Regioselective and enantioselective propargylic hydroxylations catalyzed by P450tol monooxygenases

Regioselective and enantioselective propargylic hydroxylations catalyzed by P450tol monooxygenases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Regioselective and enantioselective propargylic hydroxylations catalyzed by P450tol monooxygenases Xu Deng, Cheng-Cheng Song, Wen-Jing Gu, Yu-Jie Wang, Lu Feng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3996274/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Regioselective and enantioselective hydroxylation of propargylic C-H bonds are useful reactions but often lack appropriate catalysts. Here a green and efficient asymmetric hydroxylation of primary and secondary C–H bonds at propargylic positions has been established. A series of optically active propargylic alcohols were prepared with high regio- and enantioselectivity (up to 99% ee ) under mild reaction conditionsby using P450tol, while the C≡C bonds in the molecule remained unreacted. This protocol provides a green and practical method for constructing enantiomerically chiral propargylic alcohols. In addition, we also demonstrated that the biohydroxylation strategy was able to scaled up to 2.25 mmol scale with the production of chiral propargyl alcohol 2a at a yield of 196 mg with 96% ee , which’s an important synthetic intermediate for antifungal drug Ravuconazole. Biocatalysis Hydroxylation P450 monooxygenase Propargylic alcohols Enantioselectivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Chiral propargylic alcohols are useful and versatile motifs that can be transformed into chiral allylic alcohols, allenes, bioactive molecules and natural products (Bauer 2012 ; Greshock et al. 2008 ; Helal et al. 1996 ; Lumbroso et al. 2013 ; Nakayama et al. 2011 ; Wang and Pu 2013 ). Over the past few decades, there are three mainly standard synthetic methods for the preparation of enantiopure propargylic alcohols: (1) the asymmetric transfer hydrogenation of alkynyl ketones catalyzed by transition metal (Matsumura et al. 1997 ; Shatskiy et al. 2015 ; Zhang et al. 2018 ); (2) the asymmetric addition of alkynyl organometallic reagents to aldehydes and ketones (Corey and Cimprich 1994 ; Lu et al. 2005 ; Trost and Weiss 2009 ); (3) the deracemization of racemic propargylic alcohols catalyzed by biocatalytic reaction (González-Granda et al. 2020 ; Kawanishi et al. 2019 ; Sang et al. 2022 ; Saravanan et al. 2014 ). Although some of these strategies access to chiral propargylic alcohols have been developed, many of them involve the use of transition metal catalyst, air sensitive reagents, or deliver chiral propargylic alcohols with moderate enantioselectivity. Up to date, it is still a challenge to develop a biocatalytic method with monooxygenase for the synthesis of enantiopure propargylic alcohols. As we all known, enantioselective oxidation of propargylic C-H bond is the most direct and atom economic strategy for the preparation of enantiopure propargylic alcohols. Until now, only two examples have been reported to prepare chiral propargylic alcohols: (1) Cu(MeCN) 4 PF 6 and chiral bisoxazoline ligand catalyzed acyloxylation of propargylic C-H bond to obtained the products with 15–51% ee under an excess of oxidant and reactions took 4–5 days to proceed (Stephen Clark et al. 1998 ); (2) Hydroxylation of propargylic C-H Bond catalyzed by Chloroperoxidase (CPO) using equivalent of H 2 O 2 or TBHP as terminal oxidant to synthesize propargylic alcohols with 57–95% ee or 43–90% ee , respectively (Hu and Hager 1999 ). However, selective C-H oxidation of simple alkynes represents one of the most fundamental challenges. On the one hand, the highly active compound I species are usually undistinguishing toward similar C-H bonds, resulting in low chemoselectivity (overoxidation products) (Alvarez et al. 2007 ; Hu and Hager 1999 ) and regioselectivity, On the other hand, alkynyl and alkyl groups with relatively small steric effect are difficult to differentiate by small molecular catalysts, resulting in low stereoselectivity (Stephen Clark et al. 1998 ). Further development of higher stereoselectivity and more efficient catalysts in this field is therefore of great significance for the asymmetric catalysis. Cytochrome P450 monooxygenases (P450s) catalyzed the oxidative reactions of C-H bonds with stereo- and regioselective manner under mild reaction condition (Chakrabarty et al. 2020 ; Chen et al. 2023 ; Jiang et al. 2021 ; Li et al. 2020 ; Li and Wong 2019 ; Manning et al. 2019 ; Roiban and Reetz 2015 ; Song et al. 2023 ; Whitehouse et al. 2012 ; Zhang et al. 2022a ; Zhang et al. 2023 ; Zhang et al. 2022b ). Additionally, P450s as mild and selective catalysts have been used in the asymmetric hydroxylation of benzylic, allylic, aromatic and unactivated C-H bonds (Fig. 1 a) (Chakrabarty et al. 2020 ; Kim et al. 2022 ; Neufeld et al. 2014 ; Roiban and Reetz 2015 ; Whitehouse et al. 2012 ). However, P450s as an effective and versatile catalyst for the enantioselective hydroxylation of propargylic C-H bonds has not been available in literature. In light of our ongoing interests in P450s-catalyzed asymmetric hydroxylation reactions with a broad substrate range and widely applications (Cui et al. 2023 ; Cui et al. 2019 ; Deng et al. 2022 ; Wan et al. 2022 ; Wang et al. 2022 ; Xie et al. 2020 ; Xie et al. 2023 ), we herein describe chiral propargylic alcohols were synthesized from the simple alkynes by P450tol catalyzed asymmetric propargylic C-H bonds hydroxylation with regio- and stereoselectivity (Fig. 1 b). Additionally, molecular docking and MD simulations were carried out to provide a rationale for the enantioselectivity and regioselectivity of these reactions. As far as we know, this is the first example of asymmetric hydroxylation of propargylic C-H bonds with excellent regio- and enantionselectivity by P450 monooxygenase, while the C ≡ C bonds in the molecule remained unreacted. Materials and methods Materials and procedures Chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. Isopropyl- β -D-thiogalactopyranoside (IPTG) and Ampicillin Sodium Salt ( Amp ) and Streptomycin sulface ( Str ) were purchased from Solarbio (Beijing, China). Analytical thin layer chromatography (TLC) was performed on precoated silica gel 60 GF254 plates. Flash column chromatography was carried out with 300–400 mesh silica gel. The Alkynes and racemic products were synthesized following the reported method (Liu et al. 2022 ; Watanabe et al. 2018 ). Visualization on TLC was achieved by use of UV light (254 nm). 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) were recorded on Agilent Technologies 400 MR. Chemical shifts were reported in parts per million (ppm) relative to residual signals of the solvent. The following abbreviations are used to indicate multiplicity: s = singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublets. High-resolution mass spectra (HRMS) was recorded by ESI ionization sources. Chiral HPLC analysis was performed on Shimadzu LC-20A, equipped with Chiralpak® OJ-H, OD-H, AD-H or AS-Hcolumns. Enzyme preparation Cultivation of E. coli (P450tol-4) cells was carried out using TB medium containing 50 µ g/mL Str and Amp . After growing at 37 o C to an OD 600 of 0.6–0.8, IPTG was added to the final concentration of 0.2 mM. The culture was incubated at 25 o C with another 12–14 h for enyzme expression. Recombinant E. coli (P450tol-4) cells were harvested by centrifugation at 7000 ×g at 4 o C for 5 min. The freshly prepared E. coli (P450tol-4) cells were resuspended in reaction buffer solution to a cell density (g cdw/L) for performing biotransformation. Molecular Docking and molecular dynamics simulation Docking study of 1f in the active site of enzyme P450tol (PDB No.: 7V40) was carried out using AutoDock 4.0 software (Trott and Olson 2010 ). All the docking experiments were performed using “Genetic Algorithm” search parameters and default docking parameters. All structural illustrations were generated using the PyMOL software (Seeliger and de Groot 2010 ). Molecular dynamics simulation was carried out for P450tol to further explain the mechanism of regio- and enantioselective hydroxyation of substrate 1f . The molecule structure of 1f was docked into the active site of enzyme P450tol. The iron-oxo intermediate involved in the cytochrome catalyzed oxidative hydroxylation cycle was used to model the active form of the P450 cofactor (Narayan et al. 2015 ). Simulations were performed using the GPU code (pmemd) of the AMBER 22 software package (D.A. Case 2017). The Amber-compatible parameters developed by Cheatham et al. (Shahrokh et al. 2012 ) were used for Cpd I and its axial Cys ligand. Substrate 1f parameters for the molecular dynamics (MD) simulations were generated within the antechamber module of AMBER 22 using the general AMBER force field (GAFF) (Wang et al. 2004 ), with partial charges set to fit the electrostatic potential generated at the HF/6-31G(d) level by the restrained electrostatic potential (RESP) model (Bayly et al. 1993 ). The charges were calculated according to the Merz-Singh-Kollman scheme (Besler et al. 1990 ; Singh and Kollman 1984 ) using Gaussian 16(C.01) (M.J. Frisch 2016). Amino acid protonation states were predicted using the H + + server ( http://biophysics.cs.vt.edu/H++ ) (Anandakrishnan et al. 2012 ). Then, the enzyme was solvated in a pre-equilibrated truncated hexagonal box with a 10-Å buffer of TIP3P (Jorgensen et al. 1983 ) water molecules using the AMBER 22 leap module, resulting in the addition of ∼19,000 solvent molecules. The systems were neutralized by addition of ions Na + and Cl − , all subsequent calculations were done using the Amber ff14SB force field (Maier et al. 2015 ). The following MD simulation steps as reported in ref. (Narayan et al. 2015 ), 60 ns production trajectories MD simulations were performed after equilibrated. All the structural images and distance were performed using the VMD Software, trajectory analysis and energy was post-processed by Cpptraj module (Maier et al. 2015 ), respectively. All the structural images and distance were performed using the VMD Software. Trajectory data, root mean square deviation (RMSD), root mean square fluction (RMSF) were analyzed by Cpptraj module, respectively. General procedure for the synthesis of chiral ( S )-2 on preparation-scale To a 500-mL shake flask containing a resting cell suspension of E. coli (P450tol-4) (10 g cdw/L) and 1 (0.6 mmol) in 180 mL PB buffer (50 mM, pH 8.5). The reaction mixture was shaken at 20 ℃ for 6 h. Then the mixture was extracted with ethyl acetate (3 × 180 mL). The organic phases were separated by centrifugation (7000 × g, 15 min), combined, dried over anhydrous Na 2 SO 4 , and evaporated at reduced pressure. The resulting mixture was purified by flash chromatography using ethyl acetate/petroleum ether as eluent on silica gel to afford the desired chiral product 2 . Procedure for the synthesis of chiral ( S )-2a on 2.25 mmol-scale To a 2.0-L shake flask containing a resting cell suspension of E. coli (P450tol-4) (10 g cdw/L) and 1 (2.25 mmol) in 750 mL PB buffer (50 mM, pH 8.5). The reaction mixture was shaken at 20 ℃ for 6 h. Then the mixture was extracted with ethyl acetate (3 × 1000 mL). The organic phases were separated by centrifugation (7000 × g, 15 min), combined, dried over anhydrous Na 2 SO 4 , and evaporated at reduced pressure. The resulting mixture was purified by flash chromatography using ethyl acetate/petroleum ether as eluent on silica gel to afford the desired chiral product 2a (58% yield, 196 mg). Results and discussion Screening of reaction conditions for propargylic hydroxylation In our previous work, we have obtained several cytochrome P450 monooxygenases (P450DA, P450PL2-2) (Cui et al. 2023; Cui et al. 2019; Deng et al. 2022; Wan et al. 2022; Wang et al. 2022; Xie et al. 2020; Xie et al. 2023) and also constructed five recombinant Escherichia coli strains (P450tol-1 to P450tol-5) harboring P450tol monooxygenase from Rhodococcus coprophilus TC-2 (Chen et al. 2022; Li et al. 2014) and five pairs of redox partner Fdx-FdR (ferredoxin-ferredoxin reductase) from P. lavamentivorans DS-1 (Wu et al. 2018). With these P450 monooxygenases in hand, but-1-yn-1-ylbenzene ( 1a ) was used as a model substrate to explore the hydroxylation activity and stereoselectivity of these recombinant P450 strains. Biohydroxylation reactions were carried out using E. coli whole-cells as catalyst without additional cofactors required. After incubation at 30 o C in PB buffer (pH = 8.0) for 6 h, the ee and yield of the chiral product 4-phenylbut-3-yn-2-ol ( 2a ) were analyzed using chiral HPLC (Table 1). Strains P450DA and P450PL2-2 exhibited the opposite stereoselectivity despite a very low yield in the propargylic C-H hydroxylation reactions, which produced 2a in 70% ee for R stereoselectivity and 36% ee for S stereoselectivity, respectively (Table 1, entries 1-2). Surprisingly, the five recombinant E. coli P450tol strains could convert 1a to 2a with 96% ee and S stereoselectivity despite in different yields (Table 1, entries 3-7), which the strain P450tol-4 showed the highest yield of 58% (Table 1, entry 6). Table 1. Screening of P450 strains for asymmetric hydroxylation of 1a . a The reaction was carried out on an analytical scale in 5 mL PB buffer (50 mM, pH = 8.0) containing 10 g cdw/L E. coli (P450s) cells with 1a (2 mM) at 30 °C for 6 h b The yield and ee were measured by chiral HPLC analysis, and absolute configuration was confirmed by previously reported references (Shatskiy et al. 2015; Zhang et al. 2018). c Not detected. With the optimal strain P450tol-4 in hand, the reaction conditions including temperature, the pH of the buffer and the concentration of the biocatalyst were evaluated. The results are summarized in Fig. 2. From the results, we can find that the stereoselectivity of the reaction remains unchanged with different temperature, pH and cell concentration. The highest yield was found at 20 o C, which is about the same as 15 o C. With the increase of temperature from 20 to 40 o C, the yield significantly decreased from 69% to 54% (Fig. 2A). The results in Fig. 2B indicated that the pH influenced the yield of 2a dramatically. Increasing pH from 5.0 to 8.5 improved the yield, while further increasing pH to 9.0 with the yield decreases slightly. The results in Fig. 2C indicated that the cell density of P450tol influenced the yield of 2a dramatically. The yield of the product 2a increased with the increasing of cell concentration from 5 to 10 g cdw/L, while a higher yield was not obtained by further increasing cell density to 25 g cdw/L. To sum up, the optimal reaction conditions of propargylic hydroxylations were set at 20 o C and reaction pH = 8.5 using 10 g cdw/L cell density of recombinant E. coli cells (P450tol-4). Scope of substrates Under the optimal reaction conditions, a range of aryl alkyne subtrates 1 were transformed into propargylic alcohols 2 on preparative-scale, and the results are displayed in Fig. 3A. Alkyne containing longer alkyl chain could be converted to the corresponding propargyl alcohol 2b by P450tol-4 with 99% ee , albeit in lower isolated yield (22% yield). Besides hydroxylation of secondary C( sp 3 )-H bonds, P450tol-4 could also catalyze the hydroxylation reaction of primary C−H bond in moderate isolated yield ( 2c ). Furthermore, substrates with fluorine substitution at ortho -, and para -positions of the aromatic ring ( 2d and 2e ) were well tolerated in this reaction in 28-47% yield with 94-97% ee . In the previous reported work, P450tol monooxygenase catalyzed the benzylic C-H bonds hydroxylation of toluenes to produce benzyl alcohols (Chen et al. 2022). When a methyl substitution in the aromatic ring, there is regioselectivity competition between the benzylic and propargylic C-H bonds in the hydroxylation reaction. The methyl substitution at meta -position of the aromatic ring, the product propargyl alcohol 2f was obtained with 99:1 regioselectivity ratio ( r.r. ) and 96% ee . Alkynes containing various heteroaromatic rings, such as furan and pyridine, were transformed to propargylic alcohols 2g - 2j as well with 95-98% ee in moderate isolated yield. Unfortunately, P450tol-4 exhibited no catalytic efficiency of alkyne containing the bulky naphthalene ring ( 2k ) and tertiary propargylic C–H bond ( 2l ). To show the synthetic potential of this strategy, our catalytic reaction can be scaled up to 2.25 mmol scale with the formation of propargyl alcohol 2a in 58% isolated yield (196 mg) with 96% ee (Fig. 3B). In addition, chiral propargyl alcohol 2a was the important building blocks for synthesis of antifungal drug Ravuconazole (Xu et al. 2009). Molecular docking and molecular dynamics simulation To obtain a structure-based understanding of the excellent enantio- and regioselectivity of these reactions, molecular docking of the substrate 1f onto the X-ray structure of P450tol (PDB No.: 7V40) was carried out (Fig. 4A). In the 1f -enzyme binding pose, the distance between C10-carbon (propargylic-position) or C7-carbon (benzylic-position) of 1f and heme oxygen atom (heme-O) of P450tol is 2.8 or 9.0 Å. Meanwhile, the distance between C10 pro- S -hydrogen or pro- R -hydrogen of 1f and heme-O is 1.9 Å or 3.0 Å, respectively (Fig. 4A). The results of molecular docking demonstrated that the hydrophobic interactions were formed between substrate 1f with residues Pro114, Phe198, Phe199, Trp223, Phe329 and Phe426 located in the active pocket of P450tol, which stabilize the substrates in the catalytic pocket. Further MD simulations indicated that such binding conformations of substrate 1f are quite stable during 60 ns-MD simulation, during which the distribution of the distance between the C10-carbon of 1f and the heme-O gave most of MD frames within 2.8-6.0 Å, while the C7-carbon of 1f gave most of MD frames beyond 6.0 Å (Fig. 4B), indicating that 1f binding in pose C10 is more stable, which is consistent with the selectivity data (>99:1 regioselectivity at C10-position). Another important finding from our MD simulation study is that the C10 pro- S -hydrogen (H8) of 1f is well positioned for H-abstraction than C10 pro- R -hydrogen (H9) based on the distance of (H8( 1f )-O(heme) vs H9( 1f )-O(heme)) and the angle of (O(heme)-H8( 1f )-C10( 1f ) vs O(heme)-H9( 1f )-C10( 1f )), which is consistent with the selectivity data (96% ee for S -selectivity) (Fig. 4C, 4D). In summary, our MD simulation results provide valuable molecular insights into the excellent enantio- and regioselectivity hydroxylation of C-H bonds in alkynes catalyzed by P450tol. Conclusions In conclusion, we have developed a green and efficient platform for the asymmetric hydroxylation of primary and secondary C–H bonds at propargylic positions catalyzed by P450tol monooxygenase, while the C ≡ C bonds in the molecule remained unreacted. This protocol provides a practical and sustainable method for the preparation of enantiomerically pure propargylic alcohols with high regioselectivity (> 99:1 r.r. ) and enantioselectivity (94–99% ee ), which are valuable and versatile synthetic building blocks in organic synthesis. Additionally, molecular docking and MD simulations were performed to provide a rationale for the excellent enantio- and regioselectivity of these reactions. Efforts broadening the substrate specificity (e.g., for 1k and 1l ) and inverting the enantioselectivity of the propargylic C-H bonds hydroxylations via engineered P450tol variants are currently ongoing in our laboratory. Abbreviations HPLC High Performance Liquid chromatography P450tol P450 monooxygenase from Rhodococcus coprophilus TC-2 P450DA P450 monooxygenase from Deinococcus apachensis P450PL2 P450 monooxygenase from P. lavamentivorans DS-1 Fdx-FdR five pairs of redox partner Fdx-FdR (ferredoxin-ferredoxin reductase) from P. lavamentivorans DS-1 PB buffer consist of sodium phosphate dibasic and potassium phosphate monobasic MD molecular dynamics simulation. Declarations Acknowledgements Not applicable. Author contributions XD, CS and WG:Investigation, Methodology, Validation, Data curation. YW and LF: Data curation, Visualization. MZ and WY: Writing-review & editing. XZ and YC: Project administration, Conceptualization, Methodology, Validation, Funding acquisition. Funding We are grateful for financial support from the National Natural Science Foundation of China (No. 32271537 and 22061049), the Science and Technology Department of Guizhou province (QKHJCZK2021-036 and QKHRCPTGCC-2023-003), the Science and Technology Department of Zunyi (ZSKRPT-2020-5, ZSKH-2018-3, ZSKRPT-2021-5), Zunyi Medical University (QKH-2018-5772-014). Availability of data and materials All data generated or analyzed during this study are included in this article. Ethics approval and consent to participate Not applicable. Consent for publication All authors approved the consent for publishing the manuscript to bioresources and bioprocessing. Competing interests The authors declare that they have no competing interests. Author details 1 Key Laboratory of Biocatalysis & Chiral Drug Synthesis of Guizhou Province, Key Laboratory of Basic Pharmacology of Ministry of Education, School of Pharmacy, Zunyi Medical University, Zunyi, China. 