Article pubs.acs.org/JAFC
New Monomeric Stilbenoids from Peanut (Arachis hypogaea) Seeds Challenged by an Aspergillus flavus Strain Victor S. Sobolev,*,† Nicole M. Krausert,§ and James B. Gloer§ †
National Peanut Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 509, Dawson, Georgia 39842, United States § Department of Chemistry, University of Iowa, 230 North Madison Street, Iowa City, Iowa 52242, United States S Supporting Information *
ABSTRACT: Two new stilbene derivatives have been isolated from peanut seeds challenged by an Aspergillus flavus strain, along with chiricanine B, which has not been previously reported from peanuts, as well as a stilbenoid reported previously only as a synthetic product. The structures of these new putative phytoalexins were determined by analysis of 1H and 13C NMR, HRESIMS, MSn, and UV data. The new stilbenoids were named arahypin-13 (21), arahypin-14 (22), and arahypin-15 (23). Together with other known bioactive peanut stilbenoids that were also produced in the challenged seeds, these new compounds may play a defensive role against invasive fungi. KEYWORDS: peanuts, Arachis hypogaea, arahypin, groundnuts, stilbenes, prenylated stilbenes, stilbenoids, aflatoxin, structure elucidation, NMR, HPLC-MS
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0.063−0.200 mm) was purchased from EM Science (Gibbstown, NJ, USA). Reference Compounds. trans-Resveratrol (1) (approximately 99%) was purchased from Sigma-Aldrich (Atlanta, GA, USA). Pure peanut stilbenes [trans-arachidin-1 (2), trans-arachidin-2 (3), transarachidin-3 (4), trans-3′-isopentadienyl-3,5,4′-trihydroxystilbene (5), SB-1 (6), chiricanine A (7), arahypin-1 (9), arahypin-2 (10), arahypin3 (11), arahypin-4 (12), arahypin-5 (13), arahypin-6 (14), and arahypin-7 (15)] were obtained as previously described,8,12−14 with the exception that preparative HPLC was used in the final step instead of preparative TLC. The identities of the reference compounds were confirmed by APCI-MS/MS (MS2) and UV spectroscopy. The identities of known stilbenoids, chiricanine B (8) and arahypin-10 (18), extracted from challenged peanuts, were confirmed by means of 1 H and 13C NMR, HRESIMS, MSn, and UV. These data are given in parentheses as [M + H]+ values followed by UV absorption maxima: trans-resveratrol (1) (m/z 229; 305 and 317 nm), trans-arachidin-1 (2) (m/z 313; 339 nm), trans-arachidin-2 (3) (m/z 297; 308 and 322 nm), trans-arachidin-3 (4) (m/z 297; 335 nm), trans-3′-isopentadienyl3,5,4′-trihydroxystilbene (5) (m/z 295; 295 nm), SB-1 (6) (m/z 345; 363 nm), chiricanine A (7) (m/z 281; 311 nm), chiricanine B (8) (m/ z 297; 234, 301, and 311 nm), arahypin-1 (9) (m/z 281; 328 nm), arahypin-2 (10) (m/z 331; 306 and 317 nm), arahypin-3 (11) (m/z 331; 306 and 320 nm), arahypin-4 (12) (m/z 315; 312sh and 327sh nm), arahypin-5 (13) (m/z 295; 270 and 338 nm), arahypin-6 (14) (m/z 607; 272 and 340 nm), arahypin-7 (15) (m/z 623; 271 and 347 nm), and arahypin-10 (18) (m/z 295; 230, 274, and 315 nm). The above results were in agreement with published data.8,9,12−15 Fungal Culture. Spores of Aspergillus f lavus NRRL 18543 (AF36) were used to elicit phytoalexin production in peanuts. The spores were obtained from PDA slants after 9 days of incubation at 30 °C. Plant Material and Processing. The Tifguard (Reg. No. CV-101, PI 651853), a runner-type peanut (Arachis hypogaea L. subsp. hypogaea
INTRODUCTION Aspergillus flavus, a common soil fungus that often infects peanut seeds, is the major producer of carcinogenic aflatoxins.1 Aflatoxins are an international food safety concern for several undeveloped countries, where aflatoxins are not regulated and where frequent outbreaks of aflatoxicosis with human mortality occur. It was estimated that about 4.5 billion people living in developing countries are chronically exposed to aflatoxins.2 Many countries experience substantial economic losses due to strict aflatoxin regulations.3 Under favorable conditions, peanut seeds can resist fungal attacks by promptly producing an array of stilbene-derived phytoalexins with antifungal properties.4−7 In recent years, several new stilbenoids, the arahypins (9−20) (Figure 1), were isolated from fungus-challenged peanut seeds.8−11 Studies of biological activities of these compounds in a broad spectrum of biological assays have demonstrated that some minor arahypins display more potent biological activities, including antifungal effects, than the major stilbenoids present, such as arachidin-1 (2) and arachidin-3 (4).7 Identification and characterization of new peanut stilbenoids may help to reveal the defensive mechanisms in peanut. A better understanding of these mechanisms is a prerequisite for implementing new strategies for controlling aflatoxins. The purpose of this research was to isolate and characterize further new peanut seed stilbenoids that may serve as defensive phytoalexins and/or possess properties beneficial to human health.
