant information has now been provided in the main text figure 2.\n4. The authors should show a figure of the active site of p450BM3 and where the mutations in AAO4 are located. Are the mutations close to the heme co-factor?\nResponse: We thank the reviewer for this insightful suggestion. The relevant information has been added to the figure in the main text. As shown in the figure, amino acid residues at positions 78, 82, and 87 are located around the heme cofactor, whereas residues at positions 47 and 49 are slightly farther away.\n5. Compound 14 is not a xanthate. It is a O-thiocarbamate.\nResponse: We sincerely thank the reviewer for pointing out our mistake. We have corrected this in the manuscript: Consequently, compound 13 was converted to the O-thiocarbamate 14 and subsequent pyrolysis afforded 15.\n6. In the methods sections, lysaste should be \"lysate\".\nResponse: We sincerely thank the reviewer for the helpful suggestions. We have revised the manuscript accordingly."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoroughly addressed all my previous concerns in the revision. The revisions demonstrate a clear improvement in rigor, coherence, and presentation. I have no further questions and recommend this manuscript for acceptance and publication in its current form.", "reviewer_2": "In their response, the authors have identified that the WT p450 enzyme forms a minor product (S1, 1.6:1 dr) that is the enantiomer of the product formed from AAO4 hydroxylation (compound 2, main text). The authors do not provide the yield of S1 nor do they optimize the reaction to make it selectively. As such, the authors are unable to develop an enantiodivergent biocatalytic Riley oxidation reaction. While this is not essential for the total synthesis of pentalenene-type natural products, this does illustrate the limitations of the biocatalytic method compared with prior works. The strategy for using meso-cis-diquinanes starting materials for complex molecule synthesis is limited as only one enantiomer of the hydroxylated product can be selectively prepared with this strategy. The authors claim that the developed method is complementary to prior methods in their response, but in the manuscript they claim that their allylic hydroxylation method is \"more effective solution for structurally complex substrates\". The comparison with past methods for enantioselective allylic oxidation is not clear given that the authors report a singular example in this work that cannot be controlled to produce the two different enantiomers selectively.\nAfter the review of prior syntheses provided by the authors, this reviewer also sees that the synthesis of pentalenene described in this manuscript is not a significant advance compared to prior works. There is a large number of prior syntheses of pentalenene natural products dating back to the 1980s. The conjugate addition strategy of a cuprate to a diquinane-enone to access the all carbon stereocenter of pentalenene was reported by Paquette (JACS 1983, 105, 7358). This reviewer is not convinced that this work represents a significant advance in either reaction methodology or total synthesis. This reviewer recommends its publication in a more specialized journal.\nThe following concerns are noted.\nThe compound S1 in the supporting information is a different compound than in the response. None of the HPLC data that was provided in the response was included in the SI.\nThe compound S1 produced by WT p450 and the product produced by AAO4 are incorrectly drawn as diastereomers."}, "author_response": "Point-by-point Response Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nThe authors have thoroughly addressed all my previous concerns in the revision. The revisions demonstrate a clear improvement in rigor, coherence, and presentation. I have no further questions and recommend this manuscript for acceptance and publication in its current form.\nResponse: We sincerely thank the reviewer for the positive evaluation and recommendation. We are pleased that all previous concerns have been fully addressed in the revised manuscript.\nReviewer #2 (Remarks to the Author):\nIn their response, the authors have identified that the WT p450 enzyme forms a minor product (S1, 1.6:1 dr) that is the enantiomer of the product formed from AAO4 hydroxylation (compound 2, main text). The authors do not provide the yield of S1 nor do they optimize the reaction to make it selectively. As such, the authors are unable to develop an enantiodivergent biocatalytic Riley oxidation reaction. While this is not essential for the total synthesis of pentalenene-type natural products, this does illustrate the limitations of the biocatalytic method compared with prior works. The strategy for using meso-cis-diquinanes starting materials for complex molecule synthesis is limited as only one enantiomer of the hydroxylated product can be selectively prepared with this strategy. The authors claim that the developed method is complementary to prior methods in their response, but in the manuscript they claim that their allylic hydroxylation method is \"more effective solution for structurally complex substrates\". The comparison with past methods for enantioselective allylic oxidation is not clear given that the authors report a singular example in this work that cannot be controlled to produce the two different enantiomers selectively.\nAfter the review of prior syntheses provided by the authors, this reviewer also sees that the synthesis of pentalenene described in this manuscript is not a significant advance compared to prior works. There is a large number of prior syntheses of pentalenene natural products dating back to the 1980s. The conjugate addition strategy of a cuprate to a diquinane-enone to access the all carbon stereocenter of pentalenene was reported by Paquette (JACS 1983, 105, 7358). This reviewer is not convinced that this work represents a significant advance in either reaction methodology or total synthesis. This reviewer recommends its publication in a more specialized journal.\nResponse: We thank the reviewer for this thoughtful comment and agree that our study does not achieve an enantiodivergent allylic oxidation. As noted, the development of such a process was not the goal of this work, and the manuscript has been revised to avoid any implication to that effect.\nThe novelty of our study lies in two main aspects. First, we demonstrate that enzyme-catalyzed desymmetrization provides a concise and efficient route to enantioenriched cis-quinane scaffolds from meso precursors, which were then leveraged to achieve the asymmetric synthesis of the natural products pentalenolactones. Second, by integrating engineered enzymes, synthetic chemistry, and biosynthetic enzymes, we present a new retrosynthetic logic for the synthesis of complex natural products. This strategy provides a complementary framework for efficient access to structurally complex and biologically active natural products and has the potential to be applied to additional members of this class.\nThe compound S1 in the supporting information is a different compound than in the response. None of the HPLC data that was provided in the response was included in the SI.\nResponse: We thank the reviewer for pointing this out. The definition of compound S1 has now been made consistent between the response, the manuscript, and the Supporting Information. In addition, all relevant chiral HPLC data discussed in the response have now been included in the revised Supporting Information.\nThe compound S1 produced by WT p450 and the product produced by AAO4 are incorrectly drawn as diastereomers.\nResponse: We thank the reviewer for comments that helped improve the clarity and accuracy of the manuscript. The structures have been corrected accordingly in the manuscript and Supporting Information."}]

Decision letter