Identification and Structural Elucidation of Ergotryptamine, a New

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Identification and Structural Elucidation of Ergotryptamine, a New Ergot Alkaloid Produced by Genetically Modified Aspergillus nidulans and Natural Isolates of Epichlo# Species Katy L. Ryan, Novruz G Akhmedov, and Daniel G. Panaccione J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505718x • Publication Date (Web): 10 Dec 2014 Downloaded from http://pubs.acs.org on December 13, 2014

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Journal of Agricultural and Food Chemistry

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Identification and Structural Elucidation of Ergotryptamine, a New Ergot Alkaloid

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Produced by Genetically Modified Aspergillus nidulans and Natural Isolates of

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Epichloё Species

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Katy L. Ryan,† Novruz G. Akhmedov,§ and Daniel G. Panaccione*,†

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†

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Morgantown WV, 26505-6108

Division of Plant and Soil Sciences, West Virginia University, 1090 Agricultural Sciences,

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§

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6045

Department of Chemistry, West Virginia University, 217 Clark Hall, Morgantown WV, 26505-

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*Corresponding author (Tel: 1-304-293-8819; E-mail: [email protected])

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ACS Paragon Plus Environment


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ABSTRACT: Ergot alkaloid pathway reconstruction in Aspergillus nidulans is an approach used

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to better understand the biosynthesis of these mycotoxins. An engineered strain named A.

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nidulans WFC (expressing ergot alkaloid synthesis genes dmaW, easF, and easC) produced the

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established intermediate N-methyl-dimethylallyltryptophan, as well as an uncharacterized ergot

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alkaloid. We investigated the chemical structure of the new metabolite and its role in the ergot

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alkaloid pathway. Mass spectrometry, labeling, and NMR studies showed that the unknown

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ergot alkaloid, designated here as ergotryptamine, differed from N-methyl-

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dimethylallyltryptophan by the loss of the carboxyl group, addition of a hydroxyl group, and

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shift in position of a carbon-carbon double bond. Feeding studies with Aspergillus mutants did

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not show ergotryptamine turnover suggesting it is a pathway byproduct as opposed to an

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authentic intermediate. Several Epichloё species also produced this metabolite, and further

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investigations revealed the equivalency of ergotryptamine with an Epichloё-derived ergot

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alkaloid provisionally described as 6,7-secolysergine.

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KEYWORDS: Ergot alkaloids, mycotoxins, ergotryptamine, 6,7-secolysergine, Aspergillus,

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Epichloё, NMR

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INTRODUCTION

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Ergot alkaloids are a class of indole-derived mycotoxins with agricultural and pharmaceutical

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significance. Several fungi in the family Clavicipitaceae (order Hypocreales) and a few fungi in

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the distantly related family Trichocomaceae (order Eurotiales) produce these secondary

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metabolites.1,2 The Epichloë spp. (Clavicipitaceae) are endophytes of turf and forage grasses

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(Poaceae, subfamily Poöideae) and synthesize a variety of compounds toxic to vertebrates and

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invertebrates.3 Ergot alkaloids in these grass symbionts cause toxicoses in grazing animals and

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are responsible for significant economic loss to livestock industries.4,5 The phylogenetically

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divergent fungus Aspergillus fumigatus (Trichocomaceae) is a common saprotroph and

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opportunistic human pathogen that causes spoilage of feed in agriculture settings.1,6,7 Compared

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to the Epichloë spp., A. fumigatus produces a different subset of ergot alkaloids but can serve as

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a prominent model organism for studying ergot alkaloid biosynthesis.8-12

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There is considerable diversity in the later steps of the ergot alkaloid pathway due to

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genetic variation within ergot alkaloid gene clusters of different fungi; however, there is strong

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evidence that the early steps of the pathway leading to the formation of intermediate

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chanoclavine-I, 7, are conserved (Figure 1).1,2,13 It was recently discovered that four genes,

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dmaW, easF, easC, and easE, amplified from the A. fumigatus genome are sufficient to direct

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synthesis of 7 when transgenically expressed in Aspergillus nidulans, a model fungus that does

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not contain ergot alkaloid synthesizing genes or produce ergot alkaloids.11 DmaW performs the

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determinant step of ergot alkaloid synthesis by prenylating tryptophan, 2, to produce

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dimethylallyltryptophan, 3.9,10,14 The easF gene encodes a methyltransferase responsible for

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converting 3 into N-methyl-dimethylallyltryptophan, 4.12 The exact reactions catalyzed by EasE

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and EasC have not yet been determined, but genetic analyses demonstrate that these two genes

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are required to convert 4 to 7.8,11,15 Bioinformatics suggest that EasC is a catalase, whereas EasE

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is an oxidoreductase.8,11,15 A scheme of conversion from 4 to 7 has been proposed by

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Kozikowski et al.16 (Figure 1).

