Phomentrioloxin, a fungal phytotoxin with potential herbicidal activity, and its derivatives: A structure-activity relationship study

Article abstract of DOI:10.1021/jf4030618

Phomentrioloxin is a phytotoxic geranylcyclohexenetriol produced in liquid culture by Phomopsis sp. (teleomorph: Diaporthe gulyae), a potential mycoherbicide proposed for the control of the annual weed Carthamus lanatus. In this study, seven derivatives obtained by chemical modifications of the toxin were assayed for phytotoxic, antimicrobial, and zootoxic activities, and the structure-activity relationships were examined. Each compound was tested on nonhost weedy and agrarian plants, fungi, Gram+ and Gram- bacteria, and on brine shrimp larvae. The results provide insights into an investigation of the structural requirements for activity. The hydroxy groups at C-2 and C-4 appeared to be important features for the phytotoxicity, as well as an unchanged cyclohexentriol ring. A role seemed also to be played by the unsaturations of the geranyl side chain. These findings could be useful for understanding the mechanisms of action of new natural products, for identifying the active sites, and possibly in devising new herbicides of natural origin.

Full text of DOI:10.1021/jf4030618

Article
pubs.acs.org/JAFC
Phomentrioloxin, a Fungal Phytotoxin with Potential Herbicidal
Activity, and its Derivatives: A Structure−Activity Relationship Study
†
†
‡
‡
†
§
Alessio Cimmino, Anna Andolfi, Maria Chiara Zonno, Angela Boari, Ciro Troise, Andrea Motta,
‡
∥
,†
Maurizio Vurro, Gavin Ash, and Antonio Evidente*
†Dipartimento di Scienze Chimiche, Universita
̀
di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126
Napoli, Italy
‡
Istituto di Scienze delle Produzioni Alimentari, Consiglio Nazionale delle Ricerche, Via Amendola 122/O, 70125 Bari, Italy
§
Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Comprensorio Olivetti, Edificio 70, Via Campi Flegrei 34,
8
0078 Pozzuoli, Italy
∥
Graham Centre for Agriculture Innovation (an Alliance between Charles Sturt University and NSW DPI), Booroma Street, Locked
Bag 588, Wagga Wagga, New South Wales 2678, Australia
*
S Supporting Information
ABSTRACT: Phomentrioloxin is a phytotoxic geranylcyclohexenetriol produced in liquid culture by Phomopsis sp. (teleomorph:
Diaporthe gulyae), a potential mycoherbicide proposed for the control of the annual weed Carthamus lanatus. In this study, seven
derivatives obtained by chemical modifications of the toxin were assayed for phytotoxic, antimicrobial, and zootoxic activities, and
the structure−activity relationships were examined. Each compound was tested on nonhost weedy and agrarian plants, fungi,
Gram+ and Gram− bacteria, and on brine shrimp larvae. The results provide insights into an investigation of the structural
requirements for activity. The hydroxy groups at C-2 and C-4 appeared to be important features for the phytotoxicity, as well as
an unchanged cyclohexentriol ring. A role seemed also to be played by the unsaturations of the geranyl side chain. These findings
could be useful for understanding the mechanisms of action of new natural products, for identifying the active sites, and possibly
in devising new herbicides of natural origin.
KEYWORDS: Phomopsis sp., Diaporthe gulyae, weeds, Carthamus lanatus, phytotoxins, phomentrioloxin, SAR
INTRODUCTION
Figure 1) were prepared by chemical transformation of the
functionalities present in 1 and tested for their phytotoxic,
antimicrobial, and zootoxic activities.
■
Carthamus lanatus L. (common name saffron thistle) is a
widespread and troublesome weed belonging to the Asteraceae
family, causing severe crop and pasture losses in Australia, and a
1
,2
MATERIALS AND METHODS
Phomopsis sp. strain was proposed for its biocontrol.
■
Recently, the teleomorph of the pathogen was classified as
General Experimental Procedures. Infrared (IR) spectra were
recorded as a deposit glass film on a Perkin-Elmer (Norwalk, CT,
USA) spectrometer, and ultraviolet (UV) spectra were measured in
MeCN, unless otherwise noted, on a Perkin-Elmer spectrophotometer.