2 National Engineering Research Center of Chiral Drugs, Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu, 610041, China. References Alvarez LX, Christ ML, Sorokin AB (2007) Selective oxidation of alkenes and alkynes catalyzed by copper complexes. Appl Catal A: Gen 325(2):303-308 doi:https://doi.org/10.1016/j.apcata.2007.02.045 Anandakrishnan R, Aguilar B, Onufriev AV (2012) H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res 40(W1):W537-W541 doi:10.1093/nar/gks375 Bauer EB (2012) Transition-Metal-Catalyzed Functionalization of Propargylic Alcohols and Their Derivatives. Synthesis 44(08):1131-1151 doi:10.1055/s-0031-1290503 Bayly CI, Cieplak P, Cornell W, Kollman PA (1993) A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. The Journal of Physical Chemistry 97(40):10269-10280 doi:10.1021/j100142a004 Besler BH, Merz Jr KM, Kollman PA (1990) Atomic charges derived from semiempirical methods. J Comput Chem 11(4):431-439 doi:https://doi.org/10.1002/jcc.540110404 Chakrabarty S, Wang Y, Perkins JC, Narayan ARH (2020) Scalable biocatalytic C–H oxyfunctionalization reactions. Chem Soc Rev 49(22):8137-8155 doi:10.1039/D0CS00440E Chen C-C, Dai M, Zhang L, Zhao J, Zeng W, Shi M, Huang J-W, Liu W, Guo R-T, Li A (2022) Molecular Basis for a Toluene Monooxygenase to Govern Substrate Selectivity. ACS Catal 12(5):2831-2839 doi:10.1021/acscatal.1c05845 Chen J, Dong S, Fang W, Jiang Y, Chen Z, Qin X, Wang C, Zhou H, Jin L, Feng Y, Wang B, Cong Z (2023) Regiodivergent and Enantioselective Hydroxylation of C−H bonds by Synergistic Use of Protein Engineering and Exogenous Dual-Functional Small Molecules. Angew Chem Int Ed 62(4):e202215088 doi:https://doi.org/10.1002/anie.202215088 Corey EJ, Cimprich KA (1994) Highly Enantioselective Alkynylation of Aldehydes Promoted by Chiral Oxazaborolidines. J Am Chem Soc 116(7):3151-3152 doi:10.1021/ja00086a066 Cui H-B, Ma T, Zhang R-Y, Shan J, Wang Z-Q, Bai M, Chen Y-Z (2023) Biocatalytic Synthesis of Chiral Benzylic Alcohols via Enantioselective Hydroxylation by a Self-Sufficient Cytochrome P450 from Deinococcus gobiensis. Synthesis(EFirst) doi:10.1055/a-2076-9792 Cui H-B, Xie L-Z, Wan N-W, He Q, Li Z, Chen Y-Z (2019) Cascade bio-hydroxylation and dehalogenation for one-pot enantioselective synthesis of optically active β-halohydrins from halohydrocarbons. Green Chem 21(16):4324-4328 doi:10.1039/C9GC01802F D.A. Case DSC, T.E.C. Iii, T.A. Darden, R.E. Duke, et al. (2017) AMBER16 and AmberTools17. University of California, San Francisco. Deng G-Z, Zhou X, Yu Q-X, Mou X-Q, An M, Cui H-B, Zhou X-J, Wan N-W, Li Z, Chen Y-Z (2022) Highly Enantioselective Hydroxylation of 3-Arylpropanenitriles to Access Chiral β-Hydroxy Nitriles by Engineering of P450pyr Monooxygenase. Org Process Res Dev 26(7):2046-2051 doi:10.1021/acs.oprd.1c00444 González-Granda S, Méndez-Sánchez D, Lavandera I, Gotor-Fernández V (2020) Laccase-mediated Oxidations of Propargylic Alcohols. Application in the Deracemization of 1-arylprop-2-yn-1-ols in Combination with Alcohol Dehydrogenases. ChemCatChem 12(2):520-527 doi:https://doi.org/10.1002/cctc.201901543 Greshock TJ, Johns DM, Noguchi Y, Williams RM (2008) Improved Total Synthesis of the Potent HDAC Inhibitor FK228 (FR-901228). Org Lett 10(4):613-616 doi:10.1021/ol702957z Helal CJ, Magriotis PA, Corey EJ (1996) Direct Catalytic Enantioselective Reduction of Achiral α,β-Ynones. Strong Remote Steric Effects Across the C−C Triple Bond. J Am Chem Soc 118(44):10938-10939 doi:10.1021/ja962849e Hu S, Hager LP (1999) Highly Enantioselective Propargylic Hydroxylations Catalyzed by Chloroperoxidase. J Am Chem Soc 121(4):872-873 doi:10.1021/ja983612g Jiang Y, Peng W, Li Z, You C, Zhao Y, Tang D, Wang B, Li S (2021) Unexpected Reactions of α,β-Unsaturated Fatty Acids Provide Insight into the Mechanisms of CYP152 Peroxygenases. Angew Chem Int Ed 60(46):24694-24701 doi:https://doi.org/10.1002/anie.202111163 Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics 79(2):926-935 doi:10.1063/1.445869 Kawanishi S, Oki S, Kundu D, Akai S (2019) Lipase/Oxovanadium Co-Catalyzed Dynamic Kinetic Resolution of Propargyl Alcohols: Competition between Racemization and Rearrangement. Org Lett 21(9):2978-2982 doi:10.1021/acs.orglett.9b00334 Kim HJ, Ishida K, Ishida-Ito M, Hertweck C (2022) Sequential Allylic Alcohol Formation by a Multifunctional Cytochrome P450 Monooxygenase with Rare Redox Partners. Angew Chem Int Ed 61(26):e202203264 doi:https://doi.org/10.1002/anie.202203264 Li A, Acevedo-Rocha CG, D'Amore L, Chen J, Peng Y, Garcia-Borràs M, Gao C, Zhu J, Rickerby H, Osuna S, Zhou J, Reetz MT (2020) Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants. Angew Chem Int Ed 59(30):12499-12505 doi:https://doi.org/10.1002/anie.202003139 Li A, Wu S, Adams JP, Snajdrova R, Li Z (2014) Asymmetric epoxidation of alkenes and benzylic hydroxylation with P450tol monooxygenase from Rhodococcus coprophilus TC-2. Chem Commun 50(63):8771-8774 doi:10.1039/C4CC03491K Li Y, Wong LL (2019) Multi-Functional Oxidase Activity of CYP102A1 (P450BM3) in the Oxidation of Quinolines and Tetrahydroquinolines. Angew Chem Int Ed 58(28):9551-9555 doi:https://doi.org/10.1002/anie.201904157 Liu Z, Qin Z-Y, Zhu L, Athavale SV, Sengupta A, Jia Z-J, Garcia-Borràs M, Houk KN, Arnold FH (2022) An Enzymatic Platform for Primary Amination of 1-Aryl-2-alkyl Alkynes. J Am Chem Soc 144(1):80-85 doi:10.1021/jacs.1c11340 Lu G, Li Y-M, Li X-S, Chan ASC (2005) Synthesis and application of new chiral catalysts for asymmetric alkynylation reactions. Coord Chem Rev 249(17):1736-1744 doi:https://doi.org/10.1016/j.ccr.2004.12.020 Lumbroso A, Cooke ML, Breit B (2013) Catalytic Asymmetric Synthesis of Allylic Alcohols and Derivatives and their Applications in Organic Synthesis. Angew Chem Int Ed 52(7):1890-1932 doi:https://doi.org/10.1002/anie.201204579 M.J. Frisch GWT, et al. (2016) Gaussian 16 Revision A.03. Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C (2015) ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. Journal of Chemical Theory and Computation 11(8):3696-3713 doi:10.1021/acs.jctc.5b00255 Manning J, Tavanti M, Porter JL, Kress N, De Visser SP, Turner NJ, Flitsch SL (2019) Regio- and Enantio-selective Chemo-enzymatic C−H-Lactonization of Decanoic Acid to (S)-δ-Decalactone. Angew Chem Int Ed 58(17):5668-5671 doi:https://doi.org/10.1002/anie.201901242 Matsumura K, Hashiguchi S, Ikariya T, Noyori R (1997) Asymmetric Transfer Hydrogenation of α, β-Acetylenic Ketones. J Am Chem Soc 119(37):8738-8739 doi:10.1021/ja971570a Nakayama A, Kogure N, Kitajima M, Takayama H (2011) Asymmetric Total Synthesis of a Pentacyclic Lycopodium Alkaloid: Huperzine-Q. Angew Chem Int Ed 50(35):8025-8028 doi:https://doi.org/10.1002/anie.201103550 Narayan ARH, Jiménez-Osés G, Liu P, Negretti S, Zhao W, Gilbert MM, Ramabhadran RO, Yang Y-F, Furan LR, Li Z, Podust LM, Montgomery J, Houk KN, Sherman DH (2015) Enzymatic hydroxylation of an unactivated methylene C–H bond guided by molecular dynamics simulations. Nature Chemistry 7(8):653-660 doi:10.1038/nchem.2285 Neufeld K, Henßen B, Pietruszka J (2014) Enantioselective Allylic Hydroxylation of ω-Alkenoic Acids and Esters by P450 BM3 Monooxygenase. Angew Chem Int Ed 53(48):13253-13257 doi:https://doi.org/10.1002/anie.201403537 Roiban G-D, Reetz MT (2015) Expanding the toolbox of organic chemists: directed evolution of P450 monooxygenases as catalysts in regio- and stereoselective oxidative hydroxylation. Chem Commun 51(12):2208-2224 doi:10.1039/C4CC09218J Sang X, Tong F, Zeng Z, Wu M, Yuan B, Sun Z, Sheng X, Qu G, Alcalde M, Hollmann F, Zhang W (2022) A Biocatalytic Platform for the Synthesis of Enantiopure Propargylic Alcohols and Amines. Org Lett 24(23):4252-4257 doi:10.1021/acs.orglett.2c01547 Saravanan T, Jana S, Chadha A (2014) Utilization of whole cell mediated deracemization in a chemoenzymatic synthesis of enantiomerically enriched polycyclic chromeno[4,3-b] pyrrolidines. Org Biomol Chem 12(26):4682-4690 doi:10.1039/C4OB00615A Seeliger D, de Groot BL (2010) Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des 24(5):417-422 doi:10.1007/s10822-010-9352-6 Shahrokh K, Orendt A, Yost GS, Cheatham Iii TE (2012) Quantum mechanically derived AMBER-compatible heme parameters for various states of the cytochrome P450 catalytic cycle. J Comput Chem 33(2):119-133 doi:https://doi.org/10.1002/jcc.21922 Shatskiy A, Kivijärvi T, Lundberg H, Tinnis F, Adolfsson H (2015) Ruthenium-Catalyzed Asymmetric Transfer Hydrogenation of Propargylic Ketones. ChemCatChem 7(23):3818-3821 doi:https://doi.org/10.1002/cctc.201500821 Singh UC, Kollman PA (1984) An approach to computing electrostatic charges for molecules. J Comput Chem 5(2):129-145 doi:https://doi.org/10.1002/jcc.540050204 Song F, Zheng M, Wang J, Liu H, Lin Z, Liu B, Deng Z, Cong H, Zhou Q, Qu X (2023) Chemoenzymatic synthesis of C14-functionalized steroids. Nat Synth doi:10.1038/s44160-023-00280-z Stephen Clark J, Tolhurst KF, Taylor M, Swallow S (1998) Enantioselective propargylic oxidation. Tetrahedron Lett 39(27):4913-4916 doi:https://doi.org/10.1016/S0040-4039(98)00894-6 Trost BM, Weiss AH (2009) The Enantioselective Addition of Alkyne Nucleophiles to Carbonyl Groups. Adv Synth Catal 351(7-8):963-983 doi:https://doi.org/10.1002/adsc.200800776 Trott O, Olson AJ (2010) AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31(2):455-461 doi:https://doi.org/10.1002/jcc.21334 Wan N-W, Cui H-B, Zhao L, Shan J, Chen K, Wang Z-Q, Zhou X-J, Cui B-D, Han W-Y, Chen Y-Z (2022) Directed evolution of cytochrome P450DA hydroxylase activity for stereoselective biohydroxylation. Catal Sci Technol 12(18):5703-5708 doi:10.1039/D2CY00164K Wang J, Wang H, Wei C, Zhang L, Cui B, Wang Z, Zhang Y, Wan N, Aisa HA, Chen Y (2022) Combined photoredox/engineered P450 enzymatic direct dioxygen-functionalization of arylalkanes to chiral acyloins. Org Biomol Chem 20(46):9085-9092 doi:10.1039/D2OB01769E Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput Chem 25(9):1157-1174 doi:https://doi.org/10.1002/jcc.20035 Wang Q, Pu L (2013) Diverse Transformations of Chiral Propargylic Alcohols Generated by BINOL-Catalyzed Alkyne Addition to Aldehydes. Synlett 24(11):1340-1363 doi:10.1055/s-0033-1338781 Watanabe K, Miyazaki Y, Okubo M, Zhou B, Tsuji H, Kawatsura M (2018) Nickel-Catalyzed Asymmetric Propargylic Amination of Propargylic Carbonates Bearing an Internal Alkyne Group. Org Lett 20(17):5448-5451 doi:10.1021/acs.orglett.8b02325 Whitehouse CJC, Bell SG, Wong L-L (2012) P450BM3 (CYP102A1): connecting the dots. Chem Soc Rev 41(3):1218-1260 doi:10.1039/C1CS15192D Wu K, Tang L, Cui H, Wan N, Liu Z, Wang Z, Zhang S, Cui B, Han W, Chen Y (2018) Biocatalytical Asymmetric Sulfoxidation by Identifying Cytochrome P450 from Parvibaculum Lavamentivorans DS-1. ChemCatChem 10(23):5410-5413 doi:https://doi.org/10.1002/cctc.201801139 Xie L, Chen K, Cui H, Wan N, Cui B, Han W, Chen Y (2020) Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation. ChemBioChem 21(13):1820-1825 