Insecticidal cyclodepsipeptides from beauveria felina

Article abstract of DOI:10.1021/np100890n

A novel cyclodepsipeptide, iso-isariin B (1), and the known isaridin E (2) were isolated from the entomopathogenic fungus Beauveria felina. Their structures were elucidated using MS/MS fragmentation and extensive 2D-heteronuclear NMR. The X-ray structure of isaridin E was obtained, showing two potent intramolecular H bonds and a type-VI turn with the HyLeu ¹-Pro² amide bond in a cis conformation. Iso-isariin B (1) was active against the pest-insect Sitophilus spp. with an LD₅₀ value of 10 μg/mL. This observation also gives some clues for ecological interpretation of cyclodepsipeptide production by B. felina.

Full text of DOI:10.1021/np100890n

ARTICLE
pubs.acs.org/jnp
Insecticidal Cyclodepsipeptides from Beauveria felina
†
†
‡
§
†
^
Aude Langenfeld, Alain Blond, Sabelle Gueye, Patrick Herson, Bastien Nay, Jo €e lle Dupont, and
,†
Soizic Prado*
†
Mol ꢀe cules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS/MNHN, Mus ꢀe um National d'Histoire
Naturelle, 57 Rue Cuvier (CP 54) 75005 Paris, France
‡
Laboratoire d'Entomologie et d'Acarologie, Facult ꢀe des Sciences et Techniques, Universit ꢀe Cheikh Anta Diop de Dakar, BP5005 Dakar,
S ꢀe n ꢀe gal
§
Institut Parisien de Chimie Mol ꢀe culaire (IPCM), UMR CNRS 7201, Centre de R ꢀe solution de Structure, Universit ꢀe Pierre et Marie
Curie-Paris 6, 4 Place Jussieu, 75252, Paris Cedex 05, France
^
Laboratoire de Mycologie, UMR CNRS 7205, Mus ꢀe um National d’Histoire Naturelle, 57 Rue Cuvier, 75231 Paris Cedex 05, France
S Supporting Information
b
ABSTRACT: A novel cyclodepsipeptide, iso-isariin B (1), and
the known isaridin E (2) were isolated from the entomopatho-
genic fungus Beauveria felina. Their structures were elucidated
using MS/MS fragmentation and extensive 2D-heteronuclear
NMR. The X-ray structure of isaridin E was obtained, showing
two potent intramolecular H bonds and a type-VI turn with the
1
2
HyLeu -Pro amide bond in a cis conformation. Iso-isariin B (1)
was active against the pest-insect Sitophilus spp. with an LD50
value of 10 μg/mL. This observation also gives some clues for
ecological interpretation of cyclodepsipeptide production by B.
felina.
he chemical ecology of fungi implies the study of their
hexadepsipeptide characterized by the presence of a 3-hydroxy-4-
methyloctanoic acid moiety (HMA), so far unprecedented in the
isariin series. We also report for the first time the X-ray structure
of isaridin E (2), a cyclohexadepsipeptide previously isolated by
T
interactions with congeners and also with potential hosts. In
such studies, it is important to identify classes of metabolites that
could be mediators in these interactions. We have investigated
the metabolite production and associated insecticidal activities of
Beauveria felina (DC.) J. W. Carmich. isolated from the soil of an
Algerian cavern but also known as entomopathogenic. In culture,
B. felina produces luxurious morphological structures named
synnemata (a group of hyphae-bearing conidia) described by
7
Balaram et al. Insecticidal activity of the compounds was
demonstrated using the pest insects Sitophilus spp. and Calloso-
bruchus maculatus, giving clues for ecological interpretation of
cyclodepsipeptide production by B. felina.