3
Diaporthe gulyae, and from its culture filtrates a new phytotoxic
geranylhydroquinone named phomentrioloxin (1, Figure 1)
was isolated. It was characterized as (1S,2S,3S,4S)-3-methoxy-6-
1
H nuclear magnetic resonance (NMR) spectra were recorded at 600
(
7-methyl-3-methylene-oct-en-1-ynyl)-cyclohex-5-ene-1,2,4-
4
or 400 MHz in CDCl , on Bruker (Kalsruhe, Germany) spectrometers.
triol.
Phomentrioloxin showed interesting phytotoxic activities,
3
The same solvent was used as the internal standard. Electrospray
ionization (ESI) mass spectra (MS) were recorded on an Agilent
Technologies (Milano, Italy) 6120 Quadrupole liquid chromatography
mass spectrometry (LC/MS) instrument. Analytical and preparative
thin layer chromatography (TLC) were performed on silica gel
e.g., reduced growth and chlorophyll content of fronds of
Lemna minor and inhibited tomato rootlet elongation, but
showed no fungicidal, antibacterial, and zootoxic activities. SAR
studies on different fungal metabolites, e.g., nonenolides
putaminoxins and stagonolides), cytochalasins, oxazatricycloal-
kalenones, alternethanoxins, and agropyrenol, a phytotoxic
4
(Kieselgel 60, F254, 0.25 and 0.5 mm, respectively) (Merck, Darmstadt,
(
Germany) plates. The spots were visualized by exposure to UV
radiation (254) or by spraying first with 10% H SO in MeOH and
2
4
substituted salycilic aldehyde produced by different fungal
then with 5% phosphomolybdic acid in EtOH, followed by heating at
110 °C for 10 min. Column chromatography was performed on a
Kieselgel column, 60, 0.063−0.200 mm (3.0 × 85 cm) (Merck,
Darmstadt, Germany).
5
−8
species,
were carried out.
Considering the uncommon chemical structure of phomen-
trioloxin containing a 1,2,4-trihydroxy-3-methoxy cyclohexene
ring and a polyunsaturated geranyl moiety, and its promising
biological properties, it seemed worthwhile to further study this
metabolite, trying to ascertain its potential as a novel natural
herbicide and to identify the structural features essential for its
biological activity. For this reason, seven derivatives (2−8,
Received: July 11, 2013
Revised: September 10, 2013
Accepted: September 15, 2013
Published: September 15, 2013
©
2013 American Chemical Society
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Journal of Agricultural and Food Chemistry
Article
Figure 1. Structures of phomentrioloxin (1) and hemisynthesis and structures of its derivatives 2−8. Reagents and conditions: (a) Ac O, pyridine, rt,
2
5
or 10 min; (b) dry Me CO, dry CuSO , rt, 2 h; (c) MnO , CH Cl , rt, 5 h; (d) H , 5% Rd/C, MeOH, rt, 15 min; (e) MeOH, NaIO rt, 1 h;
2 4 2 2 2 2 4
MeOH, NaBH rt, 1 h.
4
Production, Extraction, and Purification of Phomentrioloxin
1). To produce phomentrioloxin (1), the fungus Diaporthe gulyae
4′,O-Didehydrophomentrioloxin (6). Compound 1 (9 mg)
(
dissolved in dry CH Cl (3 mL) was oxidized with dry MnO (65 mg),
2
2
2
(
anamorph Phomopsis sp.), previously isolated from naturally
under stirring at room temperature for 5 h. The mixture was then
filtered, and the solvent evaporated under reduced pressure to give an
oily residue, which was purified by preparative TLC (silica gel, eluent
symptomatic saffron thistle (C. lanatus) plants in Australia, was
used. Pure cultures were maintained on potato-dextrose-agar (PDA)
9
(
Sigma-Aldrich Chemic GmbH, Buchs, Switzerland) and stored in the
CHCl
3
/i-PrOH (95:5, v/v)) giving 6 as a homogeneous compound
collection of the Istituto di Scienze delle Produzioni Alimentari, CNR,
Italy, with the code ITEM13496. The fungus was grown on a mineral
defined liquid media named M1-D as previously reported. The
purification of the organic extract obtained from its culture filtrate (4
(R 0.56, 3 mg). Derivative 6 had IR νmax 3385, 2186, 1674, 1607, 1594
f
−
1
1
cm . UV λ nm (log ε): 283 (3.51). H NMR (Table 1). ESIMS
+) m/z: 313 [M + Na] .
