Phomentrioloxin: A phytotoxic pentasubstituted geranylcyclohexentriol produced by Phomopsis sp., a potential mycoherbicide for Carthamus lanatus biocontrol

Article abstract of DOI:10.1021/np300200j

A new phytotoxic geranylcyclohexenetriol, named phomentrioloxin, was isolated from the liquid culture of Phomopsis sp., a fungal pathogen proposed for the biological control of Carthamus lanatus, a widespread and troublesome thistle weed belonging to the Asteraceae family causing severe crop and pastures losses in Australia. The structure of phomentrioloxin was established by spectroscopic, X-ray, and chemical methods as (1S,2S,3S,4S)-3-methoxy-6-(7- methyl-3-methylene-oct-6-en-1-ynyl)- cyclohex-5-ene-1,2,4-triol. At a concentration of 6.85 mM, the toxin causes the appearance of necrotic spots when applied to leaves of both host and nonhost plants. It also causes growth and chlorophyll content reduction of fronds of Lemna minor and inhibition of tomato rootlet elongation. Finally, in preliminary bioassays, phomentrioloxin did not show any antibacterial, fungicidal, or zootoxic activities.

Full text of DOI:10.1021/np300200j

Article
pubs.acs.org/jnp
Phomentrioloxin: A Phytotoxic Pentasubstituted
Geranylcyclohexentriol Produced by Phomopsis sp., a Potential
Mycoherbicide for Carthamus lanatus Biocontrol
Alessio Cimmino,† Anna Andolfi,† Maria C. Zonno,‡ Ciro Troise,† Antonello Santini,§ Angela Tuzi,⊥
Maurizio Vurro,‡ Gavin Ash,# and Antonio Evidente*,†
†Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali (DISSPAPA), Universita
Federico II, Via Universita 100, 80055 Portici, Italy
‡Istituto di Scienze delle Produzioni Alimentari (ISPA), CNR, Via Amendola 122/O, 70125 Bari, Italy
̀
di Napoli
̀
§Dipartimento di Scienza degli Alimenti (DSA), Universita
̀
di Napoli Federico II, Via Universita
̀
100, 80055 Portici, Italy
⊥Dipartimento di Scienze Chimiche (DSC), Universita
4, 80126 Napoli, Italy
̀
di Napoli Federico II, Complesso Universitario Monte. S. Angelo, Via Cinthia
#EH-Graham Centre for Agriculture Innovation (an alliance between Charles Sturt University and NSW DPI), Booroma Street,
Locked Bag 588, Wagga Wagga NSW 2678, Australia
S
* Supporting Information
ABSTRACT: A new phytotoxic geranylcyclohexenetriol, named phomentrioloxin, was
isolated from the liquid culture of Phomopsis sp., a fungal pathogen proposed for the
biological control of Carthamus lanatus, a widespread and troublesome thistle weed
belonging to the Asteraceae family causing severe crop and pastures losses in Australia.
The structure of phomentrioloxin was established by spectroscopic, X-ray, and chemical
methods as (1S,2S,3S,4S)-3-methoxy-6-(7-methyl-3-methylene-oct-6-en-1-ynyl)-
cyclohex-5-ene-1,2,4-triol. At a concentration of 6.85 mM, the toxin causes the
appearance of necrotic spots when applied to leaves of both host and nonhost plants. It also causes growth and chlorophyll
content reduction of fronds of Lemna minor and inhibition of tomato rootlet elongation. Finally, in preliminary bioassays,
phomentrioloxin did not show any antibacterial, fungicidal, or zootoxic activities.
affron thistle (Carthamus lanatus L. ssp. lanatus) is a
widespread winter-growing annual weed of both pastures
Considering that phytopathogenic fungi are important
sources of bioactive metabolites having herbicidal potential,11,12
and considering that species of the genus Phomopsis are well
known to be producers of phytotoxic metabolites,13,14 studies
were carried out in order to ascertain the capability of this
Phomopsis sp. to produce novel bioactive metabolites.
This article reports on (a) the isolation and the chemical
characterization of the main phytotoxin produced by one strain
of Phomopsis sp. isolated from Carthamus lanatus; (b) the
preliminary studies of its biological properties in order to
evaluate its potential to be developed as a natural and safe
herbicide.
S
and crops throughout Australia, introduced from the
Mediterranean region.1 It is considered the most economically
important thistle species in New South Wales2,3 and was one of
the weeds targeted by the Australian Cooperative Research
Centre for Weed Management Systems.4 It is declared noxious
in all Australian States.5
Poor results of mechanical5 and chemical control have made
this weed a suitable target for biological control.2 In an effort to
develop a mycoherbicide against this weed, a number of
pathogenic isolates of Phomopsis have been identified from
naturally infected saffron thistle plants in Australia,6 and their
potential as mycoherbicides against the host evaluated.7
Furthermore, a recent study on genetic diversity carried out
on a large number of strains supported the hypothesis that the
Phomopsis sp. pathogenic to saffron thistle can be considered a
new species.8 Considering that the fungus causes elliptical
necrosis on the stems resulting in plant death, the involvement
of phytotoxins produced by the fungus in the disease
development process has been assumed. Previous studies
proved the inability of these strains9 to produce the powerful
mammalian mycotoxin phomopsin A, which is indeed produced
by other Phomopsis sp.,10 but did not aim to ascertain the
eventual production of other bioactive metabolites.
