Agropyrenol, a phytotoxic fungal metabolite, and its derivatives: A structure-activity relationship study

Article abstract of DOI:10.1021/jf304933z

Agropyrenol is a phytotoxic substituted salicylic aldehyde produced in liquid culture by Ascochyta agropyrina var. nana, a potential mycoherbicide proposed for the control of the perennial weed Elytrigia repens. In this study, six derivatives obtained by

Full text of DOI:10.1021/jf304933z

Article
pubs.acs.org/JAFC
Agropyrenol, a Phytotoxic Fungal Metabolite, and Its Derivatives: A
Structure−Activity Relationship Study
Alessio Cimmino,† Maria Chiara Zonno,‡ Anna Andolfi,† Ciro Troise,† Andrea Motta,§ Maurizio Vurro,‡
and Antonio Evidente*,†
†Dipartimento di Scienze Chimiche, Universita
Napoli, Italy
̀
di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126
‡Istituto di Scienze delle Produzioni Alimentari, Consiglio Nazionale delle Ricerche (CNR), Via Amendola 122/O, 70125 Bari, Italy
§Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche (CNR), Comprensorio Olivetti, Edificio 70, Via Campi
Flegrei 34, 80078 Pozzuoli, Italy
ABSTRACT: Agropyrenol is a phytotoxic substituted salicylic aldehyde produced in liquid culture by Ascochyta agropyrina var.
nana, a potential mycoherbicide proposed for the control of the perennial weed Elytrigia repens. In this study, six 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 non-host weedy and agrarian plants, fungi, Gram-
positive and Gram-negative bacteria, and brine shrimp larvae. The results provide insights into the structure−activity
relationships of agropyrenol. Both the double bond and the diol system of the 3,4-dihydroxypentenyl side chain as well as the
aldehyde group at C-1 of the phenolic ring of agropyrenol proved to be important for the phytotoxicity. The lesser polar 3′,4′-
O,O′-isopropylidene of agropyrenol also showed significant zootoxic and slight antimicrobial activities. This finding could be
useful in devising new natural herbicides for practical application in agriculture.
KEYWORDS: Ascochyta agropyrina var. nana, weeds, Elytrigia repens, phytotoxins, agropyrenol, SAR
INTRODUCTION
■
Bioactive fungal metabolites have long been considered for
their potential direct use as novel agrochemicals or as a lead for
new natural pesticides or to discover novel mechanisms of
action.1,2 More specifically, fungal toxins and their derivatives
have also been considered as sources of novel and safe natural
herbicides.3−5 Having this perspective, the fungus Ascochyta
agropyrina (Fairman) Trotter var. nana Punith., isolated from
diseased leaves of the perennial weed Elytrigia repens (L.) Desv.
ex Nevski (quack grass), was recently studied and proven to be
a good source of novel metabolites. Indeed, from the solid
culture of A. agropyrina, papyracillic acid was first isolated, a
fungal metabolite already described for its antimicrobial,
nematicidal, and cytotoxic activities6 but not yet for its
interesting phytotoxicity.7 Later, some key derivatives were
prepared by chemical modifications of the metabolite, and their
phytotoxic and antimicrobial activities were assayed against a
number of crop and weedy plants, fungi, bacteria, and
nematodes. The results obtained allowed for identification of
Figure 1. Structures of agropyrenol, agropyrenal, and agropyrenone
some structural features important for the biological activity but
also to show that one of the three monoacetyl derivatives was
very promising and could be further considered for its
herbicidal activity.7
Interestingly, when A. agropyrina was grown in liquid culture,
it produced metabolites other than papyracillic acid, the main of
which was identified as a new 6-monosubstituted salycilic
aldehyde, named agropyrenol (1; Figure 1). Together with
agropyrenol, two other novel metabolites were isolated, i.e., a
trisubstituted naphthalene carbaldehyde and a pentasubstituted
(1, 2, and 3, respectively).