1
Taber and Vining. The ecological significance of these large
structures, produced by diverse fungi including Beauveria, Asper-
gillus, and Penicillium for example, from humid habitats in tropical
rainforest to drier environments in agricultural soils and grains
’ RESULTS AND DISCUSSION
Sequence Determination by Mass Spectrometry. The
molecular weight of iso-isariin B (1) was obtained from a positive
ESI-qTOF mass spectrum, showing the protonated molecular
2
has been discussed by Samson and Seifert. B. felina was
3
previously named Isaria felina (DC.) Fr. The first depsipeptide
þ
þ
[
M þ H] ion at m/z 596.4034 and the [M þ Na] adduct ions
isolated from this fungus was reported in 1962 and named isariin
A in reference to this former nomenclature, and subsequently,
three additional cyclodepsipeptides, isariins B, C, and D, were
at m/z 618.3851 corresponding to the molecular formula
C H N O (calcd for C H N O : 596.4023). Similarly, the
30
53
5
7
30 54 5 7
4,5
molecular formula C H N O was assigned to compound 2,
35 53 5 7
described by Baute and co-workers. More recently, Balaram
and co-workers reported the X-ray crystal structures of isaridins
A and B, two new hexadepsipeptides featuring several modified
þ
taking into account a protonated molecular [M þ H] ion
þ
observed at m/z 656.4044 and a [M þ Na] adduct ion at m/z
þ
6
678.3859 as well as the [M þ K] adduct ion at m/z 694.3640
amino acid residues. The same group reported the identification
7
(calcd for C35H N O : 656.4023).
54 5 7
and characterization of six isariins and four isaridins.
We wish to report herein the isolation and sequence char-
acterization of iso-isariin B (1), based on tandem mass spectros-
copy and extensive 2D-heteronuclear NMR. This is a new cyclic
Received: December 13, 2010
Published: March 25, 2011
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
825
dx.doi.org/10.1021/np100890n |J. Nat. Prod. 2011, 74, 825–830

Journal of Natural Products
ARTICLE
1
The collision-induced dissociation (CID) spectrum of the
Table 1. NMR Spectroscopic (DMSO-d , 298 K, H 400.13
MHz; C 75.04 MHz) Data for Iso-isariin B (1)
6
þ
13
[
M þ H] ion of 1 at m/z 596 showed a main series of adjacent
b ions at m/z 497, 426, 313, 214, and 157 corresponding to the
n
position
δ
C
, mult.
H
δ , mult, J (Hz)
successive loss of Val, Ala, Leu/Ile, Ala, Gly, and a C H O
9
16 2
2
3
1
fragment, thus suggesting the sequence C H O -Gly -Val -
HMA
9
16 2
4
5
6
Leu/Ile -Ala -Val for the linear peptide ion derived from 1.
1
2
169.8, qC
37.5, CH
A second series of ions was observed at 568, 469, 398, 285, and
2
2.51, dd (14.7; 9.3)
.22, dd (14.7; 2.1)
1
86 and was assigned to the related adjacent a ion. A third series
n
2
of ions was observed at m/z 578, 479, 408, 295, 196, and 139,
having 18 mass units less than the preceding bn ion and
corresponding to the loss of a water molecule. The mass spectra
results are consistent with an isariin and suggested a provisional
3
4
5
75.5, CH
36.2, CH
31.2, CH
4.90, ddd (9.3; 3.9; 2.1)
1.66, m
2
1.35, m
7
1
2
1.01, m
structure similar to the described isariin B: cyclo(βHA -Gly -
3
4
5
6
6
28.7, CH
2
1.25, m
Val -Leu/Ile -Ala -Val ). However, the nature of the C H O
9
16 2
fragment could not be elucidated by this method, and an NMR
study was then undertaken as described below.
1.16, m
7
8
9
22.3, CH
2
1.23, m
þ
The CID spectrum of the [M þ H] ion of compound 2 at m/
13.8, CH3
14.8, CH3
0.85, m
z 656 showed a main series of adjacent b peaks at 412, 227, and
n
0.82, m
1
14 corresponding to the successive loss of (Phe-Pro), (HyLeu-
2
Gly
β-Ala), NMe-Val, and NMe-Val.