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MATERIALS AND METHODS
Reagents, Materials, and Basic Apparatus. HPLC grade solvents used in the preparation of mobile phases and separations on silica gel were obtained from Fisher (Suwanee, GA, USA). HPLC grade H2O was prepared with a ZD20 four-bowl Milli-Q water system (Millipore). Silica gel for column chromatography (silica gel 60, © 2015 American Chemical Society
Received: Revised: Accepted: Published: 579
September 29, 2015 December 10, 2015 December 16, 2015 December 16, 2015 DOI: 10.1021/acs.jafc.5b04753 J. Agric. Food Chem. 2016, 64, 579−584
Article
Journal of Agricultural and Food Chemistry
Figure 1. Structures of known and new stilbenoids from peanut seeds: trans-resveratrol (1), trans-arachidin-1 (2), trans-arachidin-2 (3), transarachidin-3 (4), trans-3′-isopentadienyl-3,5,4′-trihydroxystilbene (5), SB-1 (6), chiricanine A (7), chiricanine B (8), arahypin-1 (9), arahypin-2 (10), arahypin-3 (11), arahypin-4 (12), arahypin-5 (13), arahypin-6 (14), arahypin-7 (15), arahypin-8 (16), arahypin-9 (17), arahypin-10 (18), arahypin11 (19), arahypin-12 (20), arahypin-13 (21), arahypin-14 (22), arahypin-15 (23), and acetonide of arahypin-4 (24). Extraction and Purification. For preparative isolation of stilbenoids, 1.9 kg of inoculated and incubated peanut seeds was extracted with 7.6 L of MeOH in a high-speed blender for 1 min (600 mL for each portion of 150 g of seeds). The combined mixture was filtered through a glass-fiber filter in a Büchner-type funnel under reduced pressure. The solid residue was resuspended in 2.8 L of MeOH, and the extraction procedure was repeated twice. The combined extract solutions were filtered through a glass-fiber filter and defatted three times with 0.5 L of n-hexane. The MeOH layer was evaporated to dryness. The residue was redissolved in CHCl3 and applied to a chromatographic column (90 mm i.d.) packed with silica gel to the height of 360 mm. The column was subsequently eluted with 1.1 L of CHCl3, 1.0 L of CHCl3/EtOAc (3:1, v/v), 1.2 L of CHCl3/EtOAc (1:1, v/v), 1.0 L of EtOAc, 1.0 L of EtOAc/acetone
var. hypogaea) cultivar (resistant to peanut root-knot nematode and tomato spotted wilt virus), 2013 harvest, from the National Peanut Research Laboratory (Dawson, GA, USA) was used. Peanut seeds were allowed to imbibe distilled water for 18 h at room temperature. They were then chopped with a sharp hand cutter into 3−7 mm pieces, repeatedly washed with distilled sterile water, blotted with a paper towel, air-dried in a laminar airflow workstation (Nuaire, Plymouth, MN, USA) to the condition where sliced peanuts did not leave water spots on filter paper, and placed on aluminum trays so that the thickness of the layer did not exceed 1 cm. The trays were evenly sprayed with the fungal spores (106/mL), placed into autoclave bags, sealed, and incubated at 30 °C for 90 h. The bags were opened every 24 h to allow fresh air to the peanuts and growing fungus. 580
DOI: 10.1021/acs.jafc.5b04753 J. Agric. Food Chem. 2016, 64, 579−584
Article
Journal of Agricultural and Food Chemistry Table 1. 1H NMR Spectroscopic Data for Compounds 21−24 δH (J, Hz) position
