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. 2025 Jul 29;5(9):4196–4203. doi: 10.1021/jacsau.5c00453

Recyclable Whole-Cell Biotransformation System for the Direct Hydroxylation of Propane Catalyzed by a Robust Intracellular Wild-Type Cytochrome P450BM3 Activated by Decoy Molecules

Yuki Sugai 1, Masayuki Karasawa 1, Osami Shoji 1,*
PMCID: PMC12458037  PMID: 41001626

Abstract

We report a decoy molecule-based whole-cell biotransformation system that achieves the direct hydroxylation of propane to 2-propanol using wild-type cytochrome P450BM3 expressed in Escherichia coli. With the most effective decoy molecule N-(2-cyclopentylethyl)–prolyl–phenylalanine (2CPE–Pro–Phe), 2-propanol was produced at a final concentration of 29 mM with a high selectivity of 99%. The catalytic activity achieved using this approach was higher than that achieved using previously reported P450BM3 mutants, demonstrating the potential of decoy molecule-assisted P450BM3 for the scalable conversion of gaseous alkanes under mild conditions. The decoy molecule system enables the use of stable wild-type P450BM3, maintaining long-term activity without deactivationan advantage over engineered mutants, which often suffer from reduced stability.

Keywords: cytochrome P450, gaseous alkane, propane, ionic liquid, whole-cell biotransformation, decoy molecule

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Introduction

The direct conversion of propane to commodity chemicals is of great interest to industrial chemistry. − Of these, 2-propanol is used widely as a chemical feedstock, solvent, and antiseptic. The global consumption of 2-propanol in 2022 was approximately 2.3 million metric tons and was predicted to increase slightly in the future. While it can be produced from propane through the hydration reaction of propylene, activating the inactive C–H bond requires a large amount of energy. ,− Monooxygenases are known to catalyze various oxidation reactions under mild conditions in nature and are promising candidates for green catalysts to accomplish those reactions. , Nonheme diiron monooxygenases, such as alkane monooxygenase, , butane monooxygenase, , and soluble methane monooxygenase, , have been reported to catalyze the hydroxylation of gaseous alkanes, including propane. Particulate methane monooxygenase, which has a copper active center, is also reported to catalyze the hydroxylation of gaseous alkanes. However, the turnover frequencies (TOFs) of these enzymes are not very high, and their multicomponent regulatory systems are rather difficult to control. Cytochrome P450s, a group of heme enzymes widely distributed across all kingdoms of life, can catalyze alkane hydroxylations with high catalytic activity, especially those isolated from bacteria. The CYP52 and CYP153 families of P450s have been reported to hydroxylate liquid alkanes longer than C5, and they are more commonly used to catalyze alkane hydroxylations than other alkane hydroxylases. − Chen et al. reported gaseous alkane hydroxylation by CYP153A6 using a series of terminal oxidants. Other P450s have also been engineered to develop biocatalysts for gaseous alkane hydroxylation. − Among the engineered P450s examined, one of the most successful examples is P450PMO, which is a mutant of CYP102A1 (P450BM3) that was prepared by directed evolution. It efficiently catalyzes the hydroxylation of propane with a high initial TOF (first 20 s) of 455 min–1. , P450BM3 is a bacterial P450 isolated from Priestia megaterium. Its natural fusion with a redox partner enables efficient electron transfer, which is known to result in remarkably high catalytic activity. − In nature, P450BM3 catalyzes the hydroxylation of long-chain fatty acids at their subterminal positions, and a plethora of variants have been successfully constructed to alter its substrate specificity or improve its activity. − Various P450 variants have also been actively generated, underscoring mutagenesis as an effective strategy. − Meanwhile, we have succeeded in expanding the substrate scope of wild-type P450BM3 (without mutagenesis) using substrate analogues, the so-called decoy molecules. ,, P450BM3 generates the reactive oxidant species compound I on binding to its native substrate, a long-chain fatty acid, which triggers the dissociation of a water molecule coordinated to the heme iron, leading to the generation of compound I (Figure a, left). When a decoy moleculean analogue of a long-chain fatty acid, binds to the enzyme along with a second substrate (such as propane or benzene), the enzyme similarly forms compound I, leading to the hydroxylation of the second substrate (Figure a, right). Decoy molecules are designed to have shorter-chain lengths than long-chain fatty acids to enable the occupation of the space above the heme by a second substrate. Using this system employing decoy molecules, we have demonstrated that the substrate specificity of P450BM3 can be altered even without mutagenesis, leading to aromatic C–H and aliphatic sp3 C–H hydroxylations. The TOF for direct hydroxylation of benzene and propane by wild-type P450BM3 in the presence of one of the effective decoy molecules (N-[3-cyclopentylpropanoyl]–l-pipecolyl–l-phenylalanine) reached 405 min–1 P450BM3–1 and 615 min–1 P450BM3–1 (first 5 min), respectively. To the best of our knowledge, the initial TOFs for propane and benzene hydroxylation are the highest reported among all P450-catalyzed reaction systems. The cocrystal structure of C7–Pro–Phe bound to P450BM3 (Figure b, right) showed that the terminal alkyl group of the decoy molecule was positioned at an appropriate distance from the heme iron center (8.0 Å) and the remaining space above the heme appeared to be available for the binding of a second substrate. On the opposite side of the decoy molecule, the amino acid moiety contributed to anchoring the molecule at the entrance of the substrate-binding channel through multiple hydrogen-bonding interactions, thereby preventing the decoy molecule from penetrating deeper into the active site.

