Phytochemistry Letters 4 (2011) 79–85

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Phytochemistry Letters

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Ergosedmine, a new peptide ergot alkaloid (ergopeptine) from the ergot fungus,Claviceps purpurea parasitizing Calamagrostis arundinacea

Silvio Uhlig a,*, Dirk Petersen b, Elin Rolen a, Wolfgang Egge-Jacobsen c, Trude Vralstad a

a National Veterinary Institute, P.O. Box 750, Sentrum, N-0106 Oslo, Norwayb Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, 0315 Oslo, Norwayc Department of Molecular Biosciences, University of Oslo, P.O. Box 1033, Blindern, 0315 Oslo, Norway

A R T I C L E I N F O

Article history:

Received 25 May 2010

Received in revised form 20 September 2010

Accepted 26 September 2010

Available online 8 October 2010

Keywords:

Alkaloid

Calamagrostis arundinacea

Ergopeptine

Ergot

Purification

A B S T R A C T

A new natural ergopeptine, ergosedmine, was isolated from sclerotia of the ascomycete Claviceps

purpurea. Its structure was elucidated by 1D- and 2D-NMR spectroscopy and mass spectrometry as N-(N-

lysergyl)-cyclo(isoleucyl-phenylanalyl-prolyl). Partial epimerization of ergosedmine into ergosedmi-

nine was evident during purification and in DMSO-d6. According to common ergot alkaloid

nomenclature ergosedmine belongs to the ergoannines, one of the four subgroups of ergopeptines.

The sclerotia were obtained from reed feather grass (Calamagrostis arundinacea). Phylogenetic analyses

using ITS- and beta-tubulin sequences of pure culture isolates obtained from the C. purpurea sclerotia

demonstrated that the ergosedmine-producing strain belongs to the genotype G2, which is associated

with wet and shady habitats.

� 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

1. Introduction

Peptide ergot alkaloids (ergopeptines) are considered to beendproducts of ergot alkaloid biosynthesis (Schardl et al., 2006).They are among the most important natural pharmaceuticals andtoxins in human history (Schiff, 2006). Different analogues exerttheir effects by acting in some cases as partial agonists or asantagonists at receptors for 5-HT, dopamine and noradrenaline(Panaccione, 2005; Schardl et al., 2006). Ergot alkaloids also affectother organisms such as bacteria, nematodes and insects(Panaccione, 2005). Although the alkaloids are produced by fungirepresenting two different orders especially the sclerotia ofClaviceps species are known to contain a wide variety of ergotalkaloids (Buchta and Cvak, 1999; Flieger et al., 1997; Panaccione,2005). The multienzyme systems involved in the biosynthesis ofergot alkaloids are remarkably unspecific, and as a result a numberof natural ergopeptine analogues have been isolated and identifieddiffering in their amino acid composition of the peptide moiety(Flieger et al., 1997; Keller, 1999; Schardl et al., 2006).Traditionally, three ergopeptine subgroups, i.e. ergotamines,ergoxines and ergotoxines, according to L-alanine, L-2-aminobu-tyric acid and L-valine, respectively, as the amino acid closest to thelysergic acid part of the molecules have been defined (Flieger et al.,1997; Komarova and Tolkachev, 2001; Schardl et al., 2006). More

* Corresponding author. Tel.: +47 23216264; fax: +47 23216201.

E-mail address: silvio.uhlig@vetinst.no (S. Uhlig).

1874-3900/$ – see front matter � 2010 Phytochemical Society of Europe. Published by

doi:10.1016/j.phytol.2010.09.004

recently, the existence of a fourth subgroup containing L-isoleucineas the amino acid closest to the lysergic acid moiety has beendemonstrated and named ergoannines. One natural ergoannineanalogue has so far been reported (Buchta and Cvak, 1999; Szantayet al., 1994). The ability to produce different types of alkaloidsseems to be related to different chemoraces that are habitatspecialized rather than host specialized (Pazoutova et al., 2000). Ithas also been shown on the molecular level that habitat drivenadaptation has resulted in different C. purpurea lineages wherethere seems to be little or no gene flow between the genotypes.Three main lineages have been recognized inferred from parsimo-ny analyses of sequence data from three different loci; ITSsequences (internal transcribed spacer region of nuclear ribosomalDNA), partial beta-tubuline sequences (protein coding gene) andRAS-like sequences (putative protein coding gene). These lineages(referred to as genotype G1, G2 and G3) are more or less habitatspecific where G1 isolates are found on terrestrial grasses, G2isolates prefer wet and shady habitats while G3 isolates areadapted to salt marches (Douhan et al., 2008).

Herein, we describe the purification and characterization ofergosedmine (1) and its C-8 epimer, ergosedminine (2) (Fig. 1),from sclerotia of C. purpurea, and report the results fromgenotyping of the producing strain.

2. Results and discussion

During our study of the alkaloid composition of C. purpurea

sclerotia from wild grasses in Norway we discovered an

Elsevier B.V. All rights reserved.

[()TD$FIG]

HN

NCH3

H

NH

H

O O

N

O

N

O

H

HO

H

12

3

4

5

6

789

1011

12

13

14 1516

17

1819 3'

2'

1'

4' 5'6'

7'

8'

9'10'

11'

12'13'

15'

16'

17'

18'

19'

20'

21'

22'

14a'

14'

H

HN

NCH3

H

NH

H

O O

N

O

N

O

H

HO

H

12

3

4

5

6

789

1011

12

13

14 1516

17

1819 3'

2'

1'

4' 5'6'

7'

8'

9'10'

11'

12'13'

15'

16'

17'

18'

19'

20'

21'

22'

14a'

14'

H

1

2

Fig. 1. Structure of compounds 1 and 2.

