Tolyprolinol, a new dipeptide from Tolypocladium sp. FKI-7981

Article abstract of DOI:10.1038/s41429-018-0041-3

A new dipeptide, named tolyprolinol, was isolated from the static culture of a fungus, Tolypocladium sp. FKI-7981. The structure of tolyprolinol was elucidated as N-acetyl-l-phenylalanyl-l-prolinol. It showed moderate antimalarial activity but did not show cytotoxicity or any other antimicrobial property.

Full text of DOI:10.1038/s41429-018-0041-3

The Journal of Antibiotics
https://doi.org/10.1038/s41429-018-0041-3
BRIEF COMMUNICATION
Tolyprolinol, a new dipeptide from Tolypocladium sp. FKI-7981
Wataru Fukasawa1 Natsuki Mori1 Masato Iwatsuki1,2 Rei Hokari2 Aki Ishiyama1,2 Moe Nakajima2
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Takahito Ouchi2 Kenichi Nonaka1,2 Hiroki Kojima2 Hirotaka Matsuo1,2 Satoshi Ōmura2 Kazuro Shiomi1,2
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Received: 18 December 2017 / Revised: 5 February 2018 / Accepted: 5 February 2018
© The Author(s) under exclusive licence to the Japan Antibiotics Research Association 2018
Abstract
A new dipeptide, named tolyprolinol, was isolated from the static culture of a fungus, Tolypocladium sp. FKI-7981. The
structure of tolyprolinol was elucidated as N-acetyl-L-phenylalanyl-L-prolinol. It showed moderate antimalarial activity but
did not show cytotoxicity or any other antimicrobial property.
Fungal secondary metabolites are rich sources of unique
compounds and a lot of useful compounds have already
been discovered. However, it has been proposed that there
is an immeasurable number of microbial metabolites not yet
discovered [1]. Therefore, our group has continued to
investigate fungal metabolites, which has already resulted in
the discovery of novel compounds such as the virgaricins A
and B, cinatrins D and E, and cladomarine [2–4]. Recent
research led us to discover a new dipeptide, tolyprolinol (1),
containing a L-phenylalanine and a L-prolinol, from a cul-
ture broth of Tolypocladium sp. FKI-7981. Prolinol is a rare
moiety among natural products and this is the first report of
a natural product containing a prolinol moiety isolated from
Tolypocladium species. We detail here the taxonomy of
producing strain, as well as the fermentation, isolation,
structure elucidation, and some biological properties of 1.
The fungal strain FKI-7981 had 95.2% similarity with
the internal transcribed spacer sequence of CBS 869.73 (ex-
type of Tolypocladium album). From this information,
combined with morphological characteristics, FKI-7981
was identified to be a member of the genus Tolypocladium
family [5, 6].
The EtOAc extract of a 14-day static cultured broth was
subjected to column choromatographies and high-
performance liquid chromatography purification to afford
to 1 (22.3 mg). The detailed fermentation and isolation
procedure for 1 is summarized in Schemes S1 and S2 in the
Supplementary Information.
The physico-chemical properties of 1 are summarized in
Table 1. It is soluble in methanol with ultraviolet (UV)
absorption at 206 nm, as well as dimethyl sulfoxide
Table 1 Physico-chemical properties of tolyprolinol (1)
Tolyprolinol (1)
Appearance
Pale yellow oil
C16H22N2O3
Molecular formula
Molecular weight
ESI-MS (m/z) positive
ESI-MS (m/z) negative
HR-ESI-MS (m/z)
Calcd.
290
313 [M + Na]+
335 [M + HCOOH–H]–
These authors contributed equally: Wataru Fukasawa, Natsuki Mori.
Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41429-018-0041-3) contains supplementary
material, which is available to authorized users.
313.1528
Found
313.1521
UV λMeOH nm (ε)
206
IR νKBr (cm−1
)
3440, 3267, 1624, 1454
14.7 (c = 0.1, MeOH)
* Satoshi Ōmura
omuras@insti.kitasato-u.ac.jp
23.5
[α]D
* Kazuro Shiomi
Solubility
Soluble
shiomi@lisci.kitasato-u.ac.jp
MeOH, DMSO, CHCl3, H2O
Slightly soluble
Insoluble
MeCN
1
Graduate School of Infection Control Sciences, Kitasato
University, Tokyo, Japan
n-Hexane
2
ESI-MS electrospray ionization mass spectrometry, IR infrared, UV
Kitasato Institute for Life Sciences, Kitasato University,
ultraviolet
Tokyo, Japan

