Identification of Indole Diterpenes in Ipomoea asarifolia and Ipomoea

Article pubs.acs.org/JAFC

Identification of Indole Diterpenes in Ipomoea asarifolia and Ipomoea muelleri, Plants Tremorgenic to Livestock Stephen T. Lee,* Dale R. Gardner, and Daniel Cook Poisonous Plant Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 1150 East, 1400 North, Logan, Utah 84341, United States ABSTRACT: Ipomoea asarifolia has been associated with a tremorgenic syndrome in livestock in Brazil and was recently reported to contain tremorgenic indole diterpenes. Ipomoea muelleri has been reported to cause a similar tremorgenic syndrome in livestock in Australia. Ipomoea asarifolia and I. muelleri were investigated by high-performance liquid chromatography−highresolution mass spectometry (HPLC−HRMS) and high-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) for indole diterpene composition. The high-resolution mass spectrometric data in combination with MS/MS fragmentation mass spectral data provided valuable information for indole diterpene characterization. The previous report of indole diterpenes in I. asarifolia was confirmed and expanded; and the presence of indole diterpenes in I. muelleri is reported for the first time. Two new indole diterpenes were isolated and their structures determined by 1D and 2D NMR spectroscopy and given the names 11-hydroxy-12,13-epoxyterpendole K and 6,7-dehydroterpendole A. The presence of terpendole K and terpendole E in I. asarifolia is unequivocally demonstrated for the first time. This is the first detailed MS analysis of known indole diterpenes and possible isomers in I. asarifolia and I. muelleri. KEYWORDS: indole diterpenes, Convolvulaceae, Ipomoea asarifolia, Ipomoea muelleri, tremorgenic

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responsible for the toxicity.9 Other publications exploring the phytochemical diversity in Ipomoea species, not directly investigating the reported toxicity of these two species, have reported that both these species as well as several others contain the ergot alkaloids.16−18 Additionally, it was recently reported that I. asarifolia contains some indole diterpenes.10 The indole diterpenes are known to be tremorgenic,11 and we suspect are likely the primary cause of the tremorgenic syndrome observed in livestock that have grazed I. asarifolia and I. muelleri. The objective of this study was to further investigate the presence of indole diterpenes in I. asarifolia to determine if I. muelleri contains the indole diterpenes and to assess the structural diversity of indole diterpenes in these plants.

INTRODUCTION Ipomoea species have been reported to cause a neurologic disease with lesions characteristic of a lysosomal storage disease as well as a tremorgenic syndrome with little or no diagnostic lesions in livestock.1−3 Two species, Ipomoea asarifolia and Ipomoea muelleri, members of the Convolvulaceae plant family, are reported to be associated with a tremorgenic syndrome in livestock.1,3 Ipomoea asarifolia has been associated with a tremorgenic syndrome in goats, sheep, and cattle in Northeastern Brazil and the Island of Marajo,3−6 whereas I. muelleri has been reported to cause a similar syndrome in sheep and cattle in Western Australia.7−9 Ipomoea species have been reported to contain several bioactive alkaloids thought to be toxic to livestock including calystegines, ergot alkaloids, indole diterpene alkaloids, and swainsonine.2,10−12 Calystegines are associated with many Ipomoea species and are produced by the plant.12 In contrast, the ergot alkaloids, indole diterpenes, and swainsonine are associated with seed transmitted endophytes associated with some Ipomoea species.2,10,11 A Clavicipitaceous endophyte, Periglandula species, are symbiotic fungi of the family Clavicipitaceae and are reported to be associated with ergot alkaloid-containing Ipomoea species. Similarly a fungal symbiont belonging to the order Chaetothyriales has been reported to be associated with the swainsonine-containing species, I. carnea.13,14 To date, no Ipomoea species have been reported to contain both the ergot or indole-diterpenes and swainsonine. In one of the initial case reports describing the toxicity of I. asarifolia, swainsonine and the calystegines were not detected.3 Subsequently, it was reported that a leaf lectin may be responsible for the reported toxicity.15 In regard to I. muelleri, investigators detected the calystegines but not swainsonine leading them to speculate that the calystegines may be This article not subject to U.S. Copyright. Published 2017 by the American Chemical Society

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MATERIALS AND METHODS

Chemicals and Reagents. Terpendole E was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Paxilline was obtained from Tocris Bioscience (Bristol, U.K.). Terpendole C was obtained from BioVision Inc. (Milpitas, CA). Plant Material. Ipomoea asarifolia (Desr.) Roem & Schult. seeds were collected near the veterinary hospital of the University of Campina Grande, Campus of Patos in the city of Patos, Paraiba, Brasil (S 7° 04′ 02″ W 37° 16′ 53″) (voucher no. 21226 (PEL)). Plants derived from the above-mentioned seeds were grown in the greenhouse with a 16 h photoperiod and day/night temperatures of 25 °C/20 °C. Leaves from the plants were harvested and frozen at −80 °C. Ipomoea muelleri Benth. seed (voucher no. 3640108 (PERTH)) and leaf material (voucher no. 5709563 (PERTH)) were Received: Revised: Accepted: Published: 5266

April 20, 2017 June 1, 2017 June 2, 2017 June 2, 2017 DOI: 10.1021/acs.jafc.7b01834 J. Agric. Food Chem. 2017, 65, 5266−5277

5267

35.1 29.7 30.4 18.9 23.1 29.7 30.2 30.9

terpendole E Isomer

terpendole C, 5b

terpendole C isomer

paxilline isomer

paxilline, 6b

paxilline isomer

paxilline isomer

paxilline isomer

I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (l) I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.m. (l), I.m. (s)

I.a. (l)

I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.a. (l), I.m. (l), I.m. (s) I.a. (l)

I.a. (l), I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (l), I.m. (s)

I.a. (l) I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.m. (l), I.m. (s)

I.a. (l), I.a. (s)

I.m. (l), I.m. (s)

I.m. (s)

A,

A

A

26.8 33.6 22.2 33.6

13-desoxypaxilline isomer

paxitriol isomer paxitriol isomer

22.2

28.0 33.7

27.0

24.7

23.2

18.6

31.4

29.1



paspaline isomer paspaline, 7c

23.5

6,7-dehydroterpendole isomer 6,7-dehydroterpendole isomer 6,7-dehydroterpendole isomer 6,7-dehydroterpendole 3,a terpendole E, 4b

I.m. (s)

A

terpendole K isomer

I.m. (s)

I.a. (l), I.a. (s)

27.2

11-hydroxy,12,13epoxyterpendole K, 2a terpendole K, 1a

I.m. (l), I.m. (s)

26.1

RT min

terpendole K isomer

identity

I.m. (l), I.m. (s)

plant tissue

438.26340 438.26315

420.25332

420.25259

420.25262

422.30458 422.30450

436.24737

436.24771

436.24771

436.24723

436.24809

520.30501

520.30479

438.29953

438.29905

534.28496

534.28492

534.28421

534.28493

518.29078

518.28950

518.29093

518.29054

MH+(m/z)