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Previously Ryan et al.11 investigated the roles of EasC and EasE, as well as the

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intermediates they produced, by reconstructing the early steps of ergot alkaloid synthesis in A.

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nidulans. A mutant strain named A. nidulans WFC (containing the genes dmaW, easF, and easC

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from A. fumigatus) accumulated ergot alkaloid pathway intermediate 4 as well as an

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uncharacterized ergot alkaloid of m/z 259 (referred to here as alkaloid X) not seen in control

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strains or the nearly isogenic transformant A. nidulans WFE (expressing dmaW, easF and easE),

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which synthesized 4 but no unusual metabolites. Based on the genetic background of the

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modified A. nidulans strain that produces this compound and the compound’s chromatographic

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and fluorescence properties, alkaloid X was proposed to be a derivative of 4. In this study, we

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investigated the chemical structure of this new ergot alkaloid, its role as a pathway intermediate

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or byproduct, and whether natural ergot alkaloid producers accumulated this metabolite.

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MATERIALS AND METHODS

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Large scale extraction and isolation of ergot alkaloids. Aspergillus nidulans

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strain WFC was previously generated by Ryan et al.11 Cultures were grown on modified ST

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medium (20 g/L sucrose, 20 g/L tryptone, 1 g/L MgSO4, 2 mL/L trace element solution,17 50

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mg/L nicotinic acid, 1 g/L tryptophan, 20 mL/L corn steep liquor, and 15 g/L agar) for 7 d at 37

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°C. Ergot alkaloids were extracted by incubating fungal mats in 10% aqueous ammonium

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carbonate (25 mL solvent per one 100 × 15 mm Petri dish) for 2 h followed by centrifugation at

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5000 rpm for 10 min to pellet spores and mycelium. The extract was purified over an Isolute

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C18 SPE column (500 mg/3 mL) (Biotage, Charlotte, NC) as described previously.19 Alkaloid X

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was eluted in 3 mL 98% methanol:2% (v/v) glacial acetic acid for NMR spectroscopy or 99.95%

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methanol:0.05% (v/v) glacial acetic acid for Aspergillus feeding studies. For NMR analyses,

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alkaloid X was purified in batches by analytical high performance liquid chromatography

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(HPLC), reapplied to an Isolute C18 column and, after elution, dried under vacuum. For

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Aspergillus feeding studies, eluate was concentrated to a final volume of 10 µL.

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Feeding studies with labeled tryptophan and A. nidulans WFC. Ten mg of L-

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tryptophan-d5 (indole d5) or L-tryptophan-1-13C (both from Sigma-Aldrich, St. Louis, MO) was

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dissolved in 1 mL water, filter sterilized through a 0.22-µm filter, and added to 99 mL of SYE

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agar (20 g/L sucrose, 20 g/L yeast extract, 1 g/L MgSO4, 50 mg/L nicotinic acid, 2 mL/L trace

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element solution) after autoclaving but prior to solidification of the medium. This medium was

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inoculated with A. nidulans WFC and incubated for one week at 37 °C. No deleterious effects of

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growing A. nidulans on these labeled derivatives of tryptophan were noted. Ergot alkaloids were

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extracted by repeatedly washing the fungal culture surface with 2 mL of methanol. The

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suspended cultures were incubated in methanol for 30 min, clarified by centrifugation,

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concentrated in a vacuum centrifuge to 50 µL, and analyzed by electrospray ionization liquid

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chromatography/mass spectrometry (ESI LC/MS).