RESULTS AND DISCUSSION
■
The organic extract of Phomopsis sp., showing a high phytotoxic
activity, was purified by a combination of CC and TLC guided
by biological assays as reported in detail in the Experimental
Section. The main phytotoxic metabolite (10 mg/L) was
obtained as a homogeneous solid, which crystallized as white
needles from MeOH. The preliminary 1H and 13C NMR
investigations proved that it contains double bonds and
Received: March 20, 2012
Published: June 13, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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hydroxy groups and that it is a novel metabolite, to which the
name phomentrioloxin was assigned (1, Figure 1).
(d, J = 4.0 Hz) at the typical chemical value of δ 6.16.17 In the
COSY spectrum18 the latter coupled with a proton (H-4) of the
secondary hydroxylated carbon (C-4) resonating as doublets of
doublet doublets (J = 6.1, 4.1, and 4.0 Hz) at δ 4.51, being also
coupled with the protons of the geminal hydroxy group (a
doublet, J = 6.1 Hz) at δ 2.64 and the proton (H-3) of another
adjacent secondary oxygenated carbon (C-3). H-3, resonating
as a double doublet (J = 7.9 and 4.1 Hz) at δ 3.70, in turn
coupled with the proton (H-2) of another adjacent
hydroxylated secondary carbon (C-2) appearing as doublets
of double doublets (J = 7.9, 3.6, and 2.0 Hz) at δ 4.21. The
latter also coupled with the proton of the geminal hydroxy
group (d, J = 2 Hz) at δ 2.68 and with the proton (H-1) of the
third adjacent secondary hydroxylated carbon (C-1). H-1
1
appeared as a broad singlet at δ 4.35. The same H NMR
spectrum also showed a further doublet (J = 1.4 Hz) of a
hydroxy group and the singlet of a methoxy group linked to C-1
and C-3 at δ 2.65 and 3.55, respectively.
Figure 1. Structures of phomentrioloxin (1), its 1,2,4-O,O′,O″-triacetyl
(2) and 1,2-O,O′-isopropylidene (3) derivatives, and the 4-O-S- and
-R-MPTA esters of the 1,2-O,O′-isopropylidene derivative of
phomentrioloxin (4 and 5, respectively).
These results agreed with the presence in 1 of a 1,2,4-
trihydroxy-3-methoxycyclohexene ring. Consequently, the
polyunsaturated side chain constituted by the remaining 10
carbons should be attached to C-6 of this pentasubstituted
cyclohexene ring. This suggested a terpenoidal origin of this
second partial moiety. Indeed, the 1H NMR and COSY spectra
showed the presence of (1) two coupled doublets (J = 1.5 Hz)
at δ 5.40 and 5.31, respectively, representing typical chemical
shift values for protons of an olefinic methylene group (H2C-
8′); (2) the multiplets of two coupled methylene groups (H2C-
4′ and H2C-5′) at δ 2.22 and 1.64, respectively; and (3) singlets
of two vinylic methyl groups (Me-9′ and Me-10′) at δ 2.22 and
1.85, respectively. The remaining two carbons of the geranyl
residue attached to C-6 of the cyclohexene ring as well as the
latter carbon are quaternary carbons resonating in the 13C
NMR spectrum (Table 1) at δ 135.4, 87.3, and 92.4 (C-6, C-1′,
and C-2′), typical chemical shift values of olefinic and alkyne
carbons.19 The geranyl nature of the side chain attached was
delineated by several long-range couplings observed between
the carbons and the protons of this moiety, as observed in the
HMBC spectrum (Table 1). The presence of these two joined
moieties in 1 was confirmed by the other data of its 13C NMR
spectrum (Table 1). Indeed the latter spectrum showed the
presence of five methine carbons at δ 135.3, 79.1, 69.1, 67.9,
and 64.8 (C-5, C-3, C-1, C-2, and C-4, respectively); three
methylenes at δ 123.8, 37.9, and 18.4 (C-8′, C-4′, and C-5′,
respectively); two methyls at δ 27.4 and 26.4 (C-9′ and C-10′,
respectively); and one methoxy carbon at δ 59.3 (OMe). They
were assigned on the basis of the couplings observed in the
HSQC spectrum.
