Cipralphelin, a new anti-oxidative N-cinnamoyl tripeptide produced by the deep sea-derived fungal strain Penicillium brevicompactum FKJ-0123

Article abstract of DOI:10.1038/s41429-019-0208-6

A new N-cinnamoyl tripeptide, designated cipralphelin (1), was isolated from a cultured broth of Penicillium brevicompactum FKJ-0123 by physicochemical (PC) screening. Compound 1 was purified by silica gel and ODS column chromatography followed by preparative HPLC. The structure of 1 was determined as N-cinnamoyl-prolyl-alanyl-phenylalanine methyl ester by nuclear magnetic resonance and mass spectrometry analyses. The absolute configurations of three amino acids were determined by an advanced Marfey’s method applied to the hydrolysate of 1. Compound 1 was evaluated for its cytotoxicity, anti-microbial activity, and ability to scavenge or quench reactive oxygen species (ROS) such as superoxide anion radicals, hydroxy radicals, and singlet oxygen. Compound 1 exhibited potent scavenging activity against hydroxy radicals.

Full text of DOI:10.1038/s41429-019-0208-6

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0208-6
BRIEF COMMUNICATION
Cipralphelin, a new anti-oxidative N-cinnamoyl tripeptide produced
by the deep sea-derived fungal strain Penicillium brevicompactum
FKJ-0123
Hirotaka Matsuo1,2 Takayuki Mokudai Mayuka Higo Kenichi Nonaka
3 ●
1 ●
1,2 ●
Yuriko Nagano
4 ●
●
5 ●
6 ●
1 ●
1 ●
1,2
Takahiko Nagahama Yoshimi Niwano Yōko Takahashi Satoshi Ōmura Takuji Nakashima
Received: 25 April 2019 / Revised: 13 June 2019 / Accepted: 15 June 2019
©
The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
A new N-cinnamoyl tripeptide, designated cipralphelin (1), was isolated from a cultured broth of Penicillium
brevicompactum FKJ-0123 by physicochemical (PC) screening. Compound 1 was purified by silica gel and ODS column
chromatography followed by preparative HPLC. The structure of 1 was determined as N-cinnamoyl-prolyl-alanyl-
phenylalanine methyl ester by nuclear magnetic resonance and mass spectrometry analyses. The absolute configurations of
three amino acids were determined by an advanced Marfey’s method applied to the hydrolysate of 1. Compound 1 was
evaluated for its cytotoxicity, anti-microbial activity, and ability to scavenge or quench reactive oxygen species (ROS) such
as superoxide anion radicals, hydroxy radicals, and singlet oxygen. Compound 1 exhibited potent scavenging activity
against hydroxy radicals.
The deep sea generally refers to ocean depths of >200
meters. The deep sea is recognized as an extreme environ-
ment due to the absence of sunlight, low temperatures, and
high hydrostatic pressure. Despite these conditions, research
has shown that fungi can be abundant in deep-sea
environments [1]. Therefore, they may be adapted to deep-
sea conditions via metabolic pathways differing greatly
from those of organisms in terrestrial environments. Many
fungal strains isolated from deep-sea environments have
yielded novel and unique chemical compounds [2, 3].
Physicochemical (PC) screening, which is based on
physicochemical properties including molecular weight,
molecular formula, UV absorbance spectrum, and polarity,
has led to the discovery of several unique compounds from
a variety of microbial strains [4]. After their initial dis-
covery, many of these new compounds exhibited useful
biological activities. For example, PC screening identified
sarcopodinols A and B (Fig. S1) in a cultured broth of
Sarcopodium sp. FKJ-0025. Both of these compounds
showed cytotoxic activity against several human tumor cell
lines [5]. Pochoniolides A and B (Fig. S1), secondary
metabolites isolated from Pochonia chlamydosporia var.
spinulospora FKI-7537, exhibited strong hydroxy radical
scavenging and singlet oxygen quenching activities [6].
Recent PC screening in our laboratory has identified
several new compounds from cultured broths of 280 deep
sea-derived fungal strains. A new N-cinnamoyl tripeptide,
designated cipralphelin (1), is a secondary metabolite pro-
duced by fungal strain FKJ-0123. We report herein the
fermentation, isolation, structure, and anti-oxidative activity
of 1.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0208-6) contains supplementary
material, which is available to authorized users.
*
Takuji Nakashima
takuji@lisci.kitasato-u.ac.jp
1
2
3
4
Kitasato Institute for Life Sciences, Kitasato University, 5-9-1
Shirokane, Minato-ku, Tokyo 108-8641, Japan
Graduate School of Infection Control Sciences, Kitasato
University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Institute for Materials Research, Tohoku University, 2-1-1
Katahira, Aoba-ku, Sendai 980-8577, Japan
Deep-Sea Biodiversity Research Group, Japan Agency for Marine-
Earth Science and Technology, 2-15 Natsushima-cho,,
Yokosuka, Kanagawa 237-0061, Japan
5
6
Department of Foods and Human Nutrition, Notre Dame Seishin
University, 2-16-9 Ifuku-cho, Okayama 700-8516, Japan
Faculty of Nursing, Shumei University, 1-1 Daigaku-cho,
Yachiyo, Chiba 276-0003, Japan

