Identification and synthesis of three cyclodidepsipeptides as potential precursors of enniatin B in Fusarium sporotrichioides

Article abstract of DOI:10.1016/j.molstruc.2010.11.029

A pathogenic fungus, Fusarium sporotrichioides Sherb., was isolated from Hypericum barbatum Jacq. The volatile compounds of broth and mycelium were analyzed using GC-MS and three cyclodidepsipeptides (dioxomorpholines), 3,6-di(propan-2-yl)-4-methyl-morpholine-2,5-dione, 3-(2-methylpropyl)-6-(propan- 2-yl)-4-methyl-morpholine-2,5-dione and 3-(butan-2-yl)-6-(propan-2-yl)-4-methyl- morpholine-2,5-dione, were found for the first time in the natural products. The structures of the compounds were confirmed by comparison of the analytical data for the natural products with samples obtained via synthetic methods. The conformational features and vibrational spectra of the three cyclodidepsipeptides were characterized by density functional theory (DFT) calculations and IR spectroscopy. The cyclic hexadepsipeptide enniatin B was identified by a LC-MS/MS analysis of the non-volatile products of broth and mycelium. The above-mentioned three cyclodidepsipeptides are probably synthesized using similar biosynthetic ways to enniatin B involving a nonribosomal mechanism.

Full text of DOI:10.1016/j.molstruc.2010.11.029

Journal of Molecular Structure 985 (2011) 397–402
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Identification and synthesis of three cyclodidepsipeptides as potential precursors
of enniatin B in Fusarium sporotrichioides
Andrija Smelcerovic a, ,1, Denitsa Yancheva b, Emiliya Cherneva b, Zivomir Petronijevic c, Marc Lamshoeft a,
⇑
Diran Herebian a,2
a Institute of Environmental Research, Technical University of Dortmund, Otto-Hahn-Str. 6, 44221 Dortmund, Germany
b Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Build. 9, 1113 Sofia, Bulgaria
c Faculty of Technology, University of Nish, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2010
Received in revised form 6 November 2010
Accepted 10 November 2010
Available online 16 November 2010
A pathogenic fungus, Fusarium sporotrichioides Sherb., was isolated from Hypericum barbatum Jacq. The
volatile compounds of broth and mycelium were analyzed using GC–MS and three cyclodidepsipeptides
(dioxomorpholines), 3,6-di(propan-2-yl)-4-methyl-morpholine-2,5-dione, 3-(2-methylpropyl)-6-(pro-
pan-2-yl)-4-methyl-morpholine-2,5-dione and 3-(butan-2-yl)-6-(propan-2-yl)-4-methyl-morpholine-
2,5-dione, were found for the first time in the natural products. The structures of the compounds were
confirmed by comparison of the analytical data for the natural products with samples obtained via syn-
thetic methods. The conformational features and vibrational spectra of the three cyclodidepsipeptides
were characterized by density functional theory (DFT) calculations and IR spectroscopy. The cyclic hexad-
epsipeptide enniatin B was identified by a LC–MS/MS analysis of the non-volatile products of broth and
mycelium. The above-mentioned three cyclodidepsipeptides are probably synthesized using similar bio-
synthetic ways to enniatin B involving a nonribosomal mechanism.
Keywords:
Cyclodidepsipeptides
Synthesis
Fusarium sporotrichioides
Enniatin B
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
derivatives which are useful building blocks for the preparation of
biodegradable polymers [12–14].
The family of cyclodepsipeptides comprises natural products
and synthetic peptide lactones with at least one ester bond in their
skeleton. The 18-membered cyclodepsipeptides, the so-called enni-
Endophytes are a poorly investigated group of microorganisms
that represent an abundant and dependable source of bioactive
and chemically novel compounds with potential for exploitation
in wide variety of medical, agricultural, and industrial areas [15].