max
+
10
4
(
1
′,1′,2′,2′,3′,8′,6′,7′-Octahydrophomentrioloxin (7). Com-
4
pound 1 (8 mg) was first dissolved in MeOH (500 μL), then added
L), carried out as previously described, produced phomentrioloxin
to a presaturated 5% Rd/C (3 mg) suspension in the same solvent
(
1) as an amorphous solid in amounts (40.3 mg, 10.0 mg/L) sufficient
for the preparation of the derivatives.
-O-Acetyl- and 1,2-O,O′-Diacetyl-phomentrioloxin (2 and
). Compound 1 (12 mg) was acetylated with pyridine (80 μL) and
(500 μL), and hydrogenated at room temperature and atmospheric
pressure under stirring conditions. The reaction was stopped after 15
min by filtration, and the solvent evaporated under reduced pressure;
the residue (13.5 mg) was purified by preparative TLC (silica gel,
eluent petroleum ether/acetone (7:3, v/v)) giving 7 as a homogeneous
compound (3 mg, R 0.35). Derivative 7 had IR ν 3410, 1666, 1460
1
3
Ac O (80 μL) at room temperature for 5 min. The reaction was
stopped by the addition of MeOH, and the azeotrope, obtained by the
addition of benzene, was evaporated by an N stream. The oily residue
(
2
f
max
2
−1
1
cm . UV λ nm (log ε): < 220. H NMR (Table 1). ESIMS (+) m/
z: 339 [M + K] , 323 [M + Na] .
max
13.7 mg) was purified by preparative TLC eluted with CHCl /i-
3
+
+
PrOH (97:3, v/v) to give the 1-O-acetyl and 1,2-O,O′-diacetyl
Conversion of Phomentrioloxin into 2-Methoxy-5-(3-meth-
ylene-oct-6-en-1-ynyl)-hex-4-ene-1,3,6-triol (8). Compound 1 (7
mg) dissolved in MeOH (10.2 mL) was oxidized with an aqueous
solution (3.2 mL) of NaIO4 (8 mg) under stirring at room
temperature in the dark. After 1 h, ethylene glycol (0.6 mL) and
derivatives of phomentrioloxin (2 and 3) as homogeneous compounds
(R 0.20, 4.1 mg; R 0.40, 3.9 mg, respectively). Derivative 2 had IR
f
f
−1
νmax 3382, 1714, 1661, 1646, 1615, 1269 cm . UV λ nm (log ε):
max
1
2
3
1
74 (sh), 257 (3.13). H NMR spectrum (Table 1); ESIMS (+) m/z:
+
+
73 [M + K] . 357 [M + Na] . Derivative 3 had IR ν 3397, 1721,
max
cold acetone (3.2 mL) were added, and the precipitated NaIO was
3
−1
661,1646, 1615, 1269 cm . UV λ nm (log ε): 273 (sh), 261
removed by filtration. The solution was extracted with CH Cl (4 × 15
max
2
2
1
+
(
3.24). H NMR spectrum (Table1); ESIMS (+) m/z: 415 [M + K] ,
mL), and the extract evaporated under reduced pressure. The residue,
+
3
99 [M + Na] .
dissolved in MeOH (2 mL), was reduced with NaBH (8 mg) for 1 h
4
1,2,4-O,O′,O″-Triacetyl Derivative (4) and 1,2-O,O′-Isopro-
at room temperature. The reaction was stopped by neutralization with
1 M HCl (1 mL) and the aqueous solution extracted with CH Cl .
pylidenephomentrioloxin (5). These two compounds were
prepared by routine acetylation with acetic anhydride and pyridine,
2
2
The extract was dehydrated (Na SO ) and purified by TLC (silica gel,
2 4
and by acid ketalization with dry Me CO of 1, respectively, as
previously reported.