H. Matsuo et al.
Table 1 1H and 13C NMR chemical shifts of 1 in CDCl3
Fungal strain FKJ-0123 (JAMSTEC ID: HPD874 72)
was isolated from a deep-sea sediment sample at Ryukyu
Trench, Japan (water depth: 283 m, sampling site: 24°
Cipralphelin (1)
Position
δH
δC
5
5.783′N, 124°55.794′E), using a remotely operated
Cinnamoyl
undersea vehicle Hyper-Dolphin of JAMSTEC during
NT08-14. This strain was identified as a member of the
genus Penicillium based on its verticillate conidiophores
and conidial chains. The internal transcribed spacer (ITS)
sequence of FKJ-0123 was compared with sequences in the
GenBank database using BLASTN 2.8.1 analyses [7]. The
sequence of FKJ-0123 was in complete agreement with the
sequence of CBS 317.59 (ex-type of Penicillium brun-
neostoloniferum, GenBank accession number KC484912).
Currently, P. brunneostoloniferum is synonymous with P.
brevicompactum. Therefore, the producing strain FKJ-0123
was identified as P. brevicompactum based on its mor-
phology and DNA barcoding.
1
2
3
4
166.2
117.7
143.4
134.8
128.0
128.8
130.0
6.69 (d, J = 15.5 Hz, 1H)
7.68 (d, J = 15.5 Hz, 1H)
5
, 9
7.48 (dd, J = 7.5, 2.3 Hz, 2H)
7.36 (overlap, 2H)
6, 8
7
7.36 (overlap, 1H)
Pro
1′
2′
171.5
60.6
27.8
4.58 (dd, J = 7.5, 3.4 Hz, 1H)
3′
1.98 (m, 1H)
2
.38 (m, 1H)
1.97 (m, 1H)
.13 (m, 1H)
3.64 (overlap, 1H)
.75 (ddd, J = 8.6, 8.6, 4.0 Hz, 1H)
Strain FKJ-0123 was grown on slants of modified
Miura’s medium [LcA: consisting of glycerol 0.1%,
KH PO 0.08%, K HPO4 0.02%, MgSO ·7H O 0.02%,
4
′
′
25.1
47.5
2
2
4
2
4
2
5
KCl 0.02%, NaNO 0.2%, yeast extract 0.02%, and agar
3
3
1
.5% (adjusted to pH 6.0 before sterilization)]. A loop of
Ala
spores of the strain was inoculated into a 500-ml Erlen-
meyer flask containing 100 ml of seed medium (glucose
1′′
2′′
3′′
171.8
48.7
17.4
4.51 (dd, J = 8.0, 7.5 Hz, 1H)
1.38 (d, 7.5 Hz, 3H)
2
0
%, yeast extract 0.2%, hipolypeptone 0.5%, KH PO
2 4
.1%, MgSO ·7H O 0.05%, and agar 0.1%). The inoculated
4
2
NH
7.23 (overlap, 1H)
seed culture was shaken at 210 rpm on a rotary shaker at
7 °C for 3 days. The culture medium in four bags, each
containing 500 g of rice (Hanamasa, Tokyo, Japan) and
Phe methyl ester
2
1′′′
172.0
53.6
37.7
−
1
2′′′
