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. 2026 Sep 27;23(9):e71755. doi: 10.1002/cbdv.71755

Selective Hydroxylation of Collagen Model Peptides by Metal Catalysis

Shun Oto 1, Akihiro Marutani 1, Yukio Hamamoto 1, Arata Kawakami 1, Yasushi Obora 1,✉, Yoshiaki Hirano 1,✉
PMCID: PMC13616479  PMID: 42801772

ABSTRACT

Collagen is formed with a repeating structure of proline (Pro)‐4‐hydroxyproline‐(4‐Hyp)‐glycine (Gly). In chemical synthesis, when synthesizing collagen sequences, it is necessary to protect the 4‐Hyp side chain within them. Therefore, if (Pro‐Pro‐Gly)n could be synthesized followed by selective hydroxylation of proline residues using iron catalysts, improved synthetic efficiency could be expected. In this study, hydroxylation reactions were performed on Boc‐Pro‐OPac and Ac‐Pro‐OPac, where the N‐termnus of proline is protected by Boc and Ac groups, respectively, and on Boc‐Pro‐Pro‐OPac, a peptide containing multiple proline residues. Subsequent NMR structural analysis confirmed that hydroxyl groups were introduced at the 5‐position of proline in Boc‐Pro‐OPac and at the 5‐position of N‐terminal proline in Boc‐Pro‐Pro‐OPac. On the other hand, the reaction did not proceed for the proline at the C‐terminal end of Ac‐Pro‐OPac and Boc‐Pro‐Pro‐OPac. To investigate the reaction mechanism and whether the reaction proceeds, density functional theory (DFT) calculations were performed to clarify the optimal structure of the substrate and the charge distribution on each atom. The progress of this reaction is influenced by the electron density of the hydrogen atom at the 5‐position of proline, but it has been clarified that steric hindrance near the 5‐position of proline exerts a significant effect.

Keywords: biochemistry, biology, chemistry, organic chemistry, research methodology

Selective hydroxylation of proline: Catalytic oxidation using iron catalysts enables the selective introduction of hydroxyl groups into the (Pro‐Pro‐Gly)n sequence, facilitating low‐cost, large‐scale synthesis of collagen sequences.

graphic file with name CBDV-23-e71755-g002.webp

1. Introduction

Generally, when proteins are synthesized within living organisms, precursor proteins are first sequentially synthesized through transcription of DNA by mRNA and translation by tRNA [1]. This process is called the central dogma. Subsequently, enzymes present within each cell react selectively with specific sites, synthesizing proteins with introduced functional groups. This process is called post‐translational modification of proteins, and it has been demonstrated that post‐translational modifications such as hydroxylation [1], nitration [2], and phosphorylation [3] diversify the final structure of proteins. Furthermore, enzymes that modify proteins possess unique steric hindrance, enabling them to react selectively with specific sites on amino acids. A representative example exhibiting high selectivity is collagen containing both proline and hydroxyproline, which has a hydroxyl group introduced into proline [1, 4].

Collagen [5, 6, 7, 8, 9] is one of the most abundant proteins in living organisms, composed of repeating amino acid sequences Xaa‐Yaa‐Gly. Here, Xaa predominantly contains proline residues (Pro), while Yaa predominantly contains 4‐hydroxyproline residues (4‐Hyp) [6, 10, 11]. These peptide chains form a right‐handed triple helix structure through hydrogen bonding, imparting mechanical strength to tissues [12]. The process of collagen synthesis within living organisms involves the synthesis of the precursor Pro‐Pro‐Gly repeating sequence via the central dogma. Subsequently, post‐translational modifications selectively convert proline to 4‐hydroxyproline through enzymes such as collagen prolyl 4‐hydroxylase (P4H) [4, 13]. On the other hand, two methods exist for artificially synthesizing collagen. The first is chemical synthesis, which sequentially reacts amino acids using either liquid‐phase or solid‐phase synthesis to produce the protein. The second is biosynthesis, which uses genetically modified E. coli or yeast engineered to synthesize the desired amino acid sequence to biochemically produce the protein. However, both of these synthesis methods have issues. Consequently, the late‐stage oxidation method used in chemical synthesis has gained attention in recent years. This method mimics the biosynthetic pathway by synthesizing the proline‐proline‐glycine repeat sequence in advance and then selectively hydroxylating the proline residues [14, 15]. If the late‐stage oxidation method is established for collagen chemical synthesis, it would enable large‐scale, low‐cost production, further heightening expectations for artificial collagen. However, the C─H bond is one of the strongest interatomic bonds, presenting the challenge of being difficult to cleave [9]. Therefore, establishing the late‐stage oxidation method requires selecting catalysts and oxidants capable of achieving positional selectivity and cleaving the C─H bond [9].