CO
169.0, qC
þ
A second linear peptide was formed from the [M þ H] ion at
5
6
R
41.9, CH
2
4.10, m
m/z 656 due to the cleavage at the NMe-Val -βAla amide bond
3.45 m
level, and the resulting b ions were detected at m/z 543, 430, and
n
5
NH
7.87, dd (3.8; 6.7)
2
83, corresponding to the successive loss of NMe-Val , NMe-
Val , and Phe . A third significant series of ions was observed at
28, 515, 402, and 255, which were assigned to an adjacent a ion
3
4
3
Val
CO
R
171.6, qC
6
n
of the above bn.
58.7, CH
29.5, CH
22.9, CH3
4.03, m
A fourth series of peaks, due to the ease of cleavage of the
NMe-Val-NMe-Val amide bond, was observed at m/z 543, 396,
β
1.85, m
γ
0.88, m
2
99, 185, and 114. These ions correspond to the successive loss
0
γ
18.7, CH
3
0.82, m
of N-Me-Val, Phe, Pro, HyLeu, β-Ala, and N-Me-Val and
NH
8.06, d (7.6)
1
2
3
4
suggested the sequence cyclo(HyLeu -Pro -Phe -N-Me-Val -N-
4
Leu
5
6
Me-Val -βala ) of isaridin E, which was further established by the
D NMR studies.
NMR Studies. The H and C spectrum of iso-isariin B (1) in
CO
R
171.2, qC
2
1
13
51.8, CH
4.02, m
β
38.6, CH
24.1, CH
20.9, CH
2
1.49, m
DMSO-d solution (Table 1) displayed a single stable conforma-
6
γ
1.62, m
tional state (>95%). In agreement with a hexapeptide structure,
1
δ
3
0.78, m
0.88, m
five amide protons were seen in the H NMR spectrum, as well as
1
3
0
six carbonyl groups in the range δ 169.0ꢀ171.8 in the
C
δ
19.03, CH
3
C
1
13
NMR spectrum. Additional H NMR and C NMR signals
suggesting the presence of a linear alkyl chain were also recorded.
The assignment of the proton and carbon signals to the amino
acid residues was achieved using COSY, TOCSY, HSQC, and
HMBC experiments. Thus, the five amide NH's that resonate at
δH 7.39 (d, J = 7.7 Hz), 7.87 (dd, J = 3.8; 6.7 Hz), 7.99 (d, J = 7.9
Hz), 8.06 (d, J = 7.6 Hz), and 8.62 ppm (d, J = 6.5 Hz) correlated
to their corresponding CHR: δ 57.6 (δ 4.07, m), δ 41.9 (δ
NH
8.62, d (6.5)
5
Ala
CO
R
171.8, qC
47.9, CH
17.30, CH
4.17, m
β
3
1.20, m
NH
7.99, d (7.9)
6
Val
C
H
C
H
CO
R
170.6, qC
57.6, CH
29.5, CH
4
.10, 3.45, m), δ 47.9 (δ 4.17, m), δ 58.7 ppm (δ 4.03, m),
C H C H
4.07, m
and δ 51.8 ppm (δ 4.02, m), respectively. From the CHR
C
H
β
2.07, m
signals, further COSY and HMBC correlations led to the
identification of the amino acid side chains. The following five
standard amino acid residues were thus identified: glycine (Gly),
valine (Val), leucine (Leu), alanine (Ala), and valine (Val).
The peptide sequence determination is based on the data from
the HMBC experiments. This heteronuclear method was pre-
γ
17.5, CH
3
3
0.85, m
0
γ
18.9, CH
0.86, m
NH
7.39, d (7.7)
2
3
4
5
ferred to the dNN(i, iþ1) and d (iþ1) connectivity obtained
of the residue iþ1. Thus amide protons of Gly , Val , Leu , Ala ,
RN
6
2
with the ROESY/NOESY experiments because of the small size
of this cyclic peptide. Indeed for such small size cyclopeptides,
conformational NOE effects have been reported to interfere with
and Val were correlated respectively to the carbonyls C-1, Gly ,
3 4 5
Val , Leu , and Ala , leading to the following peptidic sequence:
2
3
4
5
6
Gly -Val -Leu -Ala -Val . The HMBC spectrum shows a corre-
lation between the additionnal diastereotopic methylene (H -2)
8
sequential correlations. The sequence of compound 1 was
2
determined by analysis of HMBC correlations between the
at δ 2.51, 2.22 and the carbonyl C-1 (δ 169.8). Moreover this
H
C
carbonyl of the residue i and the amide and/or the R-protons
diasteroisotopic methylene displays COSY and HMBC correlations
8
26
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Journal of Natural Products
ARTICLE
Figure 2. Proposed solution structure of iso-isariin B (1) in DMSO-d6
solution.
hydroxycarboxylic acid precursors, and the formation of cyclic
ester peptide occurs by unusual recursive condensation of this
10,11
monomer.