eluent CHCl /i-PrOH (85:15, v/v)) giving 8 as a homogeneous
2
3
4
compound (3 mg, R 0.57). Derivative 8 had IR ν 3390, 1643, 1615,
f
max
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Journal of Agricultural and Food Chemistry
Article
1
a
Table 1. H NMR Data of Phomentrioloxin (1) and Its Derivatives (2, 3, and 6−8)
1
2
3
6
7
8
position
1
δ H (J in Hz)
δ H (J in Hz)
5.70 (1H) d (4.1)
δ H (J in Hz)
5.71 (1H) d (3.8)
δ H (J in Hz)
δ H (J in Hz)
δ H (J in Hz)
c
4.35 (1H) br s
4.56 (1H) br s
4.24 (1H) m
4.28 (1H) br d (12.6) 4.19
1H) br d (12.6)
(
c
2
3
4
5
6
4.21 (1H) ddd (7.9, 3.6, 4.32 (1H) ddd (7.2, 4.1, 5.56 (1H) dd (7.2, 3.8) 4.05 (1H) dd
.0) 2.0) (9.2, 3.3)
4.24 (1H) m
3.82 (1H) d (11.7) 3.77
(1H) d (11.7)
2
3.70 (1H) dd (7.9, 4.1) 3.66 (1H) dd (7.2, 4.4) 3.68 (1H) dd (7.2, 4.2) 4.00 (1H) d
9.2)
4.00 (1H) dd
(10.0, 4.0)
3.29 (1H) m
(
4.51 (1H) ddd (6.1, 4.1, 4.51 (1H) m
.0)
4.44 (1H) m
4.44 (1H) dd
(5.3, 4.0)
4.73 (1H) dd (8.5, 6.6)
6.01 (1H) d (8.5)
4
6.16 (1H) d (4.0)
6.18 (1H) d (3.6)
6.16 (1H) d (3.7)
6.21 (1H) s
5.71 (1H) d
(
5.3)
d
d
d
d
d
d
d
1
2
3
4
5
6
7
8
′
′
′
′
′
′
′
′
2.22 (2H) m
1.64 (2H) m
5.13 m
2.13 (2H) m
1.58 (2H) m
5.11 (1H) m
2.15 (2H) m
1.57 (2H) m
5.07 (1H) m
2.24 (2H) m
1.54 (2H) m
5.10 (1H) m
2.20 (2H) m
1.51 (2H) m
5.11 (1H) m
5.40 (1H) d (1.5) 5.31
5.31 (1H) d (2.4) 5.25
(1H) d (2.4)
5.31 (1H) d (1.5) 5.25
(1H) d (1.5)
5.51 (1H) s 5.43 0.90 (3H) d
5.35 (1H) s 5.27 (1H) s
1.62 (3H) s
c
(1H) d (1.5)
(1H) s
(6.5)
b
9′
2.22 (3H) s
2.13 (3H) s
1.68 (3H) s
3.54 (3H) s
2.15 (3H) s
1.67 (3H) s
3.55 (3H) s
1.62 (3H) s
0.90 (3H) d
c
(
6.5)
b
10′
1.85 (3H) s
1.69 (3H) s
0.90 (3H) d
1.69 (3H) s
c
(
6.5)
OMe
3.55 (3H) s
3.64 (3H) s
2.82 (1H) s
3.54 (3H) s
3.48 (3H) s
HO-1
HO-2
HO-4
MeCO
2.65 (1H) d (1.4)
2.68 (1H) d (2.0)
2.64 (1H) d (6.1)
2.54 (1H) d (2.0)
2.56 (1H) d (2.0)
2.15 (3H) s
2.62 (1H) d (2.0)
2.08 (3H) s, 2.07 (3H) s
a
b
c
d
The chemical shifts are in δ values (ppm) from TMS. These assignments could be reversed. The signals were overlapped. These signals are
observed as complex multiplets integrating for 12 protons in the region of δ 2.0−1.0.