doi:https://doi.org/10.1002/cbic.201900691 Xie L, Zhang Y, Zhang R, Cui H, Cui B, Han W, Wan N, Li Z, Chen Y (2023) Biocatalytic stereoselective synthesis of methyl mandelates by engineering a cytochrome P450 hydroxylase. Green Synth Catal doi:https://doi.org/10.1016/j.gresc.2023.01.005 Xu L, Muller MR, Yu X, Zhu B-Q (2009) Improved Chiral Synthesis of Ravuconazole. Synth Commun 39(9):1611-1625 doi:10.1080/00397910802563420 Zhang K, Yu A, Chu X, Li F, Liu J, Liu L, Bai W-J, He C, Wang X (2022a) Biocatalytic Enantioselective β-Hydroxylation of Unactivated C−H Bonds in Aliphatic Carboxylic Acids. Angew Chem Int Ed 61(28):e202204290 doi:https://doi.org/10.1002/anie.202204290 Zhang X, Shen P, Zhao J, Chen Y, Li X, Huang J-W, Zhang L, Li Q, Gao C, Xing Q, Chen C-C, Guo R-T, Li A (2023) Rationally Controlling Selective Steroid Hydroxylation via Scaffold Sampling of a P450 Family. ACS Catal 13(2):1280-1289 doi:10.1021/acscatal.2c04906 Zhang Y-M, Yuan M-L, Liu W-P, Xie J-H, Zhou Q-L (2018) Iridium-Catalyzed Asymmetric Transfer Hydrogenation of Alkynyl Ketones Using Sodium Formate and Ethanol as Hydrogen Sources. Org Lett 20(15):4486-4489 doi:10.1021/acs.orglett.8b01787 Zhang Y, Xiong Z, Li Y, Wilson M, Christensen KE, Jaques E, Hernández-Lladó P, Robertson J, Wong LL (2022b) Enantioselective oxidation of unactivated C–H bonds in cyclic amines by iterative docking-guided mutagenesis of P450BM3 (CYP102A1). Nat Synth 1(12):936-945 doi:10.1038/s44160-022-00166-6 Supplementary Files GraphicalAbstract.jpeg SupportingInformation0226.doc Supplementary Information The online version contains supplementary material available at Additional file 1. Additional Tables S1–S3, Characterization data for the products of chiral ( S )-2a, Amino acid and DNA sequences of P450tol, and HPLC and NMR spectra. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 24 Mar, 2024 Reviewers agreed at journal 04 Mar, 2024 Reviewers invited by journal 01 Mar, 2024 Editor assigned by journal 01 Mar, 2024 First submitted to journal 28 Feb, 2024 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3996274","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275973313,"identity":"50b5e427-0016-410c-b26e-006be789fc69","order_by":0,"name":"Xu Deng","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Deng","suffix":""},{"id":275973314,"identity":"1fdfe693-0e37-45e1-a473-430d3aa97266","order_by":1,"name":"Cheng-Cheng Song","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Cheng","middleName":"","lastName":"Song","suffix":""},{"id":275973315,"identity":"c2a9a5b2-4d31-49a5-a490-574ff10eaa3e","order_by":2,"name":"Wen-Jing Gu","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Jing","middleName":"","lastName":"Gu","suffix":""},{"id":275973316,"identity":"0cee48a2-14c2-4729-a5ed-23002851c8c0","order_by":3,"name":"Yu-Jie Wang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Jie","middleName":"","lastName":"Wang","suffix":""},{"id":275973317,"identity":"a99a8747-d8e5-4b9d-b809-64a91e4c4c8d","order_by":4,"name":"Lu Feng","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Feng","suffix":""},{"id":275973318,"identity":"b74652e2-e0be-486d-a417-2751ef89f930","order_by":5,"name":"Xiao-Jian Zhou","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Jian","middleName":"","lastName":"Zhou","suffix":""},{"id":275973319,"identity":"693e446b-a57e-4e11-872f-a93d42e2a3ba","order_by":6,"name":"Ming-Qiang Zhou","email":"","orcid":"","institution":"Chengdu Institute of Organic Chemistry Chinese Academy of Sciences: Chengdu Organic Chemicals Co Ltd","correspondingAuthor":false,"prefix":"","firstName":"Ming-Qiang","middleName":"","lastName":"Zhou","suffix":""},{"id":275973320,"identity":"ce34d400-8282-40af-b486-0cc071951909","order_by":7,"name":"Wei-Cheng Yuan","email":"","orcid":"","institution":"Chengdu Institute of Organic Chemistry Chinese Academy of Sciences: Chengdu Organic Chemicals Co Ltd","correspondingAuthor":false,"prefix":"","firstName":"Wei-Cheng","middleName":"","lastName":"Yuan","suffix":""},{"id":275973321,"identity":"3ebcb90b-077a-4b4d-9353-351baa4933f0","order_by":8,"name":"Yongzheng Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYHACNiC2gTB5SNCSRrqWwyRoMTh/+NmDjzvO2+vOSGB88LaNQd6coJYDx8wNZ565zWx2I4HZcG4bg+HOBkJaDvawSfO23WYDagExGBIMDhDScpiHTfpv2zkeoBb238RpOQbUwth2QAJkCzNRWiTPsJlJ9rYlG5idedgsOeechOEGQlr4gCEm8bPNzt7sePLBD2/KbOQJ2qKAUMDYACQkCKgHAvkGwmpGwSgYBaNgpAMAFcI9XLAuYzUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1211-3486","institution":"Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yongzheng","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-02-28 09:38:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3996274/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3996274/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52075855,"identity":"55ad1e95-0f83-4562-ad01-3d8c3f45bc41","added_by":"auto","created_at":"2024-03-06 09:38:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":42502,"visible":true,"origin":"","legend":"\u003cp\u003eAsymmetric hydroxylation of C-H bonds catalyzed by P450s\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3996274/v1/a5e8d1dd9752b9a6cf81b722.png"},{"id":52075854,"identity":"6d724be4-8b1a-4aca-9345-0ae812d806e8","added_by":"auto","created_at":"2024-03-06 09:38:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24001,"visible":true,"origin":"","legend":"\u003cp\u003eConditions optimization for asymmetric hydroxylation of 1a catalyzed by P450tol-4. (A) reaction temperature; (B) reaction pH; (C) cell density.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3996274/v1/5a101ef2f70e228887937a93.png"},{"id":52076173,"identity":"a1052f24-c359-428b-b0ef-b4bf7ffbceec","added_by":"auto","created_at":"2024-03-06 09:46:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60537,"visible":true,"origin":"","legend":"\u003cp\u003eSubstrate scope and Scale-up reaction. (A) Substrate scope of \u003cem\u003eE. coli\u003c/em\u003e (P450tol-4)-catalyzed hydroxylation of alkynes \u003cstrong\u003e1\u003c/strong\u003e. General conditions: the reaction was carried out on preparation-scale in 180 mL PB buffer (50 mM, pH = 8.5) containing 10 g cdw/L \u003cem\u003eE. coli\u003c/em\u003e (P450tol-4) cells with \u003cstrong\u003e1a\u003c/strong\u003e (3 mM) at 20 \u003csup\u003eo\u003c/sup\u003eC for 6 h. \u003csup\u003e[a]\u003c/sup\u003e The \u003cem\u003eee\u003c/em\u003e values were determined by chiral HPLC, and the isolated yields were obtained by silica gel chromatography. \u003csup\u003e[b]\u003c/sup\u003e The regioselectivity ratio (\u003cstrong\u003eC1:C2\u003c/strong\u003e) of propargylic and benzylic C-H bonds hydroxylation was determined by HPLC. \u003csup\u003e[c]\u003c/sup\u003eNot detected. (B) Scale-up reaction of \u003cem\u003eE. coli\u003c/em\u003e (P450tol-4)-catalyzed hydroxylation of alkynes \u003cstrong\u003e1a\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3996274/v1/235717dd19398d9e15e5da0d.png"},{"id":52075858,"identity":"f85cd039-c65a-43e3-b663-7b098f6b68fd","added_by":"auto","created_at":"2024-03-06 09:38:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":311549,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking and molecular dynamics simulation.\u003cstrong\u003e \u003c/strong\u003e(A) The docked conformation of substrate \u003cstrong\u003e1f \u003c/strong\u003ein the active pocket of P450tol (PDB No.: 7V40); (B) The fluctuation of the distance of the C7(\u003cstrong\u003e1f\u003c/strong\u003e)-O(heme) and C10(\u003cstrong\u003e1f\u003c/strong\u003e)-O(heme) during the MD simulations; (C) The fluctuation of the distance of the H8(\u003cstrong\u003e1f\u003c/strong\u003e)-O(heme) and H9(\u003cstrong\u003e1f\u003c/strong\u003e)-O(heme) during the MD simulations; (D) The fluctuation of the angle of the O(heme)-H8(\u003cstrong\u003e1f\u003c/strong\u003e)-C10(\u003cstrong\u003e1f\u003c/strong\u003e) and O(heme)-H9(\u003cstrong\u003e1f\u003c/strong\u003e)-C10(\u003cstrong\u003e1f\u003c/strong\u003e) during the MD simulations.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3996274/v1/42807be250556aab323ffe85.png"},{"id":52076629,"identity":"170a7931-7fd0-4b7a-99f3-3f28b1ef4e78","added_by":"auto","created_at":"2024-03-06 09:54:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":771716,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3996274/v1/92efa845-e978-4269-a2c0-36a8a8ae2fd6.pdf"},{"id":52075857,"identity":"7613da3b-3a84-426a-bcba-d7c2f37a0dad","added_by":"auto","created_at":"2024-03-06 09:38:42","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":98417,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3996274/v1/081715f1db84ef303ff12ea7.jpeg"},{"id":52075860,"identity":"fe9beeb6-d81e-4d74-af34-6f929f5472f4","added_by":"auto","created_at":"2024-03-06 09:38:42","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2898432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003cbr\u003e\n \u003c/strong\u003eThe online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 1. \u003c/strong\u003eAdditional Tables S1–S3, Characterization data for the products of chiral (\u003cem\u003eS\u003c/em\u003e)-\u003cstrong\u003e2a\u003c/strong\u003e, Amino acid and DNA sequences of P450tol, and HPLC and NMR spectra.\u003c/p\u003e","description":"","filename":"SupportingInformation0226.doc","url":"https://assets-eu.researchsquare.com/files/rs-3996274/v1/c3b528d97da88e954c8a614f.doc"}],"financialInterests":"","formattedTitle":"Regioselective and enantioselective propargylic hydroxylations catalyzed by P450tol monooxygenases","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChiral propargylic alcohols are useful and versatile motifs that can be transformed into chiral allylic alcohols, allenes, bioactive molecules and natural products (Bauer \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Greshock et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Helal et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Lumbroso et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nakayama et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wang and Pu \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Over the past few decades, there are three mainly standard synthetic methods for the preparation of enantiopure propargylic alcohols: (1) the asymmetric transfer hydrogenation of alkynyl ketones catalyzed by transition metal (Matsumura et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Shatskiy et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); (2) the asymmetric addition of alkynyl organometallic reagents to aldehydes and ketones (Corey and Cimprich \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Trost and Weiss \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e); (3) the deracemization of racemic propargylic alcohols catalyzed by biocatalytic reaction (Gonz\u0026aacute;lez-Granda et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kawanishi et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saravanan et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although some of these strategies access to chiral propargylic alcohols have been developed, many of them involve the use of transition metal catalyst, air sensitive reagents, or deliver chiral propargylic alcohols with moderate enantioselectivity. Up to date, it is still a challenge to develop a biocatalytic method with monooxygenase for the synthesis of enantiopure propargylic alcohols.