The number of studies on Hypericum species has increased rapidly
over the years, mainly due to the pharmaceutical importance of
dianthrone derivatives isolated from Hypericum perforatum [16].
In this work, we report the identification and synthesis of three
cyclodidepsipeptides (morpholine-2,5-diones) as potential precur-
sors of enniatin B in the pathogenic fungi Fusarium sporotrichioides,
isolated from the stem of fresh Hypericum barbatum Jacq.
atins, typically possess an alternating sequence of
D-2-hydroxy-3-
methylbutanoic and -configured N-methylamino acid residues.
L
This class of compounds have been known to exhibit anthelmintic
[1–3], insecticidal [4,5] and phytotoxic activities [6], as well as
inhibitory activity towards acyl-CoA:cholesterol acyltransferase
[7]. Dornetshuber et al. [8] demonstrated that enniatin B also exerts
profound cytotoxic activity against several human tumour cells. The
identification of enniatins is sometimes accompanied by the identi-
fication of cyclodidepsipeptides, which are potential precursor in
the biosynthesis of the former. The reports about the biological
activities of the cyclodidepsipeptides [9,10] inspired attempts of
total synthesis of some of these compounds [11]. A number of other
publications deal with the synthesis of the non-N-methylated
2. Results and discussion
The isolated fungus, F. sporotrichioides Sherb. [W&R,G,B,J], was
cultured in a potato dextrose agar medium. After fermentation the
volatile compounds of broth and mycelium were analyzed using
GC–MS. In both, broth and mycelium ethyl acetate extracts three
cyclodidepsipeptides were found, namely: 3,6-di(propan-2-yl)
-4-methyl-morpholine-2,5-dione (1a), 3-(2-methylpropyl)-6-(pro-
pan-2-yl)-4-methyl-morpholine-2,5-dione (1b) and 3-(butan-2-
yl)-6-(propan-2-yl)-4-methyl-morpholine-2,5-dione (1c) (Scheme
1). To the best of our knowledge in the present work compounds
⇑
Corresponding author. Tel.: +381 18 4570029; fax: +381 18 4238770.
E-mail address: a.smelcerovic@yahoo.com (A. Smelcerovic).
Present address: Department of Pharmacy, Faculty of Medicine, University of Nis,
1
Bulevar Dr. Zorana Djindjica 81, 18000 Nis, Serbia.
2
Present address: Department of General Pediatrics, University Hospital Düssel-
dorf, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany.
0022-2860/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2010.11.029

398
A. Smelcerovic et al. / Journal of Molecular Structure 985 (2011) 397–402
O
O
O
O
O
O
N
N
O
N
O
O
O
N
O
N
O
1a
1b
O
O
O
O
N
O
O
2
1c
Scheme 1. Chemical structures of the compounds under study.
1a–c were found for the first time in natural products. However, due
to the complexity of the studied mixture of the fungus products,
compounds 1a–c could not be isolated. The mass spectra of 1a–c
were compared to the mass fragmentation of a series of synthetic
cyclodepsipeptides, reported by Shemyakin et al., and a perfect
match was observed [17]. Therefore, the absolute configuration of
1a–c can be predicted with very high probability to be (3S,6R).