arahypin-13 (21)a
2 4 6 7 8 2′ 3′ 4′ 5′ 6′ 1″
6.57 br s
2″ 4″ 5″ 7″ 8″
5.59 d (9.7) 1.43 s 1.43 s
6.45 6.90 7.01 7.45 7.33 7.23 7.33 7.45 6.60
d (1.2) d (16.3) d (16.3) br d (7.2) br t (7.4) br t (7.3) br t (7.4) br d (7.2) d (9.7)
arahypin-14 (22)b
arahypin-15 (23)c
6.54 6.27 6.54 6.89 6.99 7.29 6.71
d (1.9) t (1.9) d (1.9) d (16) d (16) dd (8.3, 1.6) d (8.3)
6.59 d (1.3)
6.63 s
6.48 6.85 6.99 7.40 6.83
d d d d d
7.25 2.74 3.01 3.80 1.24 1.35
br s dd (17, 8.0) dd (17, 5.4) dd (8.0, 5.4) sd sd
6.83 7.40 2.53 2.93 3.77 1.22 1.35
d (8.6) d (8.6) dd (17, 8.0) dd (17, 5.6) m se se
6.63 6.93 7.02 7.47 7.33 7.24 7.33 7.47 2.58 3.12 3.85 1.26 1.30 1.35 1.50
(1.3) (16) (16) (8.6) (8.6)
acetonide of arahypin-4 (24)a
s d (16) d (16) d (7.4) t (7.4) t (7.4) t (7.4) d (7.4) dd (15, 10.5) dd (15, 1.3) dd (10.5, 1.3) sf sf sf sf
a In CDCl3. Spectra recorded at 500 MHz. Assignments with identical superscripts (f) are interchangeable. bIn acetone-d6. Spectra recorded at 600 MHz. Assignments with identical superscripts (d) are interchangeable. cIn acetone-d6. Spectra recorded at 500 MHz. Assignments with identical superscripts (e) are interchangeable.
(3:1), 1.2 L of EtOAc/acetone (1:1), 1.2 L of acetone, and 0.9 L of MeOH. Thirty fractions were collected from the column and analyzed by HPLC. Fractions containing the same compounds of interest were combined, evaporated to dryness on a rotary evaporator, redissolved in CHCl3, and subjected to further purification on smaller chromatographic columns (45 mm i.d.) packed with silica gel to the height of 200−300 mm. Combined original fractions 3 and 4 were subjected to separation on such a column with a hexane/acetone mixture (9:1, v/ v). Twenty-one fractions were collected. Fractions A4−A6 showed the presence of 21, and fractions A17−A21 showed the presence of 8 as the major components. Sufficiently pure compounds 21 and 8 were obtained from these fractions by means of preparative HPLC as described below. Fractions A7−A12 were additionally chromatographed with the same mobile phase to give fractions B1, B2, and B3. Fraction B2 consisted of chiricanine A (7). Separation of combined fractions 13−18 with CHCl3/EtOAc (1:1, v/v) and EtOAc gave fractions C1−C66. The fractions were combined in accordance with the strength of the solvents that were used for their elution. Fractions C37−C66 eluted with CHCl3/acetone (3:2, v/v; 500 mL) and CHCl3/acetone (2:3, v/v; 1.5 L) gave fractions enriched with arahypin-6 (14) and arahypin-7 (15). Fractions C20−C25 eluted with CHCl3/acetone (4:1, v/v; 600 mL) were subjected to further separation on silica gel to yield fractions D1−D24. Combined fractions D10−D11 gave fractions E1−E4 upon additional column chromatography, of which fraction E3 showed the presence of arahypin-10 (18) as a 90−95% pure compound on the basis of peak area percent. Further purification of arahypin-10 (18) was achieved by preparative HPLC as described below. Combined fractions C26−C36 resulted in fractions F1−F11 upon elution from an additional silica gel column with CHCl3/acetone (1:1, v/v; 600 mL). Fractions F3, F4, F5, and F8 contained 22, 23, arahypin-4 (12), and 24, respectively (70−90% pure on the basis of peak area percent). Combined fractions containing partially purified compounds were evaporated to dryness, redissolved in MeOH, and further purified on a preparative HPLC column. Preparative HPLC Separations. Preparative HPLC separations were performed using a 100 mm × 19 mm i.d., 5 μm XTerra Prep RP18 OBD column (Waters, Milford, MA, USA). The column temperature was 40 °C. The following isocratic mobile phases were used: for compound 8, 35% H2O, 64% MeOH, and 0.01% HCOOH in H2O; for all other compounds, 39% H2O, 60% MeOH, and 0.01% HCOOH in H2O. The flow rate was 8.0 mL/min. Pure fractions obtained from HPLC were separately evaporated with a rotary
evaporator to a point at which almost all of the organic solvent was removed. The remaining aqueous mixtures containing the target compounds were separately extracted four times with aqueous EtOAc (1:1, v/v). The combined EtOAc layers were evaporated nearly to dryness with a rotary evaporator. The residue was transferred into a 15 mL vial with MeOH and evaporated to dryness with a stream of N2. The residues were redissolved in EtOAc, filtered, transferred into 4 mL vials, and evaporated to dryness with a stream of N2. Then the vials were placed into a lyophilizer for 4 h at room temperature to remove traces of the solvents. All manipulations with purified compounds were carried out under minimal lighting conditions to avoid any possible photoisomerization of the stilbenoid olefinic double bond. HPLC-DAD-MS Analyses. For tandem HPLC-MS analyses, a Surveyor HPLC system equipped with an MS Pump Plus, an Autosampler Plus, a PDA Plus Detector (Thermo Electron Corp., San Jose, CA, USA) covering the 200−600 nm range, and a 100 mm × 4.6 mm i.d., 3.5 μm, XSelect HSS C18 analytical column (Waters) was used. The column was maintained at 40 °C. The solvents used for the mobile phase were mixed in the following gradient: initial conditions, 59% H2O (A)/40% MeOH (B)/0.01% in H2O (C), changed linearly to 10% A/89% B/1% C in 11 min, changed to 0% A/99% B/1% C in 0.01 min, held isocratic for 4 min, and changed to initial conditions in 0.01 min. The flow rate was 1.2 mL/min. Spectroscopic Measurements. Optical rotations were measured with an Autopol III automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). 1H and 13C NMR experiments were performed on AVANCE 500 or 600 MHz instruments (Bruker, Billerica, MA, USA). HMBC data were obtained on the AVANCE 600. Chemical shift values were referenced to the solvent signals for CDCl3 (δH 7.24/δC 77.2) or acetone-d6 (δH 2.05/δC 29.8). 1H and 13C NMR data for compounds 21−24 are given in Tables 1 and 2. HRESIMS data were recorded using a Q-TOF Premier instrument (Waters) by direct infusion in negative ion mode. APCI-MSn data were obtained on a Finnigan LCQ Advantage MAX ion trap mass spectrometer equipped with an APCI interface and operated with Xcalibur version 1.4 software (Thermo Fisher Scientific, San Jose, CA, USA). All data were acquired in the full-scan positive polarity mode from m/z 100 to 1000. Capillary temperature was 175 °C, APCI vaporizer temperature 400 °C, sheath gas flow 60 units, auxiliary/sweep gas flow 10 units, source voltage 4.5 kV, and source current 5 μA. In MS2 analyses, the [M + H]+ ions observed for each chromatographic peak in full-scan analyses were isolated and subjected to source collision-induced 581
