An antifungal tetrapeptide from the culture of Penicillium canescens

Article abstract of DOI:10.1002/cbdv.200800336

A new tetrapeptide D-Phe-L-Val-D-Val-L-Tyr (1), along with three known diketopiperazines and pseurotin A, were isolated from the culture of Penicillium canescens, collected from pollen from beehives, in a screening for new antimicrobial products from unex

Full text of DOI:10.1002/cbdv.200800336

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
An Antifungal Tetrapeptide from the Culture of Penicillium canescens
Brenda V. Bertinettia), Nora I. Pen˜ab), and Gabriela M. Cabrera*a)
a
´
) Departamento de Quꢀmica Organica y UMYMFOR, FCEyN, Universidad de Buenos Aires, Ciudad
Universitaria, Pab. II (1428), Buenos Aires, Argentina
(phone/fax: þ541145763376; e-mail: gabyc@qo.fcen.uba.ar)
b) Departamento de Biologꢀa, FCEyN, UNMdP, Mar del Plata, Argentina
A new tetrapeptide d-Phe-l-Val-d-Val-l-Tyr (1), along with three known diketopiperazines and
pseurotin A, were isolated from the culture of Penicillium canescens, collected from pollen from beehives,
in a screening for new antimicrobial products from unexplored sources. The structure of the tetrapeptide,
which exhibits antifungal activity comparable with that of the commercial product benomyl against the
soybean phytopathogen Fusarium virguliforme, was determined by spectroscopic (2D-NMR, and MS
and MS/MS) and chemical methods, and the sequence was confirmed by comparison with authentic
synthetic isomeric peptides.
Introduction. – In the pharmaceutical and agrochemical industries, thousands of
chemicals must be evaluated before finding a potentially useful compound. To improve
this search, particularly when looking for natural bioactive microbial products, the
spectrum of biodiversity is being explored for new sources. Previously unexplored
environments have their own ecosphere, interactions, and evolution that might contain
new producer organisms. Environments such as the open sea and marine sediment, and
even extreme environments such as hot springs, demonstrate that the diversity of
microorganisms is far greater than anticipated, and a growing number of new natural
products are being isolated from them [1].
In this context, fungal strains in pollen from beehives were investigated for
antimicrobial metabolites [2][3]. In this study, we describe the isolation and
identification of bioactive metabolites, including a previously undescribed tetrapeptide
together with other compounds, from a strain of Penicillium canescens whose organic
extract showed antimicrobial activity.
Penicillium is known as a genus of rich metabolite producers, and, in particular,
several P. canescens strains were reported to yield the antifungals griseofulvin, canescin,
curvulinic acid, and Sch642305, as well as tryptophan-containing alkaloids [4].
Results and Discussion. – 1. Chemistry. The P. canescens isolate BAFC 3291 was
obtained from beehive pollen and cultured in malt extract medium. The medium was
solid-phase-extracted and subjected to reversed-phase (RP) chromatography and
HPLC to yield compounds 1–5. The purification process was guided by antimicrobial
assays on the collected fractions. The spectroscopic data (2D-NMR, EI-MS, and optical
rotation) of compounds 3–5 were identical to those of the diketopiperazines
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢂrich

