A new cyclohexapeptide, penitropeptide and a new polyketide, penitropone from the endophytic fungus Penicillium tropicum

Article abstract of DOI:10.1016/j.tetlet.2016.05.095

The first chemical investigation of the endophytic fungus Penicillium tropicum isolated from leaves of Sapium ellipticum afforded a new cyclohexapeptide, penitropeptide (1), and a new polyketide, penitropone (2), in addition to two known compounds, 6-hydr

Full text of DOI:10.1016/j.tetlet.2016.05.095

Accepted Manuscript
A new cyclohexapeptide, penitropeptide and a new polyketide, penitropone
from the endophytic fungus Penicillium tropicum
Yanbo Zeng, Hao Wang, Ramsay S.T. Kamdem, Raha S. Orfali, Haofu Dai,
Gamall Makhloufi, Christoph Janiak, Zhen Liu, Peter Proksch
PII:
S0040-4039(16)30628-1
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.05.095
TETL 47714
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 April 2016
23 May 2016
25 May 2016
Please cite this article as: Zeng, Y., Wang, H., Kamdem, R.S.T., Orfali, R.S., Dai, H., Makhloufi, G., Janiak, C.,
Liu, Z., Proksch, P., A new cyclohexapeptide, penitropeptide and a new polyketide, penitropone from the endophytic
fungus Penicillium tropicum, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.05.095
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A new cyclohexapeptide, penitropeptide and a new polyketide,
penitropone from the endophytic fungus Penicillium tropicum
Yanbo Zenga,b, Hao Wangb, Ramsay S. T. Kamdemb,c, Raha S. Orfalid, Haofu Daia,
Gamall Makhloufie, Christoph Janiake, Zhen Liub,*, Peter Prokschb,*
a
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture,
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural
Sciences, Haikou 571101, China
b
Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University Düsseldorf,
Universitätsstrasse 1, 40225 Düsseldorf, Germany
c Department of Organic Chemistry, Higher Teachers’ Training College, University of Yaounde I.,
P. O. Box 47, Yaounde, Cameroon
d
Department of Pharmacognosy, Faculty of Pharmacy, King Saud University, Riyadh, Saudi
Arabia
e
Institute of Inorganic and Structural Chemistry, Heinrich-Heine-University Düsseldorf,
Universitätsstrasse 1, 40225 Düsseldorf, Germany
*Corresponding authors.
Tel.: +49 211 81 14163; fax: +49 211 81 11923;
e-mail: zhenfeizi0@sina.com (Z. Liu)
proksch@uni-duesseldorf.de (P. Proksch)
1

ABSTRACT
The first chemical investigation of the endophytic fungus Penicillium tropicum
isolated from leaves of Sapium ellipticum afforded a new cyclohexapeptide,
penitropeptide (1), and a new polyketide, penitropone (2) , in addition to two known
compounds,
6-hydroxy-8-methoxy-3S,5-dimethyl-3,4-isocoumarin
(3)
and
adametizine B (4). Their structures were unambiguously elucidated on the basis of 1D
and 2D NMR spectroscopy as well as mass spectrometry, and by comparison with the
literature. The absolute configurations of these two new compounds 1 and 2 were
determined by Marfey’s method and X-ray single crystal diffraction, respectively. All
isolated compounds were tested for their cytotoxic and antibacterial activities.
However, none of them showed significant activity.
Keywords: Penicillium tropicum; cyclohexapeptide; polyketide; X-ray diffraction.
2

Introduction
Secondary metabolites of endophytes have a long and rich history as an
important source of biologically active molecules with unusual structures.1-3 The
genus Penicillium which comprises more than 300 species is one of the most
ubiquitous fungal genera and has been intensely studied since the discovery of the
penicillins.4-5 Consequently, many structurally unique and bioactive secondary
metabolites have been characterized from this genus, as exemplified by phenolic
bisabolane sesquiterpenes peniciaculins A and B from P. aculeatum,6 azaphilone
derivatives pinophilins A, B, D–F from P. pinophilum,7-8 or novel alkaloids citrinadin
A–B and perinadine A from P. citrinum.9-11 During our ongoing search for new
secondary metabolites from endophytic fungi,12-14 the ethyl acetate extract of P.
tropicum isolated from the leaves of Sapium ellipticum (Euphorbiaceae) attracted our
attention due to the presence of structurally diverse compounds that did not match our
in house LC-UV data base. Chemical examination of this fungus led to the isolation
of a new cyclohexapeptide, penitropeptide (1), a new polyketide, penitropone (2), and
two known compounds 6-hydroxy-8-methoxy-3S,5-dimethyl-3,4-isocoumarin (3) and
adametizine B (4) (Figure 1). Herein, we describe the structural elucidation of the
new compounds 1 and 2, including their absolute configurations, established by
application of Marfey’s method and by X-ray diffraction, respectively.
3