[()TD$FIG]

1 2 3 4 5 6 7 8 9 10

AC

l/F

Cl

EN

o

ES

i

EG

al/E

Si*

EC

rp

EC

rp*

EA

m

EC

rs

EC

rs*

ES

e(1

)

ES

e* (

2)

EC

rsm

EC

r pm

EG

al*

Fig. 2. Ergot alkaloid profile of an extract from the C. purpurea sclerotia collected from

Calamagrostis arundinacea using LC–MS. Major identified alkaloids are ergonovine

(ENo), agroclavine (ACl), festuclavine (FCl), ergosine/ergosinine (ESi/ESi*), ergotamine

(EAm), tentatively a-ergocryptam (ECrpm), tentatively ergocristam (ECrsm),

a-ergocryptine/a-ergocryptinine (ECrp/ECrp*), ergocristine/ergocristinine (ECrs/

ECrs*), tentatively ergogaline/ergogalinine (EGal/EGal*), and ergosedmine/

ergosedminine (1/2).

[()TD$FIG]

Fig. 3. Strict consensus tree of the 8 most parsimonious trees from the analysis of

combined ITS and beta-tubulin sequences. The numbers above branches are

bootstrap/jackknife support values obtained from the analyses in TNT. Claviceps

nigricans is used as outgroup for the C. purpurea genotype complex. The genotype

groups are indicated in green (G1), blue (G2) and red (G3). EMBL/GenBank accession

numbers for the included sequences are given in Table 2. (For interpretation of the

references to color in this figure legend, the reader is referred to the web version of

the article.)

S. Uhlig et al. / Phytochemistry Letters 4 (2011) 79–8580

ergopeptine with a molecular weight that was higher than that ofany so far described natural ergopeptine. It was found in especiallyhigh relative amounts in the sclerotia obtained from feather reedgrass (Calamagrostis arundinacea), a grass species of the Poaceae,which is common in many parts of Eurasia. The ergot alkaloidprofile of the sclerotia was dominated by ergocristine and a-ergocryptine, as well as their lactamic derivatives (Fig. 2). In the fallof 2008 we accumulated enough sclerotia from three populationsof C. arundinacea in the Nordmarka forest (Oslo, Norway;6080200800N, 01084803400E; 6080203700N, 01084902500E; 6080205700N,01084805900E) that allowed further studies including isolation of thefungus in pure culture followed by molecular studies fordetermination of the involved genotype, and finally isolationand purification of 1 in order to unequivocally determine itsprimary structure. Parsimony analyses of ITS- and beta-tubulinsequences of 10 pure culture isolates obtained from C. purpurea

sclerotia from C. arundinacea clearly demonstrated that ourergopeptine-producing isolate belonged to the G2 lineage(Fig. 3, Tables 1 and 2). This fits well with the relatively humidand shady habitat from where the infected C. arundinacea

originated, and G2 isolates have also been obtained from otherCalamagrostis spp. (Douhan et al., 2008). Earlier reports claimedthat G2 isolates primarily produce ergosine, ergocristine and minoramounts of ergocryptine (Pazoutova et al., 2000). However, the C.

arundinacea-parasitizing C. purpurea strain produced ergocristineand a-ergocryptine in comparable amounts, and produced alsoergonovine and ergotamine (Fig. 2).

Based on mass spectral and NMR data, 1 was determined as N-(N-lysergyl)-cyclo(isoleucyl-phenylanalyl-prolyl). This ergopep-tine appears to be the second known natural ergoannine derivative(Buchta and Cvak, 1999; Flieger et al., 1997; Szantay et al., 1994).

Table 2EMBL/GenBank accession numbers of ITS- and beta-tubuline sequences obtained

from pure culture isolates of C. purpurea retrieved from public sequence databases

(EMBL/GenBank).

Genotype Isolate ID Accession numbers

ITS Beta-tubuline

G1 CMD1 EU559006 EU558985

G1 NGE1 EU559010 EU558989

G1 WFA EU559017 EU558996

G1 WLS EU559019 EU558998

G1 428 EU559002 EU558982

G2 WAB1 EU559012 EU558991

G2 WCN2 EU559013 EU558992

G2 WDG EU559014 EU558993

G2 WDS EU559016 EU558995

G2 WHS EU559018 EU558997

G2 434 EU559003 EU558979

G3 ARG1 EU559004 EU558983

G3 CDE1 EU559005 EU558984

G3 CPE10 EU559007 EU558986

G3 FSA1 EU559008 EU558987

G3 IRE12 EU559009 EU558988

G3 RH2 EU559011 EU558990

G3 WDI1 EU559015 EU558994

Table 1EMBL/GenBank accession numbers of ITS- and beta-tubuline sequences obtained

from pure culture isolates of C. purpurea from populations of Calamagrostis

arundinacea.