436(8), 418(100), 400(29), 348(41), 306(34), 288(58), 182(17), 130(27) 436(13), 418(100), 400(28), 348(21), 306(16), 288(27), 182(18), 130(8) 436(35), 418(100), 400(24), 348(30), 322(24), 306(22), 288(42), 182(15), 130(13) 422(59), 407(84), 404(100), 252(22), 184(10), 130(72) 422(30), 407(100), 404(33), 257(6), 158(10), 130(57)

−0.525 −0.525

C27H34NO4 C27H34NO4

420(58), 402(100), 374(18), 360(10), 318(17), 290(12), 182(30), 130(10) 438(4), 420(100), 402(34), 182(9), 130(6) 438(8), 420(100), 402(34), 182(9), 130(4)

−0.760 −0.485 −0.735

C27H36NO4 C27H36NO4

−0.730

C27H34NO3

C27H34NO3

420(82), 402(100), 332(30), 318(52), 290(36), 182(56), 130(26) 420(20), 405(100), 402(38), 362(15), 347(6), 305(11)

−0.776 −0.856

−0.865

−1.005

−0.700

C27H34NO3

C28H40NO2 C28H40NO2

C27H34NO4

C27H34NO4

−0.165

−0.740

C32H42NO5 C27H34NO4

−0.960

C32H42NO5

416(100),

398(29),

416(100),

438(10), 420(100), 402(35), 384(8), 332(8), 182(8), 130(4) 520(9), 505(100), 502(8), 418(17), 330(18), 307(9), 182(3) 520(18), 505(72), 502(100), 484(19), 452(55), 434(28), 416(15), 198(8) 436(9), 418(100), 400(28), 348(21), 306(16), 288(27), 182(18), 130(9) 436(8), 421(100), 418(22), 378(8), 182(3), 130(2)

−1.221

−0.054

−0.089

−0.804

345(11),

−0.744

C28H40NO3

C28H40NO3

C32H40NO6

C32H40NO6

C32H40NO6

C32H40NO6 534(2), 516(22), 416(100), 398(12), 362(14), 182(7) 534(12), 516(86), 498(61), 462(14), 434(72), 398(27), 388(10), 182(22) 534(1), 516(72), 498(68), 434(54), 416(100), 388(10), 196(8) 534(12), 519(19), 516(76), 498(75), 434(54), 398(31), 388(12), 196(8) 438(5), 423(100), 420(18), 402(7), 130(10)

0.680

−0.600

518(0), 500(76), 434(100), 416(92), 398(36), 388(12), 371(25) 518(1), 503(32), 500(100), 434(38), 416(57), 398(27), 371(26) 518(1), 434(100), 416(10), 376(9)

518(4), 500(18), 434(100), 418(16), 416(22), 182(8)

CID MS/MS (m/z) (% rel abundance)

−0.084

C32H40NO5

C32H40NO5

0.830

0.445

C32H40NO5 C32H40NO5

Δ (ppm)

calcd mol formula (MH+)

438(17), 420(24), 402(12), 290(17), 182(100), 130(91) 438(19), 420(24), 402(14), 290(187), 182(100), 168(22), 130(86)

420(60), 402(24), 334(29), 182(100), 130(84)

420(35), 405(88), 347(50), 182(22), 130(100)

420(52), 290(15), 182(71), 158(16), 130(100)

422(14), 407(3), 404(2), 182(7), 130(100) 422(19), 407(21), 183(13), 182(14), 130(100)

436(28), 288(22), 182(56), 168(22), 130(100)

436(27), 306(16), 288(21), 182(75), 168(16), 130(100)

436(30), 421(100), 403(100), 363(38), 345(94), 182(61), 130(96) 436(5), 288(17), 182(38), 168(14), 130(100)

436(20), 418(9), 288(20), 182(70), 168(17), 130(100)

520(13), 505(11), 198(47), 130(100)

438(22), 420(24), 402(12), 290(18), 182(100), 168(23), 130(88) 520(4), 505(33), 182(17), 130(100)

438(18), 423(42), 405(8), 183(27), 182(30), 130(100)

534(8), 416(25), 196(68), 182(46), 168(22), 130(100)

534(5), 416(36), 196(89), 182(66), 168(26), 130(100)

534(6), 416(12), 196(28), 182(100), 130(25)

518(12), 434(76), 318(46), 276(33), 248(56), 236(60), 194(86), 182(39), 168(52), 130(100) 534(4), 416(22), 362(30), 332(12), 182(100)

518(7), 434(16), 248(15), 196(12), 194(16), 182(56), 130(100) 518(1), 416(15), 398(10), 196(34), 182(23), 168(11), 130(100) 518(1), 416(8), 398(7), 196(21), 182(15), 130(100)

HCD MS/MS (m/z) (% rel abundance)

Table 1. HPLC and Mass Spectrometry Data for Indole Diterpene Alkaloids in Isopropyl Alcohol Extracts of Ipomoea asarifolia E (+) Leaf (I.a. (l)), Ipomoea asarifolia E (+) Seed (I.a. (s)), Ipomoea muelleri Leaf (I.m. (l)), and Ipomoea muelleri Seed (I. m. (s))

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.7b01834 J. Agric. Food Chem. 2017, 65, 5266−5277

30.3 35.1

terpendole H isomer

terpendole H isomer

terpendole H isomer

terpendole H isomer terpendole H isomer

terpendole H isomer

emindole SB, 17d

I.m. (l), I.m. (s)

5268

I.a. (l), I.a. (s) I.a. (l), I.a. (s)

I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (l), I.m. (s) 406.31044

452.24314

452.24314 452.24314

452.24314

452.24314

452.24314

452.24314

452.24314

506.32648

422.26897

536.29913

522.32139

522.32139

454.25815

454.25879

454.25842

MH+(m/z)

C28H40NO

C27H34NO5

C27H34NO5 C27H34NO5

C27H34NO5

C27H34NO5

C27H34NO5

C27H34NO5

C27H34NO5

C32H44NO4

C27H36NO3

C32H42NO6

C32H44NO5

C32H44NO5

C27H36NO5

C27H36NO5

C27H36NO5

calcd mol formula (MH+)

454(27), 436(100), 418(58), 396(28), 364(41), 348(21), 306(20), 130(13) 522(26), 504(80), 562(25), 444(100), 402(34), 384(90), 374(34), 366(35), 256(26), 272(60) 522(6), 507(100), 504(10), 198(12) 536(13), 521(100), 518(13) 422(55), 407(52), 404(100), 386(34), 182(10), 130(17) 506(47), 491(65), 488(100), 470(31), 438(86), 420(87), 402(41), 250(15), 198(20), 182(25) 452(22), 434(100), 416(29), 398(12), 362(13), 182(22), 130(4) 452(4), 437(10), 434(100), 416(52), 362(20), 346(8), 182(4), 130(3) 452(13), 434(100), 416(29), 362(12), 334(12), 182(13), 130(20) 452(34), 434(100), 416(54), 380(18), 362(34), 304 (12), 182(20), 130(4) 452(4), 434(100), 416(56), 380(20), 362(40), 304 (14), 182(22), 130(4) 452(21), 434(100), 416(30), 334(15), 182(12), 130(4) 452(34), 434(95), 416(100), 398(14), 332(16), 182(6), 130(10) 452(18), 434(100), 416(48), 398(21), 358(20), 316(35), 182(6), 130(4) 406(53), 391(100), 388(53), 257(9), 130(20)