Phomentrioloxin has a molecular weight 292, due to its
molecular formula C17H24O4 deduced by its HRESIMS,
consistent with six hydrogen deficiencies. The latter were also
confirmed by the typical bands observed in the IR spectrum for
the olefinic groups and hydroxy groups. The same spectrum
also showed a typical band for the presence of at least one
alkyne group.15 The UV spectrum showed absorptions maxima
of an extended conjugated chromophore.16
1
The detailed investigation of the H NMR spectrum (Table
1) showed the presence of an olefinic proton (H-5) appearing
ab
,
1
Table 1. H and 13C NMR Data of Phomentrioloxin (1)
c
position
δC
δH (J in Hz)
4.35 (1H) br s
HMBC
1
69.1 CH
67.9 CH
79.1 CH
64.8 CH
135.3 CH
135.4 C
H-5, H-3, HO-1
2
4.21 (1H) ddd (7.9, 3.6, 2.0)
3.70 (1H) dd (7.9, 4.1)
4.51 (1H) ddd (6.1, 4.1, 4.0)
6.16 (1H) d (4.0)
3
H-5, OMe, OH-2
4
5
6
H-1
1′
2′
3′
4′
5′
6′
7′
8′
87.3 C
H-5, H-1
H2-8′
92.4 C
131.5 C
H2-8′, H2-4′
37.9 CH2
18.4 CH2
124.4 CH
123.1 C
2.22 (2H) m
1.64 (2H) m
5.13 m
H2-8′
On the basis of these results the structure of 1,2,4-triol-3-
methoxy-6-(7-methyl-3-methyleneoct-6-en-1-ynyl)cyclohex-5-
ene was assigned to the metabolite, named phomentrioloxin
(1).
Me-10′, Me-9′
H2-4′, H2-5′
123.8 CH2
5.40 (1H) d (1.5)
5.31 (1H) d (1.5)
2.22 (3H) s
d
This structure was supported by several long-range couplings
observed in the HMBC spectrum (Table 1) and by the
HRESIMS data. Indeed, the latter spectrum showed, besides
the dimeric sodium cluster at m/z 607, the potassium and the
sodium clusters at m/z 331 and 315.1561, respectively.
The structure assigned to phomentrioloxin was further
confirmed by preparing two key derivatives, which were also
used in the bioassay described below, comparing their
phytotoxicity to that of 1. By acetylation and acid-catalized
ketalization, 1 was converted into 1,2,4-O,O′,O″-triacetyl- (2)
and 1,2-O,O′-isopropylidene (3) derivatives, respectively.
9′
27.4 CH3
26.4 CH3
59.3 CH3
H-6′, Me-10′
Me-9′
d
10′
1.85 (3H) s
OMe
HO-1
HO-2
HO-4
3.55 (3H) s
2.65 (1H) d (1.4)
2.68 (1H) d (2.0)
2.64 (1H) d (6.1)
a
b
Chemical shifts are in δ values (ppm) from TMS. 2D 1H,1H
(COSY) and 13C,1H (HSQC) NMR experiments delineated the
correlations of all the protons and the corresponding carbons.
c
d
Multiplicities were assigned by DEPT spectrum. These assignments
may be reversed.
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As expected, the IR spectrum of 2 lacked bands due to
hydroxy groups. Its H NMR spectrum differed from that of 1
configuration is R*/R*/R*/R*. It was not possible to
determine the absolute configuration, because of weak
anomalous scattering. In the crystal packing all OH groups
are involved in a pattern of intermolecular OH···O hydrogen
bonds.
The relative configuration assigned to 1 was also confirmed
by the couplings observed in the NOESY spectrum.18 The
correlation between H-1 and H-2 and that between H-4 and
both H-5 and H-3 as well as the lack of correlation between H-
2 and H-3 appeared to be particularly significant. Finally, the
correlation between the methoxy group and both Me-9′ and
Me-10′, in agreement with the inspection of the Dreiding
models of 1, indicated a bending of the side chain toward the
cyclohexene ring.
The absolute configuration of 1 was determined by applying
an advanced Mosher’s method.21 By reaction with R-(−)-α-
methoxy-α-trifluoromethylphenylacetate (MTPA) and S-
(+)MTPA chlorides, the 1,2-O,O′-isopropylidene derivative
(3) was converted into the corresponding diastereomeric S-
MTPA and R-MTPA monoesters at C-4 (4 and 5,
respectively), whose spectroscopic data were consistent with
the structure assigned to 1. Comparison between 1H NMR data
(Table 2) of the S-MTPA (4) and R-MTPA (5) esters of 1
permitted assignment of the Δδ (4, 5) values of all the protons
as reported in Figure 3 and the assignment of the S-
configuration to C-4. Consequently a 1S, 2S, and 3S
configuration was assigned to C-1, C-2, and C-3, respectively,
and 1 was formulated as (1S,2S,3S,4S)-3-methoxy-6-(7-methyl-
3-methyleneoct-6-en-1-ynyl)cyclohex-5-ene-1,2,4-triol.
1
essentially by lacking hydroxy group signals, the downfield
shifts (Δδ 1.46, 1.27, and 1.22), and the multiplicity of H-1, H-
2, and H-4 resonating as a doublet (J = 4.1), a double doublet
(J = 8.9 and 4.1 Hz), and a double doublet (J = 4.4 and 4.1 Hz),
at δ 5.81, 5.48, and 5.73, respectively. Finally, the presence of
the three singlets at δ 2.14, 2.13, and 2.09, respectively, due to
the three acetyl groups was also observed. Its ESIMS spectrum
showed the sodium cluster at m/z 441.
1
The H NMR data of derivative 3 differed from those of 1,
i.e., lacking the two broad singlets of the two hydroxy group at
C-1 and C-2 and in the presence of two singlets of the
isopropylidene group at δ 1.43 and 1.40, respectively.