As a catalyst meeting these conditions, we focused on the iron complex Fe(S,S‐PDP) (Figure 1). Fe(S,S‐PDP) is a sterically hindered electrophilic metal catalyst known to selectively oxidize electron‐rich sites on reaction substrates [14, 15, 16]. Furthermore, it has been reported that using Fe(S,S‐PDP) with hydrogen peroxide as the oxidant in a catalytic oxidation reaction selectively hydroxylates the pyrrolidine ring at the 5‐position of proline, yielding 5‐hydroxyproline [17, 18, 19, 20]. Techniques for selectively 5‐hydroxyproline among the four carbons of proline have been further refined in recent years. Since the introduction of the iron catalyst developed by White and colleagues [14, 15, 16, 17], technologies capable of oxidizing only specific proline residues in a peptide chain under milder conditions have continued to evolve [21]. A synthetic method that uses only electrical energy to directly and selectively oxidize the 5‐position of proline to yield 5‐hydroxyproline derivatives, without using any oxidizing reagents is also attracting attention [22]. In recent years, attempts have also been made to carry out the hydroxylation of proline using biosynthesis involving E. coli and other microorganisms [23, 24]. However, this prior work faces the challenge that hydroxylation is difficult when the N‐terminal protecting group of proline is not a nosyl group (Ns group) [17]. Furthermore, no hydroxylation reactions have been reported for peptides containing multiple proline residues. We first focused on the tert‐butoxycarbonyl (Boc) group as a candidate protecting group, as it is thought to mimic the peptide bond better than the Ns group (Figure 2). We synthesized Boc‐Pro‐OPac and, for peptides containing multiple proline residues, Boc‐Pro‐Pro‐OPac. Therefore, the objectives of this study are to select N‐ and C‐terminal protecting groups, perform hydroxylation reactions on peptides containing multiple proline residues, and conduct structural analysis using two‐dimensional NMR heteronuclear multi‐nuclear correlation (HMBC) and heteronuclear single‐quantum coherence (HSQC) techniques. This aims to obtain fundamental insights into the hydroxylation reaction of the polycyclic proline‐proline‐glycine structure, which is a precursor to collagen [6].

FIGURE 1.

FIGURE 1

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Structural formula of Fe(s,s‐pdp).

FIGURE 2.

FIGURE 2

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Structural formula of NS group and Boc group.

2. Results and Discussion

2.1. Peptide Synthesis

Using liquid‐phase synthesis, amino acids and peptides protected at the N‐terminus with a Boc group and at the C‐terminus with a Pac group were synthesized for use in catalytic oxidation reactions (Scheme 1) [6, 20]. First, Boc‐Pro‐OH reacted with phenyl bromide under weakly basic conditions to synthesize Boc‐Pro‐OPac. Next, the Boc group was deprotected using HCl/Dioxane. The deprotected H‐Pro‐OPac reacted with Boc‐Pro‐OH under mildly basic conditions using the condensing agent DMT‐MM to synthesize Boc‐Pro‐Pro‐OPac. Confirmation of synthesis completion was performed using HPLC and MALDI‐TOF‐MS (Figure S1). In addition, the structures of each substrate were determined using NMR spectroscopy (Figure S2).

SCHEME 1.

SCHEME 1

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Scheme of dipeptide synthesis.

2.2. Hydroxylation Reaction of Boc‐Pro‐OPac

In Boc‐Pro‐OPac, the reaction was conducted under conditions similar with those for Ns‐Pro‐OMe (Figure 3a) [14]. Analysis by HPLC revealed a peak corresponding to a byproduct in addition to a peak considered to be the hydroxylated compound 2 (Figure 4a). This byproduct was isolated and structurally analyzed by NMR spectra. A new peak, likely corresponding to a carbonyl carbon, was detected in the 1 3C NMR spectrum, indicating that carbonylation had proceeded (Figure S3). Next, to control carbonylation due to excess oxidation, the reaction was performed with the iron catalyst reduced from 15 mol% to 2.5 mol% and hydrogen peroxide reduced from 1.9 equivalents to 0.6 equivalents (Figure 3b). Although the conversion rate decreased, this suppressed the formation of carbonylation and other byproducts (Figure 4b). Subsequent reactions used these reaction conditions as the optimized conditions.

