The work was first done on the four compounds belonging to the trans-δ-viniferin scaffold. For these compounds, the goal was to obtain at least 1 mg of each pure enantiomer to be able to run biological assays and determine if the anti-MRSA activity of each individual enantiomers will significantly differ from the one reported for the enantiomeric mixture. This is of particular interest for compound 3 which showed an anti-MRSA activity almost as high as vancomycin (Righi et al., 2020).

The isolation of the different enantiomers was performed using an analytical HPLC Chiralpak^® IB N-5 column (250 × 4.6 mm i. d., 5 µm; Daicel). Separation was performed in isocratic mode using a mixture of heptane and EtOH containing 0.1% diethylamine as mobile phase, and compounds were manually collected after the PDA detector. For each compound, various isocratic runs were tested to select the most efficient one. After optimization, concentrated solutions were prepared (ca. 5–15 mg/ml in dichloromethane/ethyl acetate mixtures, depending on the solubility of each compound). As the isolation was performed on an analytical column, a series of injections with increasing injected amounts of sample were performed to determine the maximum loading capacity for each pair of enantiomers. Due to the unusually high volumes of solvent injected for an analytical column, the peak shape was strongly distorted. Based on two parameters (solubility and peak resolution), the maximal injectable amount was defined for each compound. It was for example possible to inject 1.25 mg (100 μl, 12.5 mg/ml) of compound 3 and separate the two enantiomers without overlapping of the peaks. In the case of compound 1, the loading capacity of the column was lower, allowing an injection of a maximum of 0.4 mg (40 μl, 10 mg/ml). In this case, more injections were necessary to obtain enough material for the chemical and biological analysis.

Using this approach, the enantiomers of compounds 1–4 were successfully separated and obtained in sufficient amount for chemical and biological evaluation. The four enantiomeric pairs obtained were monitored by UHPLC-PDA-ELSD-MS, NMR, and chiral-HPLC-PDA to ensure their purity. Their optical properties were studied by optical rotation and electronic circular dichroism (ECD). The latter were compared to calculated ones and to the literature to determine their absolute configurations (Han et al., 2014; Gavezzotti et al., 2015). The workflow is detailed below for compound 1 as an example (Figure 2).

Analysis of compound 1 by UHPLC-PDA showed a single chromatographic peak corresponding to the compound previously isolated as a racemic mixture. Analysis using a chiral HPLC column confirmed this by the presence of two peaks of similar intensities suggesting that the two enantiomers are present in similar proportions (Figure 2A). The UV spectrum of each chromatographic peak was measured and found to be the same as expected (Figure 2B). The purified enantiomers 1a and 1b were subjected to specific rotation ([α]_D) and ECD analysis to determine the absolute configuration of the 7′ and 8′ carbons (Figure 2E). The specific rotation of compound 1a was −19.6, and that of compound 1b was 15.4, while compound 1 had a near zero value of 0.8. These observed values suggest an effective chiral separation of the two optically pure enantiomers. These compounds were then subjected to ECD analysis, and the obtained spectra showed opposite curves with identical magnitude confirming the separation of the two enantiomers, compared to the flat spectrum obtained from compound 1 (Figure 2E).

The experimental ECD spectra were compared to calculated ones, based on the relative configuration determined by NMR. Briefly, NMR analysis of the ROESY spectrum of 1 showed correlations from H-7′ to H-10′/H-14′ and from H-8′ to H-2′/H-6′ indicating a trans relative stereochemistry between H-7′/H-8′ (Figure 2C, Supplementary Figure S1). This indicates that the two carbons have the same absolute stereochemistry (R,R or S,S).