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1179
aurantiamine [5], cyclo(l-phenylalanyl-trans-4-hydroxy-l-proline), and cyclo(l-phe-
nylalanyl-l-proline) [6], respectively, while compound 2 was identical to pseurotin A
[7] (Fig. 1).
Fig. 1. Compounds isolated from Penicillium canescens
The molecular formula of compound 1 was determined as C28H38N4O6 on the basis
of its HR-MS (ESI/APCI; m/z 527.2857 [MþH]þ ). The 1H-NMR ((D6)DMSO)
spectrum displayed signals corresponding to two aromatic rings, one monosubstituted
with H-atom signals at 7.20–7.29 ppm, and the other para-disubstituted with shielded
H-atom signals at 6.59 and 6.94 ppm; three signals at 4.25, 4.14 (2 H) and 3.89 ppm; ten
other signals for aliphatic H-atoms, and three exchangeable H-atom signals at 8.31,
8.25, and 7.58 ppm. By the COSY spectrum, four partial structures were identified: two
NHꢀCHꢀCHMe2 and two CHꢀCH2. Together with the two aromatic rings, these data
suggested that 1 contained two valines, a phenylalanine, and a tyrosine. The 13C-NMR,
HSQC, and HMBC data supported these assumptions, allowing the full assignment of
the four amino acid components (Table). The sequence of the amino acids in the
peptide was revealed by ESI-MS/MS. The ESI-MS/MS of the [MþH]þ ion, used as
precursor, yielded peaks of product ions at m/z 247.1, 219.1 and 281.1 as major ions, and
at 182.1 and 120.1 for minor ones. Two of the major product ions, b2 and a2, can be
assigned to phenylalanine and valine residues, respectively, while the other three ions
corresponded to Val-Tyr (y2), Tyr (y1), and the immonium ion of phenylalanine,
suggesting the sequence Phe-Val-Val-Tyr (Fig. 2). This sequence was confirmed by the
NOESY spectrum where the a-H-atom of Phe at d 3.89 correlated with the NH-Val1 at
d 8.25, and the a-H-atom of Val1 at d 4.25 ppm correlated with the NH-Val2 at d 8.31.
Other NOE correlations are shown in Fig. 3.
To determine the absolute configuration, compound 1 was hydrolyzed with 6n HCl
to obtain the corresponding amino acids, which were analyzed by GC and GC/MS as
the N-trifluoroacetyl isopropyl ester derivatives in a Chirasil-val column. The result
showed the presence of d-Phe, l- and d-Val, and l-Tyr in tetrapeptide 1. The two
possible structures for 1, d-Phe-l-Val-d-Val-l-Tyr and d-Phe-d-Val-l-Val-l-Tyr were

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table 1. NMR Data for the Natural Tetrapeptide 1, and the Synthetic Peptides 1synth and 6. In (D6)DMSO;
d in ppm, J in Hz. Assignments based on 1H,1H-COSY, HSQC, HMBC, and NOESY experiments.
1
1synth
6a)
d(C) d(H)
d(C) d(H)
d(C)
d(H)
d-Phe
1
2
3
169.9
169.8
170.6b)
55.9
54.1 3.89 (br. t, J¼7.0)
54.0 3.90 (br. t, J¼7.1)
3.60 (br. s)
38.8 2.95 (dd, J¼13.4, 7.2), 38.5 2.96 (dd, J¼13.4, 7.1), 40.1
3.02 (dd, J¼13.6, 4.4),
2.79 (dd, J¼13.4, 7.4)
2.79 (dd, J¼13.4, 7.4)
2.69 (dd, J¼13.6, 8.4)
4
136.6
136.5
137.8
129.7
128.6
126.7
5, 9
6, 8
7
129.5 7.21–7.27 (m)
128.5 7.24–7.29 (m)
126.8 7.19–7.23 (m)
129.6 7.22–7.28 (m)
128.5 7.25–7.30 (m)
126.9 7.19–7.23 (m)
7.21–7.25 (m)
7.24–7.29 (m)
7.17–7.21 (m)
Val-1
1
2
3
4
5
NH
171.3
171.3
170.8b)
57.8
30.7
19.6
18.2
57.7 4.25 (t, J¼8.0)
30.6 1.71–1.78 (m)
19.2 0.71 (d, J¼6.7)
18.1 0.57 (d, J¼6.7)
8.25 (br. d, J¼8.0)
57.6 4.26 (d, J¼8.2)
30.9 1.72–1.79 (m)
19.2 0.70 (d, J¼6.7)
18.0 0.56 (d, J¼6.7)
8.26 (br. d, J¼8.2)
4.18 (dd, J¼8.6, 6.3)
1.97–2.04 (m)
0.83 (d, J¼6.7)
0.78 (d, J¼6.7)
8.08–8.11 (m)b)
Val-2
1
2
3
4
5
NH
170.7
170.9
170.8b)
57.5
31.5
19.6
18.2
57.3 4.12–4.17 (m)
28.9 1.91–1.96 (m)
19.8 0.73 (d, J¼6.7)
17.9 0.68 (d, J¼6.7)
8.31 (br. d, J¼8.3)
57.2 4.15–4.20 (m)
29.4 1.89–1.96 (m)
19.7 0.72 (d, J¼6.7)
17.8 0.68 (d, J¼6.7)
8.31 (br. d, J¼8.5)
4.37 (br. t, J¼7.4)
1.89–1.96 (m)
0.82 (d, J¼6.7)
0.80 (d, J¼6.7)
8.10–8.13 (m)b)
l-Tyr
1
2
3
173.5
54.8 4.12–4.17 (m)
37.0 2.89 (dd, J¼13.6, 4.8), 36.9 2.90 (dd, J¼13.8, 4.9), 36.6
173.5
54.6 4.17–4.22 (m)
173.1
54.6
4.21–4.26 (m)
2.88 (dd, J¼13.9, 5.5),
2.69 (dd, J¼13.6, 8.4)
2.68 (dd, J¼13.8, 8.5)
2.79 (dd, J¼13.9, 8.1)
4
128.6
128.7
127.9
130.3
115.2
155.9
5, 9
6, 8
7
130.2 6.94 (d, J¼8.3)
114.9 6.59 (d, J¼8.3)
155.8
130.2 6.94 (d, J¼8.5)
114.9 6.59 (d, J¼8.5)
155.8
6.98 (d, J¼8.4)
6.61 (d, J¼8.4)
NH
7.58 (br. s)
7.60 (br. s)
7.99 (br. d, J¼8.3)
a) Compound 6 did not show any NOESY correlations using different mixing times from 0.3 to 0.9 s.
b) Superimposed signals.
evaluated by spectroscopic comparison with the two synthetic tetrapeptides d-Phe-l-
Val-d-Val-l-Tyr (1synth) and d-Phe-d-Val-l-Val-l-Tyr (6), which were prepared for this
purpose. In this way, the complete structure of d-Phe-l-Val-d-Val-l-Tyr was
determined for 1 (Table).
2. Bioactivity. Compounds 1, 3, and 5 were responsible for the bioactivity of the
extract. Compound 1 showed good, although specific, antifungal activity against
Fusarium virguliforme, the causal agent of sudden-death syndrome of soybean, with an

