Generation of Antifungal Stilbenes Using the Enzymatic Secretome of

Mar 23, 2017 - Resveratrol can be considered as a good model substrate for checking the efficiency of the enzymatic reaction with B. cinerea secretome...
15 downloads 11 Views 10MB Size
Download PDF
Article pubs.acs.org/jnp

Generation of Antifungal Stilbenes Using the Enzymatic Secretome of Botrytis cinerea Katia Gindro,†,∥ Sylvain Schnee,†,∥ Davide Righi,‡ Laurence Marcourt,‡ Samad Nejad Ebrahimi,§ Josep Massana Codina,† Francine Voinesco,† Emilie Michellod,† Jean-Luc Wolfender,‡ and Emerson Ferreira Queiroz*,‡ †

Agroscope, Domaine de Recherche Protection des Végétaux, Route de Duillier 50, P.O. Box 1012, 1260 Nyon, Switzerland School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU, 1, Rue Michel Servet, 1211 Geneva 4, Switzerland § Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, ShahidBeheshti University, G. C., Evin, Tehran, Iran ‡

S Supporting Information *

ABSTRACT: The protein secretome of Botrytis cinerea was used to perform the biotransformation of resveratrol, pterostilbene, and a mixture of both. Metabolite profiling by UHPLC-HRMS revealed the presence of compounds with unusual molecular formula, suggesting the existence of new products. To isolate these products, the reactions were scaledup, and 21 analogues were isolated and fully characterized by NMR and HRESIMS analyses. The reaction with pterostilbene afforded five new compounds, while the reaction with a mixture of pterostilbene and resveratrol afforded seven unusual stilbene dimers. The antifungal properties of these compounds were evaluated using in vitro bioassays against Plasmopara viticola. The cytological effects of the isolated antifungal compounds on the ultrastructure of P. viticola were also evaluated.

F

worldwide and is considered one of the most important fungal diseases. B. cinerea has developed enzymatic mechanisms leading to the penetration of germinating tubes through the first structural and chemical barriers of plants, its further development through host plant tissues and the detoxification of host defense compounds. This implies a large panel of hydrolytic exoenzymes with cell-wall-degrading properties, including, for example, esterases such as cutinases,4,9 lipases,10 pectolytic and cellulolytic enzymes,11,12 polygalacturonases,13 pectinemethylesterases, glucosaminidases, phospholipases,14 proteases,15 peroxidases, catalases capable of breaking down hydrogen peroxide,16 and laccases.17 Stilbenic phytoalexins are key defense molecules implicated in the resistance of grapevine cultivars to the three major fungal pathogens, B. cinerea (gray mold),18,19 Plasmopara viticola (downy mildew),20 and Erysiphe necator (powdery mildew).21 Grapevine-derived stilbenes are either induced phytoalexins in green organs or constitutively present in lignified canes22−24 as hydroxylated, methylated, esterified, glycosylated, or phenylated monomers or as polymers. Previous authors25,26 have shown that a specific laccase of 32 kDa constitutive of the proteome of B. cinerea was part of the detoxication process of the highly

ungi are heterotrophic organisms that colonize all ecological niches, obtaining carbon-containing nutrients as preformed organic compounds from various substrates according to their lifestyles, which range from symbiotic to necrogenous pathogenic.1 In the case of phytopathogenic fungi, a set of diverse and complementary extracellular proteins are synthesized to permit host penetration and further successful colonization. The group of secreted proteins released in the extracellular space is defined as the secretome and constitutes the pathogenicity factors in the host−pathogen interaction mechanisms.2,3 Host tissues are solubilized by releasing macerating enzymes from fungal propagules as spores and hyphae,4,5 occasioning lesion formation followed by fungal development. As an example, species from the Botrytis genus are recognized as the most ubiquitous saprophytes, developing on a wide range of plant species. In addition, Botrytis spp., and particularly Botrytis cinerea, are defined as necrogenous saprophytes6 and are considered as the main plant pathogens on pre- and post-harvest crops.7 During the infection process, this fungus invades plant tissues, first using senescent or dead floral parts (such as dehiscent stamens, old leaves and injured fruits) as nutrient bases for germination, enzyme excretion and penetration in healthy tissues.8 Grapevine and especially mature berries are the target of choice of B. cinerea, which is known to provoke the gray mold (also called “bunch rot”) of grapes © 2017 American Chemical Society and American Society of Pharmacognosy