To determine the absolute configuration of the carbons 7′ and 8′ for 1a and 1b, the structures with the relative configuration proposed by the NMR were modelled and energetically optimized in vacuo using B3LYP hybrid function with a 6–31G (d,p) basis (Stephens et al., 1994; Nugroho and Morita, 2013; Mándi and Kurtán, 2019). All possible conformers were calculated for each molecule and subsequently optimized using the same function and basis set as before but in a SCRF (self-consistent reaction field) model in MeCN. After a cut-off of 4 kcal/mol in energy, the selected conformers were modelled using a TD-DFT (time dependent density functional theory) method with a hybrid function B3LYP and an extended basis def2svp, under a SCRF model in MeCN (Nugroho and Morita, 2013). After this step, the ECD spectra of the two enantiomers were simulated and compared with those of the compounds experimentally obtained. The calculated ECD for the 7′R, 8′R stereoisomer showed an excellent fit with the experimental data of 1a, with positive CE (Cotton Effect) around 215 and 320 nm and negative CE around 250 and 300 nm. The other stereoisomer, 7′S, 8′S, displayed a mirror image. The positive CE around 215 nm in the experimental spectrum was likely due to a π → π* transition in the phenyl moieties, while the CE around 300 nm were likely due to the extended π conjugated system. From this comparison it was possible to assign the (-)-(R,R) configuration to compound 1a (t _R 30 min) and (+)-(S,S) to compound 1b (t _R 46 min) (Figures 2D–F). These attributions are consistent with those found in the literature based on ECD spectra of ε-viniferin and 1,2-diaryldihydrobenzofuran (Lins et al., 1986; Kurihara et al., 1990; Bhusainahalli et al., 2012; Han et al., 2014; Gavezzotti et al., 2015).

The same procedure described above for compound 1 was applied for the chiral isolation and the absolute configuration determination of the enantiomeric pairs of compounds 2, 3, and 4. This allowed the characterization of compounds 2a (−)-(R,R), 2b (+)-(S,S), 3a (−)-(R,R), 3b (+)-(S,S), 4a (−)-(R,R) and 4b (+)-(S,S) (Figure 3). A summary for each compound with the chromatographic and spectroscopic details can be found on Supplementary Figure S2 (compound 1), Supplementary Figure S3 (compound 2), Supplementary Figure S4 (compound 3) and Supplementary Figure S5 (compound 4).

It is well known that enantiomers of a chiral drug can exhibit different pharmacological behavior in a chiral environment such as a receptor (McConathy and Owens, 2003). Since some of the trans-δ-viniferins investigated in this study presented remarkable antimicrobial activities against resistant strains of S. aureus (Righi et al., 2020), we decided to investigate the activities of their respective enantiomers.

For this purpose, the minimal inhibitory concentrations (MICs) of compounds 1, 2, 3, and 4 and their respective purified enantiomers (1a, 1b, 2a, 2b, 3a, 3b, 4a, and 4b) were determined against a methicillin-susceptible and a methicillin-resistant strain of S. aureus (Table 1). The trans-δ-viniferin derivatives 2 and 3 showed comparable potent activities against both S. aureus strains, in agreement with our previous results (Righi et al., 2020). The enantiomers 2a/2b and 3a/3b showed similar activities as their respective racemic mixtures. This demonstrate that the enantiomeric form of these trans-δ-viniferins has no influence on the bioactivity and that the possible targets do not require a specific chiral form.

After successfully applying this methodology to the four trans-δ-viniferin derivatives, we used it to separate the enantiomers of the other main scaffolds (Figure 1). To our knowledge, the enantiomeric separation of these four scaffolds has never been reported before. The subsequently obtained ECD spectra can be used as references for future research, including studies on plant metabolites since these families are present in nature (except acyclic dimers) (Khan et al., 1986; Lins et al., 1991; Ohyama et al., 1995; Pezet et al., 2003).

Since compounds 1–8 share common structural features, we decided to continue with the same stationary phase used in the study of the trans-δ-viniferins 1–4 to separate the enantiomers of compounds 5–8. However, the efficiency of the column was not the same for these other scaffolds and the separation of these compounds at the milligram level was not possible. Fortunately, ECD measurements require concentrations of about 20 µM for these types of compounds, which represents a mass requirement of about 20 µg (molar mass of about 500 g/mol). These quantities are compatible with small volume injections to maintain the separation achieved between the chromatographic peaks. For each compound (5–8), repeated injections with manual collection were performed to obtain the pure enantiomers. Each enantiomer was monitored with UHPLC-PDA-ELSD-MS, NMR and chiral-HPLC-PDA.