4.77 (ddd, J = 7.5, 6.9, 6.1 Hz, 1H)
3.05 (dd, J = 13.8, 6.9 Hz, 1H)
5
0 ml of seaweed tea (0.1 mg ml (ITO EN, Tokyo, Japan),
3′′′
were sterilized and inoculated a 25-ml aliquot of the seed
culture. The bags were allowed to ferment for 13 days at
3
.15 (dd, J = 13.8, 6.1 Hz, 1H)
4
5
6
7
′′′
136.3
129.2
128.3
126.8
52.1
2
5 °C. The whole cultured rice (2 kg) was extracted with 2l
′′′, 9′′′
′′′, 8′′′
′′′
7.09 (d, J = 6.9 Hz, 2H)
7.23 (overlap, 2H)
of MeOH and centrifuged to separate cells and supernatant.
The supernatant (2l) was concentrated in vacuo and
extracted with EtOAc. The EtOAc extract (2.4 g) was pur-
ified by silica gel column chromatography (n-hexane:
7.18 (d, J = 7.5 Hz, 1H)
3.63 (s, 3H)
OCH3
NH
EtOAc = 9:1, 6:4, 3:7 and CHCl :MeOH = 40:1, 20:1,
7.28 (d, J = 7.5 Hz, 1H)
3
1
0:1, 0:1). The CHCl :MeOH = 40:1 fraction (1.03 g) was
3
divided into five fractions by ODS column chromatography
10% MeCNaq. to 100% MeCN, 60 min gradient system).
(
Compound 1 was isolated as a pale yellow, amorphous
Fraction 5 (311.5 mg) was purified by ODS column chro-
matography (10% MeOHaq. to 100% MeOH with 0.1%
formic acid, 60 min gradient system). The fraction con-
taining 1 (81.1 mg) was purified by preparative high-
performance liquid chromatography (HPLC) with an
Inertsil ODS-4 column (GL Sciences, Tokyo, Japan) using
an isocratic eluent of 60% MeOH (0.1% formic acid) to
obtain 1 (10.1 mg). The physicochemical properties of 1
solid with a molecular formula of C H N O , as deter-
2
7
31
3
5
+
mined by HR-ESIMS (m/z 478.2331 [M+H] , calcd. for
1
C H N O , 478.2342). H NMR and HMQC spectra,
2
7
32
3
5
obtained in CDCl (Table 1), indicated the presence of two
3
exchangeable protons, 10 aromatic protons, including four
groups of symmetric protons, trans-olefinic protons, three
methine protons adjacent to a heteroatom, four methylene
protons, and methoxy and methyl protons. In addition, 13
C
23
were as follows; ½αŠ = 6.2 (c = 0.1, MeOH); IR (KBr) λ
NMR spectra and HMQC data (Table 1) indicated the
presence of four carbonyl carbon atoms, 12 aromatic carbon
atoms, including four groups of symmetric carbons, two
D
max
−
1
cm 2918, 1716, 1608, 1288; UV (MeOH) λ nm (log ε)
max
2
05 (4.32), 283 (4.36).