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Feeding study with alkaloid X and Aspergillus mutants. Aspergillus nidulans

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strain WFE was described in Ryan et al.11 and A. fumigatus easC and easE knockout mutants

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were previously generated by Goetz et al.8 A total of 1x106 spores was inoculated in 200 µL of

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liquid sucrose malt medium (15 g/L malt extract broth and 20 g/L sucrose) supplemented with 50

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µmol alkaloid X and incubated for 5 d at 37 °C. Ergot alkaloids were extracted by the addition

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of 200 µL methanol, vortexing briefly, and incubating for 30 min at room temperature, followed

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by centrifugation for clarification. Samples were then analyzed by HPLC.

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Yield of alkaloid X. Aspergillus nidulans strain WFC was grown on ST agar for 10 d

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at 37 °C. One square cm, surface area, of fungal culture was vortexed in 400 µL methanol and

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incubated for 30 min. The number of spores/µL suspension, which is measure of fungal growth,

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was counted on a hemacytometer (Hausser Scientific, Horsham, PA). Samples were centrifuged

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to remove fungal matter, followed by HPLC analysis. Metabolite yield was calculated by

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comparison of peak areas to an ergonovine (Sigma, St. Louis, MO) standard curve and

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normalized to the concentration of conidia per µL.

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Extraction of ergot alkaloids from Epichloë sp.-infected grasses. An lpsA

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knockout strain Epichloё festucae var. lolii × E. typhina isolate Lp1 (henceforth called E. sp.

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Lp1) was previously generated and inoculated into perennial ryegrass (Lolium perenne).18,19

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Other Epichloë spp.-infected grasses were provided by Christopher Schardl (University of

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Kentucky). The Epichloë spp. studied included several species recently realigned from the genus

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Neotyphodium and have previously been known by their Neotyphodium sp. names.20 Ergot

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alkaloids were extracted from fungal-infected grass pseudostems as previously described.21

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High Performance Liquid Chromatography (HPLC). Samples were loaded onto a

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150 mm × 4.6 mm i.d., 5-µm ODS3 Prodigy C18 column (Phenomenex, Torrance, CA) and

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subjected to a gradient of mobile phases A (5% v/v acetonitrile:95% v/v 50 mM ammonium

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acetate) and B (75% acetonitrile:25% 50 mM ammonium acetate) as previously described.21

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Ergot alkaloids were detected by their fluorescence at two detector settings: 272 nm excitation

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and 372 nm emission, and 310 nm excitation and 410 nm emission. For purification of alkaloid

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X, A. nidulans WFC extracts purified by Isolute column chromatography were applied to the

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analytical HPLC column and runoff was collected for 30 s after alkaloid X was visualized by the

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detectors. Multiple batches (>10) were processed, and runoff was concentrated together for a

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final yield of 200 µg.

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Electrospray Ionization Liquid Chromatography/Mass Spectrometry (ESI

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LC/MS). For most analyses (other than comparison of alkaloid X from A. nidulans WFC to

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compounds extracted from Epichloë sp.-infected grasses), 10 µL of concentrated fungal extract

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was injected into a Finnigan LCQ DecaXP plus mass spectrometer equipped with a Surveyor

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HPLC system. Analytes were separated on a 150 mm × 2 mm i.d., 4 µm polar-RP C18 column

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(Phenomenex) maintained at 30 °C, by combining mobile phases A (5% acetonitrile:0.1% formic

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acid:94.9% water) and B (75% acetonitrile:0.1% formic acid:24.9% water) as described by Ryan

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et al.11 The flow rate was 200 µL/min. Analytes were ionized by electrospray ionization in

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positive mode and were detected by scanning for ions between m/z 200–400. MS2 scans for

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compounds exhibiting m/z 259.0 also were conducted. For isotope incorporation, m/z intensities

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were recorded at peak retention time (alkaloid X retention time = 22 min; 4 retention time = 33

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min) for the following peaks: m/z 259.0 [alkaloid X + H]+; m/z 260.0 [alkaloid X + 1 + H]+; m/z

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263.0 [alkaloid X + 4 + H]+; m/z 287.0 [4 + H]+; m/z 288.0 [4 + 1 + H]+; and, m/z 291.0 [4 + 4 +

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H]+. Eight intensity readings for each metabolite were recorded around peak intensity. Student’s

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t-test was performed with JMP (SAS, Cary, NC) to compare sample means of m/z intensity ratios

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for alkaloid X and 4 between samples grown on labeled 2 supplemented medium and

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unsupplemented medium.