As explained above, the NMR analysis of compounds 5, 6, 7, and 8 was used to determine their relative stereochemistry. The purified enantiomers 5a, 5b, 6a, 6b, 7a, 7b, 8a, and 8b were subjected to ECD analysis to determine the absolute configuration of the chiral carbons. Analysis of the ECD spectra showed opposite curves with identical magnitude, indicating efficient chiral separation of the two optically pure enantiomers (Figure 4).

To determine the absolute configuration of the chiral carbons in each structure, the 3D structures of the most stable conformers of each enantiomer were calculated using TD-DFT at B3LYP/def2svp//B3LYP/6–31G (d,p) level and a SCRF model in MeCN. After this step, the ECD spectra of the two enantiomers were simulated and compared with the experimental ones (Figure 4).

For example, the calculated ECD spectrum for the (7R,8R,7′R,8′R) stereoisomer of compound 5 matches with the experimental data of 5a, with negative CE around 200 and 235 nm, and a positive CE around 285 nm. Inherently to this type of calculations, some bands can be wrongly predicted, as it is the case for the experimental positive CE around 215 nm, predicted as negative. The other stereoisomer, the (7S,8S,7′S,8′S) displayed a mirror image. This comparison resulted in the attribution of the (7R,8R,7′R,8′R) configuration to 5a and (7S,8S,7′S,8′S) configuration to 5b (Figure 4).

Using the same approach, it was possible to assign the absolute configuration of 6a as (7′R,8′R) and 6b as (7′S,8′S). The absolute configuration of 7a was determined as (7R,8S,7′S,8′R) and 7b as (7S,8R,7′R,8′S). Finally, the absolute configuration of 8a was assigned as (7R,8S,7′S,8′S) and 8b as (7S,8R,7′R,8′R) (Figure 4). A summary for each compound with the chromatographic and spectroscopic details can be found on Supplementary Figure S6 (compound 5), Supplementary Figure S7 (compound 6), Supplementary Figure S8 (compound 7) and Supplementary Figure S9 (compound 8).

The specific rotations were measured in MeCN on a JASCO P-1030 polarimeter (Loveland, CO, United States) in a 10 cm tube. The UV and ECD spectra were recorded on a JASCO J-815 spectrometer (Loveland, CO, United States) in MeCN, using a 1 cm cell. The scan speed was set at 200 nm/min in continuous mode between 500 and 190 nm, with a band width of 1 nm, a data pitch of 0.1 nm and 2 accumulations. NMR spectroscopic data were recorded on a Bruker Avance Neo 600 MHz NMR spectrometer equipped with a QCI 5 mm cryoprobe and a SampleJet automated sample changer (Bruker BioSpin, Rheinstetten, Germany). ¹H NMR experiments were recorded in DMSO-d ₆. Chemical shifts are reported in parts per million (δ) and coupling constants (J) in Hz. The residual DMSO-d ₆ signal (δ_H 2.50); was used as internal standard for ¹H. Chromatographic data were obtained on an Ultra-high-performance liquid chromatography system equipped with a photodiode array, an evaporative light-scattering detector, and a single quadrupole detector using heated electrospray ionization (UHPLC-PDA-ELSD-MS) (Waters, Milford, MA, United States). The ESI parameters were the following: capillary voltage 800 V, cone voltage 15 V, source temperature 120°C, and probe temperature 600°C. Acquisition was done in negative ionization mode (NI) with an m/z range of 150–1000 Da. The chromatographic separation was performed on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm i. d., 1.7 μm; Waters) at 0.6 ml/min, 40°C with H₂O (A) and MeCN (B) both containing 0.1% formic acid as solvents. The gradient was carried out as follows: 5–100% B in 7 min, 1 min at 100% B, and a re-equilibration step at 5% B for 2 min. The ELSD temperature was fixed at 45°C, with a gain of 9. The PDA data were acquired from 190 to 500 nm, with a resolution of 1.2 nm. The sampling rate was set at 20 points/s.

The compounds 1–8 used in this experiment were obtained by the biotransformation of resveratrol and pterostilbene by the enzymatic secretome of Botrytis cinerea Pers., as described in our previous article (Righi et al., 2020). Their purity was controlled by UHPLC-PDA-ELSD-MS and NMR.

