Kozupeptins, antimalarial agents produced by paracamarosporium species: Isolation, structural elucidation, total synthesis, and bioactivity

Article abstract of DOI:10.1021/acs.orglett.9b00483

Kozupeptins A and B, novel antimalarial lipopeptides, were isolated from the culture broths of Paracamarosporium sp. FKI-7019. They exhibited potent antimalarial activity against chloroquine-sensitive and-resistant Plasmodium falciparum strains in vitro.

Full text of DOI:10.1021/acs.orglett.9b00483

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Kozupeptins, Antimalarial Agents Produced by Paracamarosporium
Species: Isolation, Structural Elucidation, Total Synthesis, and
Bioactivity
Yumi Hayashi,† Wataru Fukasawa,† Tomoyasu Hirose,†,‡ Masato Iw‡atsuki,†,‡ Rei Hokari,‡‡
Aki Ishiyama,†,‡ Masahiro Kanaida,† Kenichi Nonaka,†,‡ Akira Take, Kazuhiko Otoguro,
́
Satoshi Omura,†,‡ Kazuro Shiomi,*,†,‡ and Toshiaki Sunazuka*,†,‡
̅
†Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
‡Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
S
* Supporting Information
ABSTRACT: Kozupeptins A and B, novel antimalarial lipopeptides, were
isolated from the culture broths of Paracamarosporium sp. FKI-7019. They
exhibited potent antimalarial activity against chloroquine-sensitive and
-resistant Plasmodium falciparum strains in vitro. The structural elucidation
was accomplished by a combination of spectroscopic analyses and
chemical approaches including a total synthesis of kozupeptin A. Synthetic
kozupeptin A demonstrated a therapeutic effect in vivo, and an
intermediate exhibited much higher antimalarial activity than kozupeptin
A.
alaria, caused by Plasmodium species, is one of the
M
world’s most devastating infectious diseases and
remains a serious global health problem.1 Antimalarial agents
have been continuously developed, but drug-resistant Plasmo-
dium parasites evolve quickly and are now widespread.2
Therefore, the development of novel and safe antimalarial
drugs, with new modes of action and structural features, is
urgently required.
In the course of our screening program to discover
antimalarial agents from metabolites of microorganisms
which are active against drug-resistant parasites in vitro and
in vivo, we have discovered various metabolites with potent
antimalarial properties.3
Herein, we report the isolation of new potent antimalarial
compounds, designated as kozupeptins A and B (1a, 1b), from
the culture broths of Paracamarosporium sp. FKI-7019 (Figure
1). We also describe an efficient total synthesis and evaluation
of the antimalarial activity of these new compounds. This work
shows that the kozupeptins could be a promising leading
candidate for development of a new antimalarial drug.
An antimalarial activity-guided screening program from the
fermentation broths of our microorganism library led us to
focus on a fungus strain, Paracamarosporium sp. FKI-7019
isolated from the soil around a plant in Kozu-Island, Tokyo,
Japan. This strain was grown and maintained on an agar slant
(see Supporting Information for details).
Figure 1. Structures of kozupeptins A and B (1a and 1b).
from an ethanol extracted culture broth of Paracamarosporium
sp. FKI-7019. From another fermentation lot of ethanol
extracted broth (12.0 L), 1b (7.2 mg) was obtained (see
Supporting Information for details).
The molecular formula of kozupeptin A (1a) was
determined to be C58H107N7O10 by high-resolution ESI-MS
measurement (m/z 1062.8140 calculated for C58H108N7O10
[M + H]+ 1062.8152). The structure of 1a was elucidated by
1D and 2D NMR. The peptide nature of the molecule was
The isolation procedure for the kozupeptin A (1a) is
summarized in Scheme 1. The procedure afforded 1.7 mg of 1a
Received: February 6, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b00483
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
Scheme 1. Isolation Procedure of Kozupeptin A (1a)
Figure 3. ROESY correlation and 1H coupling constant of 6-
membered N,O-acetal formed with sphingoid and alaninal.
same way as that of 1a (see Supporting Information).