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1181
Fig. 2. Product ions observed in the ESI-MS/MS spectrum of 1
Fig. 3. Important NOESY correlations for 1
inhibition halo diameter of 20 mm, whereas the commercial fungicide benomyl
exhibited an inhibition halo diameter of 30 mm, both at 25 mg/pt. Compound 1 also
showed moderate antibiotic activity against Bacillus subtilis. Compounds 3 and 5
exhibited moderate antifungal activities against the phytopathogenic fungi Colleto-
trichum truncatum and Fusarium lateritium, with inhibition halo diameters of 8 and
12 mm, respectively. The bioactivity of synthetic peptide 1synth was the same as 1, and 6
proved to be inactive in all the tested bioassays.
3. Discussion. Small peptides are known to possess a wide range of bioactivities. A
family of tetrapeptides containing a C-terminal tyrosine have shown antiopiate activity
with considerable selectivity for the m-opiate binding site. They are transported across
the blood–brain barrier and exert more than one central action at the brain [8].
Antimicrobial peptides have also been proposed as new pesticides, because they are
short-sequence peptides, produced by microorganisms, and they cover a broad
spectrum of bioactivities taking into account the increasing necessity for plant-disease
protection that satisfies the new requirements based on toxicology, traceability, and
environmental impact [9]. Synthetic peptides were prepared using natural compounds
as models, and a tetrapeptide, a pentapeptide, and several hexapeptides with antifungal
activities were reported. All these peptides have at least a basic amino acid in their
structure [9].

