Westersidones A–C: Antimicrobial Depsidones From the Deep‐Sea Cold Seep‐Derived Endozoic Fungus Westerdykella dispersa CS‐734

ABSTRACT

Eight depsidone derivatives (1–8), including three new compounds designated as westersidones A–C (1–3), were isolated from Westerdykella dispersa CS‐734, an endozoic fungus derived from the deep‐sea cold seep mussel Gigantidas platifrons. Their structures were elucidated through extensive spectroscopic analysis, including HRESIMS, NMR, and single‐crystal X‐ray diffraction. Remarkably, compound 1 represents a rare and unprecedented hybrid natural product formed by the fusion of a depsidone and a naphtho‐γ‐pyrone skeleton. The antibacterial activities against human and aquatic pathogens of the isolated compounds were assayed. Notably, compound 8 exhibited the most potent activity against several tested strains, with MICs 4–16 µg/mL, underscoring the critical role of the C‐4 aldehyde group in enhancing antimicrobial potency.

Chemical investigation of the deep‐sea cold seep‐derived fungus Westerdykella dispersa yields eight depsidones, including three new compounds westersidones A–C. Remarkably, westersidone A represents an unprecedented hybrid polyketide featuring a depsidone fused with a naphtho‐γ‐pyrone skeleton. Bioassays and structure‐activity relationship (SAR) analyses reveal their promising antibacterial potential, shedding light on chemical defenses within extreme marine ecosystems.

1. Introduction

Gigantidas platifrons, a deep‐sea mussel, is a typical dominant species and the primary consumer in cold seep chemosynthetic ecosystems. To survive in these extreme environments, G. platifrons has established complex symbiotic relationships with microorganisms [1, 2, 3]. Endozoic fungi are considered pivotal members of the mussel symbiotic microbial network, potentially facilitating biochemical communication and providing chemical defense via their secondary metabolites [4]. Depsidones are a structurally unique class of natural products characterized by a tricyclic core formed by two 2,4‐dihydroxybenzoic acid units linked via ether and lactone bonds [5]. This class of compounds possessed diverse biological activities, including anti‐tumor [6], antibacterial [7], antimalarial [8] and antioxidant properties [9]. In our ongoing search for bioactive metabolites from deep‐sea‐derived fungi, we isolated an endozoic fungal strain of Westerdykella dispersa CS‐734 from the deep‐sea mussel G. platifrons, collected at a cold seep in the South China Sea. Chemical investigation of this strain led to the isolation and identification of eight depsidone derivatives (1–8), including three new compounds, westersidones A–C (1–3), and five known compounds (4–8) [10, 11, 12]. Notably, compound 1 represents the first hybrid natural product formed by the fusion of a depsidone and a naphtho‐γ‐pyrone skeleton. The antibacterial potential of these isolates against both human and aquatic pathogens was evaluated. Herein, we report the details of their isolation, structural elucidation, and antibacterial evaluation.

2. Results and Discussion

2.1. Structure Elucidation of Compounds 1–3

The EtOAc and MeOH extract of W. dispersa CS‐734, cultured on rice solid medium, were fractionated and separated by a combination of various column chromatography to yield compounds 1–8 (Figure 1).

FIGURE 1.

Chemical structures of the characterized compounds 1–8.

Compound 1 was isolated as a yellow amorphous powder. Its molecular formula was determined as C₃₉H₃₆O₁₀ (m/z 665.2381 [M + H]⁺) on the basis of HRESIMS data (Figure S1), indicating 22 degrees of unsaturation. The ¹H NMR (Table 1 and Figure S2) spectrum showed the presence of 7 aromatic/olefinic methines, an sp³‐hybrid methylene, and eight methyls (including two methoxy groups). The ¹³C NMR and DEPT spectroscopic data (Table 1 and Figure S3) indicated the presence of 39 carbon atoms, accounting for 8 methyls (including two methoxy groups), 1 methylene, 7 aromatic/olefinic methines, and 23 quaternary carbons (including one ketone and on ester carbonyl groups). Detailed analysis of its 1D and 2D NMR data (Figures S4 and S5) enable the establishment of two structural fragments, 1A and 1B, for compound 1 (Figure 2). Fragment 1A was identified as a depsidone derivative similar to folipastatin [13], with the key difference being the replacement of the methyl group (δ _C 8.3) in folipastatin by a methylene carbon (δ _C 19.9) in 1. Fragment 1B was assigned as a naphtho‐γ‐pyrone analogous to rubrofusarin B [14]. However, the methine signals at δ _H/C 6.95/101.3 observed in the ¹H and ¹³C NMR spectra of rubrofusarin B¹⁴ was absent in those of 1. Instead, a quaternary carbon resonance at δ _C 107.6 was observed in the NMR spectra of 1. The linkage between fragments 1A and 1B via methylene C‐12 was confirmed by key HMBC correlations (Figure S6) from H‐12 to C‐3, C‐4, C‐4a, C‐21a, C‐22, and C‐22a. The E‐geometry of the double bond at C‐1′ and C‐1″ was determined based on the NOE correlations (Figure S7) from H‐2 to H‐2″ and from H‐7 to H‐2′. Thus, the structure of compound 1 was elucidated.