Consequently, 1b was found to be an analog of 1a which
lacks both sphingoid and alaninal units (Figure 1).
immediately inferred from the presence of a number of signals
To evaluate the predicted structure and determine the
absolute stereochemistry, we performed an asymmetric total
synthesis of 1a. Aiming for a practical synthesis, we decided to
synthesize the peptide unit of 1a by liquid-phase peptide
synthesis (LPPS) utilizing a hydrophobic anchor molecule,
which can combine the best characteristics of conventional
LPPS with the benefits of solid-phase peptide synthesis
(SPPS).7,8 LPPS utilizing a hydrophobic anchor molecule
was first reported by Tamiaki et al. in 2001. They reported a
protocol in which intermediates of peptide synthesis were
purified by crystallization combined with flash chromatography
using a benzyl alcohol derivative as an hydrophobic anchor
molecule.9 Afterward, Chiba et al. and our group used this
protocol to synthesize several peptides including bioactive
ones.10,11 Fluorene-derived anchor supported compounds and
a synthetic protocol called AJIPHASE were developed by
Ajinomoto Co., Inc.12 In spite of the highly efficient output of
this LPPS-based method, limited examples illustrated the
strategic use of it as described above.
1
in the amide NH and α-amino acid proton regions of its H
NMR and of amide carbonyl groups in the 13C NMR spectra
(Table S2). Furthermore, a monounsaturated fatty acid with
one double bond was thought to be present in the molecule
due to many methylene signals and an olefin signal. Combined
1
analysis of the H−1H COSY, TOCSY, and HMBC spectra
identified the structures of four amino acids including one Thr,
one 4-methylproline (4-MePro), one Val and one Asn, an
alaninal unit, a sphingoid unit, and a fatty acid unit (Figure 2).
Figure 2. Partial structures of kozupeptin A (1a) indicated by the
1
correlations of H−1H COSY, TOCSY, and HMBC.
Our synthetic approach began with the deprotection of the
Fmoc group of the known 3, a fluorene-derived anchor
supported Fmoc-Ala (Scheme 2).12a The supported amino
acid was subsequently subjected to four coupling−depro-
tection cycles to construct the linear pentapeptide 8 by
standard Fmoc peptide synthesis using N,N′-diisopropyl-
carbodiimide (DIC). Each product in peptide elongations
could be purified by direct crystallization from the reaction
mixture in high yields. Condensation with oleic acid employing
the same approach effectively afforded 9. After synthesis of all
the peptide and fatty acid units of 1a was accomplished,
removal of the anchor molecule and deprotection of t-Bu
group was achieved at the same time under acidic conditions to
give carboxylic acid 10 in 75% yield after silica gel flash
chromatography separation. The carboxyl group of 10 was
converted to Weinreb amide 11 with a slight epimerization (dr
= 97:3, α-position of Ala) using 3-(diethoxyphosphoryloxy)-
1,2,3-benzotriazin-4(3H)-one (DEPBT) as a condensation
reagent.13
Reduction of the Weinreb amide utilizing lithium aluminum
hydride (LAH) afforded the aldehyde 12,14 which was treated
with natural type phytosphingosine ((2S,3S,4R)-2-amino-1,3,4-
octadecanetriol, 13) to give 1a in moderate yield over two
steps. All the physicochemical data of the synthetic sample
were identical to those of the naturally occurring compound
and were used to help determine the absolute configuration of
1a.