The ethyl acetate extracts of broth and mycelium were analyzed
by mycotoxin LC–MS/MS multi-method [18,19]. The cyclic
hexadepsipeptide enniatin B ((3S,6R,9S,12R,15S,18R)-4,10,16-tri-
methyl-3,6,9,12,15,18-hexa(propan-2-yl)-1,7,13-trioxa-4,10,16-tri-
azacyclooctadecane-2,5,8,11,14,17-hexone; 2, Scheme 1) could be
unambiguously identified. We did not find any of the other myco-
toxins integrated in the LC–MS/MS method (neosolaniol, 15-acet-
yldeoxynival, monoacetoxyscirpenol, diacetoxyscirpenol, HT-2
toxin, T-2 toxin, verrucarin A, deoxynivalenol, nivalenol, 3-acety-
method described by Cook and Cox [22]. Namely, the synthesis
of cyclodidepsipeptides was performed via N-( -bromoacyl)-
a
a-
amino acids as non-cyclic intermediate products (Scheme 2). The
generic 2-bromo-3-methylbutanoyl chloride 4 was obtained by a
sequence of condensation reactions using the experimental proto-
cols of Harpp et al. [24]. Treatment of isovaleric acid (3-methylbu-
tanoic acid; 3) with thionylchloride, and a subsequent in situ
reaction with N-bromosuccinimide (NBS; 1-bromopyrrolidine-
2,5-dion) readily afforded 4. This one-pot synthesis is straightfor-
ward and the yields are acceptable for a variety of primary and
secondary acid chlorides and diacid chlorides. The amidation of 4
was carried out with large excess of the respective N-methylamino
acids 5a–c and gave the non-cyclic products 6a–c. In principle,
three general approaches might be envisioned for the cyclization
of the linear intermediate products [14]. In our case we converted
the 2-(2-bromo-3-methylbutanoyl(methyl)amino) acids 6a–c into
K+ salts 7a–c and after acidification and extraction procedures at
room temperature, we isolated the corresponding 6-(propan-2-
yl)-4-methyl-morpholine-2,5-diones 1a–c. Compounds 1a–c were
characterized by FTIR, NMR and MS spectra. Retention times in
GC and mass spectra of synthesized and fungus-produced com-
pounds are identical.
The most probable structures of 1a–c were further character-
ized by computational tools [25–27]. Evaluation of the molecular
structure and conformational isomerism of 1a–c was done by
DFT methods. The conformational flexibility of the heterocycle
and the presence of two stereogenic centers (C3 and C6) give rise
to a wide number of possible structures for 1a–c. Additionally,
compound 1c contains a third chiralic center in the 3-alkyl substi-
tuent, which increases further the number of the possible
structures. In order to determine the preferred geometry we
constructed the most probable diastereomers of 1a–c and then
optimized their geometries at the B3LYP/6-311GÃÃ level of theory.
For 1a and 1b molecule, 16 diastereomers were considered for
optimization, taking into account all relevant combinations of boat
and chair cycle conformations and axial and equatorial substitu-
ents positions. As a result, we found that in all cases the morpho-
line-2,5-dione ring adopts boat conformation and thus the number
of the possible isomers was reduced to 8. The eight different min-
ima found are doubly degenerated by symmetry states and corre-
spond to four enantiomeric pairs of molecules. In the case of 1c the
analysis followed the same reasoning with larger number of dia-
stereoisomer included in order to account for the third stereogenic
center.
ldeoxynivalenol, fusarenon-X, zearalenone,
a-zearalenol, alfatox-
in-B1, alfatoxin-B2, alfatoxin-G1, alfatoxin-G2, ochratoxin A,
ochratoxin B, fumonisin B1, fumonisin B2, beauvericin, altenuene,
alternariol, alternariolmethylether, moniliformin, ergocornine,
ergotamine, citrinin, patulin and gibberellic acid). An HPLC–ESI-
MS/MS chromatogram of enniatin B is depicted in Fig. 1. The mech-
anism of enniatin biosynthesis was described [20] as step-wise
condensation of dipeptidol blocks, terminated by cyclization reac-
tion of the formed linear hexapeptide. The above-mentioned three
cyclodidepsipeptides (1a–c) are probably synthesized as side prod-
ucts of cyclization in this process using similar nonribosomal bio-
synthetic mechanism to 2. According to the suggested scheme [20]
the main precursor for the formation of 2 would be a dipeptide
block, containing N-methyl-L-valine and D-2-hydroxy-3-methylbu-
tanoic acid residues, which could explain the fact that 1a was
found in prevailing amount in the broth and the mycelium. The
production of the other two cyclodidepsipeptides, 1b and 1c, pre-
sumably is due to low substrate specificity of the enniatin synthe-
tase for L-amino acids [20,21].