−0.650

−1.534 −0.730 −0.745

−0.741

−0.690

−0.790 −0.980

−0.380

−0.430

−0.600

−0.640

−0.400

−0.900

−0.660

454(72), 436(86), 418(45), 400(22), 130(100)

−0.500

CID MS/MS (m/z) (% rel abundance) 454(3), 439(100), 436(28), 418(16), 364(6), 130(4)

−0.380

Δ (ppm) HCD MS/MS (m/z) (% rel abundance)

452(20), 316(23), 212(25), 196(30), 184(28), 182(36), 130(100) 406(53), 391(15), 257(7), 183(26), 182(29), 130(100)

452(11), 434(35), 416(19), 362(14), 248(14), 194(16), 182(51), 168(26), 130(100) 452(28), 434(12), 334(38), 322(13), 304(15), 182(94), 130(100) 452(18), 434(14), 362(14), 334(14), 304(20), 250(18), 194(16), 182(100), 168(20), 130(97) 452(9), 434(11), 362(14), 334(14), 304 (16), 250(14), 194(16), 182(100), 168(19), 130(90) 452(18), 434(11), 334(32), 260(15), 182(65), 130(100) 452(14), 304(10), 196(19), 182(30), 130(100)

506(3), 420(6), 290(6), 250(7), 198(12), 182(100), 168(11), 130(23) 452(22), 434(10), 248(9), 196(10), 182(100), 130(42)

422(32), 407(20), 404(6), 386(9), 182(32), 130(100)

536(2), 521(4), 193(4), 130(100)

522 (14), 507(24), 198(39), 130(100)

522(48), 384(38), 272(76), 232(100), 130(54)

454(4), 216(6), 182(16), 130(100)

454(3), 130(100)

454(3), 439(19), 421(6), 233(7), 182(9), 130(100)

a Identity determined by 1H and 13C NMR spectroscopy. bIdentity confirmed by comparison and coinjection of standards. cIdentity confirmed by high-resolution mass spectral data, comparison with published CID MS/MS data,20 and based on correlation with published HPLC retention data.22 dTentative identification based on high-resolution mass spectral data, CID-MS/MS data, HCD-MS/MS data and correlation with published HPLC retention data.22

26.4 29.1

26.1

24.9

22.1

26.4

I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.m. (l), I.m. (s)

terpendole B, 14

d

20.4

22.1

terpendole A/M isomer

terpendole H, 16d

30.2

terpendole J, 11,d

I.m. (l), I.m. (s)

26.6

terpendole J isomer

12.6

30.2

terpendole I isomer

terpendole H Iisomer

29.7

terpendole I isomer

33.6

18.9

terpendole I, 10d

terpendole D, 15d

RT min

identity

I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (s) I.m. (s)

I.a. (s), I.m. (l), I.m. (s) I.a. (s), I.m. (l), I.m. (s) I.a. (l), I.a. (s), I.m. (l), I.m. (s) I.a. (l)

plant tissue

Table 1. continued

Journal of Agricultural and Food Chemistry Article


Journal of Agricultural and Food Chemistry

Article

Figure 1. Structures of indole diterpene alkaloids or isomers of indole diterpene alkaloids identified in isopropyl alcohol extracts of I. asarifolia and I. muelleri. method previously described include a different HPLC column, substitution of 0.1% formic acid in place of 0.1% acetic acid in the mobile phase, and detection by high-resolution mass spectrometry (HRMS) and MS/MS detection using higher energy collision induced dissociation (HCD) in addition to the collision induced dissociation (CID) MS/MS detection used by Rasmussen et al.19 In brief, plant material (100, 50, or 25 mg) was weighed into 7 mL screw-top vials (100 mg samples) or 2 mL autosample vials (50 and 25 mg) and extracted with 2 mL, 1 mL, or 0.5 mL isopropyl alcohol for the 100, 50, or 25 mg samples, respectively, by mechanical rotation for 3 h. The samples were centrifuged (5 min), the isopropyl alcohol removed and filtered. A portion (200 μL) was transferred to a 300 μL autosampler vial for HPLC−MS analysis. Samples were injected (10 μL) onto a 100 mm × 2.1 mm i.d., 5 μm, Betasil C18 with a 10 mm × 2.1 mm i.d. guard column of the same material (Keystone Scientific, Inc.

obtained from herbarium specimens at the Western Australian herbarium in Perth, Western Australia. Subsequently, the frozen leaf material was freeze-dried and all plant material was ground to pass through a 2 mm screen. Fungicide Treatment. Ipomoea asarifolia seeds were scarified and imbibed overnight in a 0.9% pyraclostrobin (BASF, Research Triangle Park, NC) solution to eliminate Periglandula ipomoeae, the seed transmitted endosymbiont. Pyraclostrobin, a strobilurin class fungicide, has a mode of action shown to be effective against a broad range of fungal species including a large number of ascomycetes. Following treatment, seeds were potted and the resulting plants were grown in the greenhouse. Indole Diterpene Analysis. High-performance liquid chromatography−mass spectrometry (HPLC−MS) analysis of plant material was based on a previously published method.19 Deviations from the 5269



Article

Table 2. 1H and 13C NMR Chemical Shifts of Terpendole K, 1, Isolated from Ipomoea asarifolia and Obtained in C2D6SO and CDCl3 terpendole K, 1 carbon no. solvent

13

C chemical shift (ppm)

152.5 50.1 43.2 28.7

C-6 C-7 C-9 C-10 C-11 C-12 C-13 C-14 C-15

105.2 144.9 73.9 70.2 58.4 64.5 74.8 28.7 20.4

C-16 C-17

49.6 26.9

C-18 C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27 C-28 C-29 C-31 C-33 C-34 C-35 C-36

114.9 124.5 117.7 118.4 119.3 111.8 139.9 16.3 19.2 74.4 16.6 27.8 92.2 122.1 137.9 18.4 25.1

13

H chemical shift (ppm)

C2D6SO

NH C-2 C-3 C-4 C-5

terpendole K, 1

1

C2D6SO


3.84 (1H, d, J = 10.0 Hz) 4.16 (1H, d, J = 10.0 Hz) 3.84 (1H, s) 4.58 (OH, 1H, s) 1.53−1.58 (2H, m) 1.53 (1H, m) 1.83 (1H, m) 2.65 (1H, m) 2.31 (1H, dd, J = 12.5, 10.5 Hz) 2.60 (1H, dd, J = 12.5 6.2 Hz)

d, J = 7 Hz) t, J = 7 Hz) t, J = 7 Hz) d, J = 7 Hz)

1.25 (3H, s) 1.04 (3H, s) 1.28 1.19 5.54 5.12

(3H, (3H, (1H, (1H,

CDCl3 7.74 (1H, s)

2.20 (1H, dd, J = 16.0, 6.6 Hz) 2.90 (1H, d, J = 16.0 Hz) 5.25 (1H, dd, J = 6.5, 2.0 Hz)

(1H, (1H, (1H, (1H,


CDCl3

10.72 (1H, s)

7.26 6.90 6.94 7.28

1

s) s) d, J = 6.5 Hz) brd, J = 6.5 Hz)