Compounds 1–4 were screened on different Daicel chiral columns to find the most efficient stationary phase: IA, IB-N5, IC, ID, IE, IF, IG, and IH (https://chiraltech.com for the details about the stationary phases). A first screening was performed with heptane/alcohol mixtures (EtOH or isopropanol) with 0.1% diethylamine. A second screening was performed with heptane/ethyl acetate, heptane/dichloromethane and heptane/tetrahydrofuran mixtures with 0.1% diethylamine. A last screening was performed with acetonitrile or methanol/ethanol mixtures with 0.1% diethylamine. The selected stationary phase was a cellulose substituted with tris (3,5-dimethylphenylcarbamate) immobilized on a 5 μm silica-gel (Chiralpak^® IB N-5 250 × 4.6 mm i. d., 5vµm; Daicel, Osaka, Japan). Compounds 1–8 were then injected on a HP 1100 Agilent High-Performance liquid chromatography equipped with a photodiode array detector (HPLC-PDA) (Agilent technologies, Santa Clara, CA, United States). The chromatographic separations were performed on the previously selected column Chiralpak^® IB N-5 column (250 × 4.6 mm i. d., 5 μm; Daicel) at 1 ml/min, with heptane (A) and EtOH (B) both containing 0.1% diethylamine as solvents. Solutions at 5–15 mg/ml of compounds 1–8 in dichloromethane/ethyl acetate mixtures were first injected to determine the best isocratic condition for each compound. Increasing volumes were injected until reaching the limit of separation. Repeated injections were finally performed with manual collection of the compounds in glass vials after the PDA detector. Separated enantiomers were controlled by UHPLC-PDA-ELSD-MS, chiral-HPLC-PDA and NMR.

The absolute configuration of the compounds was assigned according to the comparison of the calculated and experimental ECDs. The structures and the relative configurations proposed by NMR were energetically optimized through 3LYP/6–31G (d, p) in Gaussian 16 (© 2015–2022, Gaussian inc.). All possible conformers were calculated in Avogadro software (Version 1.2.0, © 2018 Avogadro Chemistry) through a Random rotor search algorithm. All conformers were energetically optimized through 3LYP/6–31G (d) in Gaussian 16 using a SCRF model in MeCN. Then, the conformers with the lowest energy (cut-off of 4 kcal/mol) were selected and checked to avoid imaginary frequencies. The conformers were selected and submitted to Gaussian16 software for ECD calculations, using TD-DFT with B3LYP/def2svp as basis set with SCRF model in MeCN. The computation in Gaussian was performed at the University of Geneva on the Baobab cluster (https://plone.unige.ch/distic/pub/hpc/baobab_en, accessed on 19 November 2021). The calculated ECD spectrum was generated in SpecDis1.71 software (Version 1.71, T. Bruhn, Berlin, Germany, 2017) based in Boltzmann weighing average.

Trans-δ-viniferin (1): [α]²⁵ _D 0.8 (c 0.06, MeCN). UV (MeCN) λmax (log ε) 227 (sh) (4.46), 285 (4.08), 312 (4.32), 333 (4.127), 350 (sh) (3.99) nm. ¹H NMR (DMSO-d ₆, 600 MHz) δ 4.45 (1H, d, J = 7.9 Hz, H-8′), 5.39 (1H, d, J = 7.9 Hz, H-7′), 6.04 (2H, d, J = 2.2 Hz, H-10′, H-14′), 6.10 (1H, t, J = 2.5 Hz, H-12′), 6.11 (1H, t, J = 2.2 Hz, H-12), 6.38 (2H, d, J = 2.1 Hz, H-10, H-14), 6.77 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.84 (1H, d, J = 16.3 Hz, H-8), 6.89 (1H, d, J = 8.3 Hz, H-5), 6.99 (1H, d, J = 16.3 Hz, H-7), 7.19 (2H, d, J = 8.6 Hz, H-2′, H-6′), 7.23 (1H, s, H-2), 7.42 (1H, dd, J = 8.2, 1.9 Hz, H-6), 9.18 (2H, s, 11OH, 13OH), 9.21 (2H, s, 11′OH, 13′OH), 9.53 (1H, s, 4′OH). HR-ESI/MS analysis: m/z 453.1348 [M-H]^-, (calcd for C₂₈H₂₁O₆, 453.1338, ∆ = 2.2 ppm). MS/MS spectrum: CCMSLIB00009918864.