Received: August 17, 2016 Published: March 23, 2017 887

DOI: 10.1021/acs.jnatprod.6b00760 J. Nat. Prod. 2017, 80, 887−898

Journal of Natural Products

Article

Figure 1. Compounds isolated from the biotransformation of resveratrol.

toxic pterostilbene produced by grapevines. Specific stilbene oxidase activities have an additional role in the oxidative dimerization of resveratrol into δ-viniferin (resveratrol dehydrodimer), which is more toxic than resveratrol itself. Because of evolutionary confrontation and adaptation, phytopathogens have developed specific proteins to counteract defense host metabolites or to activate the plant response. In particular, necrotrophic and necrogenous saprophytic fungal pathogens can exploit the dead tissues generated after enzymatic maceration. The richness and complexity of the protein secretome of B. cinerea explains its important adaptability and plasticity in the plant infection process.2 The investigation of the secretomes of various pathogens is thus the key to understanding important pathogenicity steps in the plant infection process.27−29 The exploitation of natural products (NPs) is of great interest in order to find new antifungal lead compounds for agriculture (e.g., strobilurines is a fungus metabolite),30 especially to reduce the use and the impact on human health of commercial plant protection fungicides. Plants produce a vast variety of NPs with an incredible structural diversity, some of which confer selective advantages against microbial attack.31 The exploitation of NPs of plant origin as a source of antifungal compounds to treat crop diseases could be considered an important alternative because plant extracts are shown to contain a wide variety of antifungal compounds.32−34 Bioactive NPs have also been obtained using biological methods such as biotransformation reactions. Biotransformation can be defined as the use of an intact whole organism or an isolated enzyme system to induce chemical modifications in organic compounds.35 Biotransformation has a number of advantages when compared to classical organic chemistry. The reactions can occur in mild conditions, near neutral pH, ambient temperatures, and atmospheric pressure, and protection of certain functional groups is often not necessary.36 The Botrytis species has been used for chemical transformations of a large number of NPs, such as steroids, sesquiterpenoids, monoterpenoids, flavonoids, stilbenoids, and phytoalexins.37

The classic method for performing a biotransformation using a given microorganism is by adding the compound to be transformed to the culture medium containing the microorganism.35 Even though this approach has been used successfully, the purification process at the end of the reaction can be extremely complicated because the targeted metabolites are mixed with the microorganism metabolites. Moreover, the compounds to be processed must have low toxicity against the microorganism used, otherwise the reaction cannot be achieved. Another way to perform the biotransformation is to use a purified enzyme. Enzymatic biostransformation has become an important alternative to traditional organic synthesis.38 Because of the attractive chemo-, regio- and enantioselectivities of the reactions commonly displayed by enzymes, this methodology has also been successfully used in the biotransformation of NPs. A series of natural product biotransformations have been performed by using the pure horseradish peroxidase enzyme (HRP).39 HRP is one of the less specific peroxidases and for this reason, it catalyzes a great number of different reactions with several types of substrates. HPR is capable of performing a series of reactions, such as nitration,40 sulfoxidation,41 hydroxylation42 and oxidation.43 The bottleneck in this case is the limited number of commercially available purified enzymes that can be used for those biotransformations. In the present study, the crude enzymatic extract of Botrytis cinerea was used for the biotransformation of resveratrol, pterostilbene and the combination of both compounds to generate original stilbene analogues. Among the 21 compounds obtained, 11 compounds are new (8-12, 16−21), and some presented strong antifungal activities against Plasmopara viticola.

■

RESULTS AND DISCUSSION To establish the best conditions for biotransformation with the B. cinerea secretome, trans-resveratrol (3,5,4′-trihydroxystilbene) was used as substrate. Resveratrol can be considered as a good model substrate for checking the efficiency of the 888

DOI: 10.1021/acs.jnatprod.6b00760 J. Nat. Prod. 2017, 80, 887−898

Journal of Natural Products

Article

Figure 2. Compounds isolated from the biotransformation of pterostilbene.