For identification and confirmation of the compounds the
cyclodepsipeptides were synthesized. The synthesis of 4-methyl-
morpholine-2,5-diones was previously reported as a coupling of
N-methylamino acids and 2-hydroxy-3-methylbutanoic acid [11]
or 2-bromo-3-methylbutanoyl chloride [22]. Other authors ex-
plored also the condensation of esters of secondary amines with
2-chloropropionyl chloride [23] for the preparation of morpho-
line-2,5-diones with larger N-substituents. In this work the exper-
imental conditions were adjusted in order to implement the

A. Smelcerovic et al. / Journal of Molecular Structure 985 (2011) 397–402
399
RT: 0.00 - 25.02
NL:
21.49
100
90
80
70
60
5.37E6
TIC F: + c SRM
ms2
657.40@cid-
41.00
[195.75-196.25]
Relative
Abundance
50
40
30
20
10
22.30 23.57
21.03
21.50
0
100
NL:
9.11E6
TIC F: + c SRM
90
80
70
60
50
40
30
20
10
0
ms2
657.40@cid-
23.00
[640.05-640.55]
21.04
20
22.24
22
0
2
4
6
8
10
12
14
16
18
24
Time (min)
Fig. 1. HPLC–ESI(pos)-MS/MS chromatogram of enniatin B (m/z 657, [M + NH4]+) and its corresponding product ions (m/z 196/CID 41 eV and m/z 640/CID 23 eV).
O
O
1. SOCl2
2. NBS
N-Me amino acids
O
R
0.5 M K2CO3
(5a-c)
O
OH
Cl
N
Br
Br
OH
(3)
(4)
(6a-c)
O
R
O
O
R
c
1
a
b
1
HCl
2
3
6
5
O
R =
N
4
N
O
3-isopropyl 3-isobutyl
(Val)
(Leu)
3-sec-butyl
(Ile)
Br
O-K+
(7a-c)
(1a-c)
Scheme 2. Synthesis of 6-(propan-2-yl)-4-methyl-morpholine-2,5-diones 1a–c.
For illustration of the relative stability of the diastereomers
studied, we focused our attention only on the molecules with
(3S) configuration as inherent for the naturally occurring amino
acids. The optimized energies (Ecorr), vibrational zero-point ener-
gies (VZPE), and relative energies (Erel) with respect to the most
stable forms are summarized in Table 1. The two different boat
conformations are denoted ‘‘a’’ and ‘‘b’’. Each of the diastereoiso-
mers of 1c included in Table 1 corresponds to an enantiomeric pair.
As it can be seen from the values listed in Table 1, the stability
of the diastereomers decreases in the following order:
a(3S,6R) > a(3S,6S) > b(3S,6S) > b(3S,6R). The energy difference be-
tween the most stable and the least stable diastereoisomers of 1a
and 1c falls within the range 30.1–32.9 kJ molÀ1. For 1b this value
is much lower (12.8 kJ molÀ1) due to the smaller sterical hindrance
between the isobutyl group and the morpholine-2,5-dione ring in
its isomers. According to the calculations, the most stable molecu-
lar structures of 1a–c are those with equatorial isopropyl group
and axial 3-alkyl substituent (Fig. 2). These conformations reflect
the stereostructure expected according to the possible mechanism
for the biosynthesis of 1a–c, i.e. the cycles are formed by D-2-hy-

400
A. Smelcerovic et al. / Journal of Molecular Structure 985 (2011) 397–402
Table 1
1a in KBr
Calculated total energies Ecorr and relative energies Erel of (3S) diastereoisomers of 1a–
c.
1,2
0,8
0,4
0,0
VZPE (kJ molÀ1
)
Erel (kJ molÀ1
)
a
Entry
Ecorr (a.u.)