1.66 (3H, d, J = 1 Hz) 1.67 (3H, d, J = 1 Hz)

151.9 50.9 44.0 30.8 106.2 144.7 73.4 71.4 60.6 65.0 76.4 30.6 20.8 50.2 27.5 117.8 125.4 118.8 120.8 119.2 111.7 140.0 16.4 20.2 75.1 16.9 28.1 93.0 121.8 140.3 18.9 25.9

3.20 (1H, brd, J = 16.1 Hz) 1.85 (1H, m) 5.39 (1H, dd, J = 6.6, 2.0 Hz) 4.07 (1H, d, J = 9.8 Hz) 3.93 (1H, d, J = 9.8 Hz) 3.84 (1H, s)

1.55−1.50 (2H, m) 1.95 (1H, m) 1.65 (1H, m) 2.72 (1H, m) 2.70 (1H, dd, J = 12.5, 6.5 Hz) 2.42 (1H, td, J = 12.8, 2.0 Hz)

7.43 7.06 7.08 7.29

(1H, (1H, (1H, (1H,

m) m) m) m)

1.31 (3H, s) 1.14 (3H, s) 1.33 1.31 5.47 5.27

(3H, (3H, (1H, (1H,

s) s) brd, J = 6.6 Hz) m)

1.74 (3H, d, J = 1 Hz) 1.71 (3H, d, J = 1 Hz)

temperature 300 °C, sheath gas flow 40 arbitrary units, auxiliary gas flow 5 arbitrary units, source voltage 3.5 kV, source current 100 μA. From 0 to 5 min flow from the column was diverted to waste, from 5 to 40 min the flow was directed to the ion source. MS/MS data were acquired for selected masses in a 2 scan positive ion mode. In the first scan the selected masses (Table 1; structures in Figure 1) were fragmented under CID conditions with an isolation width (m/z) 1.0, normalized collision energy 23.0, activation Q 0.250, activation time 10.0 ms. In the second scan, the selected masses were fragmented under HCD conditions with an isolation width (m/z) 1.0, normalized collision energy 38.0, activation Q 0.080, activation time 2.0 ms. Isolation of Terpendole K, 1; 11-Hydroxy-12,13-epoxyterpendole K, 2; and 6,7-Dehydroterpendole A, 3. Greenhouse grown, endophyte positive I. asarifolia leaves were harvested on September 12, 2016, freeze-dried, and ground to pass through a 2 mm screen. The leaf material (427 g) was Soxhlet extracted with ethyl acetate for 72 h. The extract was rotary evaporated to dryness resulting in a dark green residue (44 g). The residue was partitioned between equal volumes (2.4 L) of hexane and methanol/water (16:1, v/v). The methanol−water layer was collected and rotary evaporated to dryness into a dark green residue (16 g), dissolved in a minimum amount of chloroform, and applied to the head of a 32 cm × 7.5 cm i.d. silica

Bellefonte, PA). The samples were eluted from the column with a gradient flow at a flow rate of 0.300 mL/min. The mobile phase program was 0.1% formic acid/acetonitrile (80:20, v/v) for 1 min followed by a linear gradient to a composition of 100% acetonitrile at 40 min. For high-resolution mass spectrometric analysis of HPLC peaks (HPLC−HRMS), the mobile phase was delivered and samples injected using an Ultimate 3000 HPLC (Thermo Scientific, San Jose, CA) and the column eluent was connected to the heated electrospray source of an Exactive Plus Orbitrap high-resolution mass spectrometer (Thermo Scientific) calibrated as per the manufacturer’s instructions and with a scan range 100−800 Da, resolution 70 000, microscans 1, sheath gas flow 35, auxiliary gas flow 10, spray voltage 4 kV, capillary temperature 320 °C, S lens rf field 55, and auxiliary gas temperature 300 °C. For HPLC−MS/MS, the samples, HPLC column, and mobile phase program were the same as previously described herein. The mobile phase was delivered and samples injected using a 1200 series HPLC (Agilent Technologies, Santa Clara, CA) and the column eluent was connected to the heated electrospray ion source of a VELOS PRO LTQ (Thermo Scientific) mass spectrometer with the following operating parameters: capillary temperature 275 °C, source heater 5270



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Table 3. 1H and 13C NMR Chemical Shifts for 11-Hydroxy-12,13-epoxyterpendole K, 2, and 6,7-Dehydroterpendole A, 3 11-hydroxy-12,13-epoxyterpendole K, 2 carbon no. solvent

13


149.7 49.4 40.0 28.2

C-6

100.2

C-7 C-9 C-10

147.3 70.2 71.8

C2D6SO

CDCl3

1

10.72 (1H, s)

C-11 OH-11 C-12 C-13 C-14

58.9 71.2 28.1

C-15

21.9

C-16 C-17

48.3 26.7

C-18 C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27 C-28 C-29 C-31 C-33 C-34 C-35 C-36



C2D6SO

NH C-2 C-3 C-4 C-5

11-hydroxy-12,13-epoxyterpendole K, 2 13

62.8

116.1 124.3 117.8 118.6 119.6 111.9 140.2 14.9 18.3 74.4 28.1 16.6 91.7 122.4 137.7 18.4 25.1

2.38 (1H, m) 2.14 (1H, m) 5.03 (1H, dd, J = 8.0, 2.0 Hz) 4.02 (1H, d, J = 10.0 Hz) 3.79 (1H, dd, J = 10.0, 2.0 Hz) 3.90 (1H, m) 5.32 (1H, d, J = 4.4 Hz)

2.20 (1H, m) 1.53 (1H, m) 1.79 (1H, m) 1.60 (1H, m) 2.88 (1H, m) 2.70 (1H, dd, J = 13.2, 6.6 Hz) 2.38 (1H, m)

7.30 6.92 6.97 7.29

(1H, (1H, (1H, (1H,

d, J = 7.5) t, J = 7.5) t, J = 7.5) d, J = 7.5)

1.09 (3H, s) 1.06 (3H, s) 1.41 1.36 5.57 5.13

(3H, (3H, (1H, (1H,

s) s) d, J = 6.4 Hz) brd, J = 6.4 Hz)

1.72 (3H, s) 1.73 (3H, s)

6,7-dehydroterpendole A, 3 13



CDCl3

CDCl3

1

7.70 (1H, s) 149.2 50.4 40.7 30.2

2.72 (1H, brd, J = 15.1 Hz) 1.76 (1H, dd, J = 15.2, 8.2 Hz)

151.8 50.9 44.0 30.8

5.15 (1H, dd, J = 8.2, 2.2 Hz)

106.4

146.7 69.5 72.7

4.138 (1H, d, J = 10.3 Hz) 3.75 (1H, dd, J = 10.3, 3.3 Hz)

144.5 73.3 71.3

58.5 73.4 29.2 22.4

48.8 24.5

4.14 (1H, d, J = 3.3 Hz)

2.19 (1H, m) 1.55 (1H, m) 1.95 (1H, ddd, J = 13.0, 13.0, 4.3 Hz) 1.86 (1H, m) 2.94 (1H, m) 2.79 (1H, dd, J = 13.2, 6.5 Hz)