The usual isomer isariin B was not found during
this study, and the occurrence of iso-isariin B in B. felina is related
to the existence of a polyketide route leading to the HMA
residue. The HMA moiety is unusual in this kind of isariin
peptide but has been found in beauveriolide cyclopeptides
1
2,13
isolated from Beauveria sp.
1
1
Figure 1. HMA moiety of 1 with Hꢀ H COSY and HMBC correla-
tions (HfC).
Attempts to isolate the corresponding 3-hydroxy-4-methyloc-
tanoic acid by acidic hydrolysis of 1 were unsuccessful. Therefore
the absolute configurations of the oxymethine (C-3) and of the
C-4 remain undefined. It should be mentioned that Tomada et al.
have demonstrated that the absolute stereochemistry of the
with the oxymethine (δH 4.90; δ 75.5, C-3), suggesting the
attachment of this oxymethine C-3 to C-2. The absence of
correlation between the CO (Val ) and an amide proton asso-
C
6
14
HMA residue in natural beauveriolide is (3S,4R).
ciated with the shift of the oxymethine suggests that the ester
linkage is at this position.
The pattern of hydrogen bonding as shown in Figure 2 is
deduced from the thermal coefficient values of the amide proton
1
13
The rest of the H and C signals were assigned to a
measured in DMSO-d
low value of the thermal coefficient of amide protons of Ala and
(Supporting Information, Table S1). The
6
5
13
3
-hydroxy-4-methyloctanoic acid moiety. Indeed, the C spec-
trum clearly shows the presence of two additional methyls at δC
3.8 and 14.8 (apart from the four standard amino acid residues
Ala, Val, Val, Leu). A COSY correlation between the methyl at
δ 0.82 and the methine at δ 1.66 (H-4) demonstrates the
6
ꢀ1
Val (Δδ/ΔT respectively ꢀ3.4 and ꢀ0.8 ppb K ) suggests
3
1
that they are involved in intramolecular hydrogens bonds,
whereas the NH's of Leu and Val are exposed to the solvent
4
3
ꢀ
1
(Δδ/ΔT respectively ꢀ6.6 and ꢀ5.6 ppb K ). An ambiguity
H
H
3
2
existence of this methyl on C-4. This was confirmed by the
persists in the case of the amide proton of Gly , as its thermal
HMBC correlation between the carbon at δ 75.5 (C-3) and the
coefficient is intermediate. However, the NOE correlation seen
C
2
3
proton of the methyl at δ 0.82. Similarly, an HMBC correlation
between the NH (Gly ) and the NH (Val ) suggests that the NH
H
2
is observed between a methylene at δ 31.2 (C-5) and the methyl
of Gly is exposed to the solvent. The strong dNN NOE between
C
5
6
2
at δ 0.82, thus fully establishing the methyl position on C-4. As
NH (Ala ) and NH (Val ) and the NOE between NH (Gly ) and
H
3
depicted in Figure 1, the rest of this alkyl chain is easily
characterized with the COSY and HMBC correlations. All the
data are thus consistent with the structure cyclo[3-hydroxy-4-
NH(Val ) provide the location of the turn segment in the cyclic
3
D
4
5
peptide. In the case of compound 1, the Val - Leu -Ala segment
is likely to form a β-turn with an intramolecular hydrogen bond
between CO (Gly ) and NH (Ala ). A second intramolecular
hydrogen bond between CO (HMA ) and NH (Val ) would
stabilize this structure.
The H and C spectra of compound 2 in DMSO-d (Table 2)
2
3
4
5
6
2
5
methyloctanoyl-Gly -Val -Leu -Ala -Val ] for compound 1.