3H-benzofuranone, named agropyrenal and agropyrenone (2
and 3; Figure 1), respectively.8
Received: November 20, 2012
Revised: January 28, 2013
Accepted: January 29, 2013
Published: January 29, 2013
© 2013 American Chemical Society
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visualized by exposure to UV radiation (253) 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. Column
chromatography was performed on a silica-gel column (Merck,
Kieselgel 60, 0.063−0.200 mm).
Several studies were carried out to correlate the structure of
microbial phytotoxins to their biological properties and to
identify the active sites of the compounds. In particular, those
carried out on phytotoxins with potential herbicidal activity
were aimed at not only identifying the structural features
important for the phytotoxicity but also producing derivatives
with improved biological activities and biotechnological
properties. Structure−activity relationship (SAR) studies were
carried out on nonenolides (putaminoxins and stagonolides),
cytochalasins, oxazatricyclakalenones, and alternethanoxins
produced by fungi proposed for the control of Erigeron
annuus,9 Bromus sp., Cirsium arvense,10,11 and Sonchus arvensis.12
Considering the originality of the agropyrenol chemical
structure, containing a 6-substituted salycilic aldehyde ring and
a 3,4-dihydroxy-1-pentenyl residue, and its phytotoxic proper-
ties associated with the lack of antimicrobial or zootoxic
activities, it seemed of interest to further study this metabolite,
trying to ascertain its potential as a novel natural herbicide and
identify the structural features essential for its biological activity.
For this reason, six derivatives (4−9; Scheme 1) were prepared
by chemical transformation of the functionalities present in
agropyrenol (1) and tested for their phytotoxic, antimicrobial,
and zootoxic activities.
Production, Extraction, and Purification of Agropyrenol (1).
To produce agropyrenol, the fungus A. agropyrina (Fairman) Trotter
var. nana Punith, previously isolated from naturally infected leaves of
Elytrigia repens, was used. The fungus, deposited in the culture
collections of both the All-Russian Research Institute of Plant
Protection, Pushkin, St. Petersburg, Russia (strain code A-10), and
the Institute of Sciences of Food Production, Bari, Italy (strain code
ITEM 12530), was grown on a mineral-defined liquid media named
M1-D13 as previously reported.8 The purification of the organic extract
obtained from its culture filtrate (8.6 L), carried out as previously
described,8 produced agropyrenol (1) as a yellow amorphous solid in
sufficient amount (125.9 mg, 14.6 mg/L) for the preparation of the
derivatives, as described in the successive sections of the present paper.
3′,4′-O,O′-Diacetyl Derivative (4). This compound was prepared
by routine acetylation of compound 1 with acetic anhydride and
pyridine as previously reported.8
3′,4′-O,O′-Isopropylideneagropyrenol (5). Compound 1 (10
mg) was dissolved in dry Me2CO (10 mL) and kept in stirring
conditions with dry CuSO4 (400 mg) under reflux for 2 h. The mixture
was then filtered, and the solvent was evaporated under reduced
pressure to give an oily residue, which was purified by preparative TLC
(silica gel, eluent CHCl3), allowing us to obtain derivative 5 as a
homogeneous compound (11 mg, Rf = 0.66). IR νmax: 3604, 2868,
1643, 1610, 1572, 1451, 1237 cm−1. UV λmax nm (log ε): 347 (3.30),
274 (3.60), 230 (3.97). 1H NMR: see Table 1. ESI−MS (+) m/z: 285
[M + Na]+. ESI−MS (−) m/z: 261 [M − H]−.
a
Scheme 1
4′,O-Didehydroagropyrenol (6). Compound 1 (10 mg)
dissolved in dry CH2Cl2 (3 mL) was oxidized with dry MnO2 (82
mg) under stirring at room temperature for 1.5 h. The mixture was
then filtered, and the solution was evaporated under reduced pressure
to give an oily residue, which was purified by preparative TLC [silica
gel, eluent CHCl3/iPrOH (97:3, v/v)], giving derivative 6 as a
homogeneous compound (3 mg, Rf = 0.69). IR νmax: 3434, 2851, 1736,
1684, 1646, 1580, 1465 cm−1. UV λmax nm (log ε): 355 (3.05), 285
(3.40), 246 (3.62). 1H NMR: see Table 1. ESI−MS (+) m/z: 177 [M
− CH3CO]+. ESI−MS (−) m/z: 176 [M − CH3CO − H]−.