(-)-(R,R)-trans-δ-viniferin (1a): heptane:EtOH 82:18 + 0.1% DEA, t _R 30 min [α]²⁵ _D −19.6 (c 0.1, MeCN). ECD (c 22 µM, MeCN) λmax ([θ]) 214 (+33168), 233 (−11271), 272 (−6268), 299 (−4705), 335 (+3558) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 1.

(+)-(S,S)-trans-δ-viniferin (1b): heptane:EtOH 82:18 + 0.1% DEA, t _R 46 min [α]²⁵ _D +15.4 (c 0.1, MeCN). ECD (c 18 µM, MeCN) λmax ([θ]) 214 (−42728), 232 (+8421), 274 (+6444), 298 (+6205), 332 (−4617) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 1.

11′,13′-di-O-methyl-trans-δ-viniferin (2): [α]²⁵ _D 0.5 (c 0.07, MeCN). UV (MeCN) λmax (log ε) 227 (sh) (4.45), 285 (4.06), 312 (4.30), 333 (4.25), 350 (sh) (3.97) nm. ¹H NMR (DMSO-d ₆, 600 MHz) δ 3.70 (6H, s, CH₃O-11′, CH₃O-13′), 4.58 (1H, d, J = 8.1 Hz, H-8′), 5.56 (1H, d, J = 8.1 Hz, H-7′), 6.10 (1H, t, J = 2.3 Hz, H-12), 6.36 (4H, 2xd, J = 2.3 Hz, H-10, H-14, H-10′, H-14′), 6.43 (1H, t, J = 2.3 Hz, H-12′), 6.76 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.82 (1H, d, J = 16.3 Hz, H-8), 6.90 (1H, d, J = 8.4 Hz, H-5), 6.97 (1H, d, J = 16.3 Hz, H-7), 7.19 (3H, m, H-2, H-2′, H-6′), 7.42 (1H, dd, J = 8.4, 1.9 Hz, H-6), 9.16 (2H, s, 11OH, 13OH), 9.52 (1H, s, 4′OH). HR-ESI/MS analysis: m/z 481.1652 [M-H]^-, (calcd for C₃₀H₂₅O₆, 481.1651, ∆ = 0.2 ppm). MS/MS spectrum: CCMSLIB00009918871.

(-)-(R,R)-11′,13′-di-O-methyl-trans-δ-viniferin (2a): heptane:EtOH 80:20 + 0.1% DEA, t _R 23 min [α]²⁵ _D −19.7 (c 0.06, MeCN). ECD (c 17 µM, MeCN) λmax ([θ]) 216 (+31567), 231 (−20076), 272 (−4156), 298 (−2064), 326 (+8532) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 2.

(+)-(S,S)-11′,13′-di-O-methyl-trans-δ-viniferin (2b): heptane:EtOH 80:20 + 0.1% DEA, t _R 42 min [α]²⁵ _D 20.8 (c 0.06, MeCN). ECD (c 17 µM, MeCN) λmax ([θ]) 215 (−39094), 231 (+19255), 270 (+6091), 300 (+4294), 324 (−7083) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 2.

11,13-Di-O-methyl-trans-δ-viniferin (3): [α]²⁵ _D −0.8 (c 0.07, MeCN); UV (MeCN) λmax (log ε) 227 (sh) (4.48), 285 (4.10), 312 (4.34), 333 (4.30), 350 (sh) (4.00) nm. ¹H NMR (DMSO-d ₆, 600 MHz) δ 3.75 (6H, s, CH₃O-11, CH₃O-13), 4.46 (1H, d, J = 8.3 Hz, H-8′), 5.41 (1H, d, J = 8.3 Hz, H-7′), 6.04 (2H, d, J = 2.2 Hz, H-10′, H-14′), 6.10 (1H, t, J = 2.2 Hz, H-12′), 6.35 (1H, t, J = 2.2 Hz, H-12), 6.73 (2H, d, J = 2.2 Hz, H-10, H-14), 6.76 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.90 (1H, d, J = 8.3 Hz, H-5), 6.96 (1H, d, J = 16.4 Hz, H-8), 7.19 (2H, d, J = 8.6 Hz, H-2′, H-6′), 7.22 (1H, d, J = 16.4 Hz, H-7), 7.23 (1H, d, J = 1.9 Hz, H-2), 7.44 (1H, dd, J = 8.3, 1.9 Hz, H-6), 9.22 (2H, s, 11′OH, 13′OH), 9.53 (1H, s, 4′OH). HR-ESI/MS analysis: m/z 481.1654 [M-H]^-, (calcd for C₃₀H₂₅O₆, 481.1651, ∆ = 0.6 ppm). MS/MS spectrum: CCMSLIB00009918873.