1a
a(3S,6R)
a(3S,6S)
b(3S,6S)
b(3S,6R)
À710.962654
À710.960431
À710.954504
À710.951171
769.515
769.862
769.589
770.613
0.00
5.836
21.397
30.148
1b
a(3S,6R)
a(3S,6S)
b(3S,6S)
b(3S,6R)
À750.259985
À750.259249
À750.255984
À750.255080
843.378
843.596
844.400
844.426
0.00
1.932
10.504
12.878
3500 3150 2800
1800 1650 1500 1350 1200 1050 900
Wave number [cm-1]
1c
a(3S,6R)b
a(3S,6S)b
b(3S,6S)b
b(3S,6R)b
À750.256677
À750.254642
À750.247871
À750.244145
844.198
843.904
844.746
844.830
0.0
5.342
23.120
32.902
Fig. 3. Experimental IR spectrum of 1a in solid state.
a
Corrected with the zero-point vibrational energy (ZPVE).
Corresponds to an enantiomeric pair of diastereoisomers.
for both carbonyl stretching vibrations depending on the substitu-
tion and the stereostructure of the compounds [11]. From that per-
spective, and given the absence of strong electrostatic interactions
in solid state, the registered band shift for 1a–c (Fig. 3) provides
further support that the position of the two bands is very sensitive
to substitution at 3-, 4- and 6-position and the cycle conformation.
In 1a–c only alkyl substituents are present and their N-methyl
group prevents hydrogen bonding. The stretching and deformation
C–H vibrations of the aliphatic part are not affected by the forma-
tion of the heterocycle and appear in their usual ranges. The char-
b
droxy-3-methylbutanoic acid and L-N-methylamino acid residues.
In molecules 1a and 1c the sp3 C-atoms of the heterocycle (C3
and C6) are displaced by 33° and 40° out of the plane formed by
atoms O1, C2, N4 and C5. The boat in 1b is slightly more flat and
the corresponding values are 29° and 38° respectively. The exper-
imentally determined X-ray structure of the b(3R,6R) diastereoiso-
mer of 1a [28] shows an envelope conformation much closer to
flatness – C2, C3, N4, C5 and C6 lying in one plane and only O1 being
displaced by 18° out of the plane formed by the other atoms. How-
ever, this should be regarded rather as an exception since all other
X-ray studies on related morpholine-2,5-diones [10,29–32] report
boat conformations similar to those found in our molecular model-
ling study.
acteristic m(C2–O) and m(N–CH3) vibrations give rise to intensive
and strongly overlapped bands at 1217 and 1210 cmÀ1 in KBr.
In conclusion, 1a–c were found for the first time in the natural
products. For identification and confirmation, the compounds were
synthesized through N-(a-bromoacyl)-a-amino acids as non-cyclic
intermediate products. Biosynthetic ways of 1a–c are probably
similar with those of enniatin B, which was also identified in the
broth and mycelium. The stereostructure of 1a–c, expected accord-
ing to the assumed biosynthetic mechanism, was characterized by
quantum chemical calculations. Based on them, the preferred
conformations of 1a–c are those with equatorial isopropyl group
and axial 3-alkyl substituent. The role of 1a–c for the producing
organism is questionable as well as for enniatins and other
mycotoxins.
The vibrational spectra of 1a–c closely resemble each other due
to the structural similarity of the compounds. The most character-
istic IR region (3500–900 cmÀ1) is presented in Fig. 3 with the
experimental IR spectrum of 1a. The most prominent feature of
the IR spectra of 1a–c is the appearance of two very strong bands
of equal intensity between 1740 and 1620 cmÀ1. They correspond
to the stretching vibrations of the carbonyl groups in the morpho-
line-2,5-dione ring.