7,O-Dihydroagropyrenol (7). Compound 1 (5 mg) was dissolved
in MeOH (200 μL) and reduced at room temperature with NaBH4 (7
mg) for 2 h. The mixture was then diluted with water (10 mL),
neutralized to pH 7 with 0.1 N HCl, extracted with EtOAc (4 × 15
mL), dehydrated with Na2SO4, and finally evaporated under reduced
pressure, giving an oily residue. It was purified by preparative TLC
[silica gel, eluent CHCl3/iPrOH (1:1, v/v)] allowing us to obtain
derivative 7 as a homogeneous compound (5 mg, Rf = 0.74). IR νmax
:
3395, 1659, 1577, 1462 cm−1. UV λmax nm (log ε): 253 (2.84), 216
(sh). 1H NMR: see Table 1. ESI−MS (+) m/z: 263 [M + K]+, 247 [M
+ Na]+.
a
Reagents and conditions: (a) Ac2O, pyridine, room temperature (rt),
12 h; (b) dry Me2CO, dry CuSO4, rt, 18 h; (c) MnO2, CH2Cl2, 25 °C,
1.5 h; (d) NaBH4, MeOH, rt, 2 h; (e) H2, Pd/C, MeOH, rt, 1 h; and
(f) Ac2O, pyridine, rt, 1.5 h.
7,7,1′2′-Tetrahydro-7-deoxyagropyrenol (8). Compound 1 (5
mg) was first dissolved in MeOH (500 μL), then added to a
presaturated 10% Pd/C (3 mg) suspension in the same solvent (500
μL), and hydrogenated at room temperature and atmospheric pressure
under stirring conditions. The reaction was stopped after 1 h by
filtration and evaporated under reduced pressure; the residue (2.8 mg)
was purified by preparative TLC [silica gel, eluent CHCl3/iPrOH (9:1,
v/v)], giving derivative 8 as a homogeneous compound (2.5 mg, Rf =
0.53). IR νmax: 3373, 1585, 1541, 1465 cm−1. UV λmax nm (log ε): 280
MATERIALS AND METHODS
■
General Experimental Procedures. Infrared (IR) spectra were
recorded as deposit glass film on a Perkin-Elmer (Norwalk, CT)
spectrometer, and ultraviolet (UV) spectra were measured in MeCN,
unless otherwise noted, on a Perkin-Elmer spectrophotometer. 1H
nuclear magnetic resonance (NMR) spectra were recorded at 600 or
400 MHz in CDCl3 on Bruker (Kalsruhe, Germany) spectrometers.
The same solvent was used as an internal standard. Electrospray
ionization (ESI) mass spectra (MS) were recorded on an Agilent
(Milano, Italy) Technologies 6120 Quadrupole LC/MS instrument.
Analytical and preparative thin-layer chromatographies (TLCs) were
performed on silica-gel (Kieselgel 60, F254, 0.25 and 0.5 mm,
respectively, Merck, Darmstadt, Germany) plates. The spots were
1
(1.81). H NMR: see Table 1. ESI−MS (+) m/z: 249 [M + K]+, 233
[M + Na]+. ESI−MS (−) m/z: 209 [M − H]−.