Population Isolate ID Accession numbers

ITS Beta-tubuline

1 VI 04905 FN687668 FN687678

1 VI 04906 FN687669 FN687679

2 VI 04907 FN687670 FN687680

2 VI 04909 FN687671 FN687681

3 VI 04912 FN687672 FN687682

3 VI 04913 FN687673 FN687683

3 VI 04914 FN687674 FN687684

3 VI 04915 FN687675 FN687685

3 VI 04916 FN687676 FN687686

3 VI 04917 FN687677 FN687687

S. Uhlig et al. / Phytochemistry Letters 4 (2011) 79–85 81

Previously, a trace impurity in commercial dihydroergocristinewas determined to be a congener of the compound, the structuraldifference to dihydroergocristine being exchange of the amino acidvaline by isoleucine (Halada et al., 1998). The compound was giventhe name 9,10-dihydroergosedmine, and hence 1 is equivalentwith ergosedmine.

The molecular formula C36H41N5O5 was deduced from theprotonated molecular ions obtained by direct infusion of a solutionof the isolated compound into the LTQ Orbitrap MS. The massdifference to ergocristine, which was co-isolated from thesclerotia, was equivalent to CH2. Comparison of the spectraobtained from fragmentation of the [M+H]+ ions of ergocristine and1 indicated that the lysergic part was conserved in the molecule ofthe new ergopeptine as seen by the characteristic m/z 208, 223, 251and 268 series of fragment ions (Fig. 4) (Lehner et al., 2004; Uhliget al., 2007). However, the fragment from cleavage through thecyclol-ring of the peptide moiety was 14 mass units higher in theMS2 spectrum of 1 compared to ergocristine (m/z 362 vs. m/z 348)tracing the CH2-difference between the two alkaloids to the aminoacid closest to the lysergic part of the molecule (Fig. 4) (Lehneret al., 2004). This amino acid is L-valine in ergocristine, meaningthat the corresponding amino acid in 1 was either leucine orisoleucine. In order to facilitate the assignment of the 1H and 13CNMR signals for 1 a complete set of 1D- and 2D-NMR spectra wererecorded for ergocristine. As to our knowledge the full 1H and 13CNMR assignments for ergocristine and ergocristinine are notreported elsewhere these data are included as supplementarymaterial. In addition, the assignment was facilitated by compari-son with previously recorded spectra of commercial ergonovine aswell as literature data (Bach et al., 1974; Casy, 1994; Cole et al.,[()TD$FIG]

200 300 400 500 6

m/z

362.1

869

223.1

229

319.1

444

339

.17

08

20

8.0

755

251.1

180

563.2

672

180.0

804

26

8.1

447

Fig. 4. HR-MS2 from fragmentation of the protonated molecular ions of a

2003; Szantay et al., 1994; Uhlig and Petersen, 2008). Spontaneousepimerization of 8R ergopeptines into their biologically less active8S isomers has been reported to occur in many solvents, amongothers DMSO (Casy, 1994; Hafner et al., 2008). Partial epimeriza-tion of 1 into its 8S isomer 2 (ergosedminine) was also obvious inour experiments as many of the 1H NMR resonances wereaccompanied by minor signals. However, when the two C-8epimers were separated and NMR-spectra recorded for each of theepimers the sample of 1 appeared again as a mixture of twoepimers while 2 was stable and did not show any evidence ofepimerization. Most assignments were relatively straightforwardby tracing the COSY, TOCSY and 1H–13C HMBC coupling environ-ments. The molecules of 1 and 2 (as well as ergonovine andergocristine that served as spectral references) possess severalunambiguously identifiable entry points: the assignment of thelysergic A, B and C-ring was achieved by starting from the NH-proton (H-1) as entry. While the 1H NMR signals of H-1 appearedwith a chemical shift of 10.82 ppm and 10.77 ppm for 1 and 2,respectively (Table 3), only a single resonance was observed for H-1 in case of the mixture of C-8 epimers of ergocristine at10.78 ppm. The NMR resonances of the lysergic D-ring could beassigned by starting from H-9, which was observed as a broadsinglet for 1 at 6.29 ppm and as a doublet in case of 2 at 6.49 ppm

00

606.3

096

624

.32

02

563.2672

362.1869

339.1708

319.1444

268.1447

251.1180

223.1229

m/z

3.4C34H35N4O4

1.5C22H24N3O2

1.4C20H23N2O3

1.1C20H19N2O2

0.8C16H16N3O

0.3C16H15N2O

−0.3C15H15N2

Δ (ppm)Composition

mixture of 1 and 2 and elemental composition of major fragments.

Table 31H and 13C NMR data for compounds 1 and 2 (DMSO-d6).

Position 1 2

dH (J in Hz)a dCb dH (J in Hz)a dC

b

1 10.82,d (1.4) 10.77,d (1.4)

2 7.03, m 119.2 7.09, m 119.5

3 108.1 108.3

4a 2.48, m 26.4 2.48, m 26.9

4b 3.46, dd (14.6, 5.4) 3.61, dd (14.6, 5.4)