Thus, kozupeptin A (1a) was regarded as a hybrid natural
product. The assignment of the sequence was carried out by a
combination of HMBC and MS/MS analysis (see Supporting
Information for details). Consequently, both the fatty acid unit
and sphingoid unit were found to have C18, and the planar
structure of 1a was determined to be a novel linear lipopeptide
with the sequence fatty acid-Thr-4-MePro-Val-Asn-alaninal-
sphingoid. The absolute configuration of each amino acid
residue was determined by advanced Marfey’s analysis.4 Acid
hydrolysis of 1a was followed by LC-MS analysis after
derivatization of the hydrolysate with D-FDLA. Comparison
with the retention times obtained from standards revealed the
presence of L-Thr, (2S,4R)-4-MePro, L-Val, L-Asn, and L-
alaninal (detected as L-Ala by Jones oxidation of the
hydrolysate before the reaction with D-FDLA) (see Supporting
Information for details). The monounsaturated fatty acid was
1
deduced to be oleic acid by H NMR and ESI-LCMS using a
2-nitrophenylhydrazide derivative.5 This was also supported by
the GCMS fragmentation pattern of the pyrrolidine amide
derivative (see Supporting Information for details).6 Finally,
the relative stereochemistry of the six-membered N,O-acetal
which was deduced to be formed between C18-sphingoid and
alaninal was determined by a combination of the correlations
of ROESY and the values of the coupling constant in 1H NMR
(Figure 3). All substituted groups on the ring proved to be in
equatorial positions. However, the absolute configuration of
the sphingoid unit was not completely characterized before the
total synthesis of 1a was accomplished in this work (see
below). The structural elucidation of 1b was performed in the
Naturally occurring kozupeptins, synthesized 1a, and three
intermediates in the late stage of our total synthesis were
evaluated for in vitro antimalarial activity against chloroquine-
resistant and -sensitive Plasmodium falciparum strains (K1 and
B
DOI: 10.1021/acs.orglett.9b00483
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Scheme 2. Total Synthesis of Kozupeptin A (1a) Utilizing a Hydrophobic Anchor Molecule
FCR3 respectively) and cytotoxicity against a human lung
properties of the Weinreb amide 11 or carboxylic acid 10 were
much weaker.
fibroblast cell line MRC-5 (Table 1).3a Synthetic kozupeptin A
Having sufficient 1a and 12 in hand throughout our total
synthesis, the preliminary in vivo antimalarial activity of 1a, 12,
and one of the standard antimalarial drugs, artesunate, injected
i.p. (intraperitoneal), were measured in a mouse model
according to Peters’ 4-day suppressive test using the rodent
malaria P. berghei N strain, which is chloroquine-sensitive.15,3a,b
A dose of 30 mg kg−1 of 1a, 12, or artesunate for 4 days each
suppressed 52.4%, 99.8%, or 89.9% of malaria parasites (99.8%
and 89.9% should not be considered a statistically significant
difference in this time period). This initial finding that 1a and
the synthetic intermediate 12 exhibit antimalarial activity not
only in vitro but also in vivo, combined with the rare structure16
for an antimalarial agent, confirms that the kozupeptins show
promise as lead antimalarial compounds with a probable new
mode of action.
Table 1. In Vitro Antimalarial Activity
IC50 (μM) antimalarial activity
a
b
compound
K1 strain
FCR3 strain
cytotoxicity (MRC-5)
1a (natural)
1b (natural)
1a (synthetic)
0.15
1.03
0.30
0.0083
12.5
>16.0
0.17
0.019
0.29
1.46
0.55
0.013
>15.2
N/A
0.038
0.023
>23
>35
>23
>32
>30
>32
c
12
11
10
d
e
chloroquine
artemisinin
58
d
e
160
a
b
c
Chloroquine-resistant strain. Chloroquine-sensitive strain. Purified
d
by silica gel column chromatography for the evaluation. Drugs
e
commonly used to treat malaria. J. Antibiot. 2011, 64, 183.