DOI: 10.1021/acs.jafc.5b04753 J. Agric. Food Chem. 2016, 64, 579−584
Article
Journal of Agricultural and Food Chemistry
MTPA ester, δ 3.10 (dd, J = 18, 4.6 Hz, Ha-1″), 3.31 (dd, J = 18, 4.6 Hz, Hb-1″), 1.33 (s, H-4″), 1.31 (s, H-5″). On the basis of these data, the absolute configuration at C-2″ was assigned as S in both cases. Chiricanine B (8): yellowish oil; UV (in mobile phase at tR 8.8 min); λmax (nm) 234, 301, 311, 326sh; [α]D 0 (c 0.35; CHCl3); APCI-MS, m/z 297 ([M + H]+; rel int 100); APCI-MS2, m/z 297@33: 279 ([M + H − H2O)]+; rel int 90), 225 (100). Arahypin-13 (21): yellowish oil; UV (in mobile phase at tR 10.4 min); λmax (nm) 233, 267, 334; APCI-MS, m/z 279 ([M + H]+; rel int 100); APCI-MS2, m/z 279@33: 279 (100), 261 (11), 251 (55), 237 (24), 175 (12); APCI-MS3, m/z 279@35 → 251@40: 233 (16), 233 (13), 223 (100), 209 (40), 205 (20), 191 (11), 173 (12), 159 (11), 91 (11). Arahypin-14 (22): yellowish oil; UV (in mobile phase at tR 6.3 min) λmax (nm) 236, 305, 328sh; [α]D +11 (c 0.23; acetone); APCI-MS, m/ z 313 ([M + H]+; rel int 100); APCI-MS2, m/z 313@30: 295 ([M + H − H2O)]+; rel int 100), 241 (20); APCI-MS3, m/z 313@35 → 295@ 38 → 241@38: 223 (rel int 100); negative ion HRESIMS m/z 311.1288, calculated for C19H20O4 − H, 311.1286. Arahypin-15 (23): yellowish oil; UV (in mobile phase at tR 5.8 min) λmax (nm) 229,307, 321; [α]D +38 (c 0.27; acetone); APCI-MS, m/z 313 ([M + H]+; rel int 100); APCI-MS2, m/z 313@33: 295 ([M + H − H2O)]+; rel int 86), 241 (100); negative ion HRESIMS m/z 311.1283, calculated for C19H20O4 − H, 311.1286. Acetonide of arahypin-4 (24): yellowish oil; UV (in mobile phase tR 8.9 min) λmax (nm) 237, 307, 328sh; [α]D 0 (c 0.23 CHCl3); APCIMS, m/z 356 (rel int 25), 355 ([M + H]+), 312 (13), 311 (54), 298 (21), 297 (100), 279 (21); APCI-MS2, m/z 355@35: 297 (rel int 100); APCI-MS3, m/z 355@35 → 297@35: 279 (rel int 27), 225 (100); APCI-MS4, m/z 355@40 → 297@35: 279@40 → 225@40: 225 (rel int 43), 207 (53), 197 (40), 183 (17), 179 (100), 169 (10).
Table 2. 13C NMR Spectroscopic Data for Compounds 21− 24 δ (ppm) position 1 2 3 4 5 6 7 8 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″
arahypin-13 (21)a
arahypin-14 (22)b
arahypin-15 (23)b
acetonide of arahypin-4 (24)a
138.5 106.3 154.3 109.4 151.5 107.5 127.9 128.3 137.3 126.7 128.8 129.2 128.8 126.7 116.4 129.7 76.2 27.9 27.9
140.8 105.8 159.6 102.8 159.6 105.8 127.1 128.9 130.4 126.5 117.8 154.0 121.5 128.9 32.1 69.9 78.2 20.8c 26.3c
138.0 105.1 156.6 108.3 155.1 107.0 128.4 126.6 130.0 128.6 116.4 158.0 116.4 128.6 27.3 70.0 77.4 20.3d 26.1d
137.7 107.2 155.8 112.6 155.8 107.2 127.8 128.2 137.3 126.7 128.8 129.1 128.8 126.7 23.7 84.8 81.0 23.1e 25.9e 107.8 27.2e 28.6e
a In CDCl3. Spectra recorded at 125 MHz. Assignments with identical superscripts (e) are interchangeable. bIn acetone-d6. Spectra recorded at 150 MHz. Assignments with identical superscripts (c, d) are interchangeable.