\u003c/p\u003e \u003cp\u003eAs we all known, enantioselective oxidation of propargylic C-H bond is the most direct and atom economic strategy for the preparation of enantiopure propargylic alcohols. Until now, only two examples have been reported to prepare chiral propargylic alcohols: (1) Cu(MeCN)\u003csub\u003e4\u003c/sub\u003ePF\u003csub\u003e6\u003c/sub\u003e and chiral bisoxazoline ligand catalyzed acyloxylation of propargylic C-H bond to obtained the products with 15\u0026ndash;51% \u003cem\u003eee\u003c/em\u003e under an excess of oxidant and reactions took 4\u0026ndash;5 days to proceed (Stephen Clark et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e); (2) Hydroxylation of propargylic C-H Bond catalyzed by Chloroperoxidase (CPO) using equivalent of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or TBHP as terminal oxidant to synthesize propargylic alcohols with 57\u0026ndash;95% \u003cem\u003eee\u003c/em\u003e or 43\u0026ndash;90% \u003cem\u003eee\u003c/em\u003e, respectively (Hu and Hager \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). However, selective C-H oxidation of simple alkynes represents one of the most fundamental challenges. On the one hand, the highly active compound I species are usually undistinguishing toward similar C-H bonds, resulting in low chemoselectivity (overoxidation products) (Alvarez et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hu and Hager \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and regioselectivity, On the other hand, alkynyl and alkyl groups with relatively small steric effect are difficult to differentiate by small molecular catalysts, resulting in low stereoselectivity (Stephen Clark et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Further development of higher stereoselectivity and more efficient catalysts in this field is therefore of great significance for the asymmetric catalysis.\u003c/p\u003e \u003cp\u003eCytochrome P450 monooxygenases (P450s) catalyzed the oxidative reactions of C-H bonds with stereo- and regioselective manner under mild reaction condition (Chakrabarty et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li and Wong \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Manning et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Roiban and Reetz \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Whitehouse et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Additionally, P450s as mild and selective catalysts have been used in the asymmetric hydroxylation of benzylic, allylic, aromatic and unactivated C-H bonds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) (Chakrabarty et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Neufeld et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Roiban and Reetz \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Whitehouse et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, P450s as an effective and versatile catalyst for the enantioselective hydroxylation of propargylic C-H bonds has not been available in literature. In light of our ongoing interests in P450s-catalyzed asymmetric hydroxylation reactions with a broad substrate range and widely applications (Cui et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Deng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we herein describe chiral propargylic alcohols were synthesized from the simple alkynes by P450tol catalyzed asymmetric propargylic C-H bonds hydroxylation with regio- and stereoselectivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Additionally, molecular docking and MD simulations were carried out to provide a rationale for the enantioselectivity and regioselectivity of these reactions. As far as we know, this is the first example of asymmetric hydroxylation of propargylic C-H bonds with excellent regio- and enantionselectivity by P450 monooxygenase, while the C\u0026thinsp;\u0026equiv;\u0026thinsp;C bonds in the molecule remained unreacted.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and procedures\u003c/h2\u003e \u003cp\u003eChemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. Isopropyl-\u003cem\u003eβ\u003c/em\u003e-D-thiogalactopyranoside (IPTG) and Ampicillin Sodium Salt (\u003cem\u003eAmp\u003c/em\u003e) and Streptomycin sulface (\u003cem\u003eStr\u003c/em\u003e) were purchased from Solarbio (Beijing, China). Analytical thin layer chromatography (TLC) was performed on precoated silica gel 60 GF254 plates. Flash column chromatography was carried out with 300\u0026ndash;400 mesh silica gel. The Alkynes and racemic products were synthesized following the reported method (Liu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Visualization on TLC was achieved by use of UV light (254 nm). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz) and \u003csup\u003e13\u003c/sup\u003eC-NMR (100 MHz) were recorded on Agilent Technologies 400 MR. Chemical shifts were reported in parts per million (ppm) relative to residual signals of the solvent. The following abbreviations are used to indicate multiplicity: s\u0026thinsp;=\u0026thinsp;singlet, d\u0026thinsp;=\u0026thinsp;doublet, t\u0026thinsp;=\u0026thinsp;triplet, m\u0026thinsp;=\u0026thinsp;multiplet, dd\u0026thinsp;=\u0026thinsp;doublet of doublets. High-resolution mass spectra (HRMS) was recorded by ESI ionization sources. Chiral HPLC analysis was performed on Shimadzu LC-20A, equipped with Chiralpak\u0026reg; OJ-H, OD-H, AD-H or AS-Hcolumns.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme preparation\u003c/h2\u003e \u003cp\u003eCultivation of \u003cem\u003eE. coli\u003c/em\u003e (P450tol-4) cells was carried out using TB medium containing 50 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL \u003cem\u003eStr\u003c/em\u003e and \u003cem\u003eAmp\u003c/em\u003e. After growing at 37 \u003csup\u003eo\u003c/sup\u003eC to an OD\u003csub\u003e600\u003c/sub\u003e of 0.6\u0026ndash;0.8, IPTG was added to the final concentration of 0.2 mM. The culture was incubated at 25 \u003csup\u003eo\u003c/sup\u003eC with another 12\u0026ndash;14 h for enyzme expression. Recombinant \u003cem\u003eE. coli\u003c/em\u003e (P450tol-4) cells were harvested by centrifugation at 7000 \u0026times;g at 4 \u003csup\u003eo\u003c/sup\u003eC for 5 min. The freshly prepared \u003cem\u003eE. coli\u003c/em\u003e (P450tol-4) cells were resuspended in reaction buffer solution to a cell density (g cdw/L) for performing biotransformation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking and molecular dynamics simulation\u003c/h2\u003e \u003cp\u003eDocking study of \u003cb\u003e1f\u003c/b\u003e in the active site of enzyme P450tol (PDB No.: 7V40) was carried out using AutoDock 4.0 software (Trott and Olson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). All the docking experiments were performed using \u0026ldquo;Genetic Algorithm\u0026rdquo; search parameters and default docking parameters. All structural illustrations were generated using the PyMOL software (Seeliger and de Groot \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMolecular dynamics simulation was carried out for P450tol to further explain the mechanism of regio- and enantioselective hydroxyation of substrate \u003cb\u003e1f\u003c/b\u003e. The molecule structure of \u003cb\u003e1f\u003c/b\u003e was docked into the active site of enzyme P450tol. The iron-oxo intermediate involved in the cytochrome catalyzed oxidative hydroxylation cycle was used to model the active form of the P450 cofactor (Narayan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Simulations were performed using the GPU code (pmemd) of the AMBER 22 software package (D.A. Case 2017). The Amber-compatible parameters developed by Cheatham et al. (Shahrokh et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) were used for Cpd I and its axial Cys ligand. Substrate \u003cb\u003e1f\u003c/b\u003e parameters for the molecular dynamics (MD) simulations were generated within the antechamber module of AMBER 22 using the general AMBER force field (GAFF) (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), with partial charges set to fit the electrostatic potential generated at the HF/6-31G(d) level by the restrained electrostatic potential (RESP) model (Bayly et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The charges were calculated according to the Merz-Singh-Kollman scheme (Besler et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Singh and Kollman \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1984\u003c/span\u003e) using Gaussian 16(C.01) (M.J. Frisch 2016). Amino acid protonation states were predicted using the H\u0026thinsp;+\u0026thinsp;+\u0026thinsp;server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://biophysics.cs.vt.edu/H++\u003c/span\u003e\u003cspan address=\"http://biophysics.cs.vt.edu/H++\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Anandakrishnan et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Then, the enzyme was solvated in a pre-equilibrated truncated hexagonal box with a 10-\u0026Aring; buffer of TIP3P (Jorgensen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) water molecules using the AMBER 22 leap module, resulting in the addition of \u0026sim;19,000 solvent molecules. The systems were neutralized by addition of ions Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, all subsequent calculations were done using the Amber ff14SB force field (Maier et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The following MD simulation steps as reported in ref. (Narayan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), 60 ns production trajectories MD simulations were performed after equilibrated. All the structural images and distance were performed using the VMD Software, trajectory analysis and energy was post-processed by Cpptraj module (Maier et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), respectively. All the structural images and distance were performed using the VMD Software. Trajectory data, root mean square deviation (RMSD), root mean square fluction (RMSF) were analyzed by Cpptraj module, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneral procedure for the synthesis of