In conclusion, kozupeptins A and B (1a, 1b), new potent
antimalarial agents, were isolated from the culture broths of
Paracamarosporium sp. FKI-7019. An efficient total synthesis of
kozupeptin A (1a) has been achieved in a practical manner,
which confirmed the structure proposed by spectroscopic and
chemical techniques and elucidated the absolute configuration
of the molecule. It also illustrates the power of an LPPS-based
method utilizing a hydrophobic anchor molecule, a concept
that can take advantage of the characteristics of both
conventional LPPS and SPPS. Kozupeptin A (1a) and
synthetic intermediates were found to exhibit in vitro and in
(1a) was found to show a similar IC50 value (0.3 μM) to that
of natural 1a. Even more surprisingly, aldehyde 12 exhibited
significant antimalarial activity (about 20 times stronger than
that of 1a) against both the chloroquine-resistant K1 strain and
the chloroquine-sensitive FCR3 strain, which was of the same
order as that of artemisinin. In addition, 12 fortunately showed
low cytotoxicity against human MRC-5 cells. 1a would be the
prodrug of the most active compound 12. The antimalarial
C
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Activities of a Carbohydrate Antibiotic, Prumycin, against Drug-
resistant Strains of Plasmodia. J. Antibiot. 2004, 57, 400−402. (f) Ui,
H.; Ishiyama, A.; Sekiguchi, H.; Namatame, M.; Nishihara, A.;
vivo antimalarial activity, which showed promise for develop-
ment of a new antimalarial agent. Further investigation,
including a structure−activity relationship (SAR) study, of
the antimalarial potential of kozupeptins is in progress.
̅
Takahashi, Y.; Shiomi, K.; Otoguro, K.; Omura, S. Selective and
Potent In Vitro Antimalarial Activities Found in Four Microbial
Metabolites. J. Antibiot. 2007, 60, 220−222. (g) Iwatsuki, M.; Takada,
S.; Mori, M.; Ishiyama, A.; Namatame, M.; Nishihara-Tsukashima, A.;
ASSOCIATED CONTENT
* Supporting Information
■
S
̅
Nonaka, K.; Masuma, R.; Otoguro, K.; Shiomi, K.; Omura, S. In vitro
and in vivo antimalarial activity of puberulic acid and its new analogs,
viticolins A−C, produced by Penicillium sp. FKI-4410. J. Antibiot.
2011, 64, 183−188. (h) Hirose, T.; Noguchi, Y.; Furuya, Y.; Ishiyama,
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b00483.
̅
A.; Iwatsuki, M.; Otoguro, K.; Omura, S.; Sunazuka, T. Structure
Experimental details and characterization data (PDF)
NMR spectra (ZIP)
Determination and Total Synthesis of (+)-16-Hydroxy-16,22-
dihydroapparicine. Chem. - Eur. J. 2013, 19, 10741−10750.
(4) (a) Marfey, P. Determination of α-Amino Acids. II. Use of a
Bifunctional Reagent, 1,5-Difuluoro-2,4-dinitromenzene. Carlsberg
Res. Commun. 1984, 49, 591−596. (b) Fujii, K.; Ikai, Y.; Mayumi,
T.; Oka, H.; Suzuki, M.; Harada, K. A Nonempirical Method Using
LC/MS for Determination of the Absolute Configuration of
Constituent Amino Acids in a Peptide: Elucidation of Limitations
of Marfey’s Method and of Its Separation Mechanism. Anal. Chem.
1997, 69, 3346−3352.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: shiomi@lisci.kitasato-u.ac.jp (K.S.)
*E-mail: sunazuka@lisci.kitasato-u.ac.jp (T.S.)
ORCID
(5) Miwa, H.; Hiyama, C.; Yamamoto, M. High-Performance Liquid
Chromatography of Short-and Long-Chain Fatty Acids as 2-
Nitrophenylhydrazides. J. Chromatogr. A 1985, 321, 165−174.
(6) Andersson, B. A.; Holman, R. T. Pyrrolidides for mass
spectrometric determination of the position of the double bond in
monounsaturated fatty acids. Lipids 1974, 9, 185−190.