−
1
1
1
573 cm . UV λ nm (log ε): 274 (sh); 256 (3.15), 256 (3.15). H
whereas the antibiotic activity was assayed against Bacillus subtilis (a
gram positive bacterium) grown on TGYA (tryptic glucose yeast agar)
(Biolife, Bothell, WA) and Escherichia coli (a gram negative
bacterium), grown on LB agar (Sigma, St. Louis, MO, USA). Up to
60 μg of each metabolite was applied per diskette. Three replications
were performed for each compound. The eventual presence of an
inhibition halo of the microbial growth was visually assessed 1 day after
the application.
max
+
NMR (Table 1); ESIMS (+) m/z: 317 [M + Na] .
Biological Assays. A number of assays on different organisms
were performed in order to characterize prelimiarly the biological
properties of the derivatives compared to the source main metabolite 1
and thus to allow a structure−activity relationship study. Each
metabolite was first dissolved in a minimum amount of MeOH
10 M or not higher than 2% in the final solutions) and then diluted
with distilled water to the desired concentrations. The following
bioassays were performed.
−1
(
Zootoxic Activity Assay. The zootoxic activity was evaluated on
Artemia salina L. larvae (brine shrimps) by using the protocol already
1
1
−4
described. The metabolites were tested at 1.7 × 10 M, with four
replications for each compound. Forty-eight hours after performing
the assay, the number of dead larvae was counted, and toxicity was
then expressed as percentage of dead larvae in reference to the total.
Data Analysis. All of the bioassays were performed twice with at
least 3 replicates. When appropriate, standard deviation was
determined.
Leaf Disk Puncture Assay. All of the compounds (1−8) were tested
by using a leaf puncture assay on 4 plant species, namely, Chenopodium
album L., Mercurialis annua L., Sonchus oleraceus L., and Carthamus
−3
lanatus L. Pure compounds were tested at 6.85 × 10 M by applying a
droplet (20 μL−2% solution in MeOH) of solution to detached leaves
previously punctured with a needle. 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 visually
between 3 and 5 days after droplet application, by using a visual scale
from 0 (no symptoms) to 4 (necrosis wider than 1 cm). Control
treatments were carried out by applying droplets not containing the
metabolites.
Chlorophyll Degradation. Phomentrioloxin and derivatives were
tested on the aquatic plant Lemna minor for the possible capability to
degrade chlorophyll. The bioassay was carried out according to the
protocol already described in detail. All of the compounds were tested
at 6.85 × 10 M.
RESULTS AND DISCUSSION
■
Phomentrioloxin (1) was recently isolated as a new phytotoxic
geranylhydroquinone from D. gulyae culture filtrates. It was
characterized as (1S,2S,3S,4S)-3-methoxy-6-(7-methyl-3-meth-
ylene-oct-en-1-ynyl)-cyclohex-5-ene-1,2,4-triol using spectro4-
scopic and chemical methods combined with X-ray analysis.
In particular, the application of the advanced Mosher’s
12
4
method allowed assignment of the absolute configuration to
−
3
phomentrioloxin as reported below. However, due to a mistake
in the application of these rules this absolute configuration
must be corrected to 1R,2R,3R,4R.
Seven phomentrioloxin derivatives were prepared in order to
determine the relationships between the structure of phomen-
Antimicrobial Bioassay. The antimicrobial activity was tested
against three microorganisms by using an agar diffusion assay
according to the protocol already described. In particular, the
11
antifungal activity was tested on Geotrichum candidum grown on PDA,
9
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Journal of Agricultural and Food Chemistry
Article
Table 2. Biological Assays of Phomentrioloxin (1) and Its Derivatives (2−8)
a
b
c
d
e
bioassay
species
leaf puncture assay
chlorophyll
protoplasts
antibiosis
zootoxicity
C. lanatus
S. oleraceus
M. annua
C. album
L. minor
A. thaliana
G. candidum
B. subtilis
E. coli
A.salina
compd
1
2
3
4
5
6
7
8
3
4
0
0
1
0
0
0
4
3
0
0
1
0
1
0
2
2
0
0
1
0
1
0
1
1
0
0
0
0
2
0
1.79 ± 0.30
4.32 ± 0.54
4.85 ± 0.46
4.33 ± 0.02
4.70 ± 0.62
4.27 ± 0.80
2.08 ± 0.14
60.3 ± 6
41.3 ± 14
80.3 ± 8
83.0 ± 4
71.0 ± 4
84.0 ± 12
48.0 ± 3
83.0 ± 5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0
84
0
0
0
0
0
4.09 ± 0.31
0
a
b
c
Empiric scale from 0 (= inactive) to 4 (necrosis around 1 cm diameter). Total chlorophyll ± SD; control = 4.15 ± 0.35. Protoplast viability (% of
d
e
the total); control = 85.3 ± 11.02. Inhibition halo: -, absence. Larva mortality after 48 h of exposure (% of the total).