60.4 65.2 76.5 30.8 20.8

50.2 27.5

2.46 (1H, dd, J = 13.2, 10.4 Hz) 118.8 125.5 118.9 120.0 121.1 111.8 140.2 15.1 19.2 74.7 28.5 17.2 92.2 122.0 140.1 18.9 25.9

7.44 7.07 7.09 7.29

(1H, (1H, (1H, (1H,

m) m) m) m)

1.09 (3H, s) 1.06 (3H, s) 1.41 1.36 5.59 5.34

(3H, (3H, (1H, (1H,

s) s) d, J = 6.9 Hz) brd, J = 6.9 Hz)

1.72 (3H, d, J = 1 Hz) 1.73 (3H, d, J = 1 Hz)

column (70−230 mesh, 60 Å) (Sigma-Aldrich, St. Louis, MO). The column was prepared by slurry packing the silica in chloroform and capped with 2 cm sand (white quartz-50 + 70 mesh) (Sigma, St. Louis, MO). Chloroform was added to the head of the column and fractions (300 mL) were collected upon the elution of an intense yellow band. After the fourth fraction, the eluent became light yellow and the eluent composition was changed to chloroform/methanol (99:1, v/v). Light yellow fractions continued through fraction 22. The fractions were dark green from fractions 23−31 and then became gradually lighter green. The fractions were analyzed for the presence of indole diterpenes by the analytical HPLC−HRMS method described above. Fractions 23−28 contained the indole diterpenes. These fractions were combined, rotary evaporated to dryness to a dark green residue (1.3 g) and partitioned between equal volumes (765 mL) of hexane and methanol−water (16:1, v/v). The methanol/water layer was collected

H chemical shift (ppm) CDCl3

7.70 (1H, s)

101.4

63.4

1

117.8 125.4 118.8 120.9 120.0 111.7 140.0 16.4 20.3 75.1 27.9 16.7 93.0 63.0 58.0 19.8 24.8

3.21 (1H, brd, J = 16.1 Hz) 1.83 (1H, dd, J = 16.1, 6.9 Hz) 5.40 (1H, dd, J = 6.8, 2.1 Hz) 4.07 (1H, d, J = 9.5 Hz) 3.92 (1H, d, J = 9.5 Hz) 3.81 (1H, s)

1.56 (1H, m) 1.55 (1H, m) 1.96 (1H, m) 1.65 (1H, m) 2.72 (1H, m) 2.69 (1H, dd, J = 12.5, 6.3 Hz) 2.42 (1H, td, J = 12.6, 2.5 Hz)

7.42 7.08 7.06 7.29

(1H, (1H, (1H, (1H,

m) m) m) m)

1.32 (3H, s) 1.14 (3H, s) 1.35 1.25 4.60 2.84

(3H, (3H, (1H, (1H,

s) s) d, J = 6.1 Hz) d, J = 6.1 Hz)

1.31 (3H, s) 1.34 (3H, s)

and rotary evaporated to dryness resulting in a dark green residue (0.87 g). A Strata-X 33 μm polymeric reversed phase 10g/60 mL, Giga Tube (Phenomenex, Torrance, CA) was conditioned by washing first with methanol followed by water and again with methanol. The Giga Tube was then placed in a vacuum oven at 40 °C for 5 h to evaporate off residual methanol and then removed from the oven and allowed to cool to room temperature. The top frit was removed and a portion of the packing material was removed leaving approximately 1.5 cm of packing material in the tube. The top frit was then replaced. The extract (0.87 g) was dissolved in a minimum amount of methanol and applied to the remaining packing material in a small beaker and the methanol evaporated by placing the small beaker on a heat block (40 °C) in a fume hood. After the evaporation of the methanol, the packing material with the adsorbed extract was packed into the Giga Tube on top of the 1.5 cm conditioned packing material and an 5271



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Figure 2. Reconstructed HPLC−HRMS ion chromatograms from an isopropyl alcohol extract of I. asarifolia seed: (A) total ion chromatogram; (B) m/z 518.29009, terpendole K; (C) m/z 534.28501, 6,7-dehydroterpendole A and reconstructed HPLC−HRMS ion chromatograms from an isopropyl alcohol extract of I. muellleri seed; (D) total ion chromatogram; (E) m/z 518.29009, 11-hydroxy,12,13-epoxyterpendole K and isomers; (F) m/z 534.28501, 6,7-dehydroterpendole A and isomers. isomer). The compound suspected of being an isomer of terpendole K was identified as 11-hydroxy-12,13-epoxyterpendole K, 2; highresolution ESI(+)MS, m/z 518.29093 (MH+, C32H40NO5 requires 518.29009); see Table 3 for 1H and 13C NMR data. The compound with a MW = 533 was identified as 6,7-dehydroterpendole A (3): highresolution ESI(+)MS, m/z 534.28496 (MH+, C32H40NO5 requires 534.28501); see Table 3 for 1H and 13C NMR data. NMR Spectroscopy Analysis. NMR spectroscopic data (1H, 13C) were acquired using an Avance III HD spectrometer (500 MHz for 1H, 125 MHz for 13C) (Bruker Biospin, Billerica, MA) using solutions in dimethyl sulfoxide-D6 (99.96 atom % D) (δH 2.50, δC 39.5) (SigmaAldrich, St Louis, MO) and/or solutions in chloroform-D (99.8 atom % D) (δH 7.24, δC 77.23). NMR data were analyzed using Top Spin software, version 3.2 (Bruker)

additional frit placed on top. The prepared Giga Tube was washed with water (200 mL) and eluted with 18 fractions (100 mL) of methanol/water (90:10, v/v). The methanol/water fractions were analyzed for the presence of indole diterpenes by HPLC−HRMS. Methanol/water fractions 2−9 contained the indole diterpenes. These fractions were combined, rotary evaporated to dryness forming a brown residue (0.168 g). The brown residue was dissolved in isopropyl alcohol (6 mL) and aliquots (1.5 mL) manually injected onto a Prep LC2000 preparative chromatography system (Waters Corp., Milford, MA). The column used was a 150 mm × 21.20 mm i.d., 10 μm, Luna C18(2), with a 50 mm × 21.20 mm i.d. guard column of the same material (Phenomenex, Torrence, CA). The eluent was monitored by a UV/vis detector (Waters Corp.) at λ 280 nm. The column was eluted with a gradient flow at a flow rate of 20 mL/min. The mobile phase program was 0.1% formic acid/ acetonitrile, 80:20, v/v for 1 min followed by a linear gradient to a composition of 100% acetonitrile at 40 min. Fractions (20 mL) were collected at 1 min intervals. Aliquots from each fraction were analyzed for the presence of indole diterpenes by HPLC−HRMS. Fractions 33 and 34, containing the compound suspected to be terpendole K, 1, were combined and rotary evaporated to dryness resulting in a brown residue (26.3 mg). The brown residue was dissolved in 1.5 mL of isopropyl alcohol and the sample injected onto the same Waters preparative chromatography system. The mobile phase was 0.1% formic acid/acetonitrile, 40:60, v/v at a flow rate of 20 mL/min and the run time was 55 min. The compound eluted between 36 and 43 min and this fraction was collected and evaporated to dryness resulting in a brown residue (7.8 mg) identified as terpendole K, 1: high-resolution electrospray ionization (ESI)(+)MS, m/z 518.28950 (MH+, C32H40NO5 requires 518.29010). 1H and 13C NMR data (Table 2). Fractions 31−32 contained a compound suspected of being an isomer of terpendole K, 1, and another compound with a molecular weight of 533 (MH+ = 534). Fractions 31 and 32 were rotary evaporated to dryness, dissolved in 1.0 mL of isopropyl alcohol and the sample injected onto the Waters preparative chromatography system. The mobile phase was 0.1% formic acid/methanol, 20:80, v/v at a flow rate of 20 mL/min and the run time was 20 min. The compound with molecular weight of 533 (MH+ = 534) eluted between 8.5 and 10.5 min while the compound suspected of being an isomer of terpendole K, 1, eluted between 13 and 15.5 min, these fractions were collected and evaporated to dryness resulting in brown residues (2.1 mg, MH+ = 534 compound; 5.3 mg, suspected terpendole K, 1,