W. Fukasawa et al.
Table 2 1H and 13C NMR data of the major and minor conformers of tolyprolinol (1) in CDCl3
Position
Tolyprolinol (1)
Major conformer
δC (ppm)
Minor conformer
δH (ppm) (Int.,
multiplicity, J in Hz)
δC (ppm)
δH (ppm) (Int.,
multiplicity, J in Hz)
Prolinol
OH
1
—
4.38 (1H, br. s)
3.42 (1H, m)
3.48 (1H, m)
4.13 (1H, m)
1.48 (1H, m)
1.93 (1H, m)
1.65 (2H, m)
2.67 (1H, m)
3.65 (1H, m)
—
4.58 (1H, br. s)
3.32 (1H, m)
3.47 (1H, m)
3.30 (1H, m)
1.10 (1H, m)
1.50 (1H, m)
1.62 (2H, m)
3.36 (2H, m)
65.9
64.7
2
3
61.1
27.6
59.6
28.2
4
5
24.1
47.8
21.6
45.4
Phe
1′
2′
172.1
52.3
170.5
52.9
4.93 (1H, ddd, 8.6, 8.6,
5.6)
5.09 (1H, ddd, 10.0, 6.8,
6.8)
3′
39.4
3.02 (2H, m)
39.4
2.99 (2H, m)
4′
135.9
128.5
129.3
127.1
—
135.9
128.5
129.3
127.1
—
5′, 5″
6′, 6″
7′
7.2–7.3 (5H,
overlapped)
7.2–7.3 (5H,
overlapped)
NH
6.92 (1H, d, 8.0)
—
7.20 (1H)
—
8′
9′
169.7
22.9
170.8
22.8
1.98 (3H, s)
1.98 (3H, s)
NMR nuclear magnetic resonance spectroscopy
(DMSO), chloroform, and water, but insoluble in n-hexane.
The characteristic infrared absorptions at 3440, 3267, 1624,
and 1454 cm−1 suggested the presence of an amidocarbonyl
group moiety.
derivatives described below. In the major conformer of 1,
HMBC correlations observed from CH3 (δH 1.98) and Phe
NH (δH 6.92) to C-8′ (δC 169.7) indicated that the amino
group in Phe in 1 was acetylated. The planar structure of 1
was established by HMBC correlations from prolinol H2-5
(δH 2.67 and 3.65) to Phe C-1′ (δC 172.1) as N-acet-
ylphenylalanylprolinol. Compound 1 has not been pre-
viously reported and we designated it as tolyprolinol. The
minor conformer of 1 was also elucidated in the same
manner (Table 2). This minor conformer is suggested as
being derived from the regioisomeric amide bond of proli-
nol residue in 1.
The molecular formula of 1 was elucidated by HR-ESI-
MS to be C16H22N2O3 with a molecular ion peak [M + Na]+
at m/z 313.1521 (calcd. m/z 313.1528). Two conformers
1
were observed with the ratio of 3:2 in H and 13C nuclear
magnetic resonance spectroscopy (NMR) spectral data of 1
in DMSO-d6 at room temperature (Figs. S2-1 and S2-2).
The spectra did not change dramatically, even at 80 °C.
1
Amide signals and α-proton signals in H and 13C NMR
spectral data of 1 suggested 1 is a peptide compound. When
1 was measured in CDCl3, two conformers were observed
with the ratio of 5:2 at room temperature (Figs. S2-6 and
S2-7). The structure of major and minor conformers of 1 in
CDCl3 was elucidated by 1D and 2D NMR, as shown in
Table 2.
The absolute configuration of amino acids in 1 was
elucidated by Advanced Marfey’s method after acid
hydrolysis [7]. Compound 1 was hydrolyzed and deriva-
tized with FDLA and analyzed by an ultraperformance
liquid chromatography coupled with ESI-MS. As the result
of the comparison of retention time with FDLA derivatives
of standard Phe and prolinol, both Phe and prolinol were
elucidated to be the L configuration (Table S3 and Fig. S3).
Compound 1 was tested for antimalarial activity against
both a chloroquine-resistant K1 strain and chloroquine-
sensitive FCR3 strain of P. falciparum, as well as
Combined analysis of the 1H-1H correlation spectroscopy
(COSY) and heteronuclear multiple-bond correlation
(HMBC) spectra identified phenylalanine (Phe) and prolinol
residues in 1 (Fig. 1b), which were finally confirmed by
their 1-fluoro-2,4-dinitrophenyl-5-D-leucineamide (FDLA)

New dipeptide from Tolypocladium
Fig. 1 a Structure of tolyprolinol
1
(1). b Key correlations of H-1H
COSY and HMBC in 1. Bold
lines show proton spin
1
networks; arrows show H-13C
long-range correlations
References
cytotoxicity against nine human cell lines. Compound 1
showed half-maximal inhibitory concentration values of
163 and 285 µM against the K1 strain and the FCR3 strain
of P. falciparum, respectively. However, 1 did not display
any cytotoxicity against the human MRC-5 cell at 345 µM
and other eight human cancer cell lines, HL-60, Jarkat,
THP-1, HeLa S3, A549, Panc1, HT29, and H1299 at 100
µM.
The antimicrobial activity of 1 was assessed against six
microorganisms, Bacillus subtilis KB 211 (ATCC 6633),
Kocuria rhizophilia KB 212 (ATCC 9341), Escherichia
coli KB 213 (NIHJ), Xanthomonas oryzae pv. oryzae KB
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In summary, we have discovered a new dipeptide, named
tolyprolinol, consisting of L-Phe and L-prolinol, from
secondary metabolites of Tolypocladium sp. FKI-7981. We
found 1 showed moderate antimalarial activity, but did not
show cytotoxicity or other antimicrobial activity against the
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were cyclosporin-like polypeptides [15]. Therefore, the
isolation of the new dipeptide 1 suggests that Tolypocla-
dium may be a potential source of unique compounds that
could prove to be interesting lead compounds for future
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Acknowledgements This research was partially supported by the
Platform Project for Supporting Drug Discovery and Life Science
Research (Basis for Supporting Innovative Drug Discovery and Life
Science Research (BINDS)) from the Japan Agency for Medical
Research and Development (AMED) as well as by the 24th Botanical
Research Grant of the New Technology Development Foundation. We
are grateful to Dr Kenichiro Nagai, Kitasato University, and Kazunari
Sakai for useful suggestions concerning the experiments and collection
of soil samples.