5 3.07, m 62.3 3.19, m 61.8

7a 2.53, m 54.7 2.70, dd (12.5, 3.5) 53.9

7b 3.06 3.17

8 3.68, m 42.6 3.11, m 42.8

9 6.29, br s 118.4 6.49, d (5.8) 117.8

10 136.3 136.7

11 127.2 126.8

12 7.02, d (7.3) 110.7 7.13, d (7.3) 111.6

13 7.07, m 122.3 7.07, m 122.3

14 7.19, m 109.9 7.22, m 110.3

15 134.1 134.0

16 126.2 126.1

17 2.62, s 42.3 2.58, s 42.6

18 –c 176.2d

19 10.42, br s 10.12, br s

20 89.8 90.0

30 164.8 165.0

50 4.55, dd (6.4, 4.6) 55.9 4.46, dd (6.8, 4.2) 55.7

60 164.4 164.6

80a 3.36, m 45.7 3.36, m 45.7

80b 3.39, m 3.39, m

90a 1.77, m 21.8 1.77, m 21.8

90b 1.90, m 1.90, m

100a 1.89, m 26.0 1.89, m 26.0

100b 2.01, m 2.01, m

110 3.75, dd (9.5, 6.2) 63.1 3.73, dd (9.4, 6.2) 63.1

120 103.5 103.3

130 1.80, m 39.8 1.67, m 40.4

140 0.90, t (7.3) 11.6 0.94, t (7.3) 11.7

140a,a 1.14, m 21.9 1.15, m 21.9

140a,b 1.78, m 1.80, m

150 0.86, d (6.8) 12.8 0.84,d (6.6) 13.0

160a 3.10, m 38.5 3.03, dd (14.0, 4.0) 38.7

160b 3.20, dd (13.5, 6.5) 3.11, m

170 139.1 139.2

180 7.33,d (7.6) 129.9 7.26, d (7.6) 129.7

190 7.22, dd (7.6, 7.4) 127.7 7.17, dd (7.6, 7.4) 127.6

200 7.15, t (7.4) 126.1 7.06, t (7.4) 126.0

210 7.22, dd (7.6, 7.6) 127.7 7.17, dd (7.6, 7.6) 127.6

220 7.33,d (7.6) 129.9 7.26, d (7.6) 129.7

a Recorded at 600 MHz.b Assignments based on 2D 1H–13C HSQC and HMBC spectra.c Not observed.d Uncertain assignment.

S. Uhlig et al. / Phytochemistry Letters 4 (2011) 79–8582

(J = 5.8 Hz). The appearance of the H-9 1H NMR resonance as abroadened singlet for 1 indicates (1) involvement of the H-9 protonin the epimerization at C-8, and (2) supports that the epimerizationis occurring in DMSO. The observed correlation between the H-8and H-9 protons in the COSY spectrum was barely visible for 1 butwas strong in case of 2. The H-7 protons did not correlate to othernuclei in the recorded 2D-NMR spectra apart from the C-17 N-methyl carbon. However, the latter correlation could only beobserved for 2; C-7/H-7 of 1 did not show any correlations. The twodiastereotopic H-7 protons were observed at 2.53/3.06 ppm and2.70/3.17 ppm for 1 and 2, respectively (Table 3). The splittingpattern was only resolved for one of the resonances (2.70 ppm, 2).The recorded NMR data set did not allow assignment of the H-7resonances to H-7a and H-7b. However, the observed strongcoupling of 12.5 Hz for the 1H NMR signal at 2.70 ppm is likely toarise from geminal coupling between H-7a and H-7b, and the3.5 Hz coupling is likely to arise from vicinal coupling to H-8 (Casy,1994; Szantay et al., 1994). Similar coupling constants for the H-

7a/H-7b/H-8 spin system have been reported for a-ergocryptine,ergoannine, their brominated derivatives as well as ergoladinine(Cvak et al., 1996; Szantay et al., 1994). Early NMR work on ergotalkaloid conformers has shown that J7a,8a are significantly higherthan the corresponding J7a,8b, and a coupling constant around3.5 Hz would be expected for J7b,8a or J7b,8b (Pierri et al., 1982).This information suggests that the 2.70 ppm resonance in 2belongs to H-7b while the 3.17 ppm resonance belongs to H-7a.

The peptide part of the molecule could be entered from thelysergic part of the molecule via the amide proton (H-19)exhibiting 1H–13C HMBC correlations to C-20 and C-30. The 1HNMR spectra of 1 and 2 showed two terminal aliphatic methylgroups, which were part of the C-20-alkyl chain. The multiplicity ofthe methyl groups, i.e. one triplet and one doublet, indicated a sec-butyl C-20 substituent corresponding to isoleucine as the aminoacid closest to the lysergic acid part of the molecule. The couplingenvironment of the sec-butyl substituent was, however, not easy totrack down because of overlapping and contiguous signals. For

S. Uhlig et al. / Phytochemistry Letters 4 (2011) 79–85 83

example, the diastereotopic 140a methylene protons were ob-served at 1.14/1.15 ppm and 1.78/1.80 ppm (1/2) (Table 3).However, H-130 for 1 was also observed at 1.80 ppm making thetracking of correlations for the sec-butyl C-20 side-chain in theCOSY spectra ambiguous. The correlation environment was finallyclarified using the 1D-SELCOSY sequence and the isolated 8S

epimer (2). The assignment of the proline- and phenylalaninemoieties was straightforward using ergocristine as reference. Thelactam-ring and phenyl-moiety were entered via the well resolved1H NMR-signal for H-50 at 4.55 ppm and 4.46 ppm for 1 and 2,respectively (Table 3). The pyrrolidine ring was entered from H-110

(3.75 and 3.73 ppm for 1 and 2, respectively) and 1H and 13Cassignments obtained from observations in the COSY and 1H–13CHSQC spectra. The proton of the C-120 hydroxyl was not observedin the 1H NMR spectra. This might be a result of proton exchange ofthe hydroxyl-protons of 1 and 2 with those of residual water in thesample, which would broaden the resonance making it difficult toobserve or even disappear.