The higher frequency band, at 1738 cmÀ1 in solid state, is as-
signed to the ester m(C@O). It has the typical value for an aliphatic
3. Experimental
ester carbonyl group, but somewhat lower than the data reported
for N-methyl cyclodidepsipeptides, containing valine and phenyl-
alanine residues [10,11,33]. The second band in this region, at
1622 cmÀ1, is attributed to the stretching vibration of the amide
group. The band is also downshifted in comparison to the frequen-
cies reported for similar N-substituted and unsubstituted cyclodid-
epsipeptides [11,13,33,34]. The literature IR data are based only on
a few compounds of this type and suggest a broad frequency range
3.1. Plant material
H. barbatum Jacq. was collected at bloom stage in August 2005
on Rtanj mountain (Southeast Serbia). Plant material was identi-
fied by Prof. Dr. N. Randjelovic (Faculty of Occupation Safety, Nis,
Serbia) and a voucher specimen was deposited in the Herbarium
Moesicum Doljevac (Serbia) under the accession number 735.
O
O
O
O
O
O
H3C
H
H3C
H
H3C
H
O
O
CH3
CH3
O
CH3
N
N
N
(R)
(R)
(S)
(S)
(S)
(R)
CH3
CH3
CH3
H3C
H
H
H
CH3
CH3
H3C
CH3
CH3
1b
1a
1c
Fig. 2. Lowest energy (3S) diastereoisomers of 1a–c.

A. Smelcerovic et al. / Journal of Molecular Structure 985 (2011) 397–402
401
3.2. Isolation, identification and fermentation of the fungi
ane. The reaction mixture was heated for 40 min at 80 °C, then
cooled down and diluted with 15 ml of tetrachloromethane.
23.9 g (0.12 mol) of N-bromosuccinimide and 15 drops of 40%
aqueous solution of HBr were applied. After 10 min of heating at
70 °C, and 90 min at 85 °C, the solvent and the excess of SOCl2 were
removed under reduced pressure. The residue was suction filtered,
the solid was washed several times with tetrachloromethane and
the solvent was removed from the combined filtrate as before.
The obtained 2-bromo-3-methylbutanoyl chloride was used in
the next step of the reaction without further purification.
(4): GC–MS (4 min), m/z: 201, 199, 197 (M+); IR (CCl4, 0.61 mm
CaF2 cell), cmÀ1: 2972, 2939, 2871, 1794, 1559, 1464, 1391, 1373,
1225, 1191, 1127; 1H NMR (250 MHz; CDCl3): 1.92 s, 6 H (2CH3);
2.26–2.36 m, 1 H (CH); 4.30–4.33 d, 1 H, J = 7.2 (CH); 13C NMR
(250 MHz; CDCl3): 19.9, 20.1 (2 CH3), 30.6 (CH), 62.3 (CH), 173.5
(CO).
The isolation of the endophytic fungi was carried out following
the method of Strobel et al. [15]. Briefly, in the laboratory, fresh
plant materials were thoroughly surface treated with 70% ethanol.
The stems were used for the isolation of endophytic fungi. Then,
with a sterile knife blade, outer tissues were removed from the
samples and the inner tissues carefully excised and placed on the
water agar plates (WA; DIFCO, Cat. No. 214530). After 8 days of
incubation at 28 2 °C hyphal tips of the fungi were removed
and transported to potato dextrose agar (PDA; DIFCO, Cat. No.
213400). The pure cultures, thus obtained, were preserved by cryo-
preservation at 70 °C. Fungus used in the present study was iden-
tified as F. sporotrichioides Sherb. [W&R,G,B,J] according to the
protocol of Nelson et al. [35]. Shake flask fermentation was carried
out for a period of 5 days at 28 °C. After fermentation mycelium
and broth were separated by centrifugation.