The absolute configurations of the amino acid residues of
1 6
9
compound 1 were also determined using Marfey’s method. The
1
13
hydrolysate of 1 was derivatized with 1-fluoro-2,4-dinitro-5-L-
alanine amide (FDAA) and analyzed by HPLC (at 340 nm). This
showed the presence of L-Gly, L-Ala, L-Val, and D-Leu. Interest-
ingly, the composition and sequence of compound 1 is in agree-
ment with the general structures of isariin cyclodepsipeptides
6
solution also displayed a single stable conformational state
1
(>95%). The two amide protons were identified in the H
NMR spectrum, as well as the presence of six carbonyl groups
13
in the range δ
agreement with a hexapeptide structure. The two intense singlets
at δ 2.97 and 3.12 are indicative of two N-methylamino acids.
168.6ꢀ173.9 in the C NMR spectrum, in
C
7
described for Isaria, more precisely with isariin of the B type.
Nevertheless, isariins are probably synthesized by the non-
ribosomal peptide biosynthesis pathway, and the existence of two
kinds of isomers could be explained by a lack of selectivity of one
of the enzymes involved in the process for the substrate. The
biosynthesis of cyclooligomer depsipeptide bassianolide and
beauvericin is by the programmed iterative use of modules
for the assembly of dipeptidol monomers from amino acid and
H
Assignment of protons and carbons to amino acid residues was
achieved, with the help of COSY, TOCSY, HSQC, and HMBC
experiments, leading to the identification of the six amino acid
residues as leucine (Leu), proline (Pro), phenylalanine (Phe),
valine (Val), valine (Val), and β-alanine (βAla). The peptide
sequence determination was based on the HMBC experiment,
8
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Journal of Natural Products
Table 2. NMR Spectroscopic Data (CDCl -d6 298 K, H
ARTICLE
1
3
,
13
4
00.13 MHz; C 75.04 MHz) for Isaridin E (2)
position
C
δ , mult.
H
δ , mult, J (Hz)
1
HyLeu
CO
R
169.8, qC
73.4, CH
5.32, dd (11.3; 1.8)
1.95, m
β
38.8, CH
2
1
.23, m
γ
δ
24.7, CH
23.3, CH
1.95, m
3
0.98, d (3.3)
0.98, d (3.3)
0
δ
20.5, CH
3
2
Pro
CO
R
172.0, qC
60.9, CH
Figure 3. Crystal structure of isaridin E (2) (dotted lines indicate
hydrogen bonds).
4.08, dd (8.6; 2.0)
2.20, m
β
32.2, CH
21.9, CH
47.1, CH
2
compound 2 were obtained from CH CN, 65% yield at 24 ꢀC.
2
.11, m
1.75, m
.32, m
3
The values of the backbone torsion angles and the H bonds are
γ
2
1
2
summarized in Tables S2 and S3. Notably, the HyLeu -Pro and
1
3
4
the N-Me-Val - N-Me-Val amide bonds adopt a cis conforma-
δ
2
3.48, m
3
tion (ω ( 0). Two potent intramolecular H bonds, NHPhe f
3
Phe
6
6
3
COβAla and NHβAla fCOPhe , are observed with a OꢀHN
hydrogen bond distance of 2.19 and 2.08 Å, respectively. Thus,
the X-ray structure of isaridin E is closely related to that described
for isaridins A and B and also provides an example of type-VI
turns, as defined for protein, in which the turning segments have
CO
R
173.7, qC
53.8, CH
35.0, CH
136.4, CH
128.7, CH
127.2, CH
4.64, ddd (7.5; 5.1; 2.6)
2.97, m
β
2
0
Φ1
0
0
16
Φ3 , Φ5
7.25, m
the central peptide unit in a cis conformation.