The molecules of ergosedmine (1) and ergosedminine (2)contain seven stereogenic centers at positions C-5, C-8, C-20, C-50,C-110, C-120 and C-130. The absolute stereochemistry of the pair ofepimers has, however, not been possible to determine becauseonly a very small amount of purified material was available.Extensive research in ergot alkaloid chemistry over a period ofseveral decades has shown that there exists only one fixedconfiguration in all natural ergopeptines, i.e. 5R having relation tothe biosynthetic precursor L-tryptophan, and 8R as 8S is merely anartifact from spontaneous epimerization in solution (Buchta andCvak, 1999). The proof for 1 being 8R and 2 being 8S can only beobtained indirectly: (1) Using reversed-phase HPLC, 1 elutestogether with other 8R ergopeptines for which pure standardcompounds were available and 2 elutes close to other 8S

ergopeptinines, for which standard compounds were available(Fig. 2); and (2) the amount of 2 in the extract and in the NMRsample increased over time on the expense of 1, indicating that 1 isthe natural epimer, and hence 8R. Furthermore, only L-amino acidsare incorporated into the tricyclic peptide part of the ergopeptinesresulting in a 20R, 50S, 110S, 120S configuration, which has also beenconfirmed by stereoselective synthesis (Komarova and Tolkachev,2001; Schardl et al., 2006). This information suggests a stereo-chemistry for 1 and 2 as is shown in Fig. 1.

3. Experimental

3.1. General experimental procedures

HPLC solvents were of gradient grade (Romil, Cambridge, UK orRathburn, Walkerburn, Scotland). Ammonium carbonate (Fluka,Buchs, Switzerland) and dichloromethane (Merck KG, Darmstadt,Germany) were of p.a. quality. Water was distilled and furtherpurified using an ELGA Purelab Maxima system (Vivendi WaterSystems Ltd., High Wycombe, UK). NMR spectra were obtainedfrom solutions in 70 ml of DMSO-d6 (99.96% D, Sigma–Aldrich, St.Louis, MO) in 2.0 mm O.D. Norell tubes (Sigma–Aldrich) using aBruker Match clamp holder. The spectra were acquired on anAvance AVII 600 MHz NMR spectrometer (Bruker BioSpin,Silberstreifen, Germany) with a 5 mm CP-TCI (1H/13C, 15N–2H)triple-resonance inverse cryo probe, equipped with a Z-gradientcoil. The data were processed using the Bruker TOPSPIN (version1.3 or version 2.1 pl 2) software. Chemical shift values werereferenced to the residual solvent signals. HRMS spectra wererecorded by direct infusion of a diluted solution of ergosedmine inMeCN into an LTQ-Orbitrap XL mass spectrometer (Thermo FisherScientific, Waltham, MA). Ionization was achieved using electro-spray in the positive mode. LC–DAD-MS analyses of extracts andfractions were carried out using a Finnigan Surveyor HPLC

hyphenated to a LTQ linear ion trap mass spectrometer (ThermoElectron, San Jose, CA; now part of Thermo Fisher Scientific).Separations were performed on a Waters (Milford, MA) SunFire C18

(50 � 2.1 mm i.d., 3.5 mm). A mobile phase consisting of MeCN(solvent A) and 2.5 mM ammonium carbonate (solvent B) wasemployed with gradient elution (0 min, 35:65 (A:B); 14.5 min,45:55; 15 min, 100% A; 19 min, 100% A), and the flow rate was0.3 ml/min. The DAD was scanned between 195 and 600 nm. TheMS parameters were as follows: ESI positive mode; spray voltage5.0 kV; sheath gas flow rate 64 arbitrary units; auxiliary gas flowrate 20 arbitrary units; m/z range 235–700. Crude prefractionationwas performed using Phenomenex Strata silica gel (SI) columns(5 g; Torrance, CA). Semipreparative HPLC separations werecarried out on a Gilson (Middleton, WI) consisting of a model321 pump, a 232XL sampling injector and a 402 syringe pump. AHP 1100 series DAD (Hewlett-Packard, Waldbronn, Germany) wasused for the continuous monitoring of the absorption at 200, 210,and 310 nm. Semipreparative HPLC separations were also carriedout on a TSP model 4000 pump (Finnigan MAT Corporation, SanJose, CA; now part of Thermo Fisher Scientific), and a portion of theeluent split into an LCQ classic ion trap MS (Finnigan MATCorporation). The MS parameters were as follows: ESI positivemode; spray voltage 4.5 kV; sheath gas flow rate 60 arbitrary units;auxiliary gas flow rate 5 arbitrary units; m/z range 300–800. Thefollowing columns were employed for semipreparative HPLCseparations: a Waters SunFire C8 (250 � 10 mm i.d., 5 mm) and anACE Phenyl (Advanced Chromatography Technologies, Aberdeen,Scotland; 250 � 7.5 mm i.d., 5 mm). The following analyticalstandards were available: ergonovine maleate, a-ergocryptine,and lysergol (all Sigma–Aldrich, St. Louis, MO), ergotamine D-tartrate (>97%, Fluka, Buchs, Switzerland), ergotaminine, ergo-cryptinine, ergocristine, ergocristinine, ergocornine, ergocorni-nine, ergosine, ergosinine, dihydroergotamine, dihydroergosine,dihydrolysergol, isodihydrolysergol, erginine, dihydroergine, cha-noclavine, agroclavine, festuclavine, elymoclavine, and elymocla-vine fructoside (donations from Dr. Michael Sulyok, University ofNatural Resources and Applied Life Sciences, Tulln, Austria).