■

RESULTS AND DISCUSSION Ipomoea asarifolia and I. muelleri leaves and seeds were analyzed by HPLC−HRMS and HPLC−MS/MS using both CID and HCD. HRMS measurements were restricted to ±5 ppm of the calculated exact mass such that the compounds of interest and possible isomers of those compounds were selected in the resulting reconstructed ion chromatograms (RIC). CID and HCD fragmentation of HPLC peaks provided valuable complementary information. CID data allowed comparison with existing CID data available in the literature, while HCD data provided higher relative abundances of fragments of m/z 130 and 182 characteristic of the indole portion of indole diterpene structure.20,21 I. asarifolia leaves collected from plants that were grown from fungicide treated seeds were analyzed for indole diterpenes. No indole diterpenes were detected in I. asarifolia leaves derived from fungicide treated seeds. This is consistent with the indole diterpenes being produced by P. ipomoeae, the seed transmitted endophyte of I. asarifolia, rather than being produced by the host plant.10 Several indole diterpenes were detected in leaves and seeds of I. asarifolia (not fungicide-treated) and I. muelleri. The most abundant indole diterpene in I. asarifolia seeds and leaves and the major peak in the HPLC−HRMS total ion chromatogram (TIC) chromatograms of I. asarifolia seeds and leaves was 5272



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Figure 3. Reconstructed HPLC−HRMS ion chromatograms from an isopropyl alcohol extract of I. asarifolia: (A) total ion chromatogram; (B) m/z 438.30027, terpendole E and isomers; (C) m/z 436.24823, paxilline and isomers; (D) m/z 520.30574, terpendole C and isomer; (E) m/z 422.30535, paspaline and isomer; (F) m/z 420.25332, desoxypaxilline and isomer; (G) m/z 438.26388, paxitriol isomer; (H) m/z 454.25879, terpendole I and isomers; (I) m/z 522.32139, terpendole J and isomers; (J) m/z 536.30066, terpendole A or M or isomer; (K) m/z 422.26897, terpendole B; (L) m/z 506.32648, terpendole D; (M) m/z 452.24314, terpendole H; and (N) m/z 406.31044, emindole SB.

H-9,11 are completely resolved (δ 4.06 and δ 3.84) as compared to the overlapping resonances at δ 3.84 when taken in C2D6SO. Two additional compounds were isolated in sufficient quantity and purity to complete the structure elucidation by NMR analysis. One compound had similar HRMS (m/z = 518.29093) and MS/MS data to that of 1 and consistent with a molecular formula of C32H40NO5 (MH+) (Table 1) and was determined to be an isomer of terpendole K (tR = 27.2 min) when analyzed by HPLC−HRMS and HPLC−MS/MS. However, this compound was initially not present in the I. asarifolia leaves that were the source of the extract but appeared in fractions collected off the silica gravity column. This compound had the same retention time, HRMS, CID, and HCD mass spectra as the most abundant indole diterpene

initially given a tentative assignment of terpendole K, 1, with an HPLC retention time of 29.1 min (Figures 2A,B and 3A). The tentatively assigned terpendole K, 1, was subsequently isolated from freeze-dried I. asarifolia leaf material, analyzed by 1H, 13C NMR spectroscopy and found to be consistent with 1 isolated from the fungus, Albophoma yamanashiensis.22 The NMR data are presented in Table 2 with the isolated compound in both C2D6SO, for direct comparison to the literature,22 and in CDCl3, as optional data and are used for comparison to other isolated indole diterpenes. A correction in the 1H NMR data for 1 was made for the H-9 and H-11 protons from that originally published. The H-9 proton is a doublet with a J coupling of 10 Hz and not a singlet as reported previously.22 Likewise, the H11 resonance is assigned as the singlet. This assignment is supported by the 1H NMR data of 1 taken in CDCl3 where the 5273



Article

Figure 4. 1H, 13C 2D-NMR (HMBC) correlation plot for 11-hydroxy-12,13-epoxyterpendole K, 2 obtained in C2D6SO. The expanded region only displays critical long-range couplings of the OH to C-10,11, and 12 and unequivocally places the OH at C-11.

identified in I. muelleri (Figure 2D,E). Comparison of the 1H and 13C NMR data (1H, 13C- DEPT, HSQC, and HMBC) with that of 1 also confirmed a similar planar structure to that of 1. Noticeable differences in the 1H spectra included slight changes in the chemical shifts of H-5,6,9,10, and 11. The J 9,10 coupling remained at 10 Hz confirming the axial orientation of these two protons. However, the change in the coupling pattern of H-10 from a doublet (10 Hz) in 1 to a doublet of doublets (J = 10, 3 Hz) in 2 suggested a change in the dihedral angle of H-11,10. NOESY data confirmed the three-dimensional arrangement of C-9,10 and 31 as identical to that of 1 as previously described in the literature for terpendole C.23 Placement of the hydroxyl at C-11 was confirmed by NMR analysis in C2D6SO with a proton resonance appearing at δ 5.32 (d, J = 4 Hz) and a corresponding coupling to H-11 (δ 3.90, dd, J = 4, 3 Hz) with the confirmed coupling by COSY analysis. The resonance at δ 5.32 could be removed by the addition of a drop of D2O into the NMR sample tube with the resonance at δ 3.90 returning to the original doublet. In addition, long-range C−H correlation of OH-C11 was observed with C-11,12 and C-10 (Figure 4). Opening of the strained epoxy ring (C-11,12) to a less hindered OH substituent on C-11 accounts for the change in the H-11,10 dihedral angle (62° in 1 versus 43° in 2) and the increased J10,11 coupling. Therefore, the structure was assigned as 11-hydroxy-12,13-epoxyterpendole K, 2, a new indole diterpene. It is proposed that the conversion of terpendole K, 1, to 2 proceeds via acid catalyzed epoxy ring opening at C-11,12 with reformation at C-12,13. As mentioned previously, this conversion was first noticed by an increased relative concentration of 2 versus 1 after chromatography over silica gel. The conversion was confirmed on an analytical scale by HPLC−MS analysis of 1 after incubation overnight at room temperature in a mixture of silica gel and chloroform/methanol (99:1, v/v) with essentially complete conversion of 1 to 2. Energy minimized calculations (Chem 3D Pro) confirm the more stable C-12,13 epoxy structure (80 kcal/mol for 2 versus 120 kcal/mol for 1). The 6,7 double bond appears to facilitate the migration of the epoxide as evident from when the experiment was repeated with a mixture of terpendole K, 1, and terpendole C, 5, in which again 1 was converted to 2 but 5 remained unchanged. The presence of 2 as the major indole diterpene in I. muelleri establishes it as a natural product.