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RESULTS AND DISCUSSION Structure Elucidation and Possible Role of New Stilbenoids. Several known compounds were isolated, including chiricanine A (7)8,15 and arahypin-10 (18).10 Data for these, as well as other compounds of this class, were used to aid in structure assignment of the newly described stilbenes. Compound 8 was found to have a molecular mass of 296 Da through the use of APCI-MS. Analysis of the 1H and 13C NMR spectra led to the identification of 8 as the known natural product chiricanine B. Chiricanine B (8) was originally described as a metabolite of the root bark of Lonchocarpus chiricanus.15 It has not previously been reported as a peanut metabolite. The APCI-MS of 21 indicated a molecular mass of 278 Da. The 1H NMR spectrum of 21 showed similarities to that of 8, including signals for a 1,2,3,5-tetrasubstituted benzene unit as well as a phenyl group. However, it was also clear from these data that the HOCHCH2 unit found in 8 was replaced by a cisolefin in 21. Thus, 21 was identified as a dehydration product of 8. A literature search revealed that this compound is known synthetically as a precursor to pawhuskin B,16 but has not been previously reported as a natural product.
dissociation (CID). In all CID analyses, the isolation width, relative fragmentation energy, relative activation Q, and activation time were m/z 1.6, 35%, 0.25, and 30 ms, respectively. The results of MS2 experiments are represented throughout the text as m/z aaa@bb: aaa, ccc, and ddd, where aaa is parent ion, bb is normalized collision energy (%), and ccc and ddd are fragment ions. Preparation of Mosher Esters of 22 and 23. A portion of 22 (0.3 mg) was combined with 3 μL of S-(−)-α-methoxy-α(trifluoromethyl)phenylacetyl chloride (S-MTPA-Cl) and 100 μL of pyridine-d5. The resulting mixture was stirred with a Teflon-coated stir bar at room temperature for 24 h. The solution was then placed in an NMR tube for direct analysis of the crude R-MTPA ester. Analogous treatment of an additional 0.3 mg portion of 22 using R-MTPA-Cl afforded the S-MTPA ester of 22. Signals in the 1H NMR spectrum showed significant Δδ (δS − δR) values, as summarized in Figure 2: SMTPA ester, δ 3.05 (dd, J = 17, 4.8 Hz, Ha-1″), 3.34 (dd, J = 17, 4.8, Hb-1″), 1.41(s, H-4″), 1.46 (s, H-5″); R-MTPA ester, δ 3.19 (dd, J = 18, 4.5 Hz, Ha-1″), 3.36 (dd, J = 18, 4.5 Hz, Hb-1″), 1.30 (s, H-4″), 1.30 (s, H-5″). The same procedure was followed for determination of the configuration of 23. Key 1H NMR signals again showed significant Δδ values (δS − δR): S-MTPA ester, δ 3.01 (dd, J = 18, 4.8 Hz, Ha-1″), 3.22 (dd, J = 18, 4.8 Hz, Hb-1″), 1.44(s, H-4″), 1.36 (s, H-5″); R-
Figure 2. Structures of MTPA esters for 22 and 23. Key positions are labeled with the Δδ (δS − δR) values obtained by comparison of the 1H NMR spectra of the S- and R-MTPA derivatives. 582
DOI: 10.1021/acs.jafc.5b04753 J. Agric. Food Chem. 2016, 64, 579−584
Article
Journal of Agricultural and Food Chemistry The 1H and 13C NMR spectra of 22 were similar to those of the previously isolated stilbene arahypin-2 (10).8 These data indicated that both compounds contain a symmetrical 1,3,5trisubstituted aromatic ring, a 1,2,4-trisubstituted benzene unit, and a dioxygenated prenyl group. The mass of 22 was found to be 312 Da, corresponding to a difference of H2O (18 Da) relative to arahypin-2 (10) (MW 330). This difference could be accounted for by cyclization of the prenyl unit with the oxygen at the 4′-position on the 1,2,4-trisubstituted ring as shown in 22. This conclusion was supported by analysis of HMBC data and by shift comparison to other compounds containing similar structural units. APCI-MS data indicated that 23 is an isomer of 22. Analysis of the 1H NMR spectrum showed the presence of a 1,4disubstituted benzene ring and a 1,2,3,5-tetrasubstituted benzene ring, similar to those of the previously reported arahypin-5 (13).8 Upon comparison of the data for the two compounds, it was clear that the cis-olefin in the side chain of arahypin-5 was replaced by an OCHCH2 unit in 23. This, along with the 18 Da difference in mass between the two compounds, suggested the presence of a cyclized prenyl group analogous to the unit found in 21 and 