3.2. Fungal material

Ergotized heads of feather reed grass (C. arundinacea) werecollected in the Nordmarka forest in Oslo, Norway, in August 2008.The sclerotia (2.51 g) were hand-picked and cleaned from residualplant material, and stored at �26 8C until further processing.

3.3. Isolation of pure cultures of C. purpurea from sclerotia

Two to five sclerotia per population were selected for isolationof pure cultures. The sclerotia were individually surface sterilizedin subsequentially 96% EtOH (1 min), commercial bleach (2 min),30 s in 96% EtOH before 3 rinsing steps in sterile MilliQ water. Eachsclerotium was then cut into two halves, of which one was subjectfor chemical analyses and the other half was transferred to a plateof malt-yeast extract-sucrose agar (MYSA) (Skaar and Stenwig,1996) containing antibiotics. When characteristic white C.

purpurea mycelium emerged, pure cultures were transferred topotato dextrose agar (PDA). The obtained pure culture isolates(Table 1) are maintained in the culture collection ‘‘Mycoteket’’ atthe National Veterinary Institute, Norway, and are available uponrequest.

3.4. DNA extraction

About 10 mg of fresh mycelium from each culture (Table 1)were transferred to Eppendorf tubes with 700 ml of CTABextraction buffer [20 g/l cetyltrimethylammoniumbromide

S. Uhlig et al. / Phytochemistry Letters 4 (2011) 79–8584

(CTAB), purchased from Calbiochem, Darmstadt, Germany; 1.4 MNaCl, 0.1 M Tris–HCl, 20 mM Na2EDTA], crushed manually withsterile pistils and frozen at �80 8C for minimum 10 min. Totalgenomic DNA was then extracted using the CTAB miniprepextraction protocol (Gardes and Bruns, 1993; Vralstad et al., 2009).

3.5. PCR and sequencing

Two different molecular markers (loci) were PCR amplified andsequenced; internal transcribed spacer nrDNA (ITS) and a portionof a beta-tubulin (TUB) protein coding gene. The ITS region wasamplified with the universal primers ITS1 and ITS4 (White et al.,1990). The 25-ml reaction mixture consisted of 1.7 mM of eachprimer, 2 ml of genomic DNA, PuReTaq Ready-To-GoTM PCR Beads(Amersham Biosciences, UK) and milliQ water. PCR was performedon a DNA Engine Tetrad1 Peltier Thermal Cycler (PTC-225, MJResearch, Waltham, MA, USA) using initial denaturation (95 8C/10 min), 38 cycles of amplification (95 8C/1 min, 56 8C/45 s, 72 8C/1 min), and final elongation (72 8C/5 min). The partial TUB-genewas amplified with the forward primer 50-GCT CTA GAC TGC TTTCTG GCA GAC C-30 and the reverse primer 50-CGT CTA GAK GTR CCCATA CCG GCA-30 using the same reaction mix as described above(Annis and Panaccione, 1998). PCR was performed on a DNA EngineDyad1 Peltier Thermal Cycler (PTC-0220, MJ Research), usinginitial denaturation (94 8C/4 min), 35 cycles of amplification(94 8C/30 s, 55 8C/30 s, 72 8C/1 min), and final elongation (72 8C/5 min), which is a modified version of the thermocyclingconditions described elsewhere (Douhan et al., 2008). The PCRamplicons were visualized by gel electrophoresis on a 1.5% agarosegel with ethidium bromide, using pUC Mix Marker 8, ready-to-use(19–1118 bp; Fermentas, USA) for sizing of DNA fragments. PCRproducts were purified with ExoZapTM (Amersham Biosciences,Buckinghamshire, UK) according to the protocol. The productswere then sequenced in both directions with their respectiveprimers. The ITS-region was sequenced using the BigDye1

Terminator v3.1 Ready Reaction mix (Applied Biosystems, LifeTechnologies, USA) according to the manufacturers instructions.The sequencing PCR-programme consisted of initial denaturation(96 8C/1 min) and 40 cycles of 968/10 s, 56 8C/5 s and 60 8C/4 min.The partial TUB-gene was sequenced using the optimized fast cyclesequencing protocol with 1 ml of a 10 mM primer solution and4.5 ml of water in the DNA sequencing reaction (Platt et al., 2007).The thermocycling conditions consisted of initial denaturation(96 8C/1 min), 15 cycles of 968/10 s, 50 8C/5 s and 60 8C/1:15 min, 5cycles of 968/10 s, 50 8C/5 s and 60 8C/1:30 min and 5 cycles of 968/10 s, 50 8C/5 s and 60 8C/2 min. The sequencing PCR products werepurified with BigDye1 XTerminator Purification Kit (AppliedBiosystems, Life Technologies, USA) according to the manufac-turer’s instructions, and subsequently analyzed on an ABI PRISM1

3100 – Avant Genetic Analyzer (Applied Biosystems). Assemblyand manual editing of the sequence chromatograms wasconducted in BioEdit (Hall, 1999). Ambiguous positions werecoded according to IUPAC standards. All sequences were submittedto EMBL/GenBank (accession numbers in Table 1).