trioloxin and its biological properties to identify the active sites
of the compound and, possibly, increase or change its
phytotoxicity. As previously reported, 1 was converted into
spectrum of 2 (Table 1) differed from that of 1 for the
downfield shift (Δδ 1.35) of HC-1, which now appeared as a
doublet (J = 4.1 Hz) at δ 5.70, the presence of the singlet of the
acetyl group at δ 2.15, and the absence of the HO-1 signal. The
4
the corresponding 1,2,4-O,O′,O″-triacetyl derivative (4, Figure
1
1) by routine acetylation carried out with acetic anhydride and
H NMR spectrum of 3 (Table 1) differed from that of 1 for
pyridine, which showed the reversible modification of the 1,2,4-
triol system of the cyclohexene moiety. When the same reaction
was carried out for a shorter time (5 min), two partially
acetylated derivatives were obtained as the 1-O-acetyl and 1,2-
O,O′-diacetylphomentrioloxin (2 and 3, Figure 1). A different
modification of the same diol system was obtained by
conversion of 1 into the corresponding 1,2-O,O′-isopropylidene
derivative (5, Figure 1). Derivative 5 was obtained by the
reaction of 1 with dry Me CO and dry CuSO . A further
the downfield shift of both HC-1 and HC-2 (Δδ 1.36 and 1.21)
appearing as a doublet (J = 3.8 Hz) and a double doublet (J =
7.2 and 3.8 Hz) at δ 5.71 and 5.56, respectively. Furthermore,
the presence of the two singlets due to the two acetyl groups
was observed at δ 2.08 and 2.07, while the HO-1 and HO-2
signals were absent. Their ESIMS spectra showed the
+
+
potassium [M + K] and sodium [M + Na] clusters at m/z
373 and 357, and 415 and 399, respectively. The IR spectrum
of the oxidized derivative 6 differed from that of 1 essentially
due to the presence of the band of the conjugated carbonyl
2
4
different modification of the cyclohexentriol moiety was
obtained by selective oxidation to a ketone of the secondary
−1
1
group at 1674 cm . Its H NMR spectrum (Table 1) differed
from that of 1 essentially because of the absence of the HO-4
signal, while H-5 appeared as a singlet at δ 6.21. Its ESIMS
hydroxy group C-4 of 1 with MnO , obtaining derivative 6.
2
Derivative 6 also showed a marked modification of the
stereochemistry of the cyclohexene ring. Several attempts
were made using different catalysts, solvents, and time of
reaction to partially or totally hydrogenate the triple and the
two double bonds of the side chain at C-6. These reactions
aimed at differently modifying 7-methyl-3-methylene-oct-6-ene-
+
spectrum showed the sodium cluster [M + Na] at m/z 313.
The IR spectrum of the octahydroderivative 7 differed from
that of 1 for the absence of the signal of the alkyne group. Its
1
H NMR spectrum (Table 1), compared to that of 1, showed
the presence of one olefinic signal due to H-5 and the presence
of three doublets (J = 6.5 Hz) due to methyl group Me-8′, Me-
9′, and Me-10′, which gave an overlapped signal at δ 0.90. Its
1
-ynyl and at having responses on the role of its functionalities
on the biological activity. The best results were obtained using
% Rd/C in MeOH at atmospheric pressure and at room
temperature. The main derivative obtained was
′,1′,2′,2′,3′,8′,6′,7′-octahydro derivative (7), as the other
+
5
ESIMS showed the sodium cluster [M + Na] at m/z 317.