Kazuro Shiomi: 0000-0003-1264-5116
Toshiaki Sunazuka: 0000-0002-8001-081X
Notes
The authors declare no competing financial interest.
(7) For recent representative reviews of SPPS, see: (a) Amblard, M.;
Fehrentz, J.-A.; Martinez, J.; Subra, G. Methods and Protocols of
Modern Solid Phase Peptide Synthesis. Mol. Biotechnol. 2006, 33,
239−256. (b) Behrendt, R.; White, P.; Offer, J. Advances in Fmoc
solid-phase peptide Synthesis. J. Pept. Sci. 2016, 22, 4−27.
(8) For LPPS-based methods to address several problems of
conventional LPPS, see: (a) Narita, M. Liquid Phase Peptide
Synthesis by the Fragment Condensation on Soluble Polymer
Support. I. Efficient Coupling and Relative Reactivity of a Peptide
Fragment with Various Coupling Reagents. Bull. Chem. Soc. Jpn. 1978,
51, 1477−1480. (b) Pillai, V. N. R.; Mutter, M. New, Easily
Removable Poly(ethylene glycol) Supports for the Liquid-Phase
Method of Peptide Synthesis. J. Org. Chem. 1980, 45, 5364−5370.
(c) Mizuno, M.; Goto, K.; Miura, T.; Hosaka, D.; Inazu, T. A novel
peptide synthesis using fluorous chemistry. Chem. Commun. 2003,
972−973.
ACKNOWLEDGMENTS
■
We thank Dr. K. Nagai and Ms. N. Sato (School of Pharmacy,
Kitasato University) for various instrumental analyses. We
thank Dr. T. Shirahata (School of Pharmacy, Kitasato
University) for GCMS analysis. We thank Mr. T. Tokiwa
(Kitasato Institute for Life Sciences, Kitasato University) for
photographing the fungus. This study was partly supported by
JSPS KAKENHI Grant Number 17K08343. This research was
also partially supported by Platform Project for Supporting
Drug Discovery and Life Science Research (Basis for
Supporting Innovative Drug Discovery and Life Science
Research (BINDS)) from AMED under Grant Numbers
17am0101096j0001 and 18am0101096j0002.
REFERENCES
(9) Tamiaki, H.; Obata, T.; Azefu, Y.; Toma, K. A Novel Protecting
Group for Constructing Combinatorial Peptide Libraries. Bull. Chem.
Soc. Jpn. 2001, 74, 733−738.
■
(1) World Health Organization, World Malaria Report 2017.
(2) For recent representative reviews, see: (a) Sinha, S.; Medhi, B.;
Sehgal, R. Challenges of drug-resistant malaria. Parasite 2014, 21, 61.
(b) Blasco, B.; Leroy, D.; Fidock, D. A. Antimalarial drug resistance:
linking Plasmodium falciparum parasite biology to the clinic. Nat. Med.
2017, 23, 917−928.
(10) For representative reports, see: (a) Tana, G.; Kitada, S.; Fujita,
S.; Okada, Y.; Kim, S.; Chiba, K. A practical solution-phase synthesis
of an antagonistic peptide of TNF-a based on hydrophobic tag
strategy. Chem. Commun. 2010, 46, 8219−8221. (b) Okada, Y.;
Suzuki, H.; Nakae, T.; Fujita, S.; Abe, H.; Nagano, K.; Yamada, T.;
Ebata, N.; Kim, S.; Chiba, K. Tag-Assisted Liquid-Phase Peptide
Synthesis Using Hydrophobic Benzyl Alcohols as Supports. J. Org.
Chem. 2013, 78, 320−327. (c) Fujita, Y.; Fujita, S.; Okada, Y.; Chiba,
K. Soluble Tag-Assisted Peptide Head-to-Tail Cyclization: Total
Synthesis of Mahafacyclin B. Org. Lett. 2013, 15, 1155−1157.