3.6. Identification and phylogenetic analyses

Confirmation of species identity was done using the NCBInucleotide BLAST (Basic Local Alignment Search Tool) (Altschulet al., 1997). Sequences confirmed as C. purpurea were alignedmanually in BioEdit. Public available C. purpurea sequences (ITSand TUB) of the genotypes G1, G2 and G3 were included in thealignment together with one ITS and one TUB sequence of C.

nigricans that was included as outgroup (Douhan et al., 2008).Accession numbers of the public available sequences used in thisstudy are given in Table 2. The sequence alignment was transferred

to a note-pad .tnt file and imported to the program TNT version 1.1(Goloboff et al., 2008). Maximum parsimony analyses wereconducted in TNT with heuristic searches (1000 random additionsequences) and TBR branch swapping, saving ten trees perreplication. ‘‘Collapsing rule’’ was set to minimum length = 0and ‘‘random seed’’ was set to ‘‘time’’. Resampling studiesincluding standard bootstrap (Felsenstein, 1985) and Jackknife(Farris et al., 1996) were conducted with 1000 replicates(traditional search) and with a cut-off value of 50%. Jackknifewas performed using 36% deletion.

3.7. Extraction and isolation of compounds 1 and 2

The C. purpurea sclerotia (2.51 g) were crushed in a mortar andtransferred to a 25-ml Pyrex tube. The material was repeatedlyextracted with 15 ml of acetone/water (4:1). The extracts werepooled and evaporated to dryness. Residues were dissolved in 3�1 ml of CH2Cl2 and fractionated on silica gel using a stepwisegradient comprised of CH2Cl2, CH2Cl2/MeCN (19:1, 9:1, 6:1) andMeCN. The MeCN fraction contained all ergopeptines. The residuewas dissolved in 2 ml of acetone/water, and 30-ml aliquots weremanually injected onto the SunFire Prep C8 column. A mobile phaseconsisting of MeCN (solvent A) and 2.5 mM ammonium carbonate(solvent B) was employed with gradient elution (0 min, 57:42(A:B); 0.5 min, 57:42; 15 min, 67:33; 15.25 min, 100% A;18.25 min, 100% A; 18.5 min, 57:42 (A:B); 22 min, 57:42), andthe flow rate was 3 ml/min. Approximately 1/10 of the eluent wassplit into the LCQ–MS and ergocristine (10.7 min) and 1 (13.2 min)manually collected.

Combined fractions containing ergocristine or 1 were evapo-rated to dryness and dissolved in acetone/water (4:1). Aliquots ofthe ergocristine fraction were chromatographed using the ACEPhenyl column and the Gilson HPLC. A mobile phase consisting ofMeCN (solvent A) and 2.5 mM ammonium carbonate (solvent B)was employed with gradient elution (0 min, 49:51 (A:B); 0.5 min,59:51; 12 min 53:47; 12.25 min, 100% A; 14.25 min, 100% A;14.5 min, 49:51 (A:B) 19 min, 57:42), and the flow rate was 2.5 ml/min. Ergocristine eluted after 9.7 min and was collected manually.After evaporation, 1.7 mg of ergocristine was obtained as whitecrystals.

The combined fractions containing 1 were also chromato-graphed on the ACE Phenyl column, but in connection with theLCQ–MS. The same mobile phase as for ergocrisitine was used withgradient elution (0 min, 54:46 (A:B); 0.5 min, 54:46; 15 min,66:34; 15.25 min, 100% A; 18.25 min, 100% A; 18.5 min, 54:46(A:B) 22 min, 54:46), and the flow rate was 3 ml/min. Ergosedmine(1) eluted after 9.5 min, while 2 eluted after 14.3 min. Initially,both were combined for the recording of NMR spectra, but werelater separated in order to verify the NMR-assignment for each C-8epimer. After evaporation, 0.2 mg of a mixture of 1 and 2 wasobtained as white crystals.

3.8. N-(N-Lysergyl)-cyclo(isoleucyl-phenylanalyl-prolyl)

(ergosedmine, 1) and N-(N-isolysergyl)-cyclo(isoleucyl-phenylanalyl-

prolyl) (ergosedminine, 2)

White amorphous powder; HPLC-online UV lmax 205, 237,311 nm; 1H and 13C NMR data (DMSO-d6), see Table 3; positive ionHRESIMS m/z 624.3189 (calcd for C36H42N5O5 [M+H]+, 624.3181).

Acknowledgments

The authors thank Friede Støren Thanke and Frøya Wisløff forthe collection of sclerotia from the grass samples. The authorswould also like to thank Klaus Høiland (Department of Biology,University of Oslo) for the identification of the grass species.

S. Uhlig et al. / Phytochemistry Letters 4 (2011) 79–85 85

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.phytol.2010.09.004.

References

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W., Lipman,D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs. Nucleic Acids Res. 25, 3389–3402.

Annis, S.L., Panaccione, D.G., 1998. Presence of peptide synthetase gene transcriptsand accumulation of ergopeptines in Claviceps purpurea and Neotyphodiumcoenophialum. Can. J. Microbiol. 44, 80–86.

Bach, N.J., Boaz, H.E., Kornfeld, E.C., Chang, C.-J., Floss, H.G., Hagaman, E.W.,Wenkert, E., 1974. Nuclear magnetic resonance spectral analysis of the ergotalkaloids. J. Org. Chem. 39, 1272–1276.

Buchta, M., Cvak, L., 1999. Ergot alkaloids and other metabolites of the genusClaviceps. In: Kren, V., Cvak, L. (Eds.), Medicinal and Aromatic Plants – IndustrialProfiles, vol. 6. Ergot – The Genus Claviceps, Harwood Academic Publishers,Amsterdam, pp. 173–201.