22. On the basis of APCI-MS data, a molecular mass of 354 Da was assigned for 24. Analysis of the 1H NMR data indicated close similarity to arahypin-4 (12).8 In addition to signals for a symmetrical 1,3,4,5-tetrasubstituted benzene unit, a phenyl group, and a dioxy prenyl group present in the data for araphypin-4, the spectrum of 24 contained two additional methyl signals. Analysis of the 13C NMR data also showed the presence of an additional doubly oxygenated carbon. On the basis of these data, 24 was identified as an acetonide of arahypin-4 (12) and was presumed to be an artifact of the isolation process. However, attempts to obtain this compound from a sample of pure arahypin-4 (12) by meticulously reproducing the isolation and purification procedures for 24, including chromatography on silica gel in the presence of acetone and evaporation of solvents, as well as other steps, did not reveal the formation of 24 at detectable levels. This issue could possibly be resolved by repeating the isolation procedure of 24 from a crude extract without the use of acetone. Some related compounds, in which the prenyl group is not cyclized to form a free dihydrobenzopyran unit, have been reported to be produced in scalemic mixtures favoring the Risomer.8,17 Chiricanine B (8), which contains a dihydrobenzopyran unit, was shown to have the S-configuration through enantioselective synthesis and was determined to have a specific rotation of −12°.18 However, the sample of chiricanine B (8) isolated here as described above showed no optical rotation, indicating a racemic mixture. The absolute configuration at C2″ of 22 ([α]D +11) and 23 ([α]D +38) was determined using Mosher’s method. Individual samples of 22 and 23 were each treated with (R)-(−)-α- or (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride in pyridine-d5 overnight at room temperature. Analysis of the resulting products showed only a single set of 1H NMR signals in each case, indicating that each starting material was present as a single enantiomer. MS and NMR data indicated that triacylation products were obtained in each case, but protons relevant to the Mosher analysis were in proximity only to the acyl group adjacent to the stereocenter, so the key Δδ values were considered to be due only to this unit. Evaluation of the corresponding Δδ values led to assignment of the S-configuration at the C-2″ position for both compounds.
Stilbene derivatives are known for their diverse biological activities. 4,7,19,20 It is reasonable to suggest that new compounds 21−23 are likely to show biological activities on the basis of the similarity of their structures to those of stilbenoids with known bioactivities. Experiments with sufficient quantities of the new compounds and appropriate test organisms or in vitro systems would be required to verify the presence of such activities in the new stilbenoids. Considering the importance of knowledge of natural plant defense mechanisms, studies of the biological activity of these new stilbenoids are planned. The present study revealed the production of new stilbenoids 22 and 23 by fungus-challenged peanut seeds. In addition, two stilbenoids that have been reported previously from other sources (8 and 21) are reported in peanuts for the first time. Stilbenoids may play a protective role against fungi, as reflected in the literature.4−6,15,19,20
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04753. 1 H NMR and 13C NMR spectra of chiricanine B (8), arahypin-13 (21), arahypin-14 (22), arahypin-15 (23), and the acetonide of arahypin-4 (24) and HMBC spectra and tabulated correlations for arahypin-14 (22) and arahypin-15 (23) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(V.S.S.) Phone: (229) 995-7446. Fax: (229) 995-7416. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Kerestin Goodman for valuable help in the laboratory. ABBREVIATIONS USED DAD, diode array detector; APCI-MS, atmospheric pressure chemical ionization mass spectrometry; HRESIMS, highresolution electrospray ionization mass spectrometry; HMBC, heteronuclear multiple-bond correlation; Q-TOF, quadrupole time-of-flight mass spectrometer
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REFERENCES
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