Finally, the IR spectrum of derivative 8 did not substantially
differ from that of 1, while significant differences were observed
1
1
partially hydrogenated compounds were present in very low
yield in the reaction mixture. Compound 7 showed the
complete saturation of the side chain at C-6 that became a 3,7-
dimethyloctyl. Finally, the reductive opening of the cyclo-
hexentriol moiety was obtained by the selective oxidation
comparing their H NMR spectra (Table 1). In particular, the
presence of two broad doublets (J = 12.6 Hz) and two doublets
(J = 11.7 Hz), typical AB systems of the two CH O-1 and
2
CH O-2, resonated at δ 4.28 and 4.19, and 3.82 and 3.77,
2
respectively. Its ESIMS spectrum showed the sodium cluster
+
cleavage with NaIO of the diol system present between C-1
[M + Na] at m/z 317.
4
and C-2. The corresponding unstable dialdehyde was
immediately reduced with NaBH4 to the corresponding
primary hydroxy groups yielding derivative 8. The latter, 2-
methoxy-5-(3-methylene-oct-6-en-1-ynyl)-hex-4-ene-1,3,6-triol
Assayes on leaves by puncture confirmed that 1 and 2 were
equally toxic, causing the appearance of wide necrosis (Table
2); compounds 5 and 7 caused the appearance of necrosis of
modest size, whereas all of the other compounds proved to be
inactive. On protoplasts (60.3% viable protoplast obtained
testing 1), only 2 and 7 proved to be active, leaving 41.3 and
48.0% viable protoplasts after the treatment, respectively
(viability of the control 85.3%). When assayed on L. minor
for chlorophyll degradation, besides 1, only compound 7
proved to be effective, causing around 50% chlorophyll
degradation, whereas all of the other compounds were inactive.
The results obtained confirmed the preliminary ones
(8), had a structure completely different from that of the parent
compound, except for the part corresponding to the side chain.
The structures of all of the derivatives 2, 3 and 6−8 were
determined by comparing their spectroscopic data, essentially
1
IR, H NMR (see Table 1), and ESIMS with those of
phomentrioloxin.
In particular, the IR spectrum of 2 and 3 differed from that of
1
for the presence of the carbonyl and ester groups at 1714 and
−1
1
4
1
269, and 1721 and 1269 cm , respectively. The H NMR
obtained testing 1, 4, and 5. Furthermore, they showed that
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Journal of Agricultural and Food Chemistry
Article
the hydroxy groups at C-2 and C-4 are very important features
for phytotoxicity. In fact, when the 2-hydroxy or the 2- and 4-
hydroxy groups were acetylated as in 3 and 4, respectively, a
total loss of activity was observed. The role of these two groups
(2) Ash, G. J.; Stodart, B.; Sakuanrungsirikul, S.; Anschaw, E.; Crump,
N.; Hailstones, D.; Harper, D. I. Genetic characterization of a novel
Phomopsis sp., a putative biocontrol agent for Carthamus lanatus.
Mycologia 2010, 102, 54−61.
(3) Thompson, S. M.; Tan, Y. P.; Young, A. J.; Neate, S. M.; Aiteken,
(
2- and 4-hydroxy) was confirmed by the reduced activity
A. A. B.; Shivvas, R. G. Stem cankers on sunflower (Helianthus annuus)
in Australia reveal a complex of pathogenic Diaporthe (Phomopsis)
species. Persoonia 2011, 27, 80−99.
observed when the hydroxy group at C-2, together with that at
C-1, was ketalized as in 5 and by the total loss of activity when
the hydroxy group at C-4 was oxidized as in 6. The hydroxy
group at C-1 seems not to be important for activity. In fact,
when it was acetylated as in 2, this derivative proved to be as
toxic as 1. The reduced activity showed by derivative 7
indicated that the unsaturation of the geranyl side chain also
plays a role in imparting phytotoxicity. Finally, the lack of
activity of derivative 8 also meant that the presence of an
unchanged cyclohexenetriol ring is important for activity.
When assayed on brine shrimps at 1.7 × 10 M, only
compound 2 caused 45% larvae mortality after 24 h of exposure
to the toxin, which increased to 84% at 48 h. All the other
compounds were inactive at the same concentration. The
activity of derivative 2 could indicate that it can more easily
penetrate the cell membrane due to its increased lipophilicity.