1.

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(a) P450BM3 reaction mechanism. General catalytic cycle of P450BM3 (left). Predicted catalytic cycle for propane hydroxylation by P450BM3 with decoy molecules (right). (b) Cocrystal structure of P450BM3 with palmitoleic acid (left, PDB ID: 1FAG) and C7–Pro–Phe (right, PDB ID: 6K58). Black dashed lines represent hydrogen-bonding interactions. (c) Concept of this study: a whole-cell biocatalyst for propane hydroxylation activated by decoy molecules. Recently, we also demonstrated whole-cell biotransformation of benzene to phenol using P450BM3 expressed in E. coli, assisted by decoy molecules.

We also demonstrated that the decoy molecule-based system can be expanded to whole-cell biotransformation of benzene to phenol using P450BM3 expressed in Escherichia coli. , In this system, C7–Pro–Phe was identified as the most effective decoy molecule, likely due to its high uptake efficiency through the porin OmpF in E. coli. In reactions using decoy molecules, the catalytic activity is highly dependent on the structure of the decoy molecules. Thus, we hypothesized that screening the decoy molecules developed in previous studies could enable the establishment of an efficient whole-cell biocatalytic system for propane hydroxylation. Furthermore, because gaseous substrates, such as propane, can readily enter E. coli cells via passive diffusion, the reaction was expected to proceed without the need to enhance substrate uptake. In this study, we extrapolated the whole-cell biotransformation system using decoy molecules to propane hydroxylation, which is an aliphatic sp3 C–H hydroxylation of the gaseous substrate (Figure c). We have developed a microplate-based screening of decoy molecules for whole-cell propane hydroxylation using an ionic liquid (IL) to stabilize the gaseous propane concentration to ensure reproducibility. A newly synthesized decoy molecule with high membrane permeability, designed based on insights from screening, exhibited the highest activity and enabled a reusable whole-cell reaction system that produced up to 29 mM of 2-propanol in a 12 h reaction.