TABLE 1.

¹H and ¹³C NMR data of compound 1 (measured in DMSO‐d ₆).

No.	δ H (Mult., J in Hz) a	δC, Type b	No.	δ H (Mult., J in Hz) a	δC, Type b
1	—	148.1, C	17a	—	107.6, C
2	6.30, s	112.5, CH	18	—	160.6, C
3	—	161.1, C	19	6.45, s	96.8, CH
4	—	116.1, C	20	—	160.9, C
4a	—	161.1, C	21	7.02, s	94.9, CH
5a	—	141.4, C	21a	—	139.1, C
6	—	135.7, C	22	—	107.6, C
7	6.45, s	110.9, CH	22a	—	150.5, C
8	—	152.7, C	23	2.28, s	19.7, CH3
9	—	114.6, C	24	3.84, s	55.9, OCH3
9a	—	143.4, C	25	3.79, s	55.0, OCH3
11	—	163.8, CO	1′	—	133.0, C
11a	—	109.8, C	2′	5.55, dd (7.2, 1.7)	125.6, CH
12	4.36, s	19.9, CH2	3′	1.64, d (7.2)	14.0, CH3
13	2.09, s	9.0, CH3	4′	2.00, s	18.0, CH3
14	—	168.1, C	1″	—	135.4, C
15	6.18, s	106.2, CH	2″	5.27, dd (7.3, 1.7)	123.9, CH
16	—	184.2, CO	3″	1.62, d (7.3)	13.7, CH3
16a	—	103.1, C	4″	1.73, s	17.2, CH3
17	—	161.1, C	17‐OH	14.94, s	—

^a Measured at 400 MHz.

^b Measured at 100 MHz.

FIGURE 2.

Key COSY (bold lines in black), HMBC (arrows in red), and NOESY (arrows in blue) correlations for compound 1.

Compound 2 was originally obtained as a pale yellow amorphous powder. Its molecular formula was established as C₂₄H₂₆O₇ by HRESIMS (m/z 449.1563 [M + Na]⁺, Figure S8), corresponding to 12 degrees of unsaturation. The ¹³C NMR data (Table 2 and Figure S10) indicated the presence of 24 carbons, including four methyls, one methoxyl, two hydroxymethyls, four methines, and thirteen quaternary carbons (including one carbonyl carbon). Detailed analysis of 1D and 2D NMR data (Figures S9–S12) suggested that 2 is an analogue of the known compound 4 [11], characterized by a methoxy group at C‐12 and a hydroxymethyl group at C‐3″ instead of the original hydroxyl and methyl group, respectively. This above assignment was further confirmed by key HMBC correlations (Figure S13) from 12‐OCH₃ to C‐12 and from H‐3″ to C‐1″ and C‐2″, as well as the key COSY correlations (Figure S11) from H‐2″ to H‐3″. The E‐geometry of the Δ ^1′ and Δ ^1″ double bonds were determined based on NOESY correlations (Figure S14) of H‐2′/H‐7, H‐3′/H‐4′, H‐2″/H‐2 and H‐3″/H‐4″ (Figure 3). To further verify the structural assignment, compound 2 was dissolved in methanol (MeOH) and kept at 4°C for 2 weeks, affording quality crystals suitable for X‐ray crystallographic analysis, which unambiguously confirmed the complete structure of 2 (Figure 4, CCDC depositary Number: 2542472).