Furthermore, the spectrum indicated downfield shifts (Δδ
0.22 and 0.27) of the doublet (J = 5.7 Hz) and a double doublet
(J = 5.7 and 4.6 Hz) of H-1 and H-2 resonating at δ 4.57 and
4.48 as engaged in the isopropylidene ring. The ESIMS data
showed the sodium cluster of both 3 and its dimer at m/z 355
[M + Na]+, 687 [2M + Na]+.
The relative configuration of 1 was deduced from the values
measured for coupling between the protons of the cyclohexene
ring. On the basis of these values (Table 1), H-2 and H-3, and
H-1 and H-4, were pseudoaxially and pseudoequatorially
located, while the cyclohexene ring probably assumes a twisted
conformation, as also observed by an inspection of a Dreiding
model of 1.17 This relative configuration as well as the structure
assigned to 1 were confirmed by the X-ray analysis (Figure 2).
The closest phytotoxin to phomentrioloxin appears to be
foeniculoxin (Table 3), a geranyl hydroquinone isolated as the
main bioactive lipophilic metabolite from the culture filtrates of
a strain of Phomopsis phoeniculi, the causal agent of wilting of
stems and inflorescences of fennel.22 Foeniculoxin belongs to
the polyprenylated 1,4-benzoquinone and 1,4-hydroquinone
groups of natural compounds, which, like ubiquinone,
plastoquinone, and tocopherols, are widespread in plants,
animals, and marine organisms, in which they play important
roles in electron transport, in photosynthesis, and as
antioxidants.23 Some examples of geranyl-quinones and -hydro-
quinones isolated from different organisms and with interesting
biological activities, structurally related to phomentrioloxin, are
reported in Table 3.24−32 Very significant for the pathological
and taxonomical aspects appeared to be the close relation
between the structures of the toxins produced by the strain of
P. foeniculi isolated in Italy and of that produced by the strain of
Phomopsis sp. isolated in Australia from saffron thistle. A
number of Phomopsis species have been isolated from fennel in
Europe.33,34 This commonality of toxins between the Australian
and European isolates could reflect common origins for these
isolates in Europe or could indicate gene flow between species
in the region.
Figure 2. ORTEP view of phomentrioloxin (1) showing atomic
labeling. Displacement ellipsoids are drawn at the 30% probability
level.
Compound 1 crystallizes in the orthorhombic P212121 space
group. An ORTEP view of the molecule is shown in Figure 2.
Bond lengths and angles in 1 are in the normal range.20 In the
six-membered ring, the C-5−C-6 bond distance and the
geometry around C-5 and C-6 confirmed the presence of the
cyclohexene double bond. The cyclohexene ring adopts a
twisted conformation with C-2 and C-3 pointing up and down
from the plane formed by C-1/C-6/C-5/C-4. The ring
geometry is constrained by the presence of the C-5−C-6
double bond. A further structural constraint is represented by a
triple bond (C-1′−C-2′) vicinal to the ring and a double bond
between C-3′ and C-8′. The two hydroxy groups at C-1 and C-4
are axial and point in opposite directions with respect to the
plane of the cyclohexene ring. The third hydroxy group at C-2
is disposed in the equatorial position. The sequence of torsion
angles around the C3′−C4′, C4′−C5′, and C5′−C6′ bonds
[−67.8(6)°, −71.3(6)°, −172.6(5)°, respectively] of the 7-
methyl-3-methyleneoct-6-en-1-ynyl group generates the char-
acteristic V-shape of the molecule. Four stereogenic centers are
present in the molecule at C1/C2/C3/C4, whose relative
A broad number of different biological assays were
performed with the main toxin and two derivatives. The leaf
puncture assay was carried out also with the extracts and the
fractions for guiding the purification procedures. These assays,
summarized in Table 4, aimed at obtaining preliminary
information on (1) the phytotoxicity of the compound; (2)
the potential for its use as a natural herbicide; (3) the impact on
nontarget organisms; and (4) preliminary observations on
structure−activity relationships.