(-)-(R,R)-11,13-di-O-methyl-trans-δ-viniferin (3a): heptane:EtOH 80:20 + 0.1% DEA, t _R 14 min [α]²⁵ _D −15.4 (c 0.1, MeCN). ECD (c 20 µM, MeCN) λmax ([θ]) 215 (+23454), 233 (−15588), 275 (−6800), 297 (−4917), 327 (+8434) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 3.

(+)-(S,S)-11,13-di-O-methyl-trans-δ-viniferin (3b): heptane:EtOH 80:20 + 0.1% DEA, t _R 38 min [α]²⁵ _D 13.8 (c 0.1, MeCN). ECD (c 18 µM, MeCN) λmax ([θ]) 214 (−27117), 233 (+10076), 274 (+5953), 298 (+4928), 333 (−7742) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 3.

11,13,11′,13′-tetra-O-methyl-trans-δ-viniferin (4): [α]²⁵ _D -0.3 (c 0.1, MeCN); UV (MeCN) λmax (log ε) 227 (sh) (4.56), 285 (4.17), 311 (4.41), 333 (4.37), 350 (sh) (4.12) nm. ¹H NMR (DMSO-d ₆, 600 MHz) δ 3.70 (6H, s, CH₃O-11′OMe, CH₃O-13′), 3.74 (6H, s, CH₃O-11, CH₃O-13), 4.61 (1H, d, J = 8.5 Hz, H-8′), 5.59 (1H, d, J = 8.5 Hz, H-7′), 6.35 (1H, t, J = 2.3 Hz, H-12), 6.38 (2H, d, J = 2.3 Hz, H-10′, H-14′), 6.43 (1H, t, J = 2.3 Hz, H-12′), 6.72 (2H, d, J = 2.3 Hz, H-10, H-14), 6.76 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.92 (1H, d, J = 8.3 Hz, H-5), 6.95 (1H, d, J = 16.4 Hz, H-8), 7.21 (4H, m, H-2, H-2′, H-6′, H-7), 7.45 (1H, dd, J = 8.3, 1.9 Hz, H-6), 9.52 (1H, s, 4′OH). HR-ESI/MS analysis: m/z 509.1968 [M-H]^-, (calcd for C₃₂H₂₉O₆, 509.1964, ∆ = 0.8 ppm). MS/MS spectrum: CCMSLIB00009918877.

(-)-(R,R)-11,13,11′,13′-tetra-O-methyl-trans-δ-viniferin (4a): heptane:EtOH 90:10 + 0.1% DEA, t _R 23 min [α]²⁵ _D −23.3 (c 0.1, MeCN). ECD (c 18 µM, MeCN) λmax ([θ]) 215 (+33824), 232 (−25600), 275 (−6522), 293 (−6686), 322 (+10724) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 4.

(+)-(S,S)- 11, 13, 11′,13′-tetra-O-methyl-trans-δ-viniferin (4b): heptane:EtOH 90:10 + 0.1% DEA, t _R 36 min [α]²⁵ _D +19.9 (c 0.1, MeCN). ECD (c 18 µM, MeCN) λmax ([θ]) 217 (−31291), 235 (+18807), 274 (+4380), 292 (+5152), 323 (−8648) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 4.