chiral (\u003c/b\u003e \u003cb\u003eS\u003c/b\u003e \u003cb\u003e)-2 on preparation-scale\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a 500-mL shake flask containing a resting cell suspension of \u003cem\u003eE. coli\u003c/em\u003e (P450tol-4) (10 g cdw/L) and \u003cb\u003e1\u003c/b\u003e (0.6 mmol) in 180 mL PB buffer (50 mM, pH 8.5). The reaction mixture was shaken at 20 ℃ for 6 h. Then the mixture was extracted with ethyl acetate (3 \u0026times; 180 mL). The organic phases were separated by centrifugation (7000 \u0026times; g, 15 min), combined, dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and evaporated at reduced pressure. The resulting mixture was purified by flash chromatography using ethyl acetate/petroleum ether as eluent on silica gel to afford the desired chiral product \u003cb\u003e2\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProcedure for the synthesis of chiral (\u003c/b\u003e \u003cb\u003eS\u003c/b\u003e \u003cb\u003e)-2a on 2.25 mmol-scale\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a 2.0-L shake flask containing a resting cell suspension of \u003cem\u003eE. coli\u003c/em\u003e (P450tol-4) (10 g cdw/L) and \u003cb\u003e1\u003c/b\u003e (2.25 mmol) in 750 mL PB buffer (50 mM, pH 8.5). The reaction mixture was shaken at 20 ℃ for 6 h. Then the mixture was extracted with ethyl acetate (3 \u0026times; 1000 mL). The organic phases were separated by centrifugation (7000 \u0026times; g, 15 min), combined, dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and evaporated at reduced pressure. The resulting mixture was purified by flash chromatography using ethyl acetate/petroleum ether as eluent on silica gel to afford the desired chiral product \u003cb\u003e2a\u003c/b\u003e (58% yield, 196 mg).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eScreening of reaction conditions for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epropargylic hydroxylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our previous work, we have obtained several cytochrome P450 monooxygenases (P450DA, P450PL2-2) (Cui et al. 2023; Cui et al. 2019; Deng et al. 2022; Wan et al. 2022; Wang et al. 2022; Xie et al. 2020; Xie et al. 2023) and also constructed five recombinant\u003cem\u003e\u0026nbsp;Escherichia coli\u0026nbsp;\u003c/em\u003estrains (P450tol-1 to P450tol-5) harboring P450tol monooxygenase from \u003cem\u003eRhodococcus coprophilus\u0026nbsp;\u003c/em\u003eTC-2 (Chen et al. 2022; Li et al. 2014) and five pairs of redox partner Fdx-FdR (ferredoxin-ferredoxin reductase) from \u003cem\u003eP. lavamentivorans\u0026nbsp;\u003c/em\u003eDS-1 (Wu et al. 2018). With these P450 monooxygenases in hand, but-1-yn-1-ylbenzene (\u003cstrong\u003e1a\u003c/strong\u003e) was used as a model substrate to explore the hydroxylation activity and stereoselectivity of these recombinant P450 strains. Biohydroxylation reactions were carried out using \u003cem\u003eE. coli\u003c/em\u003e whole-cells as catalyst without additional cofactors required. After incubation at 30 \u003csup\u003eo\u003c/sup\u003eC in PB buffer (pH = 8.0) for 6 h, the \u003cem\u003eee\u003c/em\u003e and yield\u003cem\u003e\u0026nbsp;\u003c/em\u003eof the chiral product 4-phenylbut-3-yn-2-ol (\u003cstrong\u003e2a\u003c/strong\u003e) were analyzed using chiral HPLC (Table 1). Strains P450DA and P450PL2-2 exhibited the opposite stereoselectivity despite a very low yield in the propargylic C-H hydroxylation reactions, which produced \u003cstrong\u003e2a\u003c/strong\u003e in 70% \u003cem\u003eee\u003c/em\u003e for \u003cem\u003eR\u003c/em\u003e stereoselectivity and 36% \u003cem\u003eee\u003c/em\u003e for \u003cem\u003eS\u003c/em\u003e stereoselectivity, respectively (Table 1, entries 1-2). Surprisingly, the five recombinant \u003cem\u003eE. coli\u003c/em\u003e P450tol strains could convert \u003cstrong\u003e1a\u003c/strong\u003e to \u003cstrong\u003e2a\u003c/strong\u003e with 96% \u003cem\u003eee\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e stereoselectivity despite in different yields (Table 1, entries 3-7), which the strain P450tol-4 showed the highest yield of 58% (Table 1, entry 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Screening of P450 strains for asymmetric hydroxylation of \u003cstrong\u003e1a\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e The reaction was carried out on an analytical scale in 5 mL PB buffer (50 mM, pH = 8.0) containing 10 g cdw/L \u003cem\u003eE. coli\u003c/em\u003e (P450s) cells with \u003cstrong\u003e1a\u003c/strong\u003e (2 mM) at 30 \u0026deg;C for 6 h\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e The yield and \u003cem\u003eee\u003c/em\u003e were measured by chiral HPLC analysis, and absolute configuration was confirmed by previously reported references (Shatskiy et al. 2015; Zhang et al. 2018).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/em\u003e Not detected.\u003c/p\u003e\n\u003cp\u003eWith the optimal strain P450tol-4 in hand, the reaction conditions including temperature, the pH of the buffer and the concentration of the biocatalyst were evaluated. The results are summarized in Fig. 2. From the results, we can find that the stereoselectivity of the reaction remains unchanged with different temperature, pH and cell concentration. The highest yield was found at 20 \u003csup\u003eo\u003c/sup\u003eC, which is about the same as 15 \u003csup\u003eo\u003c/sup\u003eC. With the increase of temperature from 20 to 40 \u003csup\u003eo\u003c/sup\u003eC, the yield significantly decreased from 69% to 54% (Fig. 2A). The results in Fig. 2B indicated that the pH influenced the yield of \u003cstrong\u003e2a\u003c/strong\u003e dramatically. Increasing pH from 5.0 to 8.5 improved the yield, while further increasing pH to 9.0 with the yield decreases slightly. The results in Fig. 2C indicated that the cell density of P450tol influenced the yield of \u003cstrong\u003e2a\u003c/strong\u003e dramatically. The yield of the product \u003cstrong\u003e2a\u003c/strong\u003e increased with the increasing of cell concentration from 5 to 10 g cdw/L, while a higher yield was not obtained by further increasing cell density to 25 g cdw/L. To sum up, the optimal reaction conditions of propargylic hydroxylations were set at 20 \u003csup\u003eo\u003c/sup\u003eC and reaction pH = 8.5 using 10 g cdw/L cell density of recombinant \u003cem\u003eE. coli\u003c/em\u003e cells (P450tol-4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScope of substrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder the optimal reaction conditions, a range of aryl alkyne subtrates \u003cstrong\u003e1\u003c/strong\u003e were transformed into propargylic alcohols \u003cstrong\u003e2\u003c/strong\u003e on preparative-scale, and the results are displayed in Fig. 3A. Alkyne containing longer alkyl chain could be converted to the corresponding propargyl alcohol \u003cstrong\u003e2b\u003c/strong\u003e by P450tol-4 with 99% \u003cem\u003eee\u003c/em\u003e, albeit in lower isolated yield (22% yield). Besides hydroxylation of secondary C(\u003cem\u003esp\u003c/em\u003e\u003csup\u003e3\u003c/sup\u003e)-H bonds, P450tol-4 could also catalyze the hydroxylation reaction of primary C\u0026minus;H bond in moderate isolated yield (\u003cstrong\u003e2c\u003c/strong\u003e). Furthermore, substrates with fluorine substitution at \u003cem\u003eortho\u003c/em\u003e-, and \u003cem\u003epara\u003c/em\u003e-positions of the aromatic ring (\u003cstrong\u003e2d\u003c/strong\u003e and \u003cstrong\u003e2e\u003c/strong\u003e) were well tolerated in this reaction in 28-47% yield with 94-97% \u003cem\u003eee\u003c/em\u003e. In the previous reported work, P450tol monooxygenase catalyzed the benzylic C-H bonds hydroxylation of toluenes to produce benzyl alcohols (Chen et al. 2022). When a methyl substitution in the aromatic ring, there is regioselectivity competition between the benzylic and propargylic C-H bonds in the hydroxylation reaction. The methyl substitution at \u003cem\u003emeta\u003c/em\u003e-position of the aromatic ring, the product propargyl alcohol \u003cstrong\u003e2f\u0026nbsp;\u003c/strong\u003ewas obtained with 99:1 regioselectivity ratio (\u003cem\u003er.r.\u003c/em\u003e) and 96% \u003cem\u003eee\u003c/em\u003e. Alkynes containing various heteroaromatic rings, such as furan and pyridine, were transformed to propargylic alcohols \u003cstrong\u003e2g\u003c/strong\u003e-\u003cstrong\u003e2j\u003c/strong\u003e as well with 95-98% \u003cem\u003eee\u003c/em\u003e in moderate isolated yield. Unfortunately, P450tol-4 exhibited no catalytic efficiency of alkyne containing the bulky naphthalene ring (\u003cstrong\u003e2k\u003c/strong\u003e) and tertiary propargylic C\u0026ndash;H bond (\u003cstrong\u003e2l\u003c/strong\u003e). To show the synthetic potential of this strategy, our catalytic reaction can be scaled up to 2.25 mmol scale with the formation of propargyl alcohol \u003cstrong\u003e2a\u0026nbsp;\u003c/strong\u003ein 58% isolated yield (196 mg) with 96% \u003cem\u003eee\u003c/em\u003e (Fig. 3B).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eIn addition, chiral propargyl alcohol \u003cstrong\u003e2a\u003c/strong\u003e was the important building blocks for synthesis of antifungal drug Ravuconazole (Xu et al. 2009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking and molecular dynamics simulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain a structure-based understanding of the excellent enantio- and regioselectivity of these reactions, molecular docking of the substrate \u003cstrong\u003e1f\u003c/strong\u003e onto the X-ray structure of P450tol (PDB No.: 7V40) was carried out (Fig. 4A). In the \u003cstrong\u003e1f\u003c/strong\u003e-enzyme binding pose, the distance between C10-carbon (propargylic-position) or C7-carbon (benzylic-position) of \u003cstrong\u003e1f\u003c/strong\u003e and heme oxygen atom (heme-O) of P450tol is 2.8 or 9.0 \u0026Aring;. Meanwhile, the distance between C10 pro-\u003cem\u003eS\u003c/em\u003e-hydrogen or pro-\u003cem\u003eR\u003c/em\u003e-hydrogen of \u003cstrong\u003e1f\u003c/strong\u003e and heme-O is 1.9 \u0026Aring; or 3.0 \u0026Aring;, respectively (Fig. 4A). The results of molecular docking demonstrated that the hydrophobic interactions were formed between substrate \u003cstrong\u003e1f\u003c/strong\u003e with residues Pro114, Phe198, Phe199, Trp223, Phe329 and Phe426 located in the active pocket of P450tol, which stabilize the substrates in the catalytic pocket. Further MD simulations indicated that such binding conformations of substrate \u003cstrong\u003e1f\u003c/strong\u003e are quite stable during 60 ns-MD simulation, during which the distribution of the distance between the C10-carbon of \u003cstrong\u003e1f\u003c/strong\u003e and the heme-O gave most of MD frames within 2.8-6.0 \u0026Aring;, while the C7-carbon of \u003cstrong\u003e1f\u003c/strong\u003e gave most of MD frames beyond 6.0 \u0026Aring; (Fig. 4B), indicating that \u003cstrong\u003e1f\u003c/strong\u003e binding in pose C10 is more stable, which is consistent with the selectivity data (\u0026gt;99:1 regioselectivity at C10-position). Another important finding from our MD simulation study is that the C10 pro-\u003cem\u003eS\u003c/em\u003e-hydrogen (H8) of \u003cstrong\u003e1f\u003c/strong\u003e is well positioned for H-abstraction than C10 pro-\u003cem\u003eR\u003c/em\u003e-hydrogen (H9) based on the distance of (H8(\u003cstrong\u003e1f\u003c/strong\u003e)-O(heme) \u003cem\u003evs\u003c/em\u003e H9(\u003cstrong\u003e1f\u003c/strong\u003e)-O(heme)) and the angle of (O(heme)-H8(\u003cstrong\u003e1f\u003c/strong\u003e)-C10(\u003cstrong\u003e1f\u003c/strong\u003e) vs O(heme)-H9(\u003cstrong\u003e1f\u003c/strong\u003e)-C10(\u003cstrong\u003e1f\u003c/strong\u003e)), which is consistent with the selectivity data (96% \u003cem\u003eee\u003c/em\u003e for \u003cem\u003eS\u003c/em\u003e-selectivity) (Fig. 4C, 4D). In summary, our MD simulation results provide valuable molecular insights into the excellent enantio- and regioselectivity hydroxylation of C-H bonds in alkynes catalyzed by P450tol.