The third compound isolated from I. asarifolia leaves had a retention time of 27.0 min and HRMS consistent with a molecular formula of C32H40NO6 (MH+) (Table 1, Figure 2C). This compound exhibited MS/MS fragment ions at m/z 518 and 130 supporting an indole-diterpene structure. The fragment ion observed at m/z 434 suggested loss of the epoxy-isoprenyl fragment similar to the loss of the isoprenyl group observed in the MS/MS spectrum of 1. Comparison of the 1H and 13C NMR data with that of 1 showed the loss of the two olefinic carbon resonances (C33 and C34) at δ 121.8 and 140.3 and the appearance of two oxygen bearing carbons at δ 63.0 and 58.0 with the corresponding upfield shift of H-33 from δ 5.47 in 1 to δ 2.84 in 3. Compound 3 was thus identified as 6,7-dehydroterpendole A, 3, a new indole diterpene. As with terpendole K, 6,7-dehydroterpendole A, 3, appears to be susceptible to acid catalyzed epoxy ring opening at C-11,12 with reformation at C-12,13. Incubation of 6,7-dehydroterpendole A, 3, in a mixture of silica gel and chloroform/methanol (99:1, v/v) overnight at room temperature resulted in the formation of an indole diterpene with the same retention time, HRMS, CID, and HCD mass spectra as the 6,7-dehydroterpendole A isomer in I. muelleri (tR = 24.6 min) (Table 1, Figure 2F). We hypothesize that the 6,7-dehydroterpendole A isomer in I. muelleri (tR = 24.6 min) is the analogous isomeric compound of 3, as 2 is to 1. Energy minimized calculations (Chem 3D Pro) confirm the more stable C-12,13 epoxy structure (127 kcal/mol for the 6,7-dehydroterpendole A isomer (tR = 24.6 min) versus 171 kcal/mol for 3. However, the proposed C-12,13-expoxy derivative of 3 could not be isolated in large enough quantities to confirm its structure. The relatively low concentration of the other indole diterpenes detected prohibited their isolation and complete characterization from I. asarifolia leaf material. Terpendole K, 1, was present in I. asarifolia leaves and seeds. Terpendoles E, 4, and C, 5, were both determined to be present in leaves and seeds of I. asarifolia and I. muelleri based on comparison and coinjection of standards (Table 1 and Figures 3 and 5). Paxilline, 6, was also determined to be present in I. asarifolia leaves based on comparison and coinjection with a standard (Table 1 and Figures 3 and 5). Paspaline, 7, was determined to be present in I. asarifolia and I. muelleri leaves and seeds based on comparison of high-resolution MS data, and comparison with published CID MS/MS data20 and HPLC retention data22 (Table 1 and Figures 3 and 5). 5274



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Many of the indole diterpenes detected were found in both I. asarifolia and I. muelleri. Table 1 indicates the occurrence of the various indole diterpenes in seeds and leaves of I. asarifolia and I. muelleri. Some indole diterpenes were species specific; for example, the isomers of terpendole K, retention times 26.1, 27.2 (11-hydroxy,12,13-epoxyterpendole K, 2), and 31.4, were only detected in I. muelleri whereas two isomers of terpendole H, retention times 26.4 and 29.1, were only detected in I. asarifolia. Likewise some indole diterpenes were tissue type specific in a given species; for example, paxilline and two isomers with retention times of 23.1, 30.2, and 30.9 min, respectively, were detected in the leaves of I. asarifolia but not the seeds, while tentatively assigned terpendole I (retention time = 18.9 min) and one terpendole I isomer (retention time = 29.7 min) were detected in I. asarifolia seeds but not leaves. Species specific differences in the occurrence of the various indole diterpenes may be useful in future chemosystematic studies. The results reported herein confirm the previous report of the occurrence of paspaline, paxilline, and terpendole C in I. asarifolia.10 Schardl et al.10 tentatively reported the presence of terpendole E and K as authentic standards were unavailable. Here we report the unequivocal presence of these two terpendoles in I. asarifolia along with the structure determination of a new indole diterpene from I. asarifolia, 6,7dehydroterpendole A, 3. In addition, we report the presence of several isomers of paspaline, paxilline, terpendole C, and terpendole E in I. asarifolia. Several other indole diterpenes were tentatively reported to be present in I. asarifolia by Schardl et al. including IDT-436 and terpendoles A, I, J, M, and N.10 We tentatively assigned the presence of terpendoles I and J based upon HPLC−HRMS, HPLC−CID-MS/MS, HPLC− HCD-MS/MS data and retention time correlation with Tomoda et al.22 and detected the presence of several isomers of these compounds. We confirmed the presence of a single compound that was tentatively assigned as an isomer of terpendole A or M; however, we were unable to assign the corresponding terpendoles as these compounds are isomers. We were unable to confirm the presence of terpendole N and IDT-436 as we could not find a report of a structure or molecular formula of these compounds in the literature. We reported the presence and tentatively assigned terpendole D, terpendole H, and emindole SB based upon HPLC−HRMS, HPLC−CID-MS/MS, HPLC−HCD-MS/MS data and retention time correlation with Tomoda et al.22 In addition, we reported the presence of several isomers of terpendole H. Additional compounds that may be isomers of the terpendoles were detected; however, we only tentatively reported those with observed m/z 130 and m/z 182 MS/MS fragments as indole diterpenes. For example, compounds that may be isomers of terpendole E and terpendole J at retention times of 21.3 and 28.3 min, respectively, were detected by HPLC−HRMS (Figure 3) in I. asarifolia leaf material but did not fragment to m/z 130 and m/z 182 under CID and HCDMS/MS conditions. As a result, these compounds as well as several other compounds were not assigned as indole diterpenes. It should be noted that some of these compounds may be indole diterpenes but do not give m/z 130 and m/z 182 fragments due to substitution of the A-ring like paspalitrem A and B.20 Additionally, observed but unreported HPLC−HRMS and HPLC−MS/MS data suggest that there are several other unknown indole diterpenes in both species which have yet to be isolated and structurally characterized.