6,3′,4′-O,O′,O″-Triacetyl Derivative (9). Derivative 8 (8 mg)
was acetylated with pyridine (50 μL) and Ac2O (50 μL) at room
temperature for 1.5 h. The reaction was stopped by the addition of
MeOH. The azeotrope obtained by the addition of C6H6 was
evaporated by a N2 stream. The oily residue (12 mg) was purified by
preparative TLC [silica gel, eluent CHCl3/iPrOH (95:5, v/v)] to give
the 7,3′,4′-O,O′,O″-triacetyl derivative (9) of agropyrenol as a
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a
1
Table 1. H NMR Data of Agropyrenol and Its Derivatives (1 and 5−9)
1
5
6
7
8
9
position
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
6.83 d (7.9)
δH (J in Hz)
δH (J in Hz)
3
6.90 d (8.4)
6.97 d (8.5)
7.09 d (7.8)
6.63 d (7.8)
6.88 d (7.8)
7.12 t (7.8)
7.02 d (7.8)
2.33 s
4
7.44 dd (8.4 and 7.6)
6.93 d (7.6)
10.3 s
7.46 dd (8.5 and 7.6)
6.95 d (7.6)
7.57 t (7.8)
7.11 d (7.8)
10.42 s
7.16 t (7.9)
6.95 d (7.9)
5.00 s
6.99 t (7.8)
6.78 d (7.8)
2.21 s
5
7
1′
7.23 d (15.6)
7.22 d (15.6)
8.07 d (15.7)
6.87 d (15.6)
2.86 ddd (14.8, 10.2, and 5.3) 2.63 (2H) m
2.70 ddd (14.8, 9.9, and 6.4)
2′
6.12 dd (15.6 and 6.1) 6.07 dd (15.6 and 6.6) 6.65 dd (15.7 and 7.5) 6.01 dd (15.6 and 6.6) 1.75 m
1.68 m
1.82 (2H) m
3′
4′
5′
OH
OH′
Me2C
4.10 dd (6.4 and 6.1)
3.71 quint (6.4)
1.27 d (6.4)
4.14 dd (8.4 and 6.6)
3.90 dq (8.4 and 6.0)
1.33 d (6.0)
4.21 m
4.06 dd (6.6 and 6.4)
3.73 quint (6.4)
1.16 d (6.4)
3.93 m
5.06 (2H) m
5.06 (2H) m
1.21 d (6.1)
3.64 quint (7.2)
1.20 d (7.2)
11.91 s
1.25 s
11.90 s
11.85 s
11.92 s
9.79 d (7.4)
1.46 s
1.48 s
MeCO
MeCO
MeCO
2.11 s
2.09 s
2.07 s
a
The chemical shifts are in δ values (ppm) from TMS.
homogeneous compound (11.0 mg, Rf = 0.76). IR νmax: 1761, 1734,
1580, 1207 cm−1. UV λmax nm (log ε): 259 (2.46). 1H NMR: see Table
1. ESI−MS (+) m/z: 359 [M + Na]+.
Biological Assays. A number of assays on different organisms
were performed to preliminarily characterize the biological properties
of the derivatives compared to the “source” main metabolite and, thus,
to allow for a SAR study. Each metabolite was first dissolved in a
minimum amount of MeOH (10−1 M or not higher than 1% in the
final solution) and then diluted with distilled water to the desired
concentration. The following bioassays were performed.
Chlorophyll Degradation and Frond Growth. Agropyrenol and
derivatives were also tested on the aquatic plant Lemna minor for the
possible capability to degrade chlorophyll and inhibit frond growth.
The bioassay was carried out according to the protocol already
described in detail.14 All of the compounds were tested at 10−3 M.
Antimicrobial Bioassay. The antimicrobial activity was tested
against three microorganisms using an agar diffusion assay according
to the protocol already described.15 In particular, the antifungal activity
was tested on Geotrichum candidum grown on potato dextrose agar
(PDA), whereas the antibiotic activity was assayed against Bacillus
subtilis (a Gram-positive bacterium) grown on tryptic glucose yeast
agar (TGYA, Biolife, Bothell, WA) and Escherichia coli (a Gram-
negative bacterium) grown on Luria-Bertani (LB) agar (Sigma, St.
Louis, MO). Up to 0.5 μM 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.
Zootoxic Activity Assay. The zootoxic activity was evaluated on
Artemia salina L. larvae (brine shrimp) using the protocol already
described.15 The metabolites were tested at 4 × 10−4 M, with four
replications for each compound. At 48 h after the assay was performed,
the number of dead larvae was counted and toxicity was then
expressed as a percentage of dead larvae referred to the total.
Statistical Analysis. All of the bioassays were performed twice
with at least three replicates. When appropriate, standard deviation
(SD) was determined.