TABLE 2.

¹H and ¹³C NMR data of compounds 2 and 3 (measured in DMSO‐d ₆).

No.	2		3
	δ H (Mult., J in Hz) a	δC, Type b	δ H (Mult., J in Hz) a	δC, Type b
1	—	149.8, C	—	149.1, C
2	6.63, s	112.7, CH	6.58, s	112.7, CH
3	—	161.1, C	—	160.7, C
4	—	113.9, C	—	117.5, C
4a	—	162.8, C	—	162.0, C
5a	—	141.5, C	—	141.6, C
6	—	135.5, C	—	135.7, C
7	6.42, s	111.1, CH	6.41, s	111.0, CH
8	—	152.6, C	—	152.5, C
9	—	114.7, C	—	114.6, C
9a	—	143.1, C	—	143.2, C
11	—	163.3, CO	—	163.5, C
11a	—	110.4, C	—	110.6, C
12	4.43, s	61.8, CH2	4.53, s	52.3, CH2
13	2.06, s	9.0, CH3	2.07, s	9.1, CH3
1′	—	133.5, C	—	133.4, C
2′	5.48, qq (6.7, 1.5)	125.0, CH	5.48, qq (6.8, 3.9)	125.1, CH
3′	1.77, d (6.7)	13.8, CH3	1.76, d (6.8)	13.8, CH3
4′	1.99, s	17.6, CH3	1.99, s	17.6, CH3
1″	—	134.5, C	—	134.6, C
2″	5.38, td (6.0, 1.4)	131.1, CH	5.38, td (6.4, 3.3)	131.0, CH
3″	4.04, d (6.0)	58.1, CH3	4.04, d (6.4)	58.1, CH3
4″	1.79, s	17.6, CH3	1.79, s	17.6, CH3
12‐OCH3	3.24, s	57.2, OCH3	—	—

^a Measured at 400 MHz.

^b Measured at 100 MHz.

FIGURE 3.

Key COSY (bold lines in black), HMBC (arrows in red), and NOESY (arrows in blue) correlations for compounds 2 and 3.

FIGURE 4.

X‐ray crystal structure of compound 2 (inclusive one molecule N,N‐dimethylformamide).

Compound 3 was isolated as a white amorphous powder with the molecular formula C₂₃H₂₄O₇ (m/z 413.1595 [M + H]⁺) by HRESIMS (Figure S15), corresponding to 12 degrees of unsaturation. Its NMR data (Table 2, Figures S16, and S17) were nearly identical to compound 2, with the key exception being the absence of the methoxy group signals δ _H/C 3.24/57.2, which correspond to the 12‐OCH₃ group in 2. This indicated that 3 is the 12‐demethylated derivative of 2. The structure was further supported by key HMBC correlations (Figure S20) from the methylene protons (H₂‐12) to C‐3, C‐4, and C‐4a (Figure 3), confirming the presence of a free hydroxymethyl group at C‐12 instead of a methoxy group. The geometries of the Δ ^1′ and Δ ^1″ double bonds in 3 were established as E, consistent with those observed for 2, based on analogous NOESY correlations (Figure S21). Thus, the structure of compound 3 was assigned as the 12‐O‐demethyl analogue of 2.

2.2. Possible Biosynthetic Pathway of Compound 1

The plausible biosynthesis of compound 1 (Scheme 1) involves the assembly of a depsidone core and a naphtho‐γ $\gamma$‐pyrone core via electrophilic aromatic substitution. The two monomeric precursors are initially and independently constructed through distinct non‐reducing polyketide synthase (nrPKS) pathways, as described in literatures [15, 16]. Notably, the depsidone precursor, folipastatin, undergoes cytochrome P450 monooxygenase‐mediated benzylic oxidation, converting the C‐12 methyl group into a hydroxymethyl group to yield intermediate 4. Subsequently, the elimination of the hydroxyl group in 4 generates a highly reactive benzylic carbocation. This electrophilic species then attacks the electron‐rich C‐22 of the independently biosynthesized naphtho‐γ $\gamma$‐pyrone precursor, ultimately yielding the hybrid skeleton of compound 1.

SCHEME 1.

Possible biosynthetic pathway of compound 1.