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Analyses of alkaloid X from A. nidulans WFC in comparison to compounds extracted

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from Epichloë sp.-infected grasses were conducted with different instrumentation and methods.

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Twenty µL of concentrated sample was analyzed on a ThermoScientific Q Exactive mass

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spectrometer (ThermoScientific, Waltham, MA). Analytes were separated on a 50 × 2.1 mm i.d.,

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1.9-µm particle size, end-capped, Hypersil GOLD column (Thermo Scientific) maintained at 30

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°C, by combining mobile phases A (5% acetonitrile:0.1% formic acid:94.9% water) and B (75%

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acetonitrile:0.1% formic acid:24.9% water) over a 10 min linear gradient of 0% B to 100% B.

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The flow rate was 300 µL/min. Analytes were ionized by electrospray ionization in positive

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mode and were detected by scanning for ions between m/z 200–400. Alkaloid X had an accurate

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mass of 258.17337 Da, which deviated by only 0.62 ppm from the theoretical mass of

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C16H22N2O.

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NMR measurements. The 1D and 2D NMR spectra were recorded using a Varian

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INOVA 600 MHz spectrometer at 25 °C operating at 599.67 MHz and 150.79 MHz for proton

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and carbon, respectively, and equipped with a triple-resonance z-axis pulsed field gradient 5 mm

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probe. Sample concentration was 0.2 mg/0.7 mL (0.001 M). The FIDs of the 1- and 2-

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dimensional NMR spectra were processed using the commercially available NMR software

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package ACD/SpecManager, Product Version 12 (Advanced Chemistry Development, Inc.,

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Toronto, Ontario, Canada). 1H NMR chemical shifts are reported relative to the

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tetramethylsilane (TMS) internal standard peak at 0 ppm.

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in DMSO-d6 relative to the central peak of the septet set to 39.5 ppm. The typical parameters for

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acquiring the 1H NMR spectra were as follows: spectral widths 7807.16 Hz, acquisition time 4.0

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s, pulse width 3.16 µs (45°), relaxation time 2 s, and number of transients 128. The parameters

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for the gradient selected 1D TOCSY were applied using TOCSY1D program with spectral width

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of 7807.16 Hz, acquisition time of 4.0 s, pulse width 9.5 µs (90°), relaxation time of 1 s, mixing

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time was set to 80 ms, and the number of transients used was 128. The FIDs were processed

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with a line broadening function of 1 Hz (lb). The gradient selected gCOSY spectra were

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acquired over both F2 and F1 with a spectral width of 7807.16 Hz, pulse width 9.0 µs (90°),

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C NMR chemical shifts are reported


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relaxation delay 1 s, and 512 time increments of 64 transients each in magnitude mode. The

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FIDs were zero filled to give a 4 K × 4 K data matrix and a sine-bell function was applied in both

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dimensions (F1 and F2) prior to Fourier transformations giving a digital resolution of 3.81 Hz.

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Adiabatic version of gradient selected gHSQCAD and gHMBCAD spectra were acquired in

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phase sensitive mode with a spectral width of 7807.16 KHz for 1H (F2 dimension), a spectral

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width of 37700 Hz for 13C (F1 dimension) by using a 2048 × 512 data matrix size and 128

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transients for each t1 value. The carbon decoupler 90° pulse width was 10.4 µs, one-bond

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coupling j1xh was set to 145 Hz. For evolution of long-range correlation, the multiple bond CH

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coupling constant jnxh was set to 8 Hz and relaxation delay was set to 1.5 s. The FIDs of proton

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detected gHSQCAD were processed with a Gaussian weighting function in both dimensions,

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whereas the FIDs of proton detected gHMBCAD were processed with a sine bell weighting

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function in F2 and with a Gaussian weighting function in F1 (carbon dimension).

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The NMR parameters (chemical shifts and coupling constants) for alkaloid X were

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determined by iterative analysis using the gNMR program. As starting data the iterative process

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used 1H NMR chemical shifts and coupling constants estimated from the experimental spectra.