Results and Discussion

Initially, we investigated the applicability of the decoy system to whole-cell propane hydroxylation. E. coli BL21­(DE3) expressing wild-type P450BM3 (pET28a­[+] vector) was suspended (OD600 = 6.3) in a phosphate buffer saturated with propane, containing 100 μM C7–Pro–Phe as a decoy molecule and 40 mM glucose. This suspension was incubated at 25 °C for 5 h in a sealed reaction vessel filled with a mixed gas (propane:air at a ratio of 4:1). In the absence of C7–Pro–Phe, neither 2-propanol nor 1-propanol was detected using gas chromatography–mass spectrometry (GC–MS; Table , entry 2). In contrast, 3.50 mM of 2-propanol and a small amount of 1-propanol were produced in the presence of C7–Pro–Phe (Table , entry 5), indicating that the decoy-based reaction system was also effective for whole-cell propane hydroxylation. To enhance nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, glucose dehydrogenase (GDH), which catalyzes the regeneration of NADPH coupled with glucose oxidation, was coexpressed using several vectors with different promoter and plasmid copy numbers (pSTV29, pACYCDuet-1, and pMW119). The 2-propanol yield improved to approximately 5.5 mM when GDH was expressed using either the pSTV29 or pMW119 vector, both of which carry the lac promotera promoter generally considered weak and often associated with low levels of protein expression in E. coli (Table , strains 2 and 4). We also investigated the effect of P450BM3 expression levels by using a plasmid containing a weak promoter to reduce P450BM3 expression. The expression levels of enzymes were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Figure S1), which showed that using pSTV29 and pMW119 vectors resulted in lower enzyme expression levels than those using other expression vectors. For P450BM3, the concentration of catalytically active P450 was also estimated based on CO difference spectroscopy (Table S2). When P450BM3 was expressed from pSTV29, the yield reached 6.13 mM (Table , strain 6), indicating that lower expression levels of P450BM3 are favorable for higher yields in whole-cell reactions using decoy molecules. One possible reason for the lower yield observed at higher P450BM3 expression levels is the rapid consumption of NADPH by P450BM3, which can induce metabolic perturbations in E. coli. − Lowering the P450BM3 expression level is thought to have enabled more effective functioning of E. coli’s native metabolic pathways, thereby restoring the metabolic balance that had been disrupted by a high-level P450BM3 expression. Interestingly, when the P450BM3 expression level was reduced, the coexpression of GDH had a limited effect on the yield (Table , strain 5). Presumably, by reducing the P450BM3 expression level, a sufficient supply of NADPH was achieved without the need to promote its regeneration via GDH. To simplify the reaction system, the condition without GDH coexpression was adopted in the subsequent reactions.

1. Product Yields for Propane Hydroxylation .

        yield (mM)
entry catalysis propane/oxidant ratio in gas phase glucose (mM) 2-propanol 1-propanol
1 empty plasmid + C7–Pro–Phe 4:1 (Air) 40 trace trace
2 P450BM3 4:1 (Air) 40 trace trace
3 P450BM3 + C7–Pro–Phe 0:1 (Air) 40 0.190 ± 0.020 trace
4 P450BM3 + C7–Pro–Phe 0:1 (Air) 40 trace trace
5 P450BM3 + C7–Pro–Phe 4:1 (Air) 40 3.50 ± 0.360 8.0 × 10–2 ± 1.0 × 10–2
6 P450BM3 + C7–Pro–Phe 1:1 (Air) 40 2.65 ± 0.11 6.0 × 10–2 ± 1.0 × 10–2
7 P450BM3 + C7–Pro–Phe 1:0 40 3.57 ± 0.27 0.150 ± 1.0 × 10–2
8 P450BM3 + C7–Pro–Phe 4:1 (O2) 40 2.94 ± 0.07 0.100 ± 1.0 × 10–2
9 P450BM3 + C7–Pro–Phe 4:1 (Air) 0 0.110 ± 0.050 Trace
10 P450BM3 + C7–Pro–Phe 4:1 (Air) 20 3.51 ± 0.57 9.0 × 10–2 ± 1.8 × 10–2
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a

Reaction solution without saturated propane.

b

Reaction conditions: E. coli BL21­(DE3) expressing wild-type P450BM3 (OD600 = 6.3), decoy molecule (100 μM), glucose (40 mM), and saturated propane in 1 mL of the phosphate buffer (86 mM Na2HPO4, 16 mM KH2PO4, 85 mM NaCl, 7 mM MgSO4, 0.1 mM CaCl2, pH = 7.4), and 9 mL of propane and oxidant gas mixture at 25 °C for 5 h. The uncertainty is given as the standard deviation of at least three measurements using different batches of cell cultures. Propanol formation was determined using GC–MS.

2. Product Yields for Benzene and Propane Hydroxylation Using Various Plasmid Vectors.

strain plasmid vector for P450BM3 expression (promoter) plasmid vector for GDH expression (promoter) propane hydroxylation yield (mM 2-propanol)
1 pET28a (+) (T7)   3.50 ± 0.36
2 pET28a (+) (T7) pSTV29 (lac) 5.45 ± 0.37
3 pET28a (+) (T7) pACYCDuet-1 (T7) 2.67 ± 0.41
4 pET28a (+) (T7) pMW119 (lac) 5.57 ± 0.76
5 pSTV29 (lac)   6.72 ± 1.10
6 pSTV29 (lac) pMW119 (lac) 6.13 ± 1.42
7 pSTV29 (lac) pET28a (T7) 6.16 ± 0.21
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a

The copy numbers and promoters of each plasmid are as follows: pET28a (+): 15–20, T7 promoter; pSTV29:10–12, lac promoter; pACYCDuet-1:10–12, T7 promoter; pMW119: ∼5, lac promoter.

b

Reaction conditions: E. coli BL21­(DE3) expressing wild-type P450BM3 and GDH with the corresponding plasmid (OD600 = 6.3), C7–Pro–Phe (100 μM), glucose (40 mM), and saturated propane in 1 mL of the phosphate buffer, and 9 mL of propane and air gas mixture (4:1) at 25 °C for 5 h. The uncertainty is given as the standard deviation of at least three measurements using different batches of cell cultures. 2-Propanol formation was determined using GC–MS.