FIGURE 3.

FIGURE 3

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(a) Hydroxylation reaction prior to reaction condition investigation. (b) Hydroxylation reaction following reaction condition optimization.

FIGURE 4.

FIGURE 4

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(a) HPLC analysis results prior to reaction condition investigation. (b) HPLC analysis results after reaction condition optimization. HPLC analytical condition: A solution; 40% H2O, B solution; 60% acetonitrile at 40°C.

After isolating the product detected in Figure 4b, structural analysis was performed by NMR. The peak observed at 3.3–3.7 ppm at the fifth position of proline prior to the reaction disappeared after the reaction, as indicated by the 1H NMR results, and a new peak was observed at 5.5–5.7 ppm. In addition, a broad peak originating from a hydroxyl group was observed near 3.5 ppm (Figure 5a). The 13C NMR results showed that the peak at 46.3–46.6 ppm, which was observed before the reaction for the carbon at the 5‐position of proline, disappeared after the reaction. Furthermore, a new characteristic peak corresponding to the carbon between N and OH was observed at 82.4 ppm (Figure 5b). To investigate the direct correlation between the 1H NMR signal at 5.5–5.7 ppm and the 13C NMR signal at 82.4 ppm, analysis was performed using HSQC (Figure 5c). The HSQC results revealed a direct correlation at 5.5–5.7 ppm, confirming that a hydroxyl group was introduced at the 5‐position of proline. The MALDI‐TOF MS data for the substrates and products are summarized in Table 1. In terms of stereoselectivity, only one of the two hydrogen atoms in the fifth position has a lower positive charge and is therefore preferentially attacked by the electrophilic catalyst, Fe(S,S‐PDP) [14]. In the paper by Lee and coworkers, the oxidation of the C─H bond by Fe(S,S‐PDP) involves the insertion of O between the C and H atoms. The stereochemistry of the H atom before the reaction and that of the OH group after the reaction remain unchanged [25]. These factors result in the oxidation of the substrate being stereochemically controlled.

FIGURE 5.

FIGURE 5

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(a) Changes in Boc‐Pro‐OPac(Figure S2) and its products(Figure S4) in 1H NMR. (b) Changes in Boc‐Pro‐OPac (Figure S2) and its products (Figure S4) in 13C NMR. (c) Direct correlation diagram of newly observed peaks in 1H NMR and 13C NMR using HSQC.

TABLE 1.

The MALDI‐TOF MS data for substrates and products.

Peptide sequence Date of MALDI‐TOF‐MS
Fw (Cal.)

[M]+ or

[M+H]+

[M+Na]+ [M+K]+

Boc‐Pro‐OPac

Boc‐Hyp‐OPac

322.4

338.4

‐

‐

356.4

372.3

372.5

388.2

Boc‐Pro‐Pro‐OPac

Boc‐Hyp‐Pro‐OPac

430.5

446.5

‐

‐

452.2

469.4

468.0

483.4
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2.3. Hydroxylation Reaction of Boc‐Pro‐Pro‐OPac

In Boc‐Pro‐Pro‐OPac, the reaction was performed under the conditions optimized in Boc‐Pro‐OPac (Figure 6). HPLC analysis after the reaction detected peaks corresponding to two main products (Figure 7a). After isolating the products, they were analyzed by NMR. The 1H NMR results showed that the peak at 3.3–3.6 ppm, corresponding to the hydrogen at the 5‐position of proline on the N‐terminal side observed before the reaction, disappeared in both reaction products. Furthermore, a new peak was observed around 5.7 ppm in product 1 and around 5.5 ppm in product 2 (Figure 7b). On the other hand, it was found that the reaction had not proceeded at the 5‐position of the C‐terminal proline. The 13C NMR results showed that the peak around 46 ppm, corresponding to the carbon at the proline 5‐position on the N‐terminal side observed before the reaction, disappeared in both products after the reaction. Furthermore, a new peak corresponding to the carbon sandwiched between N and OH was observed around 82 ppm in both products (Figure 7c). Next, HSQC analysis was performed to examine the direct correlation between the 5.7 ppm region in the 1H NMR and the 82 ppm region in the 13C NMR (Figure 7d). The HSQC results confirmed the direct correlation in both products, confirming the introduction of a hydroxyl group at the 5‐position of N‐terminal proline.