Cipralphelin, a new anti-oxidative N-cinnamoyl tripeptide produced by the deep sea-derived fungal. . .
3
A
B
2
1
0
.5
2
.5
1
.5
0
0
.013
0.025
0.05
0.1
0.1
Cipralphelin (mM)
Quercetin (mM)
Fig. 1 Structural elucidation of 1: (a) Selected 2D-nuclear magnetic
resonance (NMR) correlations, (b) absolute structure of 1
Fig. 2 Scavenging activity of 1 against hydroxy radicals. Hydroxy
radicals with DMPO-OH values were measured by ESR spectrum.
Quercetin was used as a positive control. Each value represents the
mean ± S.D. (n = 3)
unsaturated carbons, three heteroatom-bonded methine
carbons, a heteroatom-bonded methylene carbon, and three
methylene carbons. These results suggest that 1 contains
two mono-substituted benzene units. The whole structure of
absolute configuration of L-proline, D-alanine, and L-phe-
nylalanine (Fig. S8). The absolute structure of 1 was
established as shown in Fig. 1b.
1
was elucidated by detailed analyses of its 2D NMR data,
1
1
including H– H COSY and HMBC spectra. As shown in
Fig. 1a, H– H COSY spectra revealed the partial structures
a–f. The connectivity of partial structures a and b was
1
1
Compound 1 was evaluated in terms of cytotoxicity, anti-
microbial activity, and its ability to scavenge or quench
reactive oxygen species (ROS), such as superoxide anion
radicals, hydroxy radicals, and singlet oxygen. Compound 1
showed potent scavenging activity against hydroxy radicals.
The hydroxy radical scavenging activity of 1 at a con-
centration of 0.1 mM was equivalent to that of 0.1 mM
quercetin (Fig. S1), a positive control for hydroxy radical
scavenging (Fig. 2). Compound 1 had no activity against
cytotoxic and anti-microbial activities.
This study describes a new compound, N-cinnamoyl
tripeptide cipralphelin (1), produced by the deep-sea fungal
strain P. brevicompactum FKJ-0123. To the best of our
knowledge, this is the first report describing a natural pro-
duct containing a cinnamic acid conjugated with proline
from fugal metabolites. In general, cinnamic acid deriva-
tives are common constituents of plants and are known for
their potent anti-oxidative activities [9]. Similarly, com-
pound 1 exhibited potent hydroxy radical scavenging
activity. Hydroxy radical has been implicated in many
diseases, including atherosclerosis, cancer, and several
neurological disorders [10, 11]. Compound 1 may be the
basis for new anti-oxidative agents. This study also verifies
deep-sea fungi as an important source of unique natural
products.
revealed by HMBC correlations between H-5 (δ 7.48) and
H
C-7 (δ 130.0) and C-3 (δ 143.4), between H-6 (δ 7.35)
C
C
H
and C-4 (δ 134.8), between H-3 (δH 7.68) and C-1 (δC
C
1
66.2) and C-5 (δ 128.0), and between H-2 (δ 6.69) and
C H
C-4 and C-1, indicating a cinnamoyl group. Partial structure
c and HMBC correlations between H-3′ (δ 1.98 and 2.36)
H
and C-1′ (δ 171.5) indicate the presence of a proline unit.
C
Partial structure d and HMBC correlations between
C-2′′-NH (δ 7.23) and C-2′′ (δ 48.7), C-3′′ (δ 17.4), and
H
C
C
C-1′′ (δ 171.8), and between C-3′′-Me (δ 1.38) and C-1′′
C
H
revealed the presence of an alanine unit. Partial structures e
and f, and HMBC correlations between H-5′′′ (δ 7.09) and
H
C-7′′′ (δ 126.8), C-9′′′ (δ 129.2) and C-3′′′ (δ 37.7),
C
C
C
between H-6′′′ (δ 7.23) and C-4′′′ (δ 136.3) and C-8′′′
H
C
(
1
δ 128.3), between H-3′′′ (δ 3.04 and 3.15) and C-5′′′ (δ
C
H
C
29.2) and C-1′′′ (δ 172.0), between C-2′′′-NH (δ 7.28)
C
H
and C-2′′′ (δ 53.6) and C-1′′′, between H-2′′′ (δ 4.77)
C
H
and C-1′′′, and between C-10′′′-Me (δH 3.63) and C-1′′′
revealed the presence of a phenylalanine methyl ester unit.
The connectivity of the partial structures was not deter-
minable with HMBC analyses alone. Therefore, the whole
structure of 1 was determined by tandem mass spectrometry
(
MS/MS) fragmentation analysis. MS/MS fragments of 1
were observed at m/z 131, 228 and 299, consistent with the
structure shown in Fig. S7. These data indicate a planar
structure, as shown in Fig. 1a, and compound 1 was named
cipralphelin.
The absolute configuration of 1 was determined using an
advanced Marfey’s analysis of the hydrolysate of 1 [8].
Compound 1 was treated with 6 M hydrochloride at 100 °C
for 12 h to obtain the hydrolysate. This process revealed the
Acknowledgements This study was supported by funds from the
Institute for Fermentation, Osaka (IFO), Japan, the JSPS KAKENHI
for Young Scientists (B) (grant number JP17K15250 and
JP15K18601) and a Kitasato University Research Grant for Young
Researchers awarded to Hirotaka, M. We thank Dr. Kenichiro Nagai,
School of Pharmacy, Kitasato University, for HRESIMS measure-
ments and the crew of the R/V Natsushima, and the operation team of

H. Matsuo et al.
the ROV Hyper-Dolphin for their cooperation in collecting deep-sea
samples.
4. Nakashima T, Takahashi Y, Ōmura S. Search for new compounds
from Kitasato microbial library by physicochemical screening.
Biochem Pharmacol 2017;134:42–55.
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. Matsuo H, et al. New metabolites, sarcopodinols A and B, isolated
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Biosci Biotechnol Biochem 2018;82:1323–6.
. Miyano R, et al. Pochoniolides A and B, new antioxidants from
the fungal strain Pchonia chlamydosporia var. spinulosporia FKI-
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
6
7
537. J Biosci Bioeng 2018;126:661–6.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
7. Altschul SF, et al. Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids
Res 1997;25:3389–402.
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