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we have developed a green and efficient platform for the asymmetric hydroxylation of primary and secondary C\u0026ndash;H bonds at propargylic positions catalyzed by P450tol monooxygenase, while the C\u0026thinsp;\u0026equiv;\u0026thinsp;C bonds in the molecule remained unreacted. This protocol provides a practical and sustainable method for the preparation of enantiomerically pure propargylic alcohols with high regioselectivity (\u0026gt;\u0026thinsp;99:1 \u003cem\u003er.r.\u003c/em\u003e) and enantioselectivity (94\u0026ndash;99% \u003cem\u003eee\u003c/em\u003e), which are valuable and versatile synthetic building blocks in organic synthesis. Additionally, molecular docking and MD simulations were performed to provide a rationale for the excellent enantio- and regioselectivity of these reactions. Efforts broadening the substrate specificity (e.g., for \u003cb\u003e1k\u003c/b\u003e and \u003cb\u003e1l\u003c/b\u003e) and inverting the enantioselectivity of the propargylic C-H bonds hydroxylations via engineered P450tol variants are currently ongoing in our laboratory.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh Performance Liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP450tol\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eP450 monooxygenase from \u003cem\u003eRhodococcus coprophilus\u003c/em\u003e TC-2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP450DA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eP450 monooxygenase from \u003cem\u003eDeinococcus apachensis\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP450PL2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eP450 monooxygenase from \u003cem\u003eP. lavamentivorans\u003c/em\u003e DS-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFdx-FdR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efive pairs of redox partner Fdx-FdR (ferredoxin-ferredoxin reductase) from \u003cem\u003eP. lavamentivorans\u003c/em\u003e DS-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePB buffer\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econsist of sodium phosphate dibasic and potassium phosphate monobasic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emolecular dynamics simulation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXD, CS and WG:Investigation, Methodology, Validation, Data curation. YW and LF: Data curation, Visualization. MZ and WY: Writing-review \u0026amp; editing. XZ and YC: Project administration, Conceptualization, Methodology, Validation, Funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for financial support from the National Natural Science Foundation of China (No. 32271537 and 22061049), the Science and Technology Department of Guizhou province (QKHJCZK2021-036 and QKHRCPTGCC-2023-003), the Science and Technology Department of Zunyi (ZSKRPT-2020-5, ZSKH-2018-3, ZSKRPT-2021-5), Zunyi Medical University (QKH-2018-5772-014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved the consent for publishing the manuscript to bioresources and bioprocessing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Key Laboratory of Biocatalysis \u0026amp; Chiral Drug Synthesis of Guizhou Province, Key Laboratory of Basic Pharmacology of Ministry of Education, School of Pharmacy, Zunyi Medical University, Zunyi, China. \u003csup\u003e2\u003c/sup\u003e National Engineering Research Center of Chiral Drugs, Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu, 610041, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlvarez LX, Christ ML, Sorokin AB (2007) Selective oxidation of alkenes and alkynes catalyzed by copper complexes. Appl Catal A: Gen 325(2):303-308 doi:https://doi.org/10.1016/j.apcata.2007.02.045\u003c/li\u003e\n\u003cli\u003eAnandakrishnan R, Aguilar B, Onufriev AV (2012) H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res 40(W1):W537-W541 doi:10.1093/nar/gks375\u003c/li\u003e\n\u003cli\u003eBauer EB (2012) Transition-Metal-Catalyzed Functionalization of Propargylic Alcohols and Their Derivatives. Synthesis 44(08):1131-1151 doi:10.1055/s-0031-1290503\u003c/li\u003e\n\u003cli\u003eBayly CI, Cieplak P, Cornell W, Kollman PA (1993) A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. The Journal of Physical Chemistry 97(40):10269-10280 doi:10.1021/j100142a004\u003c/li\u003e\n\u003cli\u003eBesler BH, Merz Jr KM, Kollman PA (1990) Atomic charges derived from semiempirical methods. J Comput Chem 11(4):431-439 doi:https://doi.org/10.1002/jcc.540110404\u003c/li\u003e\n\u003cli\u003eChakrabarty S, Wang Y, Perkins JC, Narayan ARH (2020) Scalable biocatalytic C\u0026ndash;H oxyfunctionalization reactions. Chem Soc Rev 49(22):8137-8155 doi:10.1039/D0CS00440E\u003c/li\u003e\n\u003cli\u003eChen C-C, Dai M, Zhang L, Zhao J, Zeng W, Shi M, Huang J-W, Liu W, Guo R-T, Li A (2022) Molecular Basis for a Toluene Monooxygenase to Govern Substrate Selectivity. ACS Catal 12(5):2831-2839 doi:10.1021/acscatal.1c05845\u003c/li\u003e\n\u003cli\u003eChen J, Dong S, Fang W, Jiang Y, Chen Z, Qin X, Wang C, Zhou H, Jin L, Feng Y, Wang B, Cong Z (2023) Regiodivergent and Enantioselective Hydroxylation of C\u0026minus;H bonds by Synergistic Use of Protein Engineering and Exogenous Dual-Functional Small Molecules. Angew Chem Int Ed 62(4):e202215088 doi:https://doi.org/10.1002/anie.202215088\u003c/li\u003e\n\u003cli\u003eCorey EJ, Cimprich KA (1994) Highly Enantioselective Alkynylation of Aldehydes Promoted by Chiral Oxazaborolidines. J Am Chem Soc 116(7):3151-3152 doi:10.1021/ja00086a066\u003c/li\u003e\n\u003cli\u003eCui H-B, Ma T, Zhang R-Y, Shan J, Wang Z-Q, Bai M, Chen Y-Z (2023) Biocatalytic Synthesis of Chiral Benzylic Alcohols via Enantioselective Hydroxylation by a Self-Sufficient Cytochrome P450 from Deinococcus gobiensis. Synthesis(EFirst) doi:10.1055/a-2076-9792\u003c/li\u003e\n\u003cli\u003eCui H-B, Xie L-Z, Wan N-W, He Q, Li Z, Chen Y-Z (2019) Cascade bio-hydroxylation and dehalogenation for one-pot enantioselective synthesis of optically active \u0026beta;-halohydrins from halohydrocarbons. Green Chem 21(16):4324-4328 doi:10.1039/C9GC01802F\u003c/li\u003e\n\u003cli\u003eD.A. Case DSC, T.E.C. Iii, T.A. Darden, R.E. Duke, et al. (2017) AMBER16 and AmberTools17. University of California, San Francisco. \u003c/li\u003e\n\u003cli\u003eDeng G-Z, Zhou X, Yu Q-X, Mou X-Q, An M, Cui H-B, Zhou X-J, Wan N-W, Li Z, Chen Y-Z (2022) Highly Enantioselective Hydroxylation of 3-Arylpropanenitriles to Access Chiral \u0026beta;-Hydroxy Nitriles by Engineering of P450pyr Monooxygenase. Org Process Res Dev 26(7):2046-2051 doi:10.1021/acs.oprd.1c00444\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez-Granda S, M\u0026eacute;ndez-S\u0026aacute;nchez D, Lavandera I, Gotor-Fern\u0026aacute;ndez V (2020) Laccase-mediated Oxidations of Propargylic Alcohols. Application in the Deracemization of 1-arylprop-2-yn-1-ols in Combination with Alcohol Dehydrogenases. ChemCatChem 12(2):520-527 doi:https://doi.org/10.1002/cctc.201901543\u003c/li\u003e\n\u003cli\u003eGreshock TJ, Johns DM, Noguchi Y, Williams RM (2008) Improved Total Synthesis of the Potent HDAC Inhibitor FK228 (FR-901228). Org Lett 10(4):613-616 doi:10.1021/ol702957z\u003c/li\u003e\n\u003cli\u003eHelal CJ, Magriotis PA, Corey EJ (1996) Direct Catalytic Enantioselective Reduction of Achiral \u0026alpha;,\u0026beta;-Ynones. Strong Remote Steric Effects Across the C\u0026minus;C Triple Bond. J Am Chem Soc 118(44):10938-10939 doi:10.1021/ja962849e\u003c/li\u003e\n\u003cli\u003eHu S, Hager LP (1999) Highly Enantioselective Propargylic Hydroxylations Catalyzed by Chloroperoxidase. J Am Chem Soc 121(4):872-873 doi:10.1021/ja983612g\u003c/li\u003e\n\u003cli\u003eJiang Y, Peng W, Li Z, You C, Zhao Y, Tang D, Wang B, Li S (2021) Unexpected Reactions of \u0026alpha;,\u0026beta;-Unsaturated Fatty Acids Provide Insight into the Mechanisms of CYP152 Peroxygenases. Angew Chem Int Ed 60(46):24694-24701 doi:https://doi.org/10.1002/anie.202111163\u003c/li\u003e\n\u003cli\u003eJorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics 79(2):926-935 doi:10.1063/1.445869\u003c/li\u003e\n\u003cli\u003eKawanishi S, Oki S, Kundu D, Akai S (2019) Lipase/Oxovanadium Co-Catalyzed Dynamic Kinetic Resolution of Propargyl Alcohols: Competition between Racemization and Rearrangement. Org Lett 21(9):2978-2982 doi:10.1021/acs.orglett.9b00334\u003c/li\u003e\n\u003cli\u003eKim HJ, Ishida K, Ishida-Ito M, Hertweck C (2022) Sequential Allylic Alcohol Formation by a Multifunctional Cytochrome P450 Monooxygenase with Rare Redox Partners. Angew Chem Int Ed 61(26):e202203264 doi:https://doi.org/10.1002/anie.202203264\u003c/li\u003e\n\u003cli\u003eLi A, Acevedo-Rocha CG, D\u0026apos;Amore L, Chen J, Peng Y, Garcia-Borr\u0026agrave;s M, Gao C, Zhu J, Rickerby H, Osuna S, Zhou J, Reetz MT (2020) Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants. Angew Chem Int Ed 59(30):12499-12505 doi:https://doi.org/10.1002/anie.202003139\u003c/li\u003e\n\u003cli\u003eLi A, Wu S, Adams JP, Snajdrova R, Li Z (2014) Asymmetric epoxidation of alkenes and benzylic hydroxylation with P450tol monooxygenase from Rhodococcus coprophilus TC-2. Chem Commun 50(63):8771-8774 doi:10.1039/C4CC03491K\u003c/li\u003e\n\u003cli\u003eLi Y, Wong LL (2019) Multi-Functional Oxidase Activity of CYP102A1 (P450BM3) in the Oxidation of Quinolines and Tetrahydroquinolines. Angew Chem Int Ed 58(28):9551-9555 doi:https://doi.org/10.1002/anie.201904157\u003c/li\u003e\n\u003cli\u003eLiu