Leaf Disk Puncture Assay. Agropyrenol (1) and its six derivatives
(4−9) were tested using a leaf puncture assay on five weed species, i.e.,
four dicots [namely, Chenopodium album L., Cirsium arvense (L.) Scop.,
Mercurialis annua L., and Sonchus oleraceus L.] and one monocot
[Setaria viridis (L.) P. Beauv.]. Pure compounds were tested at 2 mg/
mL by applying 20 μL 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, 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.
Seed Germination. The possible capability of agropyrenol and its
derivatives to inhibit seed germination was assayed on seeds of Setaria
viridis. Briefly, seeds were first sterilized with sodium hypochlorite (1%
× 15 min) and then washed several times with sterile distilled water.
Around 100 seeds were placed in each Petri dish on paper dishes
(Whatman No. 4) imbibed with the metabolite solution (1 mL, 10−3
M) and incubated at 25 °C in the dark. Three replications were
prepared per each compound. The number of germinated seeds were
counted 5 days after treatment and expressed as the germination
percentage in comparison to the untreated control.
Rootlet Elongation. This assay aimed at evaluating the possible
inhibition of the rootlet growth, and it was carried out using tomato
seeds. Briefly, tomato seeds were pretreated with sodium hypochlorite
(1% × 10 min.) and placed in Petri dishes on filter paper disks imbibed
with sterile distilled water for 5 days. Thus, 10 pre-germinated and
uniform length seedlings were transferred to smaller dishes each
containing the metabolite solution (2 mL, 10−3 M). Dishes were
transferred to a growth chamber under continuous light. Three
replicates were prepared for each sample. After 4 days, the rootlet
length was measured and compared to the proper untreated control.
RESULTS AND DISCUSSION
■
To determine the relationships between the structure of
agropyrenol and its biological properties, identify the active
sites of the compound, and possibly, increase its toxicity, six
derivatives of the main fungal toxin were prepared. As
previously reported,8 compound 1 was converted into the
corresponding 3′,4′-O,O′-diacetyl derivative (4; Scheme 1) by
routine acetylation carried out with acetic anhydride and
pyridine, which showed the reversible modification of the diol
system of the 3,4-dihydroxy-1-pentenyl residue. Its conversion
into the parent compound at physiological pH frequently
occurs in other natural metabolites, and it is known as “lethal
metabolism”.16 A different modification of the same diol system
was obtained by conversion of compound 1 into the
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corresponding 3′,4′-O,O′-isopropylidene derivative (5; Scheme
1). The derivative 5 was obtained by reaction of compound 1
with dry Me2CO and dry CuSO4. Further, different
modifications of the 3,4-dihydroxy-1-pentenyl residue were
obtained by oxidation of agropyrenol with MnO2. Although this
reaction yielded three oxidized derivatives, only the major one
(6, Scheme 1), showing the oxidation of the secondary hydroxy
group at C-4′, was purified. The other two oxidized derivatives
were obtained as minor products of this reaction. The ESI−MS
of their mixture proved that they are mono-oxidized at C-3′ and
dioxidized at C-3′ and C-4′, respectively. Unfortunately, they
were not purified, despite the attempts made using high-
performance liquid chromatography (HPLC). Furthermore,
opposite to what is currently reported,17 the mono-oxidized
derivative 6 was also obtained when compound 1 underwent a
reaction with NaIO4, with the aim to obtain an oxidative
cleavage of the diol system. Anyway, further mechanistic studies
would be needed to clarify the course of this latter reaction. As
expected, the reduction by NaBH4 converted compound 1 into
the corresponding 7,O-dihydro derivative (7; Scheme 1), with
the conversion of the aldehyde group into the corresponding
primary hydroxy group. The catalytic hydrogenation carried out
in MeOH with 10% Pd/C at atmospheric pressure and room
temperature allowed us to obtain the 7,7,1′,2′-tetrahydro-7-
deoxy derivative (8; Scheme 1), which has not only the
saturation of the double bond of the 3,4-dihydroxy-1-pentenyl
residue, as expected, but also the complete and unusual
reduction of the aldehyde group to the corresponding methyl
group. This latter compound was converted into the 6,3′,4′-
O,O′,O″-triacetyl derivative (9) by routine acetylation with
pyridine and acetic anhydride, already used to convert
compound 1 into derivative 4.