Next, we attempted to identify the most suitable decoy molecule for propane hydroxylation through screening of the decoy molecules developed thus far. Because N-modified prolyl–phenylalanines served as more effective decoy molecules than other backbone structures, we selected these compounds for further screening as decoy molecules (Figures d and S2). To enable rapid and highly reproducible screening of decoy molecules for reactions using gaseous alkanes as substrates, we employed an aqueous/IL biphasic system in which gaseous alkanes were dissolved in an IL, and a defined amount of this mixture was added to each well of a microplate (Figure a). In this system, propane was dissolved in the IL phase, transferred to the aqueous phase, and hydroxylated by E. coli cells present in the aqueous phase. Trihexyltetradecylphosphonium bis­(trifluoromethylsulfonyl)­imide ([P6,6,6,14]­[NTf2]), which exhibits low toxicity toward E. coli and sufficient solubility for propane, , was selected as the IL for this system. [P6,6,6,14]­[NTf2] is expected to be able to dissolve propane at mole fractions hundreds of times higher than that of water and retain it for an extended period. − Because [P6,6,6,14]­[NTf2] up to 50% (v/v) do not cause any apparent adverse effects and propane was hydroxylated most efficiently when the amount of IL was 10% of the reaction solution (Figure S3), 20 μL of propane-saturated [P6,6,6,14]­[NTf2] was added to 200 μL of the reaction solution in a microplate. To enhance the uptake of low cell-permeable decoy molecules, E. coli coexpressing the engineered porin OmpFΔ108–130which facilitates the uptake of anionic decoy molecules such as 3CCPA and C7AM containing an amide linkage at the N-modification sitewas also employed for screening (Figure b). As a result of the screening using a microplate, several decoy molecules exhibited higher reactivity than C7–Pro–Phe (Figure c). We compared the hydroxylation activity of propane with that of benzene, one of the most extensively studied non-native substrates in the decoy reaction system, and found that the top-performing decoy molecules generally exhibited higher activity toward propane. This indicates that this screening method successfully identified decoy molecules specialized for propane hydroxylation (Figure S4).

2.

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(a) Schematic of the aqueous/IL biphasic system for propane hydroxylation. (b) Top view of wild-type OmpF (PDB ID: 4GCP) and OmpFΔ108–130 (AlphaFold-predicted structure). L3-loop, corresponding to the deletion site, is shown in yellow. (c) 2-Propanol yield with each decoy molecule. Reaction conditions: E. coli BL21­(DE3) expressing wild-type P450BM3 with pET28a­(+) and OmpFΔ108–130 with pSTV29 (OD600 = 6.3), decoy molecule (100 μM), and glucose (40 mM) in 200 μL of the phosphate buffer, and saturated propane in 20 μL of [P6,6,6,14]­[NTf2] at 25 °C for 3 h. Reactions were carried out in 96-well plates sealed with aluminum seals. Propanol formation was determined by using GC–MS. (d) Structures of the screened N-substituents of decoy molecules in panel c.