2.3. Antibacterial Activities

The antimicrobial activities of compounds 1–8 were evaluated against human and aquatic pathogens, as well as cold seep‐derived Vibrio species collected in the South China Sea (Table 3). Most compounds displayed moderate antimicrobial activities, while compound 8 exhibited significantly stronger activity. A comparison between compound 8 and 7 indicated that the presence of a C‐4 aldehyde group in 8 contributes significantly to enhance antimicrobial activity. Furthermore, comparisons of matched pairs (compounds 2 vs. 3, 4 vs. 6, and 5 vs. 7) consistently revealed that methoxylation at the C‐12 position adversely affects antimicrobial activity. Compound 4 exhibited stronger activity than that of compound 3, implying that the hydroxymethylation at C‐2″ is deterimental to antimicrobial activity.

TABLE 3.

Antimicrobial activities of compounds 1–8 (MIC, µg/mL).

	401	403	406	408	MRSA	PA	AH	VH	VP	VA
1	8	32	32	—	32	16	32	16	32	16
2	—	8	—	64	16	4	32	8	4	32
3	—	64	—	64	—	—	—	—	—	—
4	—	64	64	32	8	16	8	8	16	8
5	32	—	32	32	16	2	16	16	16	32
6	—	64	64	4	8	4	16	4	8	8
7	—	64	64	32	64	16	32	32	32	16
8	—	4	64	16	4	2	4	4	8	8
Chloramphenicol	0.5	0.5	0.5	0.5	0.5	0.5	1	1	2	2

Abbreviations: 401, Vibrio fluvialis (similarity 99.857%); 403, Vibrio fluvialis (similarity 99.929%); 406, Vibrio fluvialis (similarity 99.859%); 408, Vibrio fluvialis (similarity 99.929%); MRSA, methicillin‐resistant Staphylococcus aureus; PA, Pseudomonas aeruginosa; AH, Aeromonas hydrophilia; VH, Vibrio harveyi; VP, Vibrio parahaemolyticus; VA, Vibrio alginolyticus.

– = MIC > 64 µg/mL. Values are expressed as the means of three independent experiments.

3. Conclusions

In summary, eight depsidone derivatives, including three new compounds, were characterized from the endozoic fungus W. dispersa CS‐734, which was isolated from the deep‐sea cold seep mussel G. platifrons. Among the isolated compounds, compound 1 represents a unique and unprecedented hybrid natural product formed by the fusion of a depsidone and a naphtho‐γ‐pyrone skeleton. Biological evaluation showed that compound 8 exhibited potent antibacterial activity. Furthermore, structure‐activity relationship (SAR) analysis revealed that the presence of C‐4 aldehyde group significantly enhances antimicrobial efficacy, whereas methoxylation at C‐12 and hydroxymethylation at C‐2″ adversely affect antimicrobial activity. These findings not only expand the chemical diversity of deep‐sea fungal metabolites, but also provide new insights into the possible role of endozoic fungi in enhancing host adaptability within extreme cold‐seep ecosystems.

4. Experimental Section

4.1. General Experimental Procedures

Optical rotations were measured in MeOH using an Optical Activity AA‐55 polarimeter. UV spectra were obtained with a PuXi TU‐1810 UV–visible spectrophotometer. IR spectra were acquired through a ThermoFisher‐Nicolet iS50 spectropolarimeter. NMR spectra data were recorded on a Bruker Avance 400 MHz spectrometer. HRESIMS data were measured using a Bruker maXis plus mass spectrometer. Semipreparative HPLC was performed by an Alltech HPLC system using an ODS column (Elite ODSBP column, 10 µm; 4.6 × 250 mm; 3 mL/min). Column chromatography was carried out using commercially available Si gel (200–300 mesh, Qingdao Haiyang Chemical Co.), Lobar LiChroprep RP‐18 (40–63 µm, Merck), and Sephadex LH‐20 (Pharmacia). Thin‐layer chromatography (TLC) was performed with precoated Si gel GF254 plates (Merck, Darmstadt, Germany). Solvents used for extraction and purification were distilled prior to use. Peptone from yeast extract was purchased from Sigma‐Aldrich. Rice, monosodium glutamate, and corn steep liquor were purchased from China Oil & Foodstuffs Corporation.