0
0
0
Φ2 , Φ5 , Φ4
7.20, m
The crystal structure of isaridin E seems to be consistent with
the solution structure in DMSO-d . Indeed, a strong NOE
NH
8.12, d (7.5)
6
2
4
correlation between the HR of the Pro (δ 4.08) and the HR
N-Me-Val
CO
N-CH
H
1
1
2
of the HyLeu (δ 5.32) indicates that the HyLeu -Pro amide
H
169.7, qC
29.6, CH
57.6, CH
27.6, CH
bonds are in a cis configuration. This stereochemistry is further
3
3
3.12, s
1
3
2
confirmed by the γ-carbon C chemical shifts of C of Pro at δ
2
γ
C
R
5.11, d (10.7)
2.35, m
17
1.9, in agreement with the presence of a cis proline. Moreover,
β
the relatively low thermal coefficient values of the amide proton
γ
18.8, CH
3
0.85, d (6.8)
0.86, d (6.8)
3
6
of Phe and βAla (respectively ꢀΔδ/ΔT = 0.06 and 1.90 ppb
3
0
ꢀ1
γ
20.2, CH
3
K
) confirm the presence of two potent intramolecular H
3 6 6 3
5
N-Me-Val
CO
N-CH
bonds, NHPhe fCOβAla and NHβAla fCOPhe .
168.6, qC
28.9, CH
66.5, CH
27.6, CH
19.4, CH
19.7, CH
Insecticidal Studies. B. felina is a facultative entomopathogen
fungus, as is B. bassiana, which has been used as a biopesticide
against a broad range of insects in agriculture. Since several
cyclopeptides from B. felina featured interesting insecticidal activi-
3
3
2.97, s
R
4.27, d (10.7)
2.40, m
β
4,5,17
ties,
our compounds 1 and 2 were evaluated for their insecti-
γ
3
3
0.91, d (6.4)
0.85, d (6.6)
cidal properties against Sitophilus spp. and Callosobruchus maculatus,
two pest insects responsible for seed and crop infestations. After five
days of surface contact with isaridine E (2), relatively weak lethal
doses 50 (LD ) of 300 and 220 μg/mL were found against these
0
γ
6
β-Ala
CO
173.9, qC
35.5, CH
50
R
2
4.14, m
two species. However, a remarkable LD50 of 10 μg/mL was
measured for compound 1 against Sitophilus spp. Thus, compound
1 can be considered, like bassianolide, beauvericin, and eniantins,
which have been shown to act as virulence factors for B. bassiana in
3
.12, m
2.60, dt (14.5; 3.1)
.47, m
β
35.3, CH
2
2
10
NH
7.39, d (10.1)
pathogenic context, as an agent of competition, defending the niche
of the producer fungi against competing parasites, thus providing a
possible explanation for the ecological role of these metabolites.
1
2
3
which provided the following peptide sequence: Leu -Pro -Phe -
4
5
6
N-Me-Val -N-Me-Val -β-Ala . All the data are thus consistent
’
EXPERIMENTAL SECTION
1
2
3
4
5
6
with a cyclo[HyLeu -Pro -Phe -N-Me-Val -N-Me-Val -β-Ala ]
structure for compound 2, which has been previously reported
as isaridin E. However, to go beyond the described primary
structure, we achieved a conformational analysis of isaridin E (2)
General Experimental Procedures. Optical rotations were
7
measured with a Perkin-Elmer model 341 polarimeter, and the [R]
D
2
ꢀ1
values are given in deg cm g . Melting points were determined on a
using X-ray diffraction studies (Figure 3). X-ray quality crystals of
B €u chi melting point B-545 apparatus. Mass spectra were recorded on an
8
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Journal of Natural Products
ARTICLE
API Q-STAR PULSAR i (Applied Biosystems). For the CID spectra, the
of water and treated with 0.2 mL of 1% 1-fluoro-2,4-dinitrophenyl-5-L-
alaninamide (FDAA) in acetone and 0.04 mL of 1.0 M sodium
bicarbonate. The vials were heated at 50 ꢀC for 90 min, and the contents
after cooling at room temperature were neutralized with 1 N HCl. After
degassing, an aliquot of the FDAA derivative was diluted in MeOH and
purified by chromatography using a RP C-18 column (250 ꢁ 4.6 mm)
and a linear gradient of acetonitrile and water containing 0.05%
trifluoroacetic acid from 20:80 to 50:50 in 20 min and then isocratic.