FIGURE 6.

FIGURE 6

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Boc‐Pro‐Pro‐OPac hydroxylation reaction.

FIGURE 7.

FIGURE 7

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(a) HPLC analysis after reaction with 0.6 eq hydrogen peroxide. HPLC analytical condition: A solution; 40% H2O, B solution; 60% Acetonitrile at 40°C. (b) Changes in Boc‐Pro‐Pro‐OPac (Figure S2) and its products (Figure S5) in 1H NMR. (c) Changes in Boc‐Pro‐Pro‐OPac (Figure S2) and its products (Figure S5) in 13C NMR. (d) Direct correlation diagram of newly observed peaks in 1H NMR and 13C NMR using HSQCV (Figure S5).

These results indicate that the reaction does not proceed at the C‐terminal proline. Possible causes include the influence of electron density and steric hindrance. Regarding electron density, 1H NMR results show that the hydrogen at the 5‐position of N‐terminal proline in Boc‐Pro‐Pro‐OPac is located in the higher field region compared to the hydrogen at the 5‐position of C‐terminal proline (Figure 7b). Since the iron complex used in this study attacks electron‐rich sites electrophilically, the reaction likely proceeded only at the fifth position of the N‐terminal proline. To further investigate the cause, we focused on the Ac group, which is thought to mimic the peptide bond more closely than the Boc group. We synthesized Ac‐Pro‐OPac (Figure S4), mimicking the C‐terminal proline, and reacted it with 0.6 equivalents of hydrogen peroxide (Figure 8a). HPLC analysis after the reaction confirmed a conversion rate of only 1.9 %, indicating the reaction did not proceed (Figure 8b). To elucidate why the reaction proceeded with Boc‐Pro‐OPac but not with Ac‐Pro‐OPac. Through elucidating, we determined the optimized structure and electron density using DFT calculations.

FIGURE 8.

FIGURE 8

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(a) Reaction conditions from Ac‐Pro‐OPac to Ac‐Hyp‐Opac are shown. (b) HPLC analysis after reaction with 0.6 eq. hydrogen peroxide. (internal standard substance: n,n‐dimethylbenzamide), HPLC analytical condition: A solution; 40% H2O, B solution; 60% acetonitrile at 40°C.

2.4. DFT Calculation

DFT calculations [26, 27] were performed to determine the optimized structures and NBO charges [28, 29] for Boc‐Pro‐OPac and Ac‐Pro‐OPac using Gaussian 16 (Figure 9 and S5, Tables 2 and 3) [30]. The chosen DFT function affects the reliability of reactivity and structural stability in collagenous frameworks [31]. Therefore, calculations were carried out with B3LYP‐D3 (hybrid GGA) and M06‐2X (hybrid meta‐GGA) functions [32, 33, 34, 35]. The 6‐311+G(d,p) basis set was used for all atoms. The solvent effect was concerned by the solvation model based on density (SMD) in MeCN (ε = 35.688) [36]. The NBO charge results after DFT calculations revealed no significant difference between Boc‐Pro‐OPac and Ac‐Pro‐OPac at the proline 5‐position hydrogen, where the reaction is presumed to occur. This suggests that the influence of electron density is minor as a cause for the lack of reaction progress in Ac‐Pro‐OPac. Evaluation of NBO donor‐acceptor interactions interpreted by the second‐order perturbation stabilization energy E(2) analysis of the optimized structures in Table 3. Based on the calculated interaction energy values, the σ bonding character between H7 and its bound carbon atom tends to be weaker than that of other C─H bonds within proline by donor‐acceptor interactions. This indicates that the bond between H7 and the carbon atom is a reactive site. Furthermore, the optimized structures after DFT calculations revealed the distance between the proline 5‐position hydrogen, where the reaction is thought to occur, and the methyl group hydrogen of the protecting group: 4.2 Å for Boc‐Pro‐OPac and 2.6 Å for Ac‐Pro‐OPac. This indicates that the region around the proline 5‐position hydrogen is more sterically crowded in Ac‐Pro‐OPac compared to Boc‐Pro‐OPac. The 3D molecular models were depicted using the CYL‐view [37].