The formation of 2-propanol in the whole-cell reaction under the optimal reaction conditions was examined by using the top 10 decoy molecules (Figure a). Among them, C6AM–Pro–Phe, 2CPAA–Pro–Phe, and 2CHAA–Pro–Phe afforded higher catalytic activity (Figure c, dark gray). Because these decoy molecules (N-acyl-substituted types) exhibit low membrane permeability and require the use of the engineered porins OmpFΔ108 and 130, we synthesized the corresponding N-alkyl-substituted decoy molecules with the same backbone structure to confer membrane permeability and thereby simplify the system (Figure b,c, gray). The activities of these N-alkyl-substituted decoy molecules were evaluated and compared, revealing that 2CPE–Pro–Phe, C7–Pro–Phe, C6–Pro-Phe, and 5MH–Pro–Phe exhibited the highest catalytic activities. Because 2CPE–Pro–Phe, C7–Pro–Phe, C6–Pro–Phe, and 5MH–Pro–Phe are all N-alkyl-substituted decoy molecules that can be taken up by E. coli through the native OmpF porin, the expression of the engineered porin OmpFΔ108–130 was not required in the subsequent reactions. The most effective decoy molecule2CPE–Pro–Phepossesses ring structures at its terminal ends, suggesting that the bulky terminal groups help confine propane within the active site. In this case, the decoy molecules serve as gatekeepers, preventing the release of propane from the active site and thereby enhancing the hydroxylation efficiency. Ultraviolet–visible (UV–vis) spectral changes were monitored upon titration of 2CPE–Pro–Phe into a 4 μM P450BM3 solution. When 2CPE–Pro–Phe was added alone, no spectral changes were observed; however, subsequent introduction of propane led to distinct spectral shifts (type I spectral shifts) (Figures S5 and S6). These spectral changes resembled those induced by substrates binding to P450BM3, suggesting that 2CPE–Pro–Phe and propane act cooperatively to activate P450BM3, inducing a conformational state similar to that of the substrate-bound form. While our previous study demonstrated that all tested decoy molecules are noncytotoxic, we additionally investigated the cytotoxicity of 2CPE–Pro–Phe and 2CPAA–Pro–Phe using a live/dead staining assay (Figure S7) and confirmed that these decoy molecules likewise do not exhibit cytotoxic effects.

3.

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(a) Structures and abbreviations of top 10 N-substituents of decoy molecules selected by the plate-based screening. (b) N-Alkyl substituents of decoy molecules alternative to N-acyl substituents selected by the screening. (c) Batch reaction with propane sealed in the headspace of the reaction vessel. Reaction conditions: E. coli (P450BM3 inserted into pET28a­(+) and OmpFΔ108–130 inserted into pSTV29, dark gray, or P450BM3 inserted into pSTV29, gray, OD600 = 6.3), decoy molecule (100 μM), glucose (40 mM), and saturated propane in 1 mL of the phosphate buffer, pH = 7.4, and 9 mL of propane and air gas mixture (4:1) at 25 °C for 5 h. (d) Recycling of the whole-cell biocatalyst. Reaction conditions: E. coli BL21­(DE3) expressing wild-type P450BM3 with pSTV29 (OD600 = 25.2), 2CPE–Pro–Phe (100 μM), glucose (40 mM) in 1 mL of the phosphate buffer (86 mM Na2HPO4, 16 mM KH2PO4, 85 mM NaCl, 7 mM MgSO4, 0.1 mM CaCl2, pH 7.4), and 9 mL of a propane–O2 gas mixture (1:1) at 25 °C for 4 h. After the reaction, the reaction solution was centrifuged and the collected cell pellet was resuspended in fresh reaction solution to initiate the next reaction cycle. The recycling experiment was conducted with and without the addition of 2CPE–Pro–Phe at each reaction cycle.

Finally, we carried out the whole-cell reaction using the conditions of high cell density of E. coli BL21­(DE3) (OD600 = 25.2) in the presence of the most effective decoy molecule, 2CPE–Pro–Phe, for 4 h, resulting in propanol production reaching 22.5 ± 2.71 mM (Figure d), which corresponds to the hydroxylation of 10% of the initial substrate. We found that replacing air with O2 was beneficial for preparing the mixed gas (propane:O2 = 1:1) in high-cell-density reactions (Figure S8 and Table S4). Under these conditions, the 2-propanol yield reached a plateau of 29 mM within 12 h (Figure S9), and the selectivity of 2-propanol formation reached 99.9%. A small amount of the generated 1-propanol was metabolized by E. coli, and higher selectivity for 2-propanol was achieved under prolonged reaction times. It was confirmed that E. coli cells not expressing P450BM3 were able to consume a small amount of 1-propanol, whereas 2-propanol was scarcely consumed (Figure S10).

The whole-cell reaction using wild-type P450BM3 and 2CPE–Pro–Phe, a newly developed decoy molecule, afforded a 2-propanol yield of 29 mM. While the experimental conditions, including E. coli cell density and expression level of P450BM3, differ between systems and the results are not directly comparable, this yield is notably higher than the approximately 16 mM reported for P450PMO, a 31-point mutant of P450BM3 developed through directed evolution for propane hydroxylation. The high yield is likely attributable to the advantage of the decoy molecule system, which enables the use of stable wild-type P450BM3 and allows the enzyme to retain its activity over extended reaction times without deactivationin contrast to most engineered mutants, which often exhibit reduced stability compared with the wild-type enzyme.