4.2. Fungal Material

The endozoic fungus W. dispersa CS‐734 was isolated from the gill tissue of mussel G. platifrons collected from the cold seep in the South China Sea. Based on the ITS rDNA sequencing results, this strain was identified as W. dispersa, whose sequence data is same (100%) to that of W. dispersa NTOU 4263 (accession No. MZ423069.1). The sequence data of CS‐734 have been deposited in GenBank with the accession No. PZ169650.1. The strain is preserved at the Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

4.3. Fermentation, Extraction and Isolation

The fungus was developed on potato dextrose agar (PDA) at 28°C for 7 days, and the mycelium was then cut into pieces (1 × 1 cm) to inoculate in three 1 L Erlenmeyer flasks; each contained 300 mL of potato dextrose broth (PDB). After incubation on a shaker at 180 rpm for 4 days (27°C), the culture was transferred into a rice medium in 1 L x 160 Erlenmeyer flasks; each contained 70 g rice, 0.3 g tryptone, 0.5 g yeast extract powder, 3.5% sea salt, and 100 mL of water per flask, and was incubated for 35 days at 25°C.

The fermented rice substrate was exhaustively extracted with ethyl acetate (EtOAc) and MeOH. The combined extracts were concentrated to gain crude extract (106.6 g). The crude extract was subjected to vacuum liquid chromatography (VLC) using a step‐gradient of petroleum ether (PE)/EtOAc (from 20:1 to 1:1) and dichloromethane (DCM)/MeOH (from 20:1 to 1:1) to obtain nine fractions (Frs.1–9). Fr.2 (6.0 g), eluted with PE‐EtOAc (5/1) was further isolated by gel column chromatography (CC) Sephadex LH‐20 (MeOH) combined with semi‐preparative HPLC (Elite ODS‐BP column, 10 µm; 4.6 × 250 mm; 40% MeOH‐H₂O, 3 mL/min) to afford compound 1 (2.6 mg, t_R 35.0 min). Fr.5 (15.0 g), eluted with DCM‐MeOH (20:1), was further purified by CC on Lobar LiChroprep RP‐18 using a MeOH‐H₂O gradient (from 10:90 to 100:0) to yield eight subfractions (Frs.5.1–5.8). Fr.5.3 (eluted with MeOH‐H₂O, 40:60) was further purified by CC on Si gel eluting with a DCM‐MeOH gradient (from 150:1 to 10:1) and semi‐preparative HPLC (Elite ODS‐BP column, 10 µm; 4.6 × 250 mm; 45% MeOH‐H₂O, 3 mL/min) to afford compound 4 (13.0 mg, t_R 8.6 min) and compound 6 (8.9 mg, t_R 23.0 min). Fr.5.4 (eluted with MeOH‐H₂O, 40:60–50:50) was further purified by CC on Si gel eluting with a DCM‐MeOH gradient (from 150:1 to 10:1) and semi‐preparative HPLC (Elite ODS‐BP column, 10 µm; 4.6 × 250 mm; 55% MeOH‐H₂O, 3 mL/min) to afford compound 2 (5.5 mg, t_R 8.0 min) and compound 3 (9.0 mg, t_R 11.0 min). Fr.5.5 (eluted with MeOH‐H₂O, 50:50) was further purified by Sephadex LH‐20 (MeOH) to obtain compound 5 (15.9 mg). Fr.5.7 (eluted with MeOH‐H₂O, 80:20–90:10) was further purified by CC on Si gel eluting with a DCM‐MeOH gradient (from 150:1 to 10:1) and semi‐preparative HPLC (Elite ODS‐BP column, 10 µm; 4.6 × 250 mm; 65% MeOH‐H₂O, 3 mL/min) to afford compound 7 (4.0 mg, t_R 16.0 min) and compound 8 (30.4 mg, t_R 44.0 min).

Westersidone A (1): yellow amorphous powder; UV (MeOH) λ_max (log ε) 254 (4.45), 278 (4.58) nm; IR ν _max 3304, 2925, 1732, 1652, 1614, 1587, 1424, 1322, 1253, 1173, 1118, 1026, 835 cm⁻¹; ¹H and ¹³C NMR: see Table 1. HR‐ESI‐MS m/z 665.2381 [M + H]⁺ (calc. for C₃₉H₃₇O₁₀ ⁺, 665.2387).