Assayed on leaves of several weeds at 40 μg/droplet (6.85
mM), phomentrioloxin caused the appearance of necrotic
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Table 2. 1H NMR Data (CDCl3) of 1,2,4-O,O′,O″-Triacetyl- and 1,2-O,O′-Isopropylidene and 4-O-S- and 4-O-R-MTPA Esters of
a
Phomentrioloxin (2, 3, 4 and 5, respectively)
2
3
4
5
position
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
1
5.81 (1H) d (4.1)
4.57 (1H) d (5.7)
4.609 (1H) d (6.1)
4.575 (1H) d (6.1)
2
5.48 (1H) dd (8.9, 4.1)
3.81 (1H) dd (8.9, 4.1)
5.73 (1H) dd (4.4, 4.1)
6.15 (1H) d (4.4)
4.48 (1H) dd (5.7, 4.6)
3.67 (1H) t (4.6)
4.249 (1H) dd (7.1, 6.1)
3.592 (1H) dd (7.1, 3.5)
5.835 (1H) dd (5.0, 3.5)
6.151 (1H) d (5.0)
4.317 (1H) dd (7.1, 6.1)
3.612 (1H) dd (7.1, 3.3)
5.799 (1H) dd (5.2, 3.3)
6.136 (1H) d (5.2)
3
4
4.39 (1H) ddd (8.4, 4.6, 3.3)
6.11 (1H) d (3.3)
5
1′
2′
3′
4′
5′
6′
7′
8′
1.45 (2H) m
1.32 (2H) m
5.09 (1H) m
2.20 (2H) m
1.56 (2H) m
5.11 (1H) m
2.210 (2H) m
1.547 (2H) m
5.108 (1H) m
2.203 (2H) m
1.551 (2H) m
5.105 (1H) m
5.35 (1H) d (1.7)
5.30 (1H) d (1.7)
1.70 (3H) s
5.36 (1H) d (1.5)
5.26 (1H) d (1.5)
1.69 (3H) s
5.406 (1H) d (1.6)
5.309 (1H) d (1.6)
1.688 (3H) s
5.399 (1H) d (1.0)
5.305 (1H) d (1.0)
1.686 (3H) s
b
9′
b
10′
1.62 (3H) s
1.62 (3H) s
1.620 (3H) s
1.616 (3H) s
OMe
HO-4
MeCO
Me2C
OMe
Ph
3.49 (3H) s
3.54 (3H) s
3.549 (3H) s
3.592 (3H) s
2.51 d (1H) (8.4)
2.14 (3H) s, 2.13 (3H) s, 2.09 (3H) s
1.43 (3H) s and 1.40 (3H) s
1.482 (3H) s and 1.380 (3H) s
3.386 (3H) s
1.498 (3H) s and 1.383 (3H) s
3.491 (3H) s
7.700−7.506 (5H) m
7.705−7.401 (5H) m
a
b
Chemical shifts are in δ values (ppm) from TMS. These assignments may be reversed.
content and 50% reduction of the fresh weight of the fronds
(Table 4). At a concentration 10 times lower the metabolite
had a modest and not significant toxicity, whereas at
concentrations even lower it was completely inactive (data
not shown).
Tested at 1.37 mM on germinated tomato seeds,
phomentrioloxin caused around 50% inhibition of rootlet
elongation (Table 4), whereas the two derivatives had almost
no activity (data not shown).
These results suggested that all the hydroxy groups of the
cyclohexene ring are important features for the phytotoxicity.
However, the role of the geranyl side chain and its
functionalities remain to be investigated.
When phomentrioloxin was assayed at 3.42 mM, only around
32% protoplasts of Arabidopsis thaliana remained viable (Figure
4), compared to 67% cell viability of the control cells. At 0.34
mM the toxin was still active, whereas at lower concentrations
its toxicity was progressively reduced (Figure 4).
Interestingly, phomentrioloxin and the two derivatives
proved to be inactive in the other preliminary bioassays carried
out on organisms other than plants. In particular, they proved
to be inactive when tested for antifungal activity against
Geotrichum candidum and for antibacterial activity against
Bacillus subtilis and Escherichia coli up to 100 μg/diskette.
Moreover the main toxin and the two derivatives caused no
larvae mortality when supplied to shrimps through artificial
seawater at concentrations up to 0.171 mM.
The wide range of preliminary bioassays performed seems to
show that phomentrioloxin, the main metabolite produced by
the fungus, has only phytotoxic properties and no toxicity to
other nontarget organisms at the concentrations tested. This
characteristic, to be further confirmed, could be particularly
important in the attempts of finding novel metabolites to be
used as natural and safe herbicides. The phytotoxicity is not
Figure 3. Structure of the 4-O-S- and -R-MPTA esters of the 1,2-O,O′-
isopropylidene derivative of phomentrioloxin (4 and 5, respectively)
reporting the Δδ value obtained by comparison (4 − 5) of each
proton system.
lesions one day after the application. A large area of necrosis
(around 1 cm diameter) was particularly evident on the leaves
of Cirsium arvense and Sonchus oleraceus. Slightly smaller
necrotic areas (6−8 mm) were clearly observable on leaves of
the host plant C. lanatus, but also on leaves of the other two
dicotyledonous weed species tested, i.e., Mercurialis annua and
Chenopodium album. Necrotic spots were smaller (diameter
around 3 mm) on Setaria viridis (monocot). Assayed at 3.42
mM, phomentrioloxin caused clearly observable lesions on C.
arvense, S. oleraceus, and the host as well, whereas at 1.71 mM, it
was active only on C. arvense leaves. At the highest
concentration tested the 1,2-O,O′-isopropylidene derivative 3
proved to be slightly active against the dicotyledonous weeds
and inactive against S. viridis, whereas 1,2,4-O,O′,O″-triacetyl
derivative 2 was completely inactive against all plants tested.