enzymatic reaction with B. cinerea secretome as it is known to form different oxidized metabolites after incubation with B. cinerea44 and is transformed to δ-viniferin by purified laccaselike stilbene oxidase, also from B. cinerea.25 The secretome of B. cinerea was obtained according to a modified procedure17 and used for the biotransformation of resveratrol. Resveratrol was incubated with the secretome and the reaction monitored by HPLC-UV until complete transformation of the starting material; after 5 h, the resveratrol was no longer detectable. The resulting crude reaction mixture was profiled by UHPLC-high resolution mass spectrometry (HRESIMS) in negative (NI) electrospray ionization (ESI) modes (Figure S1, Supporting Information). A comparison between the photodiode array (PDA) and MS data was obtained, and those compounds that were described in the Dictionary of Natural Products database45 from previous isolations from the Vitis genus were identified. The formation of dimeric stilbene analogues was suspected from the mass range observed for the various peaks occurring after biotransformation ([M − H]−: m/ z 347−471). To isolate these compounds for identification and characterization of their biological activity, the reaction was scaled up and 100 mg of resveratrol was used for the biotransformation. UHPLC-TOF-HRMS was used to monitor the reaction. The crude reaction mixture of resveratrol was fractionated by semipreparative HPLC-UV and seven compounds were isolated and identified by comparison with the literature as leachianol G (1),46 leachianol F (2),46 restrysol B (3),44 3β-(3′,5′dihydroxyphenyl)-2α-(4″-hydroxyphenyl)dihydrobenzofuran5-carbaldehyde (4),47 trans-δ-viniferin (5),48 cis-δ-viniferin (6),48 and 4-hydroxybenzaldehyde (7) (Figure 1). The difference between leachianol G (1),46 and leachianol F (2),46 was established from the NMR data and by the elucidation of their absolute configuration using electronic circular dichroism (ECD) spectroscopy. The ECD spectrum of 1 was measured and compared to the calculated timedependent density functional theory (TDDFT) ECD spectrum. The ECD spectrum of 1 showed a sequential positive Cotton effect (CE) around 243, 207, and 191 nm along with threes negative CE around 255, 226, and 200 nm, whereas compound 2 showed a different ECD spectrum with several positive CEs

around 267, 243, and 207 nm along with two weak CEs at 207 and 199 nm. These effects are due to π→π* transition of aromatic moieties. Based on NMR data of 1 and 2, the NOESY correlations from H-7′ and H-8 to H-10′/H-14′ and from H-8′ to H-2′/H6′ indicated that the relative configuration of C-7′/C-8′/C-8 was trans−trans. Thus, the difference between 1 and 2, should be the configuration at C-7. The comparison of the TDDFT calculation of ECD spectra for four possible stereoisomers (7′S,8′R,7S,8S, 7′S,8′R,7R,8S, 7′R,8′S,7S,8R, and 7′R,8′S,7R,8R) (Figure S2, Supporting Information) with the experimental data of 1 and 2, indicated that the most probable configuration of leachianol F (2) was 7′S,8′,R,7R,8S and leachianol G (1) was 7′S,8′R,7S,8S. The absolute configuration of leachianol F and G was thus established here for the first time. When pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) was incubated with the Botrytis secretome,49 UHPLC-TOFHRMS analysis showed the presence of a series of molecular ions, suggesting the presence of pterostilbene dimers with the following molecular formulas: C32H30O6 (2 peaks), C32H32O7 (4 peaks), and C32H32O9 (1 peak). As for resveratrol, pterostilbene was then biotransformed on a large scale (100 mg). Compounds generated were purified by reverse-phase flash chromatography.50,51 Two known compounds were isolated: β-[4-[(1E)-2-(3,5-dimethoxyphenyl)ethenyl]phenoxy]-α-(4-hydroxyphenyl)-3,5-dimethoxybenzeneethanol (13)52 and the pterostilbene trans-dehydromer (14).52 In addition to the known compounds, the purification also afforded five new compounds 8−12, described below (Figure 2). Compound 8 was isolated as an amorphous solid. The HRESIMS data suggested a formula of C32H31O9. The 1H NMR spectrum presented 14 aromatic protons, distributed according to the COSY spectrum in four aromatic rings, corresponding to the fusion of two pterostilbene units: two 4hydroxyphenyl groups and two 3,5-dihydroxymethylphenyl groups. The 1H NMR and COSY data showed that the ethylenic protons of pterostilbene were replaced by three methine protons at δ 4.61 (1H, d, J = 2.7 Hz, H-7), 4.59 (1H, d, J = 12.0 Hz, H-8′), and 3.38 (1H, dd, J = 12.0, 2.7 Hz, H-8). 889

DOI: 10.1021/acs.jnatprod.6b00760 J. Nat. Prod. 2017, 80, 887−898

Journal of Natural Products

Article

Figure 3. Compounds isolated from the biotransformation of resveratrol/pterostilbene.