The structures of all of the derivatives 4−9 were determined
1
by comparing their spectroscopic data, essentially H NMR
(see Table 1) and ESI−MS (see the Materials and Methods),
to those of agropyrenol.
Assayed by leaf puncture on detached leaves, derivatives 1, 4,
and 5 caused severe to medium necrosis to weedy dicot plants.
Derivatives 6−9 caused only moderate to nil effects on the
same plants, whereas none of the compounds tested had effects
on S. viridis.
From the analyses of results, it is possible to observe that all
of the nonreversible modifications of the diol system of the
3′,4′-dihydroxypentenyl side chain at C-2 present in derivative
6 as well as that of the aldehyde group at C-1 present in
derivatives 8 and 9 induced strong reduction or total loss of
phytotoxicity. It is interesting to note that derivative 7, in which
only the aldehyde group at C-1 was reduced, produced some
phytotoxicity on three of the five plants tested, while the
activity was completely lost when also the 1,2-double bond of
the 3,4-dihydroxypentenyl side chain was reduced, as in
derivatives 8 and 9. The activity showed by the diacetyl
derivate of agropyrenol was not surprising, as cited above; it
was probably hydrolyzed into compound 1 at physiological pH
according to the “lethal metabolism”.16 A similar mechanism of
hydrolysis could also convert derivative 5 into compound 1
and, therefore, justify its phytotoxicity. In fact, acetonide is
stable at basic pH but hydrolyzes in acid conditions.17
When tested on seeds, particularly derivatives 5 and 4 caused
a strong reduction of germination (60 and 48% reduction
compared to the control, respectively). Agropyrenol (1) and
derivative 8 had a modest effect (around 20−30%), whereas the
other derivatives had a very modest or nil effect (Table 2).
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phytotoxins from Ascochyta agropyrina var. nana, a fungal pathogen of
Elytrigia repens. Phytochemistry 2012, 79, 102−108.
(9) Evidente, A.; Capasso, R.; Andolfi, A.; Vurro, M.; Zonno, M. C.
Structure−activity relationships studies of putaminoxins and pinoli-
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Almost the same effects were observable in both the root
elongation assay and the chlorophyll assay, in which derivatives
5 and 4 again proved to be the most effective compounds,
causing a severe depletion of rootlet growth (90 and 69%
reduction compared to the control, respectively, as deducible
from Table 2) and an effective reduction in the chlorophyll
content of L. minor fronds (67 and 31% reduction in
comparison to the control, respectively; Table 2).
These results suggest that derivatives 4 and 5, which are less
polar than compound 1, could be absorbed and pass across the
cell membranes of the tested plants, before converting into
compound 1 as described above.
Derivative 5 was active also in most of the bioassays on
organisms other than plants. Indeed, it caused 88% mortality of
brine shrimp larvae in the zootoxicity bioassay and a slight
inhibition of fungal and Gram-positive bacterium growth. In
these latter assays, only derivative 8 caused a negligible
mortality to A. salina larvae, whereas all of the other derivatives
were inactive (Table 2).
Probably to justify the zootoxic and antimicrobial activities of
derivative 5 compared to compound 1, it could be possible to
invoke a similar mechanism to that proposed for its behavior in
germination, root elongation, and chlorophyll content bio-
assays.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +39-081-2539178. Fax: +39-081-2539186. E-mail:
evidente@unina.it.
■
(16) Hassal, K. A. Biochemistry and Uses of Pesticides; Verlag Chemie:
Weinheim, Germany, 1990; pp 58, 72, 304, 429, and 497.
(17) Allinger, N. L.; Cava, M. P.; De Jongh, D. C.; Johnson, C. R.;
Lebel, N. A.; Stevens, C. L. Organic Chemistry, 2nd ed.; Worth
Publishers, Inc.: New York, 1976.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
NMR spectra were recorded by Dominique Melck, who is
acknowledged, in the laboratory of Istituto di Chimica
Biomolecolare del CNR, Pozzuoli, Italy. Antonio Evidente is
associated with “Istituto di Chimica Biomolecolare del CNR”,
Pozzuoli, Italy.
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