Westersidone B (2): initially obtained as pale yellow amorphous powder, then dissolved in MeOH and kept at 4°C to yield pale crystals; UV (MeOH) λ_max (log ε) 269 (5.22) nm; IR ν _max 3304, 2925, 1732, 1614, 1587, 1424, 1322, 1253, 1173, 1118, 1026, 835 cm⁻¹; ¹H and ¹³C NMR: see Table 2. HR‐ESI‐MS m/z 449.1563 [M + Na]⁺ (calc. for C₂₄H₂₆O₇Na⁺, 449.1571).

Westersidone C (3): white amorphous powder; UV (MeOH) λ_max (log ε) 278 (4.99) nm; IR ν _max 2955, 2917, 2849, 1659, 1632, 1471, 1421, 1264, 1119, 1008, 699 cm⁻¹; ¹H and ¹³C NMR: see Table 2. HR‐ESI‐MS m/z 413.1595 [M + H]⁺ (calc. for C₂₃H₂₅O₇ ⁺, 413.1600).

4.4. X‐ray Crystallographic Analysis

The crystallographic data of compound 2 were collected on a Bruker Smart‐1000 diffractometer equipped with a graphite‐monochromatic CuKα radiation (λ = 1.54178) Å at 273.15 K. Using Olex2 [17], the structure was solved with the SHELXS [18] structure solution program using Direct Methods and refined with the SHELXL [19] refinement package using Least Squares minimization.

Crystal data for compound 2: C₂₄H₂₆O₇·C₃H₇NO, F.W. = 499.54, M.P. = 147–149 °C, Triclinic, space group P‐1, unit cell dimensions a = 8.1577(7) Å, b = 11.5501(11) Å, c = 14.0457(11) Å, V = 1262.21(19) ų, α = 93.543(5)°, β = 95.803(5)°, γ = 105.669(5)°, Z = 2, d _calc = 1.314 mg/m³, crystal dimensions 0.15 × 0.12 × 0.01 mm³, μ = 0.801 mm⁻¹, F(000) = 532.0. The 4465 measurements yielded 4465 independent reflections after equivalent data were averaged. The final refinement gave R ₁ = 0.1526 and wR ₂ = 0.3857 [I≥2σ (I)]. The crystallographic data have been deposited in the Cambridge Crystallographic Data Centre (CCDC 2542472).

4.5. Antibacterial Assays

The tested compounds and the positive control (chloramphenicol) were dissolved in DMSO to give a stock solution. All microbial strains were obtained from IOCAS (Institute of Oceanology, Chinese Academy of Sciences). The antibacterial activities against two human pathogenic bacterium (methicillin‐resistant Staphylococcus aureus EMBLC‐4 and Pseudomonas aeruginosa QDIO‐4), four aquatic pathogens (Aeromonas hydrophilia QDIO‐1, Vibrio alginolyticus QDIO‐5, Vibrio harveyi QDIO‐7 and Vibrio parahaemolyticus QDIO‐8) and four vibrio species (401, 403, 406, and 408) collected from the cold seep in the South China Sea (Vibrio fluvialis strain NBRC 103150 with different similarities), were examined by a serial dilution technique using 96‐well microtiter plates [20]. Use LB liquid medium to activate the target strains in 30°C (use 2216E medium for South China Sea cold seep vibrio in 28°C). Dilute the bacterial suspension to 0.5 McFarland turbidity (5 × 10⁵ CFU/mL) with sterilized normal saline, and then perform the second dilution of the bacterial solution to 1000 fold using LB medium. Take 95 µL of the bacterial solution and add it to a 96 ‐ well plate, and add 5 µL of the test compound and positive control solution at gradient concentrations. After shaking and mixing evenly, place it in 30°C for 16–18 h to observe the growth of bacteria (place South China Sea cold seep vibrio in 28°C). Set up 3 parallel controls for each group of experiments.

Author Contributions

B.‐N.X. performed the experiments for the isolation, structure elucidation, antibacterial assays, and prepared the manuscript; X.‐M.L. performed the 1D and 2D NMR experiments; S.‐Q.Y. and X.L. collected the mussel Gigantidas platifrons from the cold seep area in the South China Sea and isolated the fungus Westerdykella dispersa CS‐734. B.‐G.W. and X.L. supervised the research work and revised the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71341‐sup‐0001‐SuppMat.docx

Supporting File 2: cbdv71341‐sup‐0002‐Data.zip

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article.