Based on the coupling constants, a cis/trans configuration was expected for the C-7/C-8/C-8′ chain. The HMBC correlations between H-7 and the aromatic carbons at δ 127.9 (C-2, C-6) suggested the attachment of one of the 4-hydroxyphenyl moieties to the oxygenated carbon at δ 72.5 (C-7). The correlations from H-8 to the aromatic carbons at δ 109.6 (C-10, C-14) and from H-8′ to the aromatic carbons at δ 107.8 (C10′/C-14′) allowed the determination of the positions of the two trisubstituted aromatic rings: the first one at C-8 (δ 58.3) and the second at C-8′ (δ 55.4). The HMBC spectrum showed a correlation between the proton H-8′ and the carbonyl at δ 173.7 (C-7′), suggesting the presence of an ester moiety. The remaining hydroxybenzoyls were at this ester group by the chemical shift of C-1′ (δC 144.3), which could be an oxygenated aromatic carbon and by the lack of correlation between H-2′/H-6′ at δ 6.29 (2H, d, J = 8.9 Hz) and C-7′. The ECD spectrum of 8 presented two positive CEs around 243, 206 nm, and a negative CE at 199 nm (Figure S2, Supporting Information). The TDDFT calculated ECD spectrum for 8′R,7S,8S showed the most closely related pattern with experimental data where the other isomer showed an opposite sign. Therefore, the absolute configuration of 8 was determined as 8′R,7S,8S. Based on the HRMS and NMR data, 8 was established to be a new dimeric form of pterostilbene, named pterodimer A (Figure 2). The HRESIMS spectrum of 9 showed a molecular ion at m/z 527.2069 (ca. for C32H31O7, 527.2070, Δ = −0.1 ppm). The NMR data of 9 was closely related to the leachianol F (2), previously described from Sophora leachiana,46 except by the presence of four methoxy groups at δ 3.55 (3H, s, OCH3-11), 3.60 (6H, s, OCH3-11′, OCH3-13′), and 3.74 (3H, s, OCH313). The NOESY correlations observed from H-7′ (δH 4.11, 1H, d, J = 4.0 Hz) and H-8 (δH 3.22, 1H, dd, J = 8.0, 4.4 Hz) to H-10′/H-14′ (δH 5.83, 2H, d, J = 2.3 Hz) and from H-8′ (δH 2.88, 1H, dd, J = 4.4, 4.0 Hz) to H-2′/H-6′ (δH 6.66, 2H, d, J =

8.6 Hz) indicated that the relative orientation at the C-5 ring is the same as that of leachianol F (2) and G(1). The experimental ECD spectrum of 9 showed three positive CEs around 241, 208, and 198 nm along with two negative CEs at 228 and 191 nm. The TDDFT ECD calculation indicated that the absolute configuration of compound 9 was 7′S,8′R,7S,8S (Figure S2, Supporting Information). Compound 9 was identified as a new tetramethoxylated form of leachianol F (2), named 11, 11′, 13, 13′-tetramethoxyleachianol F. The ESI-HRMS of 10 displayed an [M − H]+ ion at m/z 527.2068, in agreement with the molecular formula C32H32O7. The 1H NMR of 10 demonstrated a close resemblance to that of 9. As for 9, the absolute configuration of 10 was established as 7′S,8′R,7S,8S by comparison of experimental and calculated ECD data (Figure S2, Supporting Information). Compound 10 was identified as the new tetramethoxylated form of leachianol G, named 11, 11′, 13, 13′-tetramethoxyleachianol G. The molecular formula of 11 was determined to be C32H32O7 by ESI-HRMS. The NMR data of 11 is closely related to that of restrytisol B (3), previously described from the dimerization of resveratrol,44 except for the presence of four methoxy groups at δH 3.54 (6H, s, OCH3-11, OCH3-13) and 3.64 (6H, s, OCH311′, OCH3-13′). As for restrytisol B, 11 presented the same relative stereochemistry as that determined by the NOESY correlations from H-7 to H-7′ and H-8, from H-7′ to H-8 and from H-8′ to H-2′/-6′. Compound 11 was identified as the new tetramethoxylated form of restrytisol B, named 11,11′,13,13′tetramethoxyrestrytisol B. Compound 12 was isolated as an amorphous powder. The ESI-HRMS displayed an [M − H]− ion at m/z 509.1984, in agreement with the molecular formula C32H29O6. The NMR data of 12 are closely related to those of pallidol (15), previously described from Cissus pallida,53 except for the presence of four methoxy groups at δH 3.60 (6H, s, OCH3-11, OCH3-11′) and 3.84 (6H, s, OCH3-13, OCH3-13′). The 890