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RESULTS AND DISCUSSION

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Insights on the structure of alkaloid X. Aspergillus nidulans WFC cultures grown

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on ST medium or ST medium supplemented with indole d5 labeled tryptophan, 2, were analyzed

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by ESI LC/MS for the incorporation of deuterated 2 into alkaloid X as well as into the known

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ergot alkaloid intermediate N-methyl-dimethylallyltryptophan, 4. During the first step of ergot

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alkaloid synthesis, DmaW replaces a deuterium on indole-carbon 5 with a prenyl group resulting

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in accumulation of ions with m/z values consistent with [M + 4 + H]+ rather than [M + 5 + H]+ in

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cultures supplemented with indole d5 labeled 2. Fungal cultures grown on medium

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supplemented with d5 labeled 2 contained a significantly higher ratio of intensity values of m/z

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291.0 [4 + 4 + H]+ to m/z 287.0 [4 + H]+ compared to control cultures grown on medium lacking

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d5 labeled 2 (Figure 2). Similarly, extracts from fungi grown on medium supplemented with d5

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labeled 2 had a significantly higher ratio of intensity values of m/z 263.0 [alkaloid X + 4 + H]+ to

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m/z 259.0 [alkaloid X + H]+, compared to controls (Figure 2). In conjunction with genetic data

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that demonstrated accumulation of alkaloid X is dependent on expression of three ergot alkaloid

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biosynthesis genes,11 the incorporation of deuterated 2 into alkaloid X confirms the new alkaloid

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is a derivative of 2, and the accretion of m/z values consistent with [M + 4 + H]+ rather than [M +

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5 + H]+, similar to what was observed with 4, supports its membership in the ergot alkaloid

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family.

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Based on high-resolution ESI LC/MS analyses, alkaloid X has a molecular formula of

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C16H22N2O. When considered in relation to 4, the molecular formula of alkaloid X is consistent

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with a loss of the carboxyl group and addition of an oxygen. The absence of the carboxyl group

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in alkaloid X was tested by culturing A. nidulans WFC on medium supplemented with L-

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tryptophan-1-13C. The presence of 4 in these cultures provided a positive control for the

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incorporation of L-tryptophan-1-13C. ESI LC/MS analyses of extracts from these cultures

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showed a significantly greater ratio of intensity values of m/z 288.0 [4 + 1 + H]+ to m/z 287.0 [4

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+ H]+ compared to extracts from non-supplemented control cultures (Figure 3), consistent with

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presence of the 2-derived carboxyl group in 4. However, there was no difference in the ratio of

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intensity values of m/z 260.0 [alkaloid X + 1 + H]+ to m/z 259.0 [alkaloid X + H]+ in cultures

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grown on supplemented medium compared to controls (Figure 3). The lack of increased 13C

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isotope incorporation into alkaloid X demonstrated the absence of the carboxyl group derived

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from 2 in the compound.

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Considering genetic data on the origin of alkaloid X11 along with mass spectrometric,

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labeling, and fluorescence data, certain aspects of the structure of the compound can be

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proposed. The compound appears to be a derivative of 4, containing a double bond conjugated

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to the indole ring, and lacking the carboxyl group. The compound also has an additional oxygen,

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which we hypothesized was located on the dimethylallyl-derived carbon 16 (customarily called

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carbon 8 in ergot alkaloids) as seen in 8 (Figure 4). Oxidation of 4 at this position would result

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in the double bonding shifting from the 15,16 position to the 14,15 position (conjugated to the

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indole ring), accounting for the fluorescent properties of the molecule.

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Structural elucidation of alkaloid X by NMR techniques. The 1D and 2D NMR

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techniques including 1H, 1D TOCSY, gradient selected 1H-1H gCOSY, and adiabatic versions of

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proton detected gHSQCAD and gHMBCAD experiments were used to investigate the chemical

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structure of alkaloid X, more specifically to identify the positions of the hydroxyl group and

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carbon double bond. In order to support the structural integrity of the new metabolite, 1H and 13C

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NMR spectra of 2 acquired in DMSO-d6 were accomplished with the aid of 2D NMR techniques

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including gCOSY, gHSQCAD and gHMBCAD. The lack of downfield proton H5 (δ 7.57) of

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the indole moiety of 2 confirms H5 substitution by the prenyl group (Figure 5). The 13C NMR

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spectrum of alkaloid X was not acquired due to insufficient quantities (concentration 0.001 M).