FIGURE 9.

FIGURE 9

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Optimized structure and NBO charges after DFT calculation (a) with B3LYP‐D3/6‐311+G(d.p)/SMD(MeCN) (b) M06‐2X/6‐311+G(d.p)/SMD(MeCN) in Boc‐Pro‐OPac, and (c) with B3LYP‐D3/6‐311+G(d.p)/SMD(MeCN) (d) M06‐2X/6‐311+G(d.p)/SMD(MeCN) in Ac‐Pro‐OPac.

TABLE 2.

NBO charges after DFT calculations for Boc‐Pro‐OPac and Ac‐Pro‐OPac. a

Proline H1 H2 H3 H4 H5 H6 H7
Boc‐Pro‐OPac

0.243

(0.249)

0.227

(0.233)

0.217

(0.223)

0.214

(0.219)

0.219

(0.227)

0.213

(0.218)

0.197

(0.203)
Ac‐Pro‐OPac

0.237

(0.240)

0.227

(0.233)

0.217

(0.224)

0.213

(0.219)

0.221

(0.227)

0.217

(0.223)

0.201

(0.206)
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a

NBO charges calculated with B3LYP‐D3/6‐311 + G(d.p)/SMD(MeCN).

Values in the parentheses show NBO charges calculated with M06‐2X/6‐311 + G(d.p)/SMD(MeCN).

TABLE 3.

Total second‐order perturbation energy E (2) (kJ/mol) of n‐σ*C−H and σ‐σ*C−H charge‐transfer interactions. a

Proline H1 H2 H3 H4 H5 H6 H7
Boc‐Pro‐OPac

0.243

(0.249)

0.227

(0.233)

0.217

(0.223)

0.214

(0.219)

0.219

(0.227)

0.213

(0.218)

0.197

(0.203)
Ac‐Pro‐OPac

0.237

(0.240)

0.227

(0.233)

0.217

(0.224)

0.213

(0.219)

0.221

(0.227)

0.217

(0.223)

0.201

(0.206)
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a

E (2) calculated with B3LYP‐D3/6‐311+G(d.p)/SMD(MeCN).

Values in the parentheses show E (2) calculated with M06‐2X/6‐311 + G(d.p)/SMD(MeCN).

DFT calculations provide only an optimized structure, so the actual stereochemistry was analyzed using NOESY in two‐dimensional NMR (Figure 10). NOESY analysis results showed no correlation between the proline 5‐position hydrogen and the methyl hydrogen of the Boc group in Boc‐Pro‐OPac. Conversely, a correlation was observed between the proline 5‐position hydrogen and the methyl hydrogen of the Ac group in Ac‐Pro‐OPac. This indicates that, based on the experimental data, the region around the 5‐position of proline in Ac‐Pro‐OPac is sterically congested. The iron complex used in this study is bulky and susceptible to steric hindrance, suggesting that the reaction does not proceed with Ac‐Pro‐OPac.

FIGURE 10.

FIGURE 10

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(a) NOESY analysis of Boc‐Pro‐OPac (b) NOESY analysis of ac‐Pro‐OPac.

In addition, to evaluate the proposed steric interaction, non‐covalent interactions (NCI) analysis was conducted [38]. NCI analysis was performed with NCIplot 4.2 [39]. The results were shown by visual molecular dynamics (VMD) using following settings: s = 0.5 a.u., cut‐off ρ = 0.05 a.u., and color scale ‐0.03 ≤ sign(λ 2)ρ ≤ 0.03 [40]. As a result, interactions between H7 and other groups were not observed significantly in Boc‐Pro‐OPac (Figure 11a,b). In contrast, the methyl group of the acetyl group interacts with H7 in Ac‐Pro‐OPac (Figure 11c,d). These results showed that Ac‐Pro‐OPac have more steric interactions between H7 and the protective group than Boc‐Pro‐OPac.

FIGURE 11.

FIGURE 11

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Interatomic NCI images using the electron density from the optimized structures of Boc‐Pro‐OPac at (a) B3LYP‐D3/6‐311 + G(d.p) (b) M06‐2X/6‐311 + G(d.p) level and from the optimized structures of Ac‐Pro‐OPac at (c) B3LYP‐D3/6‐311 + G(d.p) (d) M06‐2X/6‐311 + G(d.p) level.