Pallidol (5): UV (MeCN) λmax (log ε) 228 (sh) (4.29), 283 (3.58) nm. ¹H NMR (DMSO-d ₆, 600 MHz) δ 3.60 (2H, s, H-8, H-8′), 4.30 (2H, s, H-7, H-7′), 6.05 (2H, d, J = 2.0 Hz, H-12, H-12′), 6.38 (2H, d, J = 2.0 Hz, H-10, H-10′), 6.60 (4H, d, J = 8.7 Hz, H-2, H-2′, H-6, H-6′), 6.84 (4H, d, J = 8.7 Hz, H-3, H-3′, H-5, H-5′), 8.88 (2H, s, 13′OH, 13OH), 9.02 (2H, s, 11′OH, 11OH), 9.05 (2H, s, 4′OH, 4OH). HR-ESI/MS analysis: m/z 453.1346 [M-H]^-, (calcd for C₂₈H₂₁O₆, 453.1338, ∆ = 1.7 ppm). MS/MS spectrum: CCMSLIB00009918856.

(7S,8S,7′S,8′S)-pallidol (5a): heptane:EtOH 82:18 + 0.1% DEA, t _R 14 min. ECD (c 50 µM, MeCN) λmax ([θ]) 204 (+106533), 219 (−22928), 237 (77036), 289 (−7203) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 5.

(7R,8R,7′R,8′R)-pallidol (5b): heptane:EtOH 82:18 + 0.1% DEA, t _R 18 min. ECD (c 50 µM, MeCN) λmax ([θ]) 202 (−105030), 214 (25694), 236 (−75882), 288 6358) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 5.

Threo-resveratrol acyclic dimer (6): UV (MeCN) λmax (log ε) 228 (sh) (4.25), 285 (4.00), 306 (4.14), 323 (4.15), 340 (sh) (3.85) nm. ¹H NMR (DMSO-d ₆, 600 MHz) δ 4.66 (1H, d, J = 6.5 Hz, H-7′), 5.00 (1H, d, J = 6.5 Hz, H-8′), 5.41 (1H, s, 7′OH), 5.97 (1H, t, J = 2.2 Hz, H-12′), 6.03 (2H, d, J = 2.2 Hz, H-10′, H-14′), 6.10 (1H, t, J = 2.2 Hz, H-12), 6.35 (2H, d, J = 2.2 Hz, H-10, H-14), 6.58 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.81 (1H, d, J = 16.2 Hz, H-8), 6.83 (2H, d, J = 8.8 Hz, H-3, H-5), 6.89 (1H, d, J = 16.2 Hz, H-7), 6.99 (2H, d, J = 8.6 Hz, H-2′, H-6′), 7.38 (2H, d, J = 8.8 Hz, H-2, H-6). HR-ESI/MS analysis: m/z 471.1451 [M-H]^-, (calcd for C₂₈H₂₃O₇, 471.1444, ∆ = 1.5 ppm). MS/MS spectrum: CCMSLIB00009918857.

(7′R,8′R)-threo-resveratrol acyclic dimer (6a): heptane:EtOH 70:30 + 0.1% DEA, t _R 13 min. ECD (c 39 µM, MeCN) λmax ([θ]) 201 (31309), 230 (20861), 315 (−4865) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 6.

(7′S,8′S)-threo-resveratrol acyclic dimer (6b): heptane:EtOH 70:30 + 0.1% DEA, t _R 19 min. ECD (c 39 µM, MeCN) λmax ([θ]) 202 (−28697), 230 (−18357), 316 (5003) nm. UV (MeCN) and ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 6.

7-O-isopropyl-11′,13′-di-O-methylleachianol G (7): UV (MeCN) λmax (log ε) 228 (sh) (4.42), 283 (3.75), 323 (3.44) nm. ¹H NMR (DMSO-d ₆, 600 MHz) δ 0.65 (3H, d, J = 6.1 Hz, CH₃-7c), 0.85 (3H, d, J = 6.1 Hz, CH₃-7b), 3.07 (1H, dd, J = 8.5, 2.4 Hz, H-8), 3.11 (1H, hept, J = 6.1 Hz, 7H-a), 3.43 (1H, t, J = 2.7 Hz, H-8′), 3.66 (6H, s, CH3O-11′, CH3O-13′), 4.02 (1H, d, J = 8.5 Hz, H-7), 5.31 (1H, d, J = 2.1 Hz, H-14), 6.08 (1H, d, J = 2.1 Hz, H-12), 6.11 (2H, d, J = 2.3 Hz, H-10′, H-14′), 6.30 (1H, t, J = 2.3 Hz, H-12′), 6.63 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.66 (2H, d, J = 8.4 Hz, H-3, H-5), 6.80 (2H, d, J = 8.5 Hz, H-2′, H-6′), 6.90 (2H, d, J = 8.4 Hz, H-2, H-6). HR-ESI/MS analysis: m/z 541.2222 [M-H]⁻, (calcd for C₃₃H₃₃O₇, 541.2226, ∆ = 0.7 ppm). MS/MS spectrum: CCMSLIB00009918869.