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Although the cyclic peptides cyclo(l-phenylalanyl-trans-4-hydroxy-l-proline) and
cyclo(l-phenylalanyl-l-proline) were previously reported to be both antibacterial and
antifungal [10][11], the fungal strains tested were different from the current work. It is
noteworthy that the antifungal activity of cyclo(l-Phe-l-Pro) in bacteria has been
proposed to be a secondary effect, and the primary reason for its presence might be
related to quorum sensing [10][12]. Other bioactivities such as antiviral and
antitumoral properties have been reported for these cyclic peptides. In particular,
cyclo(Phe-Pro) has been shown to inhibit cancer cell growth and induce apoptosis in
HT-29 colon cancer cells [13].
More than 700 metabolites have been reported from the genus Penicillium [14],
many of them having useful properties that can be exploited for developing new
pharmaceuticals [4]. The presence of some metabolites has also assisted on the fungal
strain classification process. An extensive analysis of extrolites of the Penicillium
subgenus Penicillium has been performed [15], and the Penicillium subgenus Furcatum
has been revised with the aid of secondary metabolites.
Griseofulvin, canescin, trytophan-containing alkaloids (fellutamines, isorugulosu-
vine, meleagrin, roquefortine), curvulinic acid, and compound Sch642305 were
previously detected in P. canescens strains [4]. Although the compounds isolated in
this work were not previously reported from this species, pseurotin A and related
compounds were isolated and identified from a Penicillium sp. isolated from driftwood
on the foreshore of a beach in Canada [7], and diketopiperazines, including
aurantiamine, were isolated from related Penicillium strains.
The tetrapeptide 1 might have been identified here and missed in previous analyses,
because the usual extraction method does not allow the extraction and analysis of very
polar molecules like amino acids and peptides, so small bioactive peptides can be
overlooked [15].
Conclusions. – In summary, a previously undescribed tetrapeptide, d-Phe-l-Val-d-
Val-l-Tyr, with antifungal activity, three bioactive diketopiperazines, i. e., cyclo(l-Phe-
trans-4-hydroxy-l-Pro), cyclo(l-Phe-l-Pro), and aurantiamine, together with pseurotin
A, were isolated and identified from a culture of the fungus P. canescens. This is the first
report on the isolation of these compounds from this species.
We thank Universidad de Buenos Aires, CONICET, and ANPCYT for partial financial support.
Experimental Part
General. Optical rotations: Perkin Elmer polarimeter 343. The UV Spectra: Hewlett Packard 8451A
diode-array spectrophotometer (lmax in nm (log e)). NMR Spectra: Bruker Avance II instrument (1H:
500.13 and 13C: 125.13 MHz). EI-MS: Trio-2 VG Masslab (Manchester, UK) mass spectrometer. FAB-
MS: ZAB BEqQ (VG, Manchester, UK) mass spectrometer. ESI/APCI-MS and ESI-MS/MS: Agilent
LC-TOF 6210 with dual probe ESI/APCI (UCR Mass Spectrometry Facility, Riverside, CA, USA) or a
Bruker MicrOTOF-Q II; in m/z.
d-Phe-l-Val-d-Val-l-Tyr and d-Phe-d-Val-l-Val-l-Tyr were purchased from Instituto de Quꢀmica y
´
Fisicoquꢀmica Biologicas (UBA-CONICET) and prepared for this work purpose, using a peptide
synthesizer (Applied Biosystems model 431A). The Fmoc-amino acids activated with HOBt/DCC (1-
hydroxybenzotriazole/dicyclohexylcarbodiimide) and an AB HMP resin ([p-(hydroxymethyl)phenoxy]-
methyl polystyrene) were employed. The synthesized peptides were purified by HPLC (RP C4, Vydac
i
214TP510, gradient elution from 10 to 70% B in 40 min, 1.5 ml/min, A: H2O, B: PrOH/H2O 8 :2).