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Aspergillus nidulans WFC cultures produced a low concentration of alkaloid X, with an average

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yield of 58 fg/spore.

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The 1H NMR spectrum (Figure 5) of alkaloid X in DMSO-d6 exhibited four distinct

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downfield aromatic protons of the indole moiety at δ 7.10 (doublet, J = 2.4 Hz), δ 7.20 (doublet

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of doublets, J = 7.5, 0.8 Hz), δ 6.99 (doublet of doublets, J = 7.9, 7.5 Hz), and δ 7.01 (doublet of

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doublets, J = 7.9, 0.8 Hz) assigned as H2, H6, H7, and H8, respectively. Proton-proton

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connectivities (H6-H7-H8) through three bonds deduced from gCOSY spectrum was supported

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by 1D TOCSY experimental subspectra. Similarly, the contour plot of the gCOSY spectrum

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revealed cross peaks between the doublets integrating for one proton each at δ 6.20 and δ 7.17

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with a coupling constant of 3Jtrans= 15.8 Hz which were assigned to H14 and H15, respectively.

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Two upfield singlets integrating for six and three protons at δ 1.31 and δ 2.33, respectively were

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assigned to H13, H17 and H18. In the 1H NMR spectrum two aliphatic methylene (CH2) protons

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appeared at δ 2.77 (triplet, J = 7.5 Hz) and δ 2.98 (triplet, J = 7.5 Hz). The chemical shift

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assignments of these methylene protons (10-CH2 and 11-CH2) were based on the observed two

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and three bond gHMBCAD correlations. The methylene protons at δ 2.98 showed long-range

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correlations to the carbon resonances C2, C3, and C4 of the indole moiety at δ 123.75, δ 113.80,

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and δ 124.24, respectively, which was immediately assigned to the 10-CH2. Due to unresolved

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coupling between 16-CH3 and H15, the doublet lines at δ 7.17 were broadened which was

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assigned to H15. Similarly, the doublet of doublets at δ 7.01 is broadened due to unresolved

250

small couplings (5JHH) and was assigned to H8. Unambiguous assignments of H6 and H8 can be

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also accomplished using DPFGSENOE spectra. Since the concentration of alkaloid X was very

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low [0.001M (or 0.2 mg/0.7 mL)] this experiment was not performed. NH (position 12) and OH

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protons are exchangeable with water in solution and consequently did not appear in the 1H NMR

254

spectrum. However, the NH (position 1) proton of the indole ring showed broad singlet at δ

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10.80. The assigned NMR parameters [1H and 13C chemical shifts (δ/ppm) and coupling

256

constants (J/Hz)] are listed in Table 1.

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Utilization of gradient selected 1D TOCSY experiment permitted a sequential assignment

258

of the spin systems for the different structural segments [(C10-C11), (C6-C7-C8), and (C14-C15)] of

259

alkaloid X by means of bond connectivities. The use of the 1D TOCSY technique removed all

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undesired proton frequencies and gave a clean, selective excitation 1H NMR subspectrum. All

261

selective excitation subspectra showed only those hydrogens which are in the same 1H coupling

262

network.

263

Once all proton connectivities were established, an iterative procedure was used to

264

extract precise values of coupling constants for resonances of each proton (Figure 5). The

265

determined values of couplings derived from the experimental 1H NMR spectrum were used as

266

starting couplings for an iteration of multiplicity patterns for each proton peak, generating the

267

accurate values of coupling constants. A good match between calculated and experimental

268

splitting patterns of these protons confirmed their chemical shift assignments are correct.

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A gradient version of proton-detected gHSQCAD spectrum was recorded to identify

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proton-bearing carbons (2-CH, 6-CH, 7-CH, 8-CH, 10-CH2, 11-CH2, 13-CH3, 14-CH, 15-CH,

271

17-CH3, and 18-CH3) on the basis of one bond correlations. The assignment of five quaternary

272

carbons (C3, C4, C5, C9, C16) was extracted on the basis of two (2JH-C) or three (3JH-C)

273

gHMBCAD correlations. The contour plot of the gHMBCAD spectrum revealed a two bond

274

correlation between 16-CH3 at δ 1.31 and carbon peak at δ 69.62 which is a typical chemical

275

shift for tertiary carbon atom connected to OH group.