6-(Propan-2-yl)-4-methyl-morpholine-2,5-diones: 0.006 mol of
N-methylamino acid was suspended in 15 ml dry chloroform
cooled in ice, and 0.003 mol of 2-bromoisovaleryl chloride was
added. The mixture was stirred for 1 h, and then the temperature
was allowed to rise to 20 °C. The chloroform was removed under
reduced pressure, the residue was dissolved in 20 ml ether and
the solution was filtered to remove the solid. The filtrate was ex-
tracted with 30 ml of 0.5 M aqueous solution of potassium carbon-
ate (three portions of 10 ml) and washed with water (10 ml). The
combined alkaline solution was immediately acidified with HCl
(pH ꢀ 2) and twice re-extracted with 30 ml ether (three portions
of 10 ml). The combined ether layers from the re-extraction were
dried over sodium sulfate and the solvent was removed under re-
duced pressure. The evaporation yielded small amount of light yel-
low oil which crystallized on being kept. The residual crude
material was purified by column chromatography (d = 1 cm;
l = 7 cm) on silica gel eluting with mixture of cyclohexane and
ethyl acetate (1:1) to afford the pure title compound. After recrys-
tallization in mixture of diethyl ether and petroleum ether colour-
less crystals as needles were obtained;
3.3. GC–MS analysis
GC–MS analyses of the broth and mycelium ethyl acetate ex-
tracts were performed on a Thermo Finnigan Trace Gas Chromato-
graph and Trace MSPLUS detector, equipped with a fused silica
column (DB-5 30 m  0.25 mm  0.25
lm); carrier gas helium
(1 ml/min). The operating conditions were: temperature program,
60–320 °C at 10 °C/min and 320 °C (4 min); injector temperature,
310 °C; detector temperature, 320 °C. Ionization was performed
at 70 eV.
3.4. Mycotoxin analysis
The analysis of mycotoxins from ethyl acetate extracts of the
broth and mycelium was performed using a LC–MS/MS multi-
method. The detailed procedure has been reported earlier [18,19]
and is briefly described in this work. A Thermo-Finnigan Surveyor
HPLC system was interfaced to the mass spectrometer for auto-
mated LC–MS and LC–MS/MS analyses. The Surveyor HPLC system
included a quaternary, low-pressure mixing pump with vacuum
degassing, an autosampler with temperature-controlled tray
(10 °C), a column oven (25 °C). A C18(2) Luna Phenomenex column
3,6-Di(propan-2-yl)-4-methyl-morpholine-2,5-dione (1a): C11H19
NO3, M = 213.27; yield = 35%; RI (retention index) 1558; MS
[60–500 m/z]: 213 (M+), 171, 142, 100, 83, 71, 69; IR (KBr), cmÀ1
:
2969, 2938, 2876, 1737, 1622, 1489, 1471, 1420, 1389, 1371,
1355, 1314, 1281, 1259, 1209, 1139, 1121, 1105, 1001, 977, 930,
861, 847, 770, 752, 661, 636, 548; 1H NMR (600 MHz; CDCl3):
0.91–0.94 m, 3 H (CH3); 0.97–1.01 m, 3 H (CH3); 1.07–1.12 m, 3
H (CH3); 1.17–1.19 dd, 3 H, J = 3.5, 3.6 (CH3); 2.09–2.11 t, 1 H,
J = 5.1, 4.8 (CH); 2.39–2.42 m, 1 H (CH); 3.10 s, 3 H (N–CH3);
4.17–4.18 d, 1 H, J = 9.5 (CH); 4.85–4.86 d, 1 H, J = 10.4 (CH); 13C
NMR (600 MHz; CDCl3): 18.8, 19.1, 20.1, 21.1 (4 CH3), 27.9 (CH),
29.7 (CH), 31.9 (N–CH3), 64.4 (CH), 70.0 (CH), 170.5 (COO), 171.4
(CON).