Z, Qin Z-Y, Zhu L, Athavale SV, Sengupta A, Jia Z-J, Garcia-Borr\u0026agrave;s M, Houk KN, Arnold FH (2022) An Enzymatic Platform for Primary Amination of 1-Aryl-2-alkyl Alkynes. J Am Chem Soc 144(1):80-85 doi:10.1021/jacs.1c11340\u003c/li\u003e\n\u003cli\u003eLu G, Li Y-M, Li X-S, Chan ASC (2005) Synthesis and application of new chiral catalysts for asymmetric alkynylation reactions. Coord Chem Rev 249(17):1736-1744 doi:https://doi.org/10.1016/j.ccr.2004.12.020\u003c/li\u003e\n\u003cli\u003eLumbroso A, Cooke ML, Breit B (2013) Catalytic Asymmetric Synthesis of Allylic Alcohols and Derivatives and their Applications in Organic Synthesis. Angew Chem Int Ed 52(7):1890-1932 doi:https://doi.org/10.1002/anie.201204579\u003c/li\u003e\n\u003cli\u003eM.J. Frisch GWT, et al. (2016) Gaussian 16 Revision A.03. \u003c/li\u003e\n\u003cli\u003eMaier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C (2015) ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. Journal of Chemical Theory and Computation 11(8):3696-3713 doi:10.1021/acs.jctc.5b00255\u003c/li\u003e\n\u003cli\u003eManning J, Tavanti M, Porter JL, Kress N, De Visser SP, Turner NJ, Flitsch SL (2019) Regio- and Enantio-selective Chemo-enzymatic C\u0026minus;H-Lactonization of Decanoic Acid to (S)-\u0026delta;-Decalactone. Angew Chem Int Ed 58(17):5668-5671 doi:https://doi.org/10.1002/anie.201901242\u003c/li\u003e\n\u003cli\u003eMatsumura K, Hashiguchi S, Ikariya T, Noyori R (1997) Asymmetric Transfer Hydrogenation of \u0026alpha;,\u0026thinsp;\u0026beta;-Acetylenic Ketones. J Am Chem Soc 119(37):8738-8739 doi:10.1021/ja971570a\u003c/li\u003e\n\u003cli\u003eNakayama A, Kogure N, Kitajima M, Takayama H (2011) Asymmetric Total Synthesis of a Pentacyclic Lycopodium Alkaloid: Huperzine-Q. Angew Chem Int Ed 50(35):8025-8028 doi:https://doi.org/10.1002/anie.201103550\u003c/li\u003e\n\u003cli\u003eNarayan ARH, Jim\u0026eacute;nez-Os\u0026eacute;s G, Liu P, Negretti S, Zhao W, Gilbert MM, Ramabhadran RO, Yang Y-F, Furan LR, Li Z, Podust LM, Montgomery J, Houk KN, Sherman DH (2015) Enzymatic hydroxylation of an unactivated methylene C\u0026ndash;H bond guided by molecular dynamics simulations. Nature Chemistry 7(8):653-660 doi:10.1038/nchem.2285\u003c/li\u003e\n\u003cli\u003eNeufeld K, Hen\u0026szlig;en B, Pietruszka J (2014) Enantioselective Allylic Hydroxylation of \u0026omega;-Alkenoic Acids and Esters by P450 BM3 Monooxygenase. Angew Chem Int Ed 53(48):13253-13257 doi:https://doi.org/10.1002/anie.201403537\u003c/li\u003e\n\u003cli\u003eRoiban G-D, Reetz MT (2015) Expanding the toolbox of organic chemists: directed evolution of P450 monooxygenases as catalysts in regio- and stereoselective oxidative hydroxylation. Chem Commun 51(12):2208-2224 doi:10.1039/C4CC09218J\u003c/li\u003e\n\u003cli\u003eSang X, Tong F, Zeng Z, Wu M, Yuan B, Sun Z, Sheng X, Qu G, Alcalde M, Hollmann F, Zhang W (2022) A Biocatalytic Platform for the Synthesis of Enantiopure Propargylic Alcohols and Amines. Org Lett 24(23):4252-4257 doi:10.1021/acs.orglett.2c01547\u003c/li\u003e\n\u003cli\u003eSaravanan T, Jana S, Chadha A (2014) Utilization of whole cell mediated deracemization in a chemoenzymatic synthesis of enantiomerically enriched polycyclic chromeno[4,3-b] pyrrolidines. Org Biomol Chem 12(26):4682-4690 doi:10.1039/C4OB00615A\u003c/li\u003e\n\u003cli\u003eSeeliger D, de Groot BL (2010) Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des 24(5):417-422 doi:10.1007/s10822-010-9352-6\u003c/li\u003e\n\u003cli\u003eShahrokh K, Orendt A, Yost GS, Cheatham Iii TE (2012) Quantum mechanically derived AMBER-compatible heme parameters for various states of the cytochrome P450 catalytic cycle. J Comput Chem 33(2):119-133 doi:https://doi.org/10.1002/jcc.21922\u003c/li\u003e\n\u003cli\u003eShatskiy A, Kivij\u0026auml;rvi T, Lundberg H, Tinnis F, Adolfsson H (2015) Ruthenium-Catalyzed Asymmetric Transfer Hydrogenation of Propargylic Ketones. ChemCatChem 7(23):3818-3821 doi:https://doi.org/10.1002/cctc.201500821\u003c/li\u003e\n\u003cli\u003eSingh UC, Kollman PA (1984) An approach to computing electrostatic charges for molecules. J Comput Chem 5(2):129-145 doi:https://doi.org/10.1002/jcc.540050204\u003c/li\u003e\n\u003cli\u003eSong F, Zheng M, Wang J, Liu H, Lin Z, Liu B, Deng Z, Cong H, Zhou Q, Qu X (2023) Chemoenzymatic synthesis of C14-functionalized steroids. Nat Synth doi:10.1038/s44160-023-00280-z\u003c/li\u003e\n\u003cli\u003eStephen Clark J, Tolhurst KF, Taylor M, Swallow S (1998) Enantioselective propargylic oxidation. Tetrahedron Lett 39(27):4913-4916 doi:https://doi.org/10.1016/S0040-4039(98)00894-6\u003c/li\u003e\n\u003cli\u003eTrost BM, Weiss AH (2009) The Enantioselective Addition of Alkyne Nucleophiles to Carbonyl Groups. Adv Synth Catal 351(7-8):963-983 doi:https://doi.org/10.1002/adsc.200800776\u003c/li\u003e\n\u003cli\u003eTrott O, Olson AJ (2010) AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31(2):455-461 doi:https://doi.org/10.1002/jcc.21334\u003c/li\u003e\n\u003cli\u003eWan N-W, Cui H-B, Zhao L, Shan J, Chen K, Wang Z-Q, Zhou X-J, Cui B-D, Han W-Y, Chen Y-Z (2022) Directed evolution of cytochrome P450DA hydroxylase activity for stereoselective biohydroxylation. Catal Sci Technol 12(18):5703-5708 doi:10.1039/D2CY00164K\u003c/li\u003e\n\u003cli\u003eWang J, Wang H, Wei C, Zhang L, Cui B, Wang Z, Zhang Y, Wan N, Aisa HA, Chen Y (2022) Combined photoredox/engineered P450 enzymatic direct dioxygen-functionalization of arylalkanes to chiral acyloins. Org Biomol Chem 20(46):9085-9092 doi:10.1039/D2OB01769E\u003c/li\u003e\n\u003cli\u003eWang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput Chem 25(9):1157-1174 doi:https://doi.org/10.1002/jcc.20035\u003c/li\u003e\n\u003cli\u003eWang Q, Pu L (2013) Diverse Transformations of Chiral Propargylic Alcohols Generated by BINOL-Catalyzed Alkyne Addition to Aldehydes. Synlett 24(11):1340-1363 doi:10.1055/s-0033-1338781\u003c/li\u003e\n\u003cli\u003eWatanabe K, Miyazaki Y, Okubo M, Zhou B, Tsuji H, Kawatsura M (2018) Nickel-Catalyzed Asymmetric Propargylic Amination of Propargylic Carbonates Bearing an Internal Alkyne Group. Org Lett 20(17):5448-5451 doi:10.1021/acs.orglett.8b02325\u003c/li\u003e\n\u003cli\u003eWhitehouse CJC, Bell SG, Wong L-L (2012) P450BM3 (CYP102A1): connecting the dots. Chem Soc Rev 41(3):1218-1260 doi:10.1039/C1CS15192D\u003c/li\u003e\n\u003cli\u003eWu K, Tang L, Cui H, Wan N, Liu Z, Wang Z, Zhang S, Cui B, Han W, Chen Y (2018) Biocatalytical Asymmetric Sulfoxidation by Identifying Cytochrome P450 from Parvibaculum Lavamentivorans DS-1. ChemCatChem 10(23):5410-5413 doi:https://doi.org/10.1002/cctc.201801139\u003c/li\u003e\n\u003cli\u003eXie L, Chen K, Cui H, Wan N, Cui B, Han W, Chen Y (2020) Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation. ChemBioChem 21(13):1820-1825 doi:https://doi.org/10.1002/cbic.201900691\u003c/li\u003e\n\u003cli\u003eXie L, Zhang Y, Zhang R, Cui H, Cui B, Han W, Wan N, Li Z, Chen Y (2023) Biocatalytic stereoselective synthesis of methyl mandelates by engineering a cytochrome P450 hydroxylase. Green Synth Catal doi:https://doi.org/10.1016/j.gresc.2023.01.005\u003c/li\u003e\n\u003cli\u003eXu L, Muller MR, Yu X, Zhu B-Q (2009) Improved Chiral Synthesis of Ravuconazole. Synth Commun 39(9):1611-1625 doi:10.1080/00397910802563420\u003c/li\u003e\n\u003cli\u003eZhang K, Yu A, Chu X, Li F, Liu J, Liu L, Bai W-J, He C, Wang X (2022a) Biocatalytic Enantioselective \u0026beta;-Hydroxylation of Unactivated C\u0026minus;H Bonds in Aliphatic Carboxylic Acids. Angew Chem Int Ed 61(28):e202204290 doi:https://doi.org/10.1002/anie.202204290\u003c/li\u003e\n\u003cli\u003eZhang X, Shen P, Zhao J, Chen Y, Li X, Huang J-W, Zhang L, Li Q, Gao C, Xing Q, Chen C-C, Guo R-T, Li A (2023) Rationally Controlling Selective Steroid Hydroxylation via Scaffold Sampling of a P450 Family. ACS Catal 13(2):1280-1289 doi:10.1021/acscatal.2c04906\u003c/li\u003e\n\u003cli\u003eZhang Y-M, Yuan M-L, Liu W-P, Xie J-H, Zhou Q-L (2018) Iridium-Catalyzed Asymmetric Transfer Hydrogenation of Alkynyl Ketones Using Sodium Formate and Ethanol as Hydrogen Sources. Org Lett 20(15):4486-4489 doi:10.1021/acs.orglett.8b01787\u003c/li\u003e\n\u003cli\u003eZhang Y, Xiong Z, Li Y, Wilson M, Christensen KE, Jaques E, Hern\u0026aacute;ndez-Llad\u0026oacute; P, Robertson J, Wong LL (2022b) Enantioselective oxidation of unactivated C\u0026ndash;H bonds in cyclic amines by iterative docking-guided mutagenesis of P450BM3 (CYP102A1). Nat Synth 1(12):936-945 doi:10.1038/s44160-022-00166-6\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biocatalysis, Hydroxylation, P450 monooxygenase, Propargylic alcohols, Enantioselectivity","lastPublishedDoi":"10.21203/rs.3.rs-3996274/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3996274/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRegioselective and enantioselective hydroxylation of propargylic C-H bonds are useful reactions but often lack appropriate catalysts. Here a green and efficient asymmetric hydroxylation of primary and secondary C–H bonds at propargylic positions has been established. A series of optically active propargylic alcohols were prepared with high regio- and enantioselectivity (up to 99% \u003cem\u003eee\u003c/em\u003e) under mild reaction conditionsby using P450tol, while the C≡C bonds in the molecule remained unreacted. This protocol provides a green and practical method for constructing enantiomerically chiral propargylic alcohols. In addition, we also demonstrated that the biohydroxylation strategy was able to scaled up to 2.25 mmol scale with the production of chiral propargyl alcohol \u003cstrong\u003e2a\u003c/strong\u003e at a yield of 196 mg with 96% \u003cem\u003eee\u003c/em\u003e, which’s an important synthetic intermediate for antifungal drug Ravuconazole.\u003c/p\u003e","manuscriptTitle":"Regioselective and enantioselective propargylic hydroxylations catalyzed by P450tol monooxygenases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-06 09:38:37","doi":"10.21203/rs.3.rs-3996274/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-03-24T05:50:12+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-04T11:45:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-01T09:18:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-01T07:37:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2024-02-28T08:37:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d125943-da99-4ae9-947f-76f4cdfa05c4","owner":[],"postedDate":"March 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-10T01:26:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-06 09:38:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3996274","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3996274","identity":"rs-3996274","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}