Figure 1. Structures of compounds 1-4.
Results and discussion
Compound 1 was isolated as white, amorphous solid. Its HR-ESIMS spectrum
gave a peak [M + H]+ at m/z 698.3661 and the molecular formula C38H47N7O6,
indicating 19 degrees of unsaturation. The 1H NMR spectrum (Table 1) exhibited five
amide protons at H 10.83 (1H, d, J = 2.0 Hz, Trp5-1'NH), 8.81 (1H, dd, J = 7.5, 4.8
Hz, Gly1-NH), 8.60 (1H, d, J = 4.4 Hz, Leu6-NH), 8.57 (1H, d, J = 9.0 Hz, Trp5-NH),
and 7.36 (1H, d, J = 6.8 Hz, Phe2-NH), as well as -amino protons between H 3.40
and 4.50, suggesting a peptide structure for 1. Detailed interpretation of the COSY
and HMBC spectra (Table 1 and Figure 2) revealed the presence of six amino acid
residues in 1, including a Gly (glycine), a Phe (phenylalanine), two Pro (proline), a
Trp (tryptophan), and a Leu (leucine). The HMBC correlations from the amide
protons Trp5-NH, Leu6-NH, Gly1-NH, and Phe2-NH to their adjacent carbonyls
Pro4-CO, Trp5-CO, Leu6-CO, and Gly1-CO, respectively, as well as key ROESY
correlations (Table 1 and Figure 2) between Pro4-H/Trp5-NH, Trp5-H/Leu6-NH,
Gly1-NH/Phe2-NH, and Gly1-H/Phe2-NH, revealed Pro4-Trp5-Leu6-Gly1-Phe2 as
amino acid sequence. In addition, the HMBC correlations from Pro3-H to Phe2-CO
and the NOE correlations between Phe2-H/Pro3-H, and Pro3-H/Pro4-H indicated
the presence of a trans peptide bond between Phe2-Pro3 and a cis peptide bond
between Pro3-Pro4, which was also supported by the carbon resonances of  and  in
Pro3 (C- 28.0, C- 24.3) and Pro4 (C- 30.6, C- 21.1).15 Thus, 1 was determined to
be the new cyclo(-Gly-Phe-Pro-Pro-Trp-Leu-), for which the name penitropeptide is
proposed.
4

Table 1. 1H, 13C, HMBC and ROESY NMR data of penitropeptide (1).a
residue position
δC
δH (J in Hz)
HMBC
ROESY
Gly1
NH
CO

8.81, dd (7.5, 4.8)
Leu6-CO, Gly1-
Leu6-, Leu6-, Phe2-NH
168.8, C
43.4, CH2 4.01, dd (16.5, 7.5); Leu6-CO, Gly1-CO
Phe2-NH
3.40, dd (16.5, 4.8)
Phe2
NH
CO

7.36, d (6.8)
4.50, m
Gly1-CO, Phe2-CO, Phe2- Gly1-NH, Gly1-, Pro3-
167.5, C
53.0, CH
Pro3-
Phe2-CO, Phe2-, Phe2-1',
Phe2-2'/6'
36.4, CH2 3.08, dd (13.1, 9.3);
2.86, dd (13.1, 3.8)
137.7, C

1'
2'
3'
4'
5'
6'
CO

Phe2-, Phe2-4', Phe2-6'
Phe2-1', Phe2-5'
Phe2-2'/6'
Phe2-1', Phe2-3'
Phe2-, Phe2-2', Phe2-4'
129.1, CH 7.28, m
128.3, CH 7.28, m
126.4, CH 7.20, m
128.3, CH 7.28, m
129.1, CH 7.28, m
169.5, C
Pro3
Pro4
Trp5
Pro3-CO, Pro3-, Pro4-CO
Pro3-CO
Pro4-
Pro4-
59.1, CH
4.37, dd, (8.9, 7.3)
28.0, CH2 2.19, m; 1.62, m
24.3, CH2 1.94, m; 1.78, m
46.1, CH2 3.48, m; 3.29, m
170.8, C


Phe2-CO

Phe2-NH, Phe2-
CO

Pro4-CO, Pro4-
Pro3-, Pro3-, Trp5-NH
60.2, CH
4.22, dd (7.1, 2.3)