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Complementary to the MS data, the analysis of the 1D and 2D NMR spectra confirm that

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alkaloid X is compound 8 (Figure 4). This unique ergot alkaloid produced by A. nidulans WFC,

278

(3E)-2-methyl-4-{3-[2-(methylamino)ethyl]-1H-indol-4-yl}but-3-en-2-ol, was given the trivial

279

name ergotryptamine due to its origin in the ergot alkaloid pathway and its structural similarity

280

to tryptamine.

281 282

Role of ergotryptamine (alkaloid X) in the ergot alkaloid pathway. Ergotryptamine, 8, is a new member of the ergot alkaloid family. Genetic data11 indicate that 8

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is derived from 4. Structural data reveal that 8 is a bicyclic ergot alkaloid lacking the carboxyl

284

group found in precursor 4. The biochemical scheme proposed for chanoclavine-I, 7, synthesis

285

(Figure 1) suggests that decarboxylation coincides with closure of the third ergoline ring.16 Here

286

we show that decarboxylation can occur without ring closure, revealing that under some

287

conditions these events may occur via independent reactions. Through comparison of ergot

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alkaloid profiles of A. nidulans WFE and A. nidulans WFC (which differ only in the presence of

289

EasC versus EasE and the accumulation of 8 exclusively in A. nidulans WFC), we propose that

290

decarboxylation of 4 or its oxidized derivative requires EasC. Enzymatic studies need to be

291

performed to test the hypothesis that EasC can act as a decarboxylase.

292

The role of 8 as a pathway intermediate or byproduct was tested by feeding the

293

metabolite to Aspergillus mutants followed by HPLC analyses for the detection of downstream

294

pathway products. Aspergillus nidulans WFE transformants and A. fumigatus easC knockout

295

(ko) and A. fumigatus easE ko strains, all of which contain downstream pathway enzymes, failed

296

to turn over 8. These data indicate that 8 accumulates as a pathway byproduct (or spur product)

297

that is incapable of being enzymatically converted to 7 or other pathway products.

298

Accumulation of ergotryptamine in Epichloë species and equivalency with

299

provisional 6,7-secolysergine. HPLC analyses revealed that ergotryptamine, 8, displayed

300

the same retention time (41 min) and fluorescence properties as a molecule previously observed

301

in E. sp. Lp1 and provisionally named 6,7-secolysergine, 9.19 (Figures 4 and 6). This molecule

302

accumulated twice as abundantly in E. sp. Lp1 lpsA ko as compared to wild-type E. sp. Lp1,19 so

303

extracts of that mutant were studied to test the identity of 8 with provisional 9. Extracts from E.

304

sp. Lp1 lpsA ko-infected grasses were analyzed by ESI LC/MS to further investigate potential

305

accumulation of 8. The Q Exactive high resolution ESI LC/MS revealed co-eluting peaks from

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A. nidulans WFC and E. sp. Lp1 lpsA ko-infected grasses with near identical m/z values of

307

259.18065 and 259.18066 (Figure 7). Ionization of 8 from A. nidulans WFC and the co-eluting

308

molecule from E. sp. Lp1 lpsA ko led to an abundance of ionized fragment of m/z 241.17

309

corresponding to [8 - H2O + H]+ (Figure 7) and also to the apparent molecular ion previously

310

observed for 9.19 This fragment was also visualized during analyses of 8 on the Finnigan LCQ

311

LC/MS used previously and was found to be the dominant ion formed from selective

312

fragmentation of 8. Extracts from E. sp. Lp1 lpsA ko-infected grasses displayed similar ratios of

313

m/z 259 parent ion to m/z 241 fragment ion (3.5:1) when compared to 8 from A. nidulans WFC

314

(3.7:1). These results demonstrate the accumulation of 8 as a natural product in the fungal

315

endophyte E. sp. Lp1 and provide support for the identity of 8 with the provisionally identified 9.

316

We propose that the apparent molecular ion detected by Panaccione et al.19 was the dehydrated

317

fragment of ergotryptamine, 10 (Figure 4), and that a provisional structure was proposed for the

318

m/z 241 compound without considering bicyclic ergot alkaloids.

319

Several other Epichloë spp. contained a metabolite that co-eluted with 8 when analyzed

320

by fluorescence HPLC (Table 2). Fungal extracts from two asexual hybrid species, E.