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1183
Fungal Strain. The fungus Penicillium canescens Sopp was isolated from pollen and classified by NP
and deposited with the BAFC culture collection (Facultad de Ciencias Exactas y Naturales, Universidad
de Buenos Aires) under the accession number BAFC 3291. The pollen was collected from beehives of the
apiary of Unidad Integrada INTA (Balcarce, Buenos Aires), Facultad de Ciencias Agrarias (Universidad
de Mar del Plata), Argentina, and diluted with a sterile physiological soln. Dilutions were seeded on agar
malt Petri dishes, from which individual colonies were isolated.
Fermentation. Awell-grown agar slant of P. canescens was used to inoculate 250-ml Erlenmeyer flasks
containing 75 ml of malt extract medium containing malt extract (30 g) and peptone (5 g/l). After a
week, one Erlenmeyer media was employed to inoculate 1 l of the above media in 3-l Erlenmeyer flasks.
The fermentation was carried out at 258 for 23 d under static conditions.
Extraction and Isolation. The fermentation broth (3 l) was filtered, and Amberlite XAD-16 was
added to the filtrate. After 10 h, the suspension was filtered, and the phase was washed with H2O and
then eluted with MeOH. The MeOH eluate was evaporated and subjected to vacuum chromatography on
RP-C18 using H2O, and mixtures of H2O and MeOH of decreasing polarity. Fraction 3, eluted with H2O/
MeOH 1:1, yielded on HPLC (column: YMC C18, 5 mm, 22.5ꢁ2.5 cm; H2O/MeOH 65 :35) the
diketopiperazines cyclo(l-phenylalanyl-trans-4-hydroxy-l-proline) (4; 8.0 mg) and cyclo(l-phenylalanyl-
l-proline) (5, 16 mg). Fr. 4, eluted with H2O/MeOH 25 :75, yielded on HPLC (column: YMC C 18, 5 mm,
22.5ꢁ2.5 cm; H2O/MeOH 45 :55) pseurotin A (2, 9.0 mg), aurantiamine (3, 6.7 mg), and d-Phe-l-Val-d-
Val-l-Tyr (1; 11.6 mg).
Antibiotic Assay. The antibiotic activity was determined by the agar diffusion method using 50 mg of
sample/6 mm disk against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, and
Escherichia coli ATCC 25922. Compound 1 caused an inhibition zone of 20 mm against B. subtilis, whilst
4 showed an inhibition zone of 16 mm against S. aureus. Gentamicin, which was used as the positive test
compound, showed inhibition halos of 30 mm at a concentration level of 25 mg/disk.
Antifungal Activity. Direct bioautography on TLC was employed as the method for detecting
fungitoxic substances [16]. A concentration level of 25 mg/spot of each assayed compound was used,
except for compounds 3–5, where 50 mg/spot was tested. Benomyl, which was used as test compound,
showed an inhibition zone of 30 mm at a conc. level of 25 mg/spot (0.086 mmol/spot) and 22 mm at 14 mg/
spot (0.048 mmol/spot). Benomyl, which is used in several countries, is a systemic benzimidazole
fungicide selectively toxic to microorganisms and to invertebrates with known concerns about health and
environment protection. Compound 1 showed a diameter inhibition zone of 20 mm at 25 mg (0.048 mmol/
spot) and a minimum inhibitory concentration (MIC) of 9.5ꢁ10ꢀ3 mmol, against Fusarium virguliforme,
whilst 5 showed a halo of 12 mm against Fusarium lateritium, and 3 showed a halo of 8 mm against
Colletotrichum truncatum.
1
Data of 1. Amorphous powder. [a]2D5 ¼ ꢀ124 (c¼0.05, EtOH). UV (EtOH): 208 (3.03). H- and
13C-NMR: see Table 1. FAB-MSþ (RA; glycerol matrix): 527 ([MþH]þ , 25), 72 (100). FAB-MSꢀ (RA;
glycerol matrix): 525 ([MꢀH]ꢀ , 100); HR-ESI/APCI-MS: 527.2857 ([MþH]þ , C28H39N4Oþ6 ; calc.
527.2864, D 0.2 ppm), 549.2675 ([MþNa]þ , C28H38N4NaOþ6 ; calc. 549.2684, D 1.6 ppm). ESI-MS/MS
(527 u): 281.1 ([y2]þ
, , , , 13), 120.1
22), 247.1 ([b2]þ 95), 219.1 ([a2]þ 100), 182.1 ([y1]þ
([H2N¼CHCH2Ph]þ , 5).
Absolute Configuration of 1. Compound 1 (0.5 mg) was dissolved in MeOH, and 6n HCl (0.3 ml) was
added, heated at 1208 for 16 h and evaporated to dryness. Identification of the amino acids was
accomplished by GC after derivatization [17]. Retention times of the N-trifluoroacetyl isopropyl ester
derivatives were compared with those of standards.
The GC analyses of N-trifluoroacetyl isopropyl ester derivatives were carried out on a Hewlett
Packard 5890 gas chromatograph on a Chirasil-val cap. column (Alltech, 25-m length, 0.25-mm i.d.) with
N2 as carrier gas and a temp. programme: 608 (1 min) to 1808, 48/min and 1808 (10 min) to 2008, 108/min.
tR (min): d-Val (11.0), l-Val (11.4), d-Phe (25.3), l-Phe (29.4), d-Tyr (29.8), l-Tyr (33.9), 1 (11.0, 11.4,
25.3, 33.9). Co-injections and GC/MS were realized to confirm the identities. GC/MS was recorded on a
gas chromatograph GC 17 A coupled to a mass spectrometer QP 5000 (Shimadzu).
1
Data of 1synth. Amorphous powder. [a]D25 ¼ ꢀ137 (c¼0.05, EtOH). H- and 13C-NMR: see Table.
HR-ESI-MS: 527.2858 ([MþH]þ , C28H39N4O6þ ; calc. 527.2864, D 1.1 ppm), 549.2710 ([MþNa]þ
C28H38N4NaOþ6 ; calc. 549.2684, D 4.9 ppm).
,

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1
Data of 6. Amorphous powder. [a]2D5 ¼ ꢀ31 (c¼0.06, EtOH). H-NMR and 13C-NMR: see Table.
HR-ESI-MS: 527.2865 ([MþH]þ , C28H39N4O6þ ; calc. 527.2864, D 0.2 ppm), 549.2688 ([MþNa]þ
C28H38N4NaOþ6 ; calc. 549.2684, D 0.8 ppm).
,
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Received November 24, 2008