3-(2-Methylpropyl)-6-(propan-2-yl)-4-methyl-morpholine-2,5-
dione (1b): C12H21NO3; M = 227.30; yield = 32%; RI 1631; MS [60–
500 m/z]: 227 (M+), 185, 170, 142, 129, 112, 100, 83, 69; IR (film),
cmÀ1: 2964, 2934, 2874, 1730, 1645, 1472, 1410, 1388, 1370, 1323,
1258, 1187, 1135, 1097, 1058, 928, 851, 651; 1H NMR (600 MHz;
CDCl3): 0.90–0.99 m, 6 H (2CH3); 1.06–1.17 m, 6 H (2CH3); 1.79–
1.80 m, 1 H (CH); 2.08–2.10 m, 2 H (CH2); 2.36–2.42 m, 1 H
(CH); 3.03 s, 3H (N–CH3); 4.18–4.20 d, 1 H, J = 9.6 (CH); 5.36–
5.39 dd, 1 H, J = 7.1, 6.7 (CH); 13C NMR (600 MHz; CDCl3): 19.7,
20.1, 20.9, 21.3 (4CH3), 23.5 (CH), 24.8 (CH), 31.8 (N–CH3), 36.8
(CH2), 51.0 (CH), 69.5 (CH), 170.1 (COO), 176.2 (CON).
(150 mm  2 mm  3
column held at 25 °C was used with a solvent flow rate of 0.2 ml/
min and an injection volume of 10 l. The mobile phase for ESI-
lm) attached to a Phenomenex C18 Guard
l
pos consisted of eluent A containing methanol/water/acetic acid
(97:2:1 v/v/v), and eluent C consisting of methanol/water/acetic
acid (10:89:1 v/v/v) containing 10 mM ammonium acetate. The
chromatographic conditions were as follows: 0–2 min, 10% A; 2–
5 min, 10–60% A; 5–20 min, 60–100% A; followed by a hold time
of 8 min (20–28 min, 100% A). Finally, column re-equilibration
was carried out at 10% A for 5 min. Mobile phase for ESI-neg con-
sisted of eluent A containing methanol/water/acetic acid (97:2:1 v/
v/v), and eluent B composed of water/acetic acid (99:1 v/v). The
solvent gradient was as follows: 0–1.5 min, 5% A; 1.5–5.5 min, 5–
70% A; 5.5–13 min, 70–100% A; The final hold time was set at
10 min (13–23 min 100% A) and column re-equilibration was car-
ried out at 5% A for 5 min. Identification and quantification, in
which ammonium adduct of enniatin B [M+NH4]+ (m/z 657) was
fragmented in the collision cell to the product ions (m/z 196 at
41 eV as quantifier ion and m/z 640 at 23 eV as qualifier ion) were
performed.
3-(Butan-2-yl)-6-(propan-2-yl)-4-methyl-morpholine-2,5-dione
(1c): C12H21NO3; M = 227.30; yield = 36%; RI 1645; MS [60–500
m/z]: 227 (M+), 185, 171, 156, 142, 129, 126, 100, 88, 83, 71, 69;
IR (film), cmÀ1: 2969, 2935, 2877, 1733, 1650, 1464, 1436, 1418,
1387, 1258, 1220, 1160, 1104, 1064, 960, 930, 899, 851, 776,
669.
3.5. Synthesis
2-Bromo-3-methylbutanoyl chloride (4): 21.8 ml (35.7 g; 0.3 mol)
of thionyl chloride were added dropwise to a stirred solution of
11 ml (10.2 g; 0.1 mol) isovaleric acid in 20 ml tetrachlorometh-

402
A. Smelcerovic et al. / Journal of Molecular Structure 985 (2011) 397–402
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the most probable conformational isomers were fully optimized
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3.7. IR spectra measurements
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Acknowledgments
We thank Prof. Dr. N. Randjelovic, Faculty of Occupation Safety,
Nis, Serbia, for taxonomic identification of plant material. We also
thank BMBF, Germany (Project MOE 06/R05) and National Science
Fund of Bulgaria (Young Researchers Project DO-02-272) for finan-
cial support.
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