321

coenophiala in tall fescue (Lolium arundinaceum) and E. funkii in sleepy grass (Achnatherum

322

robustum) contained an analyte characteristic of 8. The sexual nonhybrid E. elymi produced 8 in

323

its host Canadian wild rye (Elymus canadensis). Although the genes necessary for synthesis of 8

324

are common to all ergot alkaloid producers, the accumulation of this metabolite appears to be

325

species specific, as we did not detect 8 in multiple extracts of E. gansuensis in drunken horse

326

grass (A. inebrians) or E. festucae var. lolii in perennial ryegrass (L. perenne).

327 328

Agricultural perspective. Ergot alkaloids cause toxicoses in livestock by interacting with multiple neurotransmitter receptors. The biological activities of ergotryptamine, 8, are

15



329

unknown, yet they remain of interest due to the accumulation of this metabolite in prominent

330

grass endophytes in the genus Epichloё. Feeding preference studies utilizing various mutant E.

331

sp. Lp1-infected grasses have shown that rabbits prefer to feed on non-ergot alkaloid producing

332

E. sp. Lp1 dmaW ko infected grasses rather than E. sp. Lp1 lpsA ko-infected grasses which

333

contained significant quantities of 7 and 8.22 The relative contributions of 8 relative to 7 in this

334

feeding deterrence is unknown. Further research into the biological effects of this metabolite are

335

needed to assess its bioactivity.

336

ASSOCIATED CONTENT

337

Supporting Information

338

Table S1: Exact mass of ergotryptamine and dehydrated fragment; Figure S1: NMR parameters

339

(chemical shifts and coupling constants) of tryptophan in DMSO-d6; Figure S2: gCOSY

340

spectrum of alkaloid X; Figure S3: Experimental 1D TOCSY subspectra of alkaloid X; Figure

341

S4: gHSQCAD and gHMBCAD specta of alkaloid X; Figure S5: Ergotryptamine fragment from

342

MS2. This material is available free of charge via the Internet at http://pubs.acs.org.

343

AUTHOR INFORMATION

344

Corresponding Author

345

*(D.G.P.) Telephone (304) 293-8819. E-mail: [email protected]

346

Funding

347

This study was supported by grant 2012-67013-19384 from USDA NIFA.

348

Notes

349

The authors declare no competing financial interest.

350

ACKNOWLEDGMENTS

351

This work was published with permission of the West Virginia Agriculture and Forestry

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Experiment Station as scientific article number 3224. We thank Simona Florea, Li Chen, Padma

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Nagabhyru, and Christopher Schardl (University of Kentucky) for generously providing samples

354

of endophyte-infected grasses. We also thank Brian Tapper and Geoff Lane (AgResearch, New

355

Zealand) for confirmatory analyses and helpful comments on the manuscript. We gratefully

356

acknowledge the use of the WVU Shared Research Facilities and the assistance of Huiyuan Li.

357

REFERENCES

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(1) Panaccione, D.G. Ergot Alkaloids. In The Mycota, Industrial Applications; 2nd ed.;

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Hofrichter, M., Ed.; Springer-Verlag: Berlin, Germany, 2010; Volume X, pp. 195–214.

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Machado, C.; Seidel, G.E. Effects of ergot alkaloids of food preference and satiety in rabbits, as

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FIGURE CAPTIONS

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Figure 1. Early steps of ergot alkaloid synthesis (modified from Kozikowski et al.16): 1,

430

dimethlyallypyrophosphate; 2, L-tryptophan; 3, dimethylallyltryptophan; 4, N-

431

methyldimethylallyltrptophan; 5, dienyl tryptophan intermediate; 6, hypothetical epoxide-

432

containing intermediate; and, 7, chanoclavine-I.

433

Figure 2. Derivation of alkaloid X, 8, from L-tryptophan, 2. Incorporation of four 2-derived

434

deuteriums into alkaloid X and N-methyl-dimethylallytryptophan, 4, was measured by recording

435

intensity values for compounds with m/z 259.0 and 263.0 (for alkaloid X) and m/z 287.0 and

436

291.0 (for 4). Error bars represent the standard error. Statistical significance in Student’s t-test

437

is represented by * (P

Identification and Structural Elucidation of Ergotryptamine, a New

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