(7S,8R,7′R,8′S)-O-isopropyl-11′,13′-di-O-methylleachianol G (7a): heptane:EtOH 83:17 + 0.1% DEA, t _R 8 min. UV (MeCN) λmax (log ε) 228 (sh) (4.49), 283 (3.78) nm. ECD (c 25 µM, MeCN) λmax ([θ]) 228 (17470), 238 (28892), 288 (−5405) nm. ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 7.

(7R,8S,7′S,8′R)-O-isopropyl-11′,13′-di-O-methylleachianol G (7b): heptane:EtOH 83:17 + 0.1% DEA, t _R 9 min. UV (MeCN) λmax (log ε) 228 (sh) (4.49), 283 (3.78) nm. ECD (c 28 µM, MeCN) λmax ([θ]) 230 (−15943), 239 (−22974), 288 (4648) nm. ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 7.

11.11′,13.13′-tetra-O-methylrestrytisol B (8): UV (MeCN) λmax (log ε) 228 (sh) (4.43), 283 (3.88), 323 (3.84) nm. ¹H NMR (DMSO-d ₆, 600 MHz) δ 3.53 (6H, s, CH₃O-11′OMe, CH₃O-13′), 3.57 (1H, t, J = 10.5 Hz, H-8), 3.64 (6H, s, CH₃O-11, CH₃O-13), 4.12 (1H, overlapped, H-8′), 4.99 (1H, d, J = 10.0 Hz, H-7), 5.43 (1H, d, J = 9.0 Hz, H-7′), 6.04 (1H, t, J = 2.2 Hz, H-12′), 6.10 (2H, d, J = 2.2 Hz, H-10′, H-14′), 6.25 (1H, t, J = 2.2 Hz, H-12), 6.42 (2H, d, J = 2.2 Hz, H-10, H-14), 6.52 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.73 (2H, d, J = 8.5 Hz, H-3, H-5), 6.96 (2H, d, J = 8.5 Hz, H-2′, H-6′), 7.25 (2H, d, J = 8.5 Hz, H-2, H-6). HR-ESI/MS analysis: m/z 527.2075 [M-H]^-, (calcd for C₃₂H₃₁O₇, 527.2070, ∆ = 0.9 ppm). MS/MS spectrum: CCMSLIB00009918870.

(7S,8R,7′R,8′R)-11.11′,13.13′-tetra-O-methylrestrytisol B (8a): heptane:EtOH 81:19 + 0.1% DEA, t _R 14 min. UV (MeCN) λmax (log ε) 228 (sh) (4.43), 283 (3.64) nm. ECD (c 15 µM, MeCN) λmax ([θ]) 207 (−32706), 218 (−87507) nm. ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 8.

(7R,8S,7′S,8′S)- 11.11′,13.13′-tetra-O-methylrestrytisol B (8b): heptane:EtOH 81:19 + 0.1% DEA, t _R 18 min. UV (MeCN) λmax (log ε) 228 (sh) (4.43), 283 (3.64) nm. ECD (c 15 µM, MeCN) λmax ([θ]) 208 (37443), 217 (94041) nm. ¹H NMR (DMSO-d ₆, 600 MHz): identical to compound 8.

Staphylococcus aureus strains Newman (MSSA) and COL (MRSA) was grown in Muller-Hinton broth (MHB) for 7 h at 37°C with agitation. Microtiter plate wells containing two-fold dilutions of the test compounds in MHB medium were inoculated with approximately 10⁵ bacteria/well. After static incubation at 37°C for 16 h, MICs were determined as the first well without visible growth.