We conducted research aimed at modeling post‐translational modifications in living organisms and achieving the selective hydroxylation of collagen model peptides using a metal catalyst. In nature, proline hydroxylation is known to occur only at the 3‐ and 4‐positions. The 5‐position is stereochemically inaccessible and is also a difficult site for C–H activation. Although position‐selective C–H oxidation of amino acids using transition metal catalysts has been studied, no specific examples of research focused on the 5‐position of proline have been identified. The results revealed that the fifth proline, rather than the fourth, was selectively oxidized. 5‐Hydroxyproline is not generally found in natural proteins and is being studied as a product of organic chemical synthesis or as a new form of proline modification resulting from oxidative stress. Furthermore, while “4‐hydroxyproline” found in collagen has a stable structure, 5‐hydroxyproline is chemically unstable; it is prone to degradation and ring opening because a hydroxyl group with a non‐bonding electron pair is present on the carbon atom immediately adjacent to the nitrogen atom (at the 5‐position). Consequently, methods are being explored to selectively synthesize this unstable 5‐hydroxyproline skeleton by intentionally targeting it using special protecting groups or unique catalytic reactions. These findings could serve as a key component in the creation of specialized pharmaceuticals (chiral building blocks) and raw materials for functional synthetic peptides that do not exist in nature.

3. Conclusion

In this study, to obtain fundamental insights into the hydroxylation of the collagen precursor (Pro‐Pro‐Gly)n, we investigated the hydroxylation of Boc‐Pro‐OPac and Boc‐Pro‐Pro‐OPac, which contains multiple proline residues. The results of this study revealed that in Boc‐Pro‐OPac, a hydroxyl group is selectively introduced at the 5‐position of proline. Furthermore, in Boc‐Pro‐Pro‐OPac, the hydroxyl group was introduced only at the 5‐position of N‐terminal proline. To investigate the difference in reactivity between the N‐terminal proline and the C‐terminal proline, Ac‐Pro‐OPac, considered to mimic the peptide bond better than Boc‐Pro‐OPac, was synthesized and subjected to catalytic oxidation. However, the reaction did not proceed. To further investigate the cause, DFT calculations were performed to determine the optimal structure of the substrate and the charge distribution on each atom. Additionally, NOESY analysis was conducted to examine actual stereochemistry. The results indicated that steric hindrance significantly influences the catalytic oxidation reaction. Therefore, by investigating iron catalysts with smaller, more steric‐restricted ligands or catalysts with different metal centers, the effects of steric hindrance may be mitigated, potentially allowing the reaction to proceed with Ac‐Pro‐OPac or proline residues other than the N‐terminal one.

Author Contributions

S. Oto, A. Marutani, A. Kawakami, Y. Hamamoto: synthesis, characterization, experimental, and validation. Y. Hirano, Y. Obora: conceptualization and funding acquisition. S. Oto, Y. Hamamoto, Y. Obora: DFT analysis, validation, software, data curation. Y. Hirano, Y. Obora, Y. Hamamoto, S. Oto: writing, and – Writing – original draft reviewing editing the manuscript. Y. Hirano, Y. Obora: project administration, supervision, resources,– review and editing of the manuscript.

Funding

This research was supported by Kansai University and the Organization for Research and Development of Innovative Science and Technology Kansai University. The authors would like to express their sincere gratitude.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

The authors have cited additional references within the Supporting Information [41].

Supporting File 1: cbdv71755‐sup‐0001‐SuppMat.docx

CBDV-23-e71755-s001.docx (600.7KB, docx)

Acknowledgments

VMD was developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana‐Champaign (http://www.ks.uiuc.edu/Research/vmd/). This research partly used computational resources under Collabora‐tive Research Program for Young Women Scientists provided by Academic Center for Computing and Me‐dia Studies, Kyoto University.

Contributor Information

Yasushi Obora, Email: obora@kansai-u.ac.jp.

Yoshiaki Hirano, Email: yhirano@kansai-u.ac.jp.

Data Availability Statement

The data are available in the article's Supporting Information.

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Associated Data

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Supplementary Materials

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Supporting File 1: cbdv71755‐sup‐0001‐SuppMat.docx

CBDV-23-e71755-s001.docx (600.7KB, docx)

Data Availability Statement

The data are available in the article's Supporting Information.

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