The flow rate was 1 mL/min, and the absorbance detection was at
340 nm. The chromatogram was compared with those of amino acid
standards treated in the same conditions.
1
3
collision energy was 40 eV and the collision gas was nitrogen. The
C
NMR spectra were recorded on a Bruker AC 300 spectrometer operat-
13
1
ing at 75.47 MHz (for C). The H and 2D NMR spectra were recorded
at 298 K on a Bruker AVANCE 400 spectrometer operating at 400.13
1
13
MHz. H and C chemical shifts were reported with respect to the
residual signal of dimethylsulfoxide, whose shifts relative to TMS were
taken at 2.49 ppm and at 39.5 ppm, respectively. For the HMBC
experiments the delay (1/2J) was 70 ms, and for the ROESY
experiments the mixing time was 150 ms. TOCSY experiments were
acquired with 40 and 80 ms spin-lock time using a MLEV-17 sequence.
Temperature coefficients of amide protons were obtained in the range
Amino acids in iso-isariin B (1): Gly (8.7 min), L-Ala (9.8 min), L-Val
1
2
98ꢀ318 K by the acquisition of five 1D H spectra using 5 K temperature
(13.0 min), D-Leu (18.7). t of standards: Gly (8.6 min), L-Ala (9.8 min),
R
increments.
L-Val (13.1 min), D-Val (15.6 min), L-Leu (16.2 min), D-Leu (18.6).
Insecticidal Activity. The insecticidal effect of substances was
determined in Petri dishes of diameter 90 mm. Products diluted in
acetone were tested at five concentrations (100, 200, 400, 800, and 1000
mg/mL). A 50 μL aliquot of each solution was distributed in a dish
before allowing the solvent to evaporate for 3 min. A batch of 20 insects
between 24 and 48 h old from the mass rearing was introduced into each
box and then left at room temperature. Adult mortality was monitored at
1 h after treatment and then every 12 h for 120 h (five days). Each treatment
was repeated three times; control experiments with insects treated only with
the solvent dilution and with untreated insects were also performed.
X-ray Crystal Structure Determination. A colorless crystal,
3
0
.25 ꢁ 0.11 ꢁ 0.08 mm was glued to a glass fiber. Intensity data were
collected at 200 K with a Bruker-Enraf Kappa-CCD diffractometer
equipped with a CCD two-dimensional detector [ λ Mo KR = 0.71073
Å]. Data reduction was performed with EVALCCD software. Data were
corrected for Lorentz and polarization effects, and a semiempirical
absorption correction based on symmetry equivalent reflections was
applied by using the SADABS program. Lattice parameters were obtai-
ned from least-squares analysis of 109 reflections. The structure was
solved by direct method and refined by full matrix least-squares, based
2
53 5 7 3 2
Iso-isariin B (1): C30H N O ; white needles (CH CN/H O, 80:20);
on F , using the Crystals software package. All non-hydrogen atoms were
2
0
1
13
mp 265ꢀ270 ꢀC; [R]
ꢀ10 (c 0.1, MeOH); H and C data, see
refined with anisotropic displacement parameters. All hydrogen atoms
were located with geometrical restraints in the riding mode.