Probably, the conversion of acetyl derivative 2 into the parent
compound occurs at physiological pH as frequently observed in
(4) Cimmino, A.; Andolfi, A.; Zonno, M. C.; Troise, C.; Santini, A.;
Tuzi, A.; Vurro, M.; Ash, G.; Evidente, A. Phomentrioloxin: a
phytotoxic pentasubstituted geranylhexentriol produced by Phomopsis
sp., a potential mycoherbicide for Carthamus lanatus biocontrol. J. Nat.
Prod. 2012, 75, 1130−1137.
(5) Evidente, A.; Capasso, R.; Andolfi, A.; Vurro, M.; Zonno, M. C.
Structure-activity relationships studies of putaminoxins and pinolidox-
ins phytotoxic nonenolides produced by phytopathogenic Phoma and
Ascochyta species. Nat. Toxins 1998, 6, 183−188.
−4
(6) Evidente, A.; Andolfi, A.; Cimmino, A. Fungal phytotoxins for
control of Cirsium arvense and Sonchus arvensis. Pest Technol. 2011, 5,
1
−17 and references therein cited.
(7) Evidente, A.; Punzo, B.; Andolfi, A.; Berestetskiy, A.; Motta, A.
Alternethanoxins A and B, polycyclic ethanones produced by
Alternaria sonchi, potential mycoherbicides for Sonchus arvensis
biocontrol. J. Agric. Food Chem. 2009, 57, 6656−6660.
(8) Cimmino, A.; Zonno, M. C.; Andolfi, A.; Troise, C.; Motta, A.;
13
Vurro, M.; Evidente, A. Agropyrenol, a phytotoxic fungal metabolite,
and its derivatives: A structure-activity relationship study. J. Agric. Food
Chem. 2013, 61, 1779−1783.
other natural metabolites and is known as lethal metabolism.
This may not occur completely for the analogues diacetyl and
triacetyl derivatives 3 and 4. None of the compounds was toxic
when assayed on Gram+ and Gram− bacteria, and G. candidum
using concentrations up to 60 μg/diskette.
(9) Crump, N. S.; Ash, G. J.; Nickandrow, A. Phomopsis spp.
associated with die-back of saffron thistle (Carthamus lanatus).
Australas. Plant Pathol. 1996, 25, 143.
These results could be useful when studying the mode of
action of phomentrioloxin and in devising new natural products
with potential application as herbicides in agriculture. Indeed,
although the monoacetyl derivatives (2) has the same level of
phytotoxicity of phomentrioloxin, this latter remains the best
potential herbicide as 2 exhibits a significant increase of
zootoxicity.
(10) Pinkerton, F.; Strobel, G. A. Serinol as an activator of toxin
production in attenuated cultures of Helminthosporium sacchari. Proc.
Natl. Acad. Sci. U.S.A. 1976, 73, 4007−4011.
(
11) Bottalico, A.; Capasso, R.; Evidente, A.; Randazzo, G.; Vurro, M.
Cytochalasins structure-activity relationship. Phytochemistry 1990, 29,
3−96.
12) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. High-field FT
9
(
NMR application of Mosher’s method. The absolute configuration of
marine terpenoids. J. Am. Chem. Soc. 1991, 113, 4092−4096.
(13) Hassal, K. A. Biochemistry and Uses of Pesticides; Verlag Chemie:
Weinheim, Germany, 1990; pp 58, 72, 304, 429, and 497.
ASSOCIATED CONTENT
■
*
S
Supporting Information
IR, UV, 1D 1H NMR, and HRESI MS spectra of
phomentrioloxin derivatives (2−8). This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Phone: +39 081 2539178. Fax: +39 081 6741330. E-mail:
evidente@unina.it.
■
*
Funding
C.T. is supported by a scholarship from Charles Sturt
University, Australia. A.E. is associated with the Istituto di
Chimica Biomolecolare, CNR, Pozzuoli, Italy.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
NMR spectra were recorded by Dominique Melck in the
laboratory of Istituto di Chimica Biomolecolare del CNR,
Pozzuoli, Italy.
REFERENCES
■
(
1) Shorten, C. Note Number LC0225. http://www.dpi.vic.gov.au/
agriculture/pests-diseases-and-weeds/weeds/other-declared-weeds/
saffron-thistle (accessed in 2013).
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