DOI: 10.1021/acs.jnatprod.6b00760 J. Nat. Prod. 2017, 80, 887−898

Journal of Natural Products

Article

relative configuration of 18, which was different from that of pallidol (Figure S3, Supporting Information). Compound 18 was thus identified as the new dimethoxylated form of pallidol, named 11, 13-dimethoxyisopallidol. Compound 19 was isolated as an amorphous solid. The HRESIMS spectrum showed a molecular ion at m/z 527.2070 [M − H]−, (ca. for C32H31O7, 527.2070, Δ = 0.0 ppm). The 1H NMR spectrum presented 14 aromatic protons, distributed according to the COSY spectrum into four aromatic rings, corresponding to the fusion of two pterostilbene units. The presence of four methoxy groups at δ 3.53 (6H, s, CH3-11OMe, 13OMe) and 3.60 (6H, s, CH3-11′OMe, 13′OMe) confirm this hypothesis. The 1H NMR and COSY spectra showed the sequence of four methine protons at δ 3.74 (1H, dd, J = 7.5, 3.2 Hz, H-8′), 4.09 (1H, d, J = 11.8 Hz, H-7), 4.64 (1H, dd, J = 11.8, 7.5 Hz, H-8) and 9.26 (1H, d, J = 3.2 Hz, H-7′). This latter corresponded to an aldehyde, as indicated by the HSQC correlation at δC 198.1. The HMBC correlations from H-7 to the aromatic carbons at δ 128.6 (C-2/C-6) and 129.5 (C-2′/C6′), from H-8 to the aromatic carbons at δ 108.2 (C-10/C-14) and from H-8′ to the aromatic carbons at δ 107.9 (C-10′/C14′) allowed the positioning of all the aromatic rings. On the basis of the HRMS and NMR data, 19 was established as a new dimeric form of pterostilbene, named pterodimer B. Compound 20 showed a molecular ion at m/z 481.1629 [M − H]− (for C30H25O6, ca. 481.1651, Δ = −4.5 ppm) by HRESIMS. The NMR data were very similar to those of transδ-viniferin, except for the presence of two methoxy groups at δH 3.70 (6H, s, OCH3-3′, 5′). The HMBC correlations from H-2′/ H-6′ (δH 6.37, 4H, m, H-2, 6, 2′, 6′) to C-4′ (δC 98.5) and C-7′ (δC 55.7) and the NOESY correlations from H-4′ (δH 6.43, 1H, t, J = 2.2 Hz) to OCH3-3′/OCH3-5′ and from H-2′/H-6′ to H8′ (δH 5.56, 1H, d, J = 8.1 Hz) allowed the positioning of the two methoxy groups and the establishment of the relative configuration of 20. Compound 20 was thus identified as the new dimethoxylated form of trans-δ-viniferin, named 3′,5′dimethoxy-trans-δ-viniferin. The NMR data of 21 was closely related to that of 20, except for the position of the two methoxy groups at δH 3.75 (6H, s, OCH3-3, 5). Compound 21 was thus identified as the new dimethoxylated form of trans-δ-viniferin, named 3,5-dimethoxy-trans-δ-viniferin. As presented in Figures 1−3, the biotransformation of resveratrol, pterostilbene and the mixture of both afforded different dimeric compounds. As expected, the use of the secretome of Botrytis was able to generate a wide diversity of stilbene analogues. All compounds obtained by the biotransformation of pterostilbene and the mixture of resveratrol/pterosilbene were obtained by dimerization of the stilbene units by coupling of phenoxyl radicals generated by oxidation.55,56 This oxidations are most probably performed by laccase or peroxidases produced by B. cinerea. The dimerization occurs in three regioisomeric modes: 3−8′, 8−8′ and 8−4′ coupling.56 The 3− 8′ coupling originate compounds 5, 6, 14, 20, and 21, while the 8−8′ coupling afforded compounds 1−3, 8−12, 15−17, and 19. Finally, the 8−4′ coupling originate compound 13. In the case of the new compounds 8 and 19, the authors proposed their mechanism of synthesis (Figure S3, Supporting Information). It was possible to identify seven types of products (types A to B and D to H), some of which have already been described,52 while types G and H represent new patterns of dimeric stilbenes generated specifically by the enzymatic pool of Botrytis