(d) Matsumoto, E.; Fujita, Y.; Okada, Y.; Kauppinen, E. I.; Kamiya,
H.; Chiba, K. Hydrophobic benzyl amines as supports for liquid-phase
C-terminal amidated peptide synthesis: application to the preparation
of ABT-510. J. Pept. Sci. 2015, 21, 691−695.
(3) (a) Otoguro, K.; Kohana, A.; Manabe, C.; Ishiyama, A.; Ui, H.;
̅
Shiomi, K.; Yamada, H.; Omura, S. Potent Antimalarial Activities of
Polyether Antibiotic, X-206. J. Antibiot. 2001, 54, 658−663.
(b) Otoguro, K.; Ishiyama, A.; Ui, H.; Kobayashi, M.; Manabe, C.;
̅
Yan, G.; Takahashi, Y.; Tanaka, H.; Yamada, H.; Omura, S. In Vitro
and In Vivo Antimalarial Activities of the Monoglycoside Polyether
Antibiotic, K-41 against Drug Resistant Strains of Plasmodia. J.
Antibiot. 2002, 55, 832−834. (c) Otoguro, K.; Ui, H.; Ishiyama, A.;
Arai, N.; Kobayashi, M.; Takahashi, Y.; Masuma, R.; Shiomi, K.;
̅
Yamada, H.; Omura, S. In Vitro Antimalarial Activities of the
(11) Hirose, T.; Kasai, T.; Akimoto, T.; Endo, A.; Sugawara, A.;
Nagasawa, K.; Shiomi, K.; Omura, S.; Sunazuka, T. Solution-phase
̅
Microbial Metabolites. J. Antibiot. 2003, 56, 322−324. (d) Otoguro,
K.; Ui, H.; Ishiyama, A.; Kobayashi, M.; Togashi, H.; Takahashi, Y.;
total synthesis of the hydrophilic natural product argifin using 3,4,5-
tris(octadecyloxy)benzyl tag. Tetrahedron 2011, 67, 6633−6643.
(12) (a) Takahashi, D.; Yamamoto, T. Development of an efficient
liquid-phase peptide synthesis protocol using a novel fluorene-derived
anchor support compound with Fmoc chemistry; AJIPHASE.
Tetrahedron Lett. 2012, 53, 1936−1939. (b) Takahashi, D.; Yano,
̅
Masuma, R.; Tanaka, H.; Tomoda, H.; Yamada, H.; Omura, S. In
Vitro and in Vivo Antimalarial Activities of a Non-glycosidic 18-
Membered Macrolide Antibiotic, Borrelidin, against Drug-resistant
Strains of Plasmodia. J. Antibiot. 2003, 56, 727−729. (e) Otoguro, K.;
Ishiyama, A.; Kobayashi, M.; Sekiguchi, H.; Izuhara, T.; Sunazuka, T.;
̅
Tomoda, H.; Yamada, H.; Omura, S. In Vitro and In Vivo Antimalarial
D
DOI: 10.1021/acs.orglett.9b00483
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
T.; Fukui, T. Novel diphenylmethyl-Derived Amide Protecting Group
for Efficient Liquid-Phase Peptide Synthesis: AJIPHASE. Org. Lett.
2012, 14, 4514−4517. (c) Takahashi, D.; Inomata, T.; Fukui, T.
AJIPHASE®: A Highly Efficient Synthetic Method for One-Pot
Peptide Elongation in the Solution Phase by an Fmoc Strategy. Angew.
Chem., Int. Ed. 2017, 56, 7803−7807.
(13) Li, H.; Jiang, X.; Ye, Y.-h.; Fan, C.; Romoff, T.; Goodman, M.
3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT):
A New Coupling Reagent with Remarkable Resistance to
Racemization. Org. Lett. 1999, 1, 91−93.
(14) Nahm, S.; Weinreb, S. M. N-Methoxy-N-methylamides as
Effective Acylating Agents. Tetrahedron Lett. 1981, 22, 3815−3818.
(15) Peters