Notably, the yield remained above half of its initial value even after five cycles of recycling (Figure d), suggesting that the advantage of using stable wild-type P450BM3 also contributes to the recyclability of the reaction system. When the reaction was conducted without readdition of the decoy molecules, the yield decreased sharply. This result supports the necessity of decoy molecules for the reaction and indicates that 2CPE–Pro–Phe does not accumulate inside E. coli cells. In the second cycle, a small residual amount of 2CPE–Pro–Phe was sufficient for the reaction. When focusing on recyclability, further optimization of reaction conditions is warranted, and improvements in the number of successful recycling cycles can be expected. Although the screening was conducted using propane as the substrate, other substrates with similar molecular sizes, such as butane and propylene, were also tested. The butane hydroxylation reaction yielded 15.2 ± 0.075 mM of 2-butanol with 96.9% selectivity, and propylene epoxidation reaction yielded 1.41 ± 0.081 mM of propylene oxide in 8 h reactions (Figures S11 and S12). These results suggest that the cavity formed by the decoy molecule possesses enough flexibility to accommodate the induced fit.

Conclusions

We demonstrated whole-cell biotransformation of propane to propanol using wild-type P450BM3 expressed in E. coli through the addition of a decoy molecule, 2CPE–Pro–Phe. For the rapid and reliable screening of decoy molecules, we developed a simple plate-based screening method using a water/IL biphasic system in which propane is dissolved in the IL, resulting in reproducible screening. The developed water/IL biphasic system appears to be adaptable to other gaseous substrates for screening of other enzymatic systems. Because the most effective decoy molecule identified was an N-alkyl-substituted type with high cellular uptake, native porin, OmpF, was sufficient for its transport. Moreover, by reducing the P450BM3 expression level, GDH coexpression was also not required. Thus, E. coli expressing wild-type P450BM3 alone was sufficient to carry out the reaction. Simply adding the top-performing screened decoy molecule resulted in the highest catalytic activity for propane hydroxylation among all of the reported P450BM3 mutant systems. Using the decoy molecule system, which enabled the use of stable wild-type P450BM3, unlike most engineered mutants with reduced stability, allowed a whole-cell reaction that was recyclable several times without significant activity loss.

Supplementary Material

au5c00453_si_001.pdf (1.1MB, pdf)

Acknowledgments

The authors thank Dr. Yuichiro Aiba and Dr. Shinya Ariyasu for their assistance with the experiments and for valuable discussions.

Glossary

Abbreviations

2CPE–Pro–Phe

N-(2-cyclopentylethyl)-prolyl-phenylalanine

TOFs

turnover frequencies

P450BM3

CYP102A1

GDH

glucose dehydrogenase

IL

ionic liquid

GC–MS

gas chromatography–mass spectrometry

NADPH

nicotinamide adenine dinucleotide phosphate

[P6,6,6,14]­[NTf2]

trihexyltetradecylphosphonium bis­(trifluoromethylsulfonyl)­imide

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c00453.

  • Materials, methods, experimental details, scheme of synthesis of decoy molecules, chemical structures of N-substituents, and GC–MS analysis of the reaction mixture (PDF)

Conceptualization, Y.S. and O.S.; methodology, Y.S.; investigation, Y.S. and M.K.; visualization, Y.S.; writingoriginal draft, Y.S. and O.S.; writingreview and editing, Y.S. and O.S.; funding acquisition, Y.S. and O.S.; and supervision, O.S. CRediT: Yuki Sugai conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing - original draft, writing - review & editing; Masayuki Karasawa investigation; Osami Shoji conceptualization, data curation, formal analysis, funding acquisition, methodology, project administration, resources, supervision, validation, writing - original draft, writing - review & editing.

This work was supported by JSPS KAKENHI Grant Numbers JP22H05129 and JP25H00910 awarded to O.S. Y.S. would like to express gratitude to the “THERS Make New Standards Program for the Next Generation Researchers”, supported by JST SPRING (Grant Number JPMJSP2125). Y.S. also acknowledges support from the “Graduate Program of Transformative Chem-Bio Research” at Nagoya University funded by MEXT (WISE Program).

The authors declare no competing financial interest.

Published as part of JACS Au special issue “Advances in Small Molecule Activation Towards Sustainable Chemical Transformations”.

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