overlapping of NMR signals belonging to each dimer indicated a symmetrical structure. Thus, the relative configuration of 12 was the same as that of pallidol. Compound 12 was identified as the new tetramethoxylated form of pallidol, named 11, 11′, 13, 13′-tetramethoxypallidol. To further check the type of chemodiversity that can be generated from simple stilbenes by the Botrytis secretome, a mixture of resveratrol and pterostilbene was biotransformed using the same approach. Based on the retention time and HRMS information on the previously described reactions, it was possible to identify restrysol B (3), trans-δ-viniferin (5), and pterostilbene trans-dehydromer (14), as previously described (Figure S1, Supporting Information). However, the UHPLC-TOF-HRMS metabolite profiling revealed the presence of additional molecular ions with m/z 499.1819 [M − H]− and 499.1766 [M − H]−, corresponding to the presence of new dimers potentially resulting from the fusion of resveratrol and pterostilbene (Figure S1, Supporting Information). The reaction was scaled up (100 mg of each compound) and the crude reaction mixture was separated by medium pressure liquid chromatography (MPLC) after direct chromatography transfer.54 This provided, in one step, six known compounds: leachianol G (1), leachianol F (2), restrytisol B (3), trans-δviniferin (5), pterostilbene trans-dehydromer (14), and pallidol (15),53 and the seven new products described below (Figure 3). Compound 16 has been isolated as a mixture (16a and 16b). The HRESIMS spectrum of compounds 16a/16b showed a molecular ion at m/z 499.1751. This molecular formula suggest the fusion of resveratrol with pterostilbene. The NMR data of 16a/16b were most closely related to leachianol G/F (1/2) or compound 9/10, except for the presence of two methoxy groups at δ 3.59 (6H, s, OCH3-11′, OCH3-13′) for 16a and 3.65 (6H, s, OCH3-11′, OCH3-13′) for 16b. The relative stereochemistry of 16a was determined to be the same as that of leachianol F (2) or compound 9, whereas 16b had the same configuration as that of leachianol G (2) or 10. Compounds 16a/16b were identified as the new dimethoxylated forms of leachianol F/G, named resvepterol A (16a) and resvepterol B (16b). The molecular formula of 17 was determined by ESI-HRMS to be C30H28O7. The NMR data of 17 was close to that of restrytisol B (3) or 11, except for the presence of two methoxy groups at δ 3.64 (6H, s, OCH3-11′, OCH3-13′). The NOESY correlations from H-10′/H-14′ (δH 6.34, 2H, d, J = 2.3 Hz) to OCH3-11′/OCH3-13′, H-7′ (δH 4.93, 1H, d, J = 9.8 Hz) and H-8 (δH 3.94, 1H, dd, J = 9.8, 9.0 Hz) and from H-7′ and H-7 (δH 5.38, 1H, d, J = 9.0 Hz) allowed the positioning of the methoxy groups and the establishment of the relative configuration as that of restrytisol B (3) or 11. Compound 17 was identified as a new dimethoxylated form of restrytisol B, named 11′,13′-dimethoxyrestrytisol B. The ESI-HRMS of 18 displayed an [M − H]− ion at m/z 481.1666, which is in agreement with the molecular formula C30H26O6. The NMR data of 18 is closely related to that of pallidol (15), except by the presence of two methoxy groups at δH 3.57 (3H, s, OCH3-11) and 3.80 (3H, s, OCH3-13). The NOESY correlations from H-14 (δH 6.75, 1H, d, J = 2.0 Hz) to OCH3-13, from H-12 (δH 6.31, 1H, d, J = 2.0 Hz) to OCH3-11, from H-8 (δH 3.70, 1H, d, J = 6.3 Hz) to H-2′/H-6′ (δH 6.82, 2H, d, J = 8.5 Hz) and from H-8′ (δH 3.64, 1H, d, J = 6.3 Hz) to H-2/H-6 (δH 6.90, 2H, d, J = 8.5 Hz) allowed the positioning of the methoxy group and the establishment of the 891

DOI: 10.1021/acs.jnatprod.6b00760 J. Nat. Prod. 2017, 80, 887−898

Journal of Natural Products

Article

Figure 4. Fungitoxic activitiy on the mobility of Plasmopara viticola zoospores of resveratrol, pterostilbene and compounds obtained after enzymatic biotransformation of each molecule or in combination, at 1 mM and 10 μM according to the availability of each compound. Compound 4: 3β-(3′,5′dihydroxyphenyl)-2α-(4″-hydroxyphenyl)dihydrobenzofuran-5-carbaldehyde. Significant differences are highlighted by different letters on each column (a−j) according to Tukey’s test (P-value