Crystal structure analysis: monoclinic, space group P2 ; dimensions
1
D
þ
Table 1; ESI-qTOF MS/MS on [M þ H] (ce 40 eV) m/z (%) 596
(
4
(
3
(
2
(
8
15), 578 (4), 568 (10), 551 (7), 523 (2), 507 (1), 497 (43), 483 (8),
79 (25), 469 (3), 465 (1), 455 (6), 438 (3), 426 (65), 412 (4), 408
100), 401 (13), 398 (9), 384 (20), 366 (4), 356 (5), 338 (4), 327 (5),
13 (92), 309 (5), 302 (20), 295 (99), 285 (13), 284 (56), 277 (1), 273
7), 270 (11), 267 (83), 257 (15), 239 (22), 231 (8), 228 (3), 225 (10),
17 (8), 214 (7), 213 (96), 211 (11), 203 (11), 196 (12), 189 (84), 186
6), 185 (99), 183 (2), 168 (11), 157 (11), 143 (46), 139 (1), 118 (7),
a = 13.0206(5) Å, b = 9.8032(7) Å, c = 15.4303(15) Å, β = 94.550(6)ꢀ;
3
V = 1963.4(2) Å ; Z = 2; the data were collected in the h k l range ꢀ18 to
1
7, ꢀ13 to 13, ꢀ21 to 20. Total reflections collected: 20 570; indepen-
dent reflections: 6006 (4409 I > 2σ(I)); data was collected up to a 2θ
max
value of 60ꢀ. Number of variables: 452; R[I > 2σ(I)] = 0.0519, wR
2
(all) =
ꢀ
3
0
.144, S = 0.953; highest residual electron density 0.47e Å . Crystal-
6 (58), 72 (85).
lographic data have been deposited with the Cambridge Crystallographic
Data Centre, on quoting the depository number CCDC-795741. Copies of
the data can be obtained free of charge on application to CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK (fax: (þ44)1223-336-033; e-mail:
deposit@ccdc.cam.ac.uk).
Fungal Materiel. The fungus used in this study was isolated from
the soil of an Algerian cavern in 2006 and is now maintained at the LCP
culture collection (culture collection of the Mus ꢀe um National d’Histoire
Naturelle) under the number LCP 5299.
53 5 7 3 2
Isaridin E (2): C35H N O ; colorless crytal (CH CN/H O, 80:20);
2
0
1
13
mp 140ꢀ145 ꢀC; [R] ꢀ166 (c 0.1, MeOH); H and C see Table 2;
D
þ
ESI-qTOF MS/MS on [M þ H] (ce 40 eV) m/z (%). 656 (28), 628
(
1), 612 (34), 543 (59), 515 (42), 499 (1), 472 (4), 430 (8), 412 (1),
4
2
8
02 (18), 396 (2), 368 (13), 331 (2), 299 (9), 287 (2), 283 (5), 255 (9),
27(4), 212 (9), 203 (6), 194 (100), 185 (17), 169 (1), 142 (6), 114 (2),
6 (13), 69 (1).
Fermentation and Isolation. The fungus Beauveria felina was
maintained in potato dextrose agar at 25 ꢀC. The agar was cut into small
plugs and inoculated into 16 Erlenmeyer flasks (750 mL) containing
’ ASSOCIATED CONTENT
Supporting Information. This material is available free
S
b
18
of charge via the Internet at http://pubs.acs.org.
medium defined by Riley (30.0 g glucose (Sigma Aldrich), 5.0 g tryp-
tone (Difco), 3.0 g yeast extract (Merck), 0.3 g KH PO , 0.3 g K HPO
and 0.3 g MgSO4 7H O for 1 L). After incubation at 25 ꢀC for 10 days
2
4
2
4
,
’ AUTHOR INFORMATION
3
2
on a rotary shaker (200 rpm), the culture was centrifuged (7000 rpm, 20
min) to separate the mycelium and the filtrate. After lyophilization, the
mycelium (66.4 g) was macerated in MeOH (4 ꢁ 800 mL, 2 days each)
and filtered. The MeOH phase was concentrated under reduced
pressure to leave a brown gum (9.40 g).
Corresponding Author
*
Tel: 33 1 40793119. E-mail: sprado@mnhn.fr.
’
ACKNOWLEDGMENT
This crude extract was passed through a Sephadex LH-20 column
with MeOH as eluant to obtain 12 fractions, F1ꢀF12. Fraction F3
mainly contained iso-isariin B (1) (100 mg). Fraction F5 was subjected
to column chromatography on silica gel (cyclohexane/EtOAc fom 50:50
to 100), and subfraction 7 contained isaridin E (2) (75 mg).
Hydrolysis of Amino Acid Residue. The iso-isariin B (1) sample
The French “Minist ꢁe re de la Recherche” is acknowledged for a
fellowship for one of us (A.L.). The authors wish to thank L.
Dubost for the mass spectra.
’ REFERENCES
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