Assayed on fronds of Lemna minor at 6.85 mM the main
toxin caused approximately 90% reduction of the chlorophyll
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Table 3. Natural Compounds Structurally Related to Phomentrioloxin
particularly strong, considering that clear necrosis or effects on
cells and chlorophyll could be observed only when using
relatively high concentrations of toxin. On the hand, the lack of
antifungal and antibacterial activities seems to be quite evident,
as the amount of toxin used in those assays was biologically
high. Moreover, the preliminary studies on structure−activity
relationships showed that structural modifications could modify
biological activities and phytotoxicity. These aspects will be
addressed in the future for a better assessment of the potential
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Table 4. Biological Activities of Phomentrioloxin (see Experimental Section for details)
organism
bioassay/application
toxin concentration
type of effect measured
activity
Carthamus lanatus
Chenopodium album
Cirsium arvense
leaf puncture
leaf puncture
leaf puncture
leaf puncture
leaf puncture
leaf puncture
protoplasts
6.85 × 10−3
6.85 × 10−3
6.85 × 10−3
6.85 × 10−3
6.85 × 10−3
6.85 × 10−3
3.42 × 10−3
6.85 × 10−3
6.85 × 10−3
1.37 × 10−3
M
M
M
M
M
M
M
M
M
M
necrosis
6−8 mm
6−8 mm
1 cm
necrosis
necrosis
Mercurialis annua
Sonchus oleraceus
Setaria viridis
necrosis
6−8 mm
1 cm
necrosis
necrosis
2−3 mm
51% ( 3)
94% ( 3)
47% ( 2)
50% ( 9)
inactive
Arabidopsis thaliana
Lemna minor
cell death
frond immersion
frond immersion
germinated seeds
agar diffusion
agar diffusion
agar diffusion
larvae
chlorophyll reduction
rootlet growth inhibition
rootlet growth inhibition
inhibition halo
inhibition halo
inhibition halo
mortality
Lemna minor
Lycopersicon esculentum
Escherichia coli
up to 100 μg/disk
up to 100 μg/disk
up to 100 μg/disk
1.71 × 10−4
Bacillus subtilis
inactive
Geotrichum candidum
Artemia salina
inactive
M
inactive
the Istituto di Scienze delle Produzioni Alimentari, CNR, Italy, with
the code ITEM13496.
Production, Extraction, and Purification of Phomentrioloxin
(1). The fungus was grown in 1 L Erlenmeyer flasks containing 300
mL of a defined mineral.35 Each flask was seeded with 5 mL of a
mycelia suspension and then incubated at 25 °C for 4 weeks in the
dark. After mycelial removal by filtration, the culture filtrates (4 L)
were lyophilized and then dissolved in 400 mL of distilled H2O,
acidified to pH 2.5 with HCOOH, and extracted with EtOAc (4 × 400
L). The organic extracts were combined, dried by Na2SO4, and
evaporated under reduced pressure, giving a brownish-red, oily residue
(937 mg) showing high phytotoxic activity. It was fractionated by a
silica gel column eluted with CHCl3−i-PrOH (9:1), obtaining 11
groups of homogeneous fractions. Groups 5, 6, 9, and 10 proved to be
phytotoxic and were further purified. The residues of fractions 5 and 6
were combined (170 mg) and purified by preparative TLC, eluted with
CHCl3−i-PrOH (9:1), producing four bands. The third band (81 mg)
was purified under the same conditions to afford phomentrioloxin (1,
Rf 0.32, 40.3 mg, 10.0 mg/L) as a homogeneous solid. It was
crystallized by slow evaporation from a MeOH−H2O (3:1) solution.
Figure 4. Effect of phomentrioloxin (1) on protoplasts of Arabidopsis
thaliana (see Experimental Section for the experimental details).
Phomentrioloxin (1). [α]25 −23 (c 0.4, CHCl3); IR νmax 3385,
of phomentrioloxin as a natural and environmentally friendly
herbicide.
D
2186, 1666, 1628, 1604 cm−1; UV λmax nm (log ε) 259 (4.25), 240
(4.25), 230 (4.25); 1H and 13C NMR spectra, see Table 1; ESIMS (+)
m/z 607 [2 M + Na]+, 331 [M + K]+, 315.1561 [calcd for
C17H24NaO4 315.1572, M + Na]+.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation was
measured on a Jasco P-1010 digital polarimeter, IR spectra were
recorded as glassy film on a Perkin-Elmer Spectrum One FT-IR
spectrometer, and UV spectra were recorded in MeCN solution on a
Perkin-Elmer Lambda 25 UV/vis spectrophotometer. 1H and 13C
NMR spectra were recorded at 400/100 MHz in CDCl3 on Bruker
spectrometers. The same solvent was used as an internal standard.
Carbon multiplicities were determined by DEPT spectra.18 DEPT,
COSY-45, HSQC, HMBC, and NOESY experiments18 were
performed using Bruker microprograms. HRESI and ESIMS spectra
were recorded on Waters Micromass Q-TOF Micro and Agilent
Technologies 6120 Quadrupole LC/MS instruments, respectively.
Analytical and preparative TLC were performed on silica gel plates
(Merck, Kieselgel 60 F254, 0.25); the spots were visualized by exposure
to UV light and/or by spraying first with 10% H2SO4 in MeOH and
then with 5% phosphomolybdic acid in EtOH, followed by heating at
110 °C for 10 min. CC: silica gel (Merck, Kieselgel 60, 0.063−0.200
mm).