Pro4-CO, Pro4-, Pro4-
30.6, CH2 1.84, m
21.1, CH2 1.39, m; 0.50, m
46.1, CH2 3.13, m
8.57, d (9.0)


Pro4-CO
Pro4-, Trp5-2', Trp5-4'
NH
CO

173.2, C
54.1, CH
4.48, m
Leu6-NH
Leu6-NH

Trp5-, Trp5-2', Trp5-3',
Trp5-3'a
26.8, CH2 3.35, m; 3.13, m
1' NH
10.83, d (2.0)
Trp5-2', Trp5-3', Trp5-7',
Trp5-3'a, Trp5-7'a
2'
123.0, CH 7.16, d (2.0)
111.0, C
Trp5-3', Trp5-3'a, Trp5-7'a
Trp5-NH
Trp5-NH
3'
4'
118.5, CH 7.92, d (7.9)
117.9, CH 6.98, t (7.4)
120.5, CH 7.06, t (7.4)
110.6, CH 7.33, d (8.0)
127.5, C
Trp5-3', Trp5-6', Trp5-7'a
Trp5-7', Trp5-3'a
Trp5-4', Trp5-7'a
5'
6'
7'
Trp5-5', Trp5-3'a
3'a
7'a
NH
135.7, C
Leu6
8.60, d (4.4)
Trp5-CO, Leu6-, Leu6-
Trp5-, Trp5-
5

CO

173.1, C
53.6, CH
4.01, m
Leu6-CO
39.0, CH2 1.55, m
Gly1-NH
Gly1-NH

Leu6-CO, Leu6-, Leu6-,
Leu6-/'
Leu6-, Leu6-/'
Leu6-, Leu6-, Leu6-'
Leu6-, Leu6-, Leu6-
24.0, CH
1.62, m

22.3, CH3 0.96, d (6.5)

22.3, CH3 0.90, d (6.5)
'
a Recorded at 600 MHz for 1H and 150 MHz for 13C in DMSO-d6
Figure 2. Key COSY, HMBC and NOE correlations of 1.
The absolute configurations of the amino acid residues were determined
following acidic hydrolysis of 1 and subsequent derivatization according to Marfey’s
method.16 Comparison of the resulting derivatives with those of standard amino acids
using LC-MS led to the assignment of the L-configuration for all amino acid residues.
The molecular formula of compound 2 was established as C12H16O4 on the basis
of HR-ESIMS data (m/z 225.1122 [M + H]+), implying 5 degrees of unsaturation. The
1H NMR spectrum of 2 (Table 2) showed the presence of an olefinic methine proton
at δH 5.60 (1H, s, H-7), an oxygenated methine proton at δH 4.45 (1H, ddq, J = 6.1,
1.8, 6.7 Hz, H-3), an oxygenated methylene group at δH 3.98 (1H, d, J = 11.9 Hz,
H-1a) and 3.54 (1H, d, J = 11.9 Hz, H-1b), a methylene group at δH 3.07 (1H, ddq, J
= 13.4, 6.1, 1.0 Hz, H-4a) and 2.57 (1H, dd, J = 13.4, 1.8 Hz, H-4b), three methyl
groups, including a methoxy signal at δH 3.78 (3H, s, H3-13), a vinyl methyl signal at
δH 1.86 (3H, d, J = 1.0 Hz, H3-12), and an aliphatic methyl signal at δH 1.12 (3H, d, J
6

= 6.7 Hz, H3-11). The 13C NMR spectrum of 2 exhibited a total of 12 carbons (Table
2), including a carbonyl signal at δC 188.7 (C-6), three olefinic quaternary carbons at
δC 176.5 (C-8), 149.2 (C-10) and 131.0 (C-5), an olefinic methine at δC 101.9 (C-7),
an oxygenated methine at δC 72.9 (C-3), an oxygenated quaternary carbon at δC 70.2
(C-9), an oxygenated methylene at δC 70.1 (C-1), a methoxy group at δC 56.7 (C-13),
a methylene at δC 33.4 (C-4), and two methyls at δC 16.4 (C-11) and 10.7 (C-12). The
HMBC correlations from H2-1 to C-8, C-9 and C-10, from H2-4 to C-5, C-9 and C-10,
from H3-12 to C-5, C-6 and C-10, from H-7 to C-5, C-6, C-8 and C-9, and from H3-13
to C-8 indicated the presence of the cyclohexadienone ring A and the attachment of
methyl, methoxy and two methylene groups at C-5, C-8, C-9 and C-10, respectively
(Figure 3). Moreover, the COSY correlations between H-3/H2-4 and H-3/H3-11, as
well as the HMBC correlations from H2-1 to C-3 and from H-3 to C-1 revealed the
presence of the tetrahydropyran ring B, and consequently the attachment of a hydroxy
group at C-9 to satisfy its molecular formula. Thus, the planar structure of 2 was
elucidated as shown in Figure 1 and named penitropone.
The relative configuration of 2 was deduced from the ROESY spectrum in
CD3COCD3. The NOE correlations between H-1a (δH 3.93) and 9-OH (δH 4.81), and
between H-1b (δH 3.53) and H3-11 (δH 1.10) suggested a trans orientation of 9-OH
and CH3-11 (Figure 3). Finally, the absolute configuration of 2 was determined as 3S,
9S according to X-ray diffraction of a single crystal using the Flack parameter of
–0.08(7) with Cu-K radiation (Figure 4).17
7