Casy, A.F., 1994. Rapid identification of ergot derivatives by 1H NMR spectroscopy. J.Pharm. Biomed. Anal. 12, 27–40.

Cole, R.J., Jarvis, B.B., Schweikert, M.A., 2003. Secondary Fungal Metabolites, vol. 1.Academic Press, San Diego.

Cvak, L., Minar, J., Pakhomova, S., Ondracek, J., Kratochil, B., Sedmera, P., Havlicek,V., Jegorov, A., 1996. Ergoladinine, an ergot alkaloid. Phytochemistry 42, 231–233.

Douhan, G.W., Smith, M.E., Huyrn, K.L., Westbrook, A., Beerli, P., Fisher, A.J., 2008.Multigene analysis suggests ecological speciation in the fungal pathogen Cla-viceps purpurea. Mol. Ecol. 17, 2276–2286.

Farris, J.S., Albert, V.A., Kallersjo, M., Lipscomb, D., Kluge, A.G., 1996. Parsimonyjackknifing outperforms neighbor-joining. Cladistics 12, 99–124.

Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using thebootstrap. Evolution 4, 783–791.

Flieger, M., Wurst, M., Shelby, R., 1997. Ergot alkaloids—sources, structures andanalytical methods. Folia Microbiol. 42, 3–30.

Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidio-mycetes—application to the identification of mycorrhizae and rust. Mol. Ecol. 2,113–118.

Goloboff, P.A., Farris, J.S., Nixon, K.C., 2008. TNT, a free program for phylogeneticanalysis. Cladistics 24, 774–786.

Hafner, M., Sulyok, M., Schumacher, R., Crews, C., Krska, R., 2008. Stability andepimerisation behaviour of ergot alkaloids in various solvents. World Myco-toxin J. 1, 67–78.

Halada, P., Sedmera, P., Havlicek, V., Jegorov, A., Cvak, L., Ryska, M., 1998. Massspectrometric amino acid structure determination in ergopeptines. Eur. MassSpectrom. 4, 385–392.

Hall, T.A., 1999. BioEdit: a user friendly biological sequence alignment editor andanalysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98.

Keller, U., 1999. Biosynthesis of ergot alkaloids. In: Kren, V., Cvak, L. (Eds.),Medicinal and Aromatic Plants – Industrial Profiles, vol. 6. Ergot – The GenusClaviceps, Harwood Academic Publishers, Amsterdam, pp. 94–164.

Komarova, E.L., Tolkachev, N.O., 2001. Medicinal plants: the chemistry of peptideergot alkaloids: Part 1. Classification and chemistry of ergot peptides. Pharm.Chem. J. 35, 504–513.

Lehner, A.F., Craig, M., Fannin, N., Bush, L., Tobin, T., 2004. Fragmentation patterns ofselected ergot alkaloids by electrospray ionization tandem quadrupole massspectrometry. J. Mass Spectrom. 39, 1275–1286.

Panaccione, D.G., 2005. Origins and significance of ergot alkaloid diversity in fungi.FEMS Microbiol. Lett. 251, 9–17.

Pazoutova, S., Olsovska, J., Linka, M., Kolinska, R., Flieger, M., 2000. Chemoraces andhabitat specialization of Claviceps purpurea populations. Appl. Environ. Micro-biol. 66 (12), 5419–5425.

Pierri, L., Pitman, I.H., Winkler, D.A., Andrews, P.R., 1982. Conformational analysis ofthe ergot alkaloid ergotamine and ergotaminine. J. Med. Chem. 25, 937–942.

Platt, A.R., Woodhall, R.W., George, A.L., 2007. Improved DNA sequencing qualityand efficiency using an optimized fast cycle sequencing protocol. Biotechniques43, 58–62.

Schardl, C.L., Panaccione, D.G., Tudzynski, P., 2006. Ergot alkaloids—biology andmolecular biology. Alkaloids Chem. Biol. 63, 1099–4831.

Schiff, P.L., 2006. Ergot and its alkaloids. Am. J. Pharm. Educ. 70, 1–10.Skaar, I., Stenwig, H., 1996. Malt-yeast extract-sucrose agar, a suitable medium for

enumeration and isolation of fungi from silage. Appl. Environ. Microbiol. 62,3614–3619.

Szantay Jr., C., Bihari, M., Brlik, J., Csehi, A., Kassai, A., Aranyi, A., 1994. Structuralelucidation of two novel ergot alkaloid impurities in a-ergokryptine andbromokryptine. Acta Pharm. Hung. 64, 105–108.

Uhlig, S., Petersen, D., 2008. Lactam ergot alkaloids (ergopeptams) as predominantalkaloids in sclerotia of Claviceps purpurea from Norwegian wild grasses.Toxicon 52, 175–185.

Uhlig, S., Vikøren, T., Ivanova, I., Handeland, K., 2007. Ergot alkaloids in Norwegianwild grasses: a mass spectrometric approach. Rapid Commun. Mass Spectrom.21, 1651–1660.

Vralstad, T., Knutsen, A.K., Tengs, T., Holst-Jensen, A., 2009. A quantitative TaqManMGB real-time polymerase chain reaction based assay for detection of thecausative agent of crayfish plague Aphanomyces astaci. Vet. Microbiol. 137, 146–155.

White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencingof fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand,D.H., Skinsky, J.J., White, T.J. (Eds.), PCR Protocols: A Guide to Methods andApplications. Academic Press, San Diego, pp. 315–322.