1,2,4-O,O′,O″-Triacetylphomentrioloxin (2). Phomentrioloxin
(15.0 mg) was acetylated with pyridine (50 μL) and Ac2O (50 μL) at
room temperature for 10 min. The reaction was stopped by addition of
MeOH, and the azeotrope, obtained by the addition of benzene, was
evaporated by an N2 stream. The oily residue (13.7 mg) was purified
by preparative TLC, eluted with CHCl3−i-PrOH (97:3), to give the
1,2,4-O,O′,O″-triacetyl derivative 2 of phomentrioloxin as a homoge-
neous compound (Rf 0.63, 3.2 mg). Derivative 2: [α]25 −132 (c 0.3,
D
CHCl3); IR νmax 2335, 1747, 1637,1599, 1220 cm−1; UV λmax nm (log
1
ε) 273 (sh), 261 (4.56); H NMR, see Table 2; ESIMS (+) m/z 441
[M + Na]+.
1,2-O,O′-Isopropylidene of Phomentrioloxin (3). To phomen-
trioloxin (12.0 mg) dissolved in dry acetone (12.0 mL) was added
anhydrous CuSO4 (480.0 mg). The reaction was carried out by reflux
under stirring for 2 h and then stopped by filtration. The solution
obtained was evaporated under reduced pressure. The residue (13.4
mg) was purified by preparative TLC, eluted with CHCl3−i-PrOH
(97:3), yielding 3 as a homogeneous oil (Rf 0.53, 6.3 mg): [α]25D +21
(c 0.2); IR νmax 3450, 2280, 1725, 1631, 1604 cm−1; UV λmax nm (log
Fungal Strain. The fungal strain of Phomopsis sp. used in this study
was isolated from symptomatic saffron thistle (C. lanatus) plants in
Australia in 1994.6 The fungus was identified by Dr. Micheal Priest
(New South Wales Department of Primary Industries, ASCU Orange
Agricultural Orange, Australia) and deposited as DAR Herb 73822
saffron thistle (C. lanatus) plants in Australia in 1994.6 Pure cultures
were maintained on potato-dextrose-agar (PDA, Sigma-Aldrich
Chemic GmbH, Buchs, Switzerland) and stored in the collection of
1
ε) 260 (5.01); H NMR, see Table 2; ESIMS (+) m/z 687 [2M +
Na]+, 355 [M + Na]+.
4-O-(S)-α-Methoxy-α-trifluoromethyl-α-phenylacetate
(MTPA) Ester of Derivative 3 (4). (R)-(−)-MPTA-Cl (10 μL) was
added to 3 (2.0 mg) dissolved in dry pyridine (20 μL). The mixture
was kept at room temperature for 1 h, and then the reaction stopped
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Journal of Natural Products
Article
by adding MeOH. Pyridine was removed by an N2 stream. The residue
(2.5 mg) was purified by preparative TLC, eluted with CHCl3, yielding
4 as a homogeneous oil (Rf 0.69, 2.1 mg): IR νmax 2389, 1750, 1451,
1381, 1244 1169 cm−1; UV λmax nm (log ε) 260 (4.56); 1H NMR, see
Table 2; ESIMS (+) m/z 571 [M + Na]+.
and chlorophyll contents were determined by using the protocol
described.39
(c) For the assay on protoplasts, callus cultures of Arabidopsis
thaliana L. Heynh. Ecotype Landsberg (kindly provided by S. Caretto
and G. Colella, ISPA CNR Lecce) were maintained at 25 °C under
continuous fluorescent white light on MS (Sigma) basal medium
supplemented with 30 g/L sucrose, 0.5 mg/L naphthaleneacetic acid,
0.05 mg/L kinetin, pH 5.5, and 0.6% (w/v) agar. The callus cultures
were subcultured at 4-week intervals by transferring approximately 50
mg of callus tissue in Magenta boxes containing 100 mL of the same
agar medium. Extraction and assay on protoplasts were performed
according to the protocol described by Zonno et al. (2008).40 Four
replications were prepared for each concentration. The experiment
was repeated twice. The number of viable cells was expressed as a
percentage of the total number of cells.
The bioassay for the inhibition of rootlet elongation was carried out
on tomato (Lycopersicon esculentum) seeds (var. Marmande). Briefly,
seeds were surface sterilized by NaOCl (1%), washed, and allowed to
germinate in Petri dishes. Ten germinated seeds were then transferred
to small plates containing 1 mL of the test solution (1.37 mM). Plates
were kept in a incubator at 25 °C in the dark for 4 days and then
rootlets measured. Their length was compared to that of the controls.
Three replications were prepared for each compound tested. The
experiment was repeated twice.
Antimicrobial Activity. The antifungal activity of phomentriolox-
in and the two derivatives was tested on Geotrichum candidum, whereas
the antibacterial activity was assayed against Bacillus subtilis (Gram +)
and Escherichia coli (Gram −), according to the protocols already
described, by using up to 100 μg of each metabolite/diskette.41 Three
replications were performed for each compound. The zootoxic activity
of the three metabolites was tested on larvae of Artemia salina L. (brine
shrimp) up to 1.71 × 10−4 M, as previously described,41 with four
replications.
4-O-(R)-α-Methoxy-α-trifluoromethyl-α-phenylacetate
(MTPA) Ester of Derivative 3 (5). (S)-(+)-MPTA-Cl (10 μL) was
added to 3 (2.0 mg) dissolved in dry pyridine (20 μL). The reaction
was carried out under the same conditions used for preparing 4. The
purification of the crude residue (2.2 mg) by preparative TLC (solvent
system D) gave 5 as a homogeneous oil (Rf 0.69, 1.6 mg): IR νmax
2377, 1750, 1451, 1377, 1237, 1168 cm−1; UV λmax nm (log ε) 261
1
(4.85); H NMR, see Table 2; ESIMS (+) m/z 571 [M + Na]+.