Table 2. 1H, 13C, and HMBC NMR data of penitropone (2).
a
position
δC
δH (J in Hz)a
HMBCa
δH (J in Hz)b
3.93, d (11.8)
1
70.1, CH2
3.98, d (11.9)
C-3, C-8, C-9, C-10
3.54, d (11.9)
3.53, d (11.8)
3
4
72.9, CH
33.4, CH2
4.45, ddq (6.1, 1.8, 6.7)
C-1, C-10
4.40, ddq (6.1, 2.0, 6.7)
3.07, ddq (13.4, 6.1, 1.0) C-3, C-5, C-9, C-10, C-11 3.05, ddq (13.4, 6.1, 1.0)
2.57, dd (13.4, 1.8)
2.50, dd (13.4, 2.0)
5.49, s
5
6
131.0, C
188.7, C
7
101.9, CH 5.60, s
176.5, C
C-5, C-6, C-8, C-9
8
9
70.2, C
10
11
12
13
9-OH
149.2, C
16.4, CH3
10.7, CH3
56.7, CH3
1.12, d (6.7)
C-3, C-4
C-5, C-6, C-10
C-8
1.10, d (6.7)
1.80, d (1.0)
3.74, s
1.86, d (1.0)
3.78, s
4.81, s
a Recorded at 300 MHz for 1H and 75 MHz for 13C in CD3OD;
b Recorded at 600 MHz in CD3COCD3
Figure 3. Key COSY, HMBC and NOE correlations of 2.
Figure 4. Molecular structure of 2 from single-crystal X-ray diffractometry (70%
thermal ellipsoids, H atom of arbitrary radii).
The two known compounds were identified as 6-hydroxy-8-methoxy-3S,5-
8

dimethyl-3,4-isocoumarin (3),18 and adametizine B (4).19,20
Compounds 1–4 were tested for their cytotoxicity against the human ovarian cell
line A2780. However, none of them exhibited significant inhibitory activity at the
concentration of 10.0 µM. Also, the compounds showed no inhibitory activity against
Staphylococcus aureus ATCC 25923 and Acinebacter baumannii ATCC BAA1605
even at the highest test concentration of 64 µg/mL.
In summary, four compounds were isolated during the first chemical examination
of endophytic fungus P. tropicum. Penitropeptide (1) is a new cyclic hexapeptide and
the configurations of the amino acid residues were established using Marfey’s method.
Penitropone (2) and 6-hydroxy-8-methoxy-3S,5-dimethyl-3,4- isocoumarin (3) are
polyketides while the absolute configuration of the new compound penitropone (2)
was determined by X-ray single crystal diffraction. Adametizine B (4) is novel
epidithiodiketopiperazine derivative, which was first isolated from the hyper saline
lake derived fungus Penicillium sp.19 and later obtained from the sponge-derived
fungus Penicillium adametzioides.20
Acknowledgements
Financial support by China Scholarship Council to Y. Z. is gratefully
acknowledged. P. P. wants to thank the Manchot Stiftung for support.
Supplementary data
Supplementary data (UV, MS and NMR spectra of 1 and 2 as well as X-ray data
of 2) associated with this article can be found in the online version.
References and notes
9

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11

Graphical abstract
12

Highlights
 Four secondary metabolites were obtained during the first chemical examination
of endophytic fungus Penicillium tropicum.
 One new cyclohexapeptide, namely penitropeptide, of which the absolute
configuration was determined using Marfey’s method.
 One new polyketide derivative, penitropone whose sterostructure was confirmed
by single crystal X-ray diffraction.
13