Figure 5. Reversed-phase HPLC retention time correlation with Tomoda et al.22

Isomers of terpendole K, 6,7-dehydroterpendole A, terpendole C, terpendole E, paxilline, and paspaline were detected in I. asarifolia and/or I. muelleri plant material based on HPLC− HRMS, HPLC−CID-MS/MS, and HPLC−HCD-MS/MS data (Figures 2 and 3). The CID and HCD MS/MS mass spectra for the chromatographic peaks of these isomers included MS/ MS fragments at m/z 130 and 182 characteristic of indole diterpenes (Figure 1 and Table 1).20,21 Three isomers of terpendole K eluting at 26.1, 27.2 (11-hydroxy,12,13epoxyterpendole K, 2), and 31.4 min were detected in I. muelleri plant material. Three isomers of 6,7-dehydroterpendole A, 3, eluting at 18.6, 23.2, and 24.6 min were detected in I. muelleri seeds. Paspalitrem B, an indole diterpene, is an isomer of terpendole K; however, the CID mass spectra of paspalitrem B reported by Uhlig et al.20 are not consistent with the CID mass spectra of the terpendole K isomers detected in I. asarifolia and I. muelleri. An isomer of terpendole C and an isomer of terpendole E that eluted at 30.4 and 35.1 min, respectively, were detected in I. asarifolia and in I. muelleri plant material. Isomers of paxilline eluting at 18.9, 29.7, and 30.2 min were detected in I. muelleri plant material. These isomers and an additional paxilline isomer eluting at 30.9 min were detected in I. asarifolia plant material. I. asarifolia and I. muelleri leaves and seeds were also analyzed for additional known indole diterpenes for which standards and complete HPLC retention and CID MS/MS data were unavailable. Using HPLC−HRMS, HPLC−CID-MS/MS, and HPLC−HCD-MS/MS, 22 different compounds with exact mass data and MS/MS fragments of m/z 130 and 182 that correspond to isomers of nine known indole diterpenes, including 13-desoxypaxilline, 8; paxitriol, 9; terpendole I, 10; terpendole J, 11; terpendole A, 12; or M, 13; terpendole B, 14; terpendole D, 15; terpendole H, 16; and emindole SB, 17, were detected. Tentative assignments for terpendoles I, H, J, D, B, and emindole SB with retention times of 18.9, 20.4, 30.2, 33.6, 26.4, 35.1, respectively, were made based on HPLC−HRMS, HPLC−CID-MS/MS, and HPLC−HCD-MS/MS data and retention time correlation with Tomoda et al.22 (Figure 5). 5275



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preliminary analysis of the Ipomoea asarifolia leaf material. We thank Charles Hailes and Jessie Roper for technical assistance.

We suspect that the indole diterpenes are primarily responsible for the tremorgenic syndrome in livestock poisoned by I. asarifolia and I. muelleri. Indole diterpenes such as terpendole C, 5; paxilline, 6; and terpendole M, 13, have been shown to be tremorgenic while others are apparently not tremorgenic as pure compounds.21,24,25 It is possible that some of the other structurally uncharacterized indole diterpenes detected in both these taxa may be tremorgenic as well. However, both these species contain other bioactive metabolites from the Convolvulaceae or their symbionts that may contribute to toxicity or potentiate the toxicity of the indole diterpenes; for example, both species contain the ergot alkaloids,17 and I. muelleri contains the calystegines9 but I asarifolia does not.3 This is the first detailed MS analysis of known indole diterpenes and possible isomers in these two Ipomoea species. The high-resolution mass spectrometric data in combination with the complementary CID and HCD fragmentation data provided valuable information for indole diterpene characterization. Two new indole diterpenes, 11-hydroxy-12,13-epoxyterpendole K, 2, and 6,7-dehydroterpendole A, 3, were isolated and their structures determined by 1D and 2D NMR spectroscopy. We confirmed the previous report of the indole diterpenes in I. asarifolia; to our knowledge this is the first report of terpendole K in plants and terpendole E in I. asarifolia. This is the first report of the indole diterpenes being present in I. muelleri. In addition, we demonstrate that there are several isomers of the terpendoles in Ipomoea species previously not reported. We suspect that the indole diterpenes in these species are likely responsible for the tremorgenic syndrome observed in livestock grazing these species.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: 435-752-2941. Fax: 435-753-5681. E-mail: stephen. [email protected]. ORCID

Stephen T. Lee: 0000-0002-0597-8353 Funding

We thank Utah State University and the National Science Foundation (Grant CHE-1429195) for use of and funding for the Bruker 500 MHz NMR. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Franklin Riet-Correa, National Institute for Agricultural Research, LaEstanzuela, Colonia, Uruguay, for providing the Ipomoea asarifolia seed for analysis and from which plants were grown. We thank Jeremy Allen, Department of Agriculture and Food, Western Australia, South Perth, Western Australia, Australia, for discussions regarding Ipomoea muelleri and the observed tremorgenic syndrome in livestock. We thank the Western Australian Herbarium, Kensington, Western Australia, for providing Ipomoea muelleri plant material for analysis. We thank Wade Mace, Grasslands Research Centre, AgResearch, Palmerstown, North, New Zealand, for a 5276



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(18) Beaulieu, W. T.; Panaccione, D. G.; Ryan, K. L.; Kaonongbua, W.; Clay, K. Phylogenetic and chemotypic diversity of Periglandula species in eight new morning glory hosts (Convolvulaceae). Mycologia 2015, 107, 667−678. (19) Rasmussen, S.; Lane, G. A.; Mace, W.; Parsons, A. J.; Fraser, K.; Xue, H. The use of genomic and metabolomics methods to quantify fungal endosymbionts and alkaloids in grasses. In Plant Metabolomics: Methods and Protocols; Methods in Molecular Biology, Vol. 860; Hardy, N. W., Hall, R. D., Eds.; Springer: New York, 2012; pp 213− 226. (20) Uhlig, S.; Botha, C. J.; Vralstad, T.; Rolen, E.; Miles, C. O. Indole-diterpenes and ergot alkaloids in Cynodon dactylon (Bermuda grass) infected with Claviceps cynodontis from an outbreak of tremors in cattle. J. Agric. Food Chem. 2009, 57, 11112−11119. (21) Gatenby, W. A.; Munday-Finch, S. C.; Wilkins, A. L.; Miles, C. O.; Terpendole, M. a novel indole-diterpenoid isolated from Lolium perenne infected with the fungus Neotyphodium lolii. J. Agric. Food Chem. 1999, 47, 1092−1097. (22) Tomoda, H.; Tabata, N.; Yang, D.-J.; Takayanagi, H.; Omura, S. Terpendoles, novel ACAT inhibitors produced by Albophoma yamanashiensis III. Production, isolation and structure elucidation of new compounds. J. Antibiot. 1995, 48, 793−804. (23) Huang, X.-H.; Nishida, H.; Tomoda, H.; Tabata, N.; Shiomi, K.; Yang, D.-J.; Takayanagi, H.; Omura, S. Terpendoles, novel ACAT inhibitors produced by Albophoma yamanashiensis II. Structure elucidation of terpendoles A, B, C and D. J. Antibiot. 1995, 48, 5−11. (24) Munday-Finch, S. C.; Wilkins, A. L.; Miles, C. O.; Tomoda, H.; Omura, S. Isolation and structure elucidation of lolilline, a possible biosynthetic precursor of the lolitrem family of tremorgenic mycotoxins. J. Agric. Food Chem. 1997, 45, 199−204. (25) Miles, C. O.; Wilkins, A. L.; Gallagher, R. T.; Hawkes, A. D.; Munday, S. C.; Towers, N. R. Synthesis and tremorgenicity of paxitriols and lolitriol: possible biosynthetic precursors of lolitrem B. J. Agric. Food Chem. 1992, 40, 234−238.

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Identification of Indole Diterpenes in Ipomoea asarifolia and Ipomoea

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