Crystal Structure Determination of Phomentrioloxin (1).
Colorless, block-shaped single crystals of 1 were obtained at ambient
temperature by slow evaporation of a MeOH−H2O (3:1) solution. X-
ray data collection was performed at 173 K under N2 flow on a Bruker-
Nonius KappaCCD diffractometer equipped with graphite-monochro-
mated Mo Kα radiation (λ = 0.71073 Å, CCD rotation images, thick
slices, φ and ω scans to fill asymmetric unit). Cell parameters were
obtained from a least-squares fit of the θ angles of 44 reflections in the
range 3.818° ≤ θ ≤ 18.589°. A semiempirical absorption correction
(multiscan, SADABS) was applied. The structure was solved by direct
methods and anisotropically refined by the full matrix least-squares
method on F2 against all independent measured reflections (SIR97
package)36 and refined by the full matrix least-squares method on F2
against all independent measured reflections (SHELXL program of the
SHELX97 package).37 The position of hydroxy H atoms was
determined from a difference Fourier map and refined according to
a riding model. In the absence of significant anomalous scatters, the
absolute configuration cannot be determined. Friedel pairs were
therefore merged before the final refinement. The final refinement
converged to R1 = 0.0546 for 1627 observed reflections having I >
2σ(I). Minimum and maximum residual electronic density was −0.211
and 0.236 e Å−3. Crystal data: formula C17H24O4, formula weight
292.36 g mol−1, orthorhombic P212121, a = 4.632(3) Å, b = 11.982(7)
Å, c = 29.48(2) Å, α = β = γ = 90°, 6405 collected reflections, 1627
unique reflections.
Phytotoxic Activity. (a) For the bioassay-guided purification of
the phytotoxic compounds, culture filtrates, organic extracts, and
chromatographic fractions were assayed by using a leaf puncture assay
as described below. Extracts and fractions were dissolved in MeOH
(20 μg extract/μL) and then diluted with distilled H2O (final
concentration of MeOH = 2%). Phomentrioloxin (1) was tested at
concentrations of 6.85, 3.42, and 1.71 mM, by applying 20 μL of
solution to detached leaves previously punctured with a needle. It was
tested against Carthamus lanatus (the host plant of the pathogen,
family Asteraceae), Mercurialis annua L. (Euphorbiaceae), Chenopo-
dium album L. (Chenopodiaceae), Cirsium arvense (L.) Scop.
(Asteraceae), Sonchus oleraceus L. (Asteraceae), and Setaria viridis
(L.) P. Beauv (Poaceae). Five replications (droplets) on separate
leaves were used for each metabolite and for each plant species tested.
Leaves were kept in a moistened chamber under continuous
fluorescent lights. Symptoms were estimated five days after droplet
application, by using a visual empiric scale from 0 (no symptoms) to 4
(wide necrosis, around 1 cm diameter). The two derivatives were
assayed by using the same protocol.
ASSOCIATED CONTENT
■
S
* Supporting Information
Spectra of 1 and a cif data file are available free of charge via the
Internet at http://pubs.acs.org. Crystallographic data for the
structure have also been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication
number CCDC 872237. These data can be obtained free of
charge at www.ccdc.cam.ac.uk/conts/retrieving.html or from
the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (internat.) +44-1223/336-033.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +39 081 2539178. Fax: +39 081 2539186. E-mail:
evidente@unina.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(b) Pure compounds were tested against Lemna minor at
concentrations between 6.85 mM and 68.5 μM, by adapting a
protocol already described.38 Briefly, the wells of sterile, polystyrene
96-well microtiter plates were filled with a 50 μL aliquot of solutions
containing the metabolites to be tested, at the concentration reported
above. One frond of actively growing axenic L. minor was placed into
each well. Control wells were included in each plate. Four replications
were prepared for each compound. The plates were incubated in a
growth chamber with 12/24 h fluorescent lights and observed daily up
to 4 days. One day after the application of the test solution, 100 μL of
distilled H2O was added to each well. The appearance of necrosis or
chlorosis was assessed visually by comparison of the treated plants with
the control appearance. Moreover, plantlet fresh weight was measured,
The NMR spectra were recorded in the laboratory of the
CERMANU Centre, Universita
Italy, by Mr. P. Mazzei, whose contribution is gratefully
̀
di Napoli Federico II, Portici,
acknowledged. Authors are also grateful to “Centro Regionale
̀
di Competenza−Nuove Tecnologie per le Attivita Produttive”
(CRdC-NTAP) of the Campania Governorate, and to “Centro
Interdipartimentale di Metodologie Chimico Fisiche”
̀
(CIMCF) of the Universita di Napoli Federico II, for X-ray
facilities. This is Contribution DISSPAPA No. 263. A.E. is
associated with “Istituto di Chimica Biomolecolare del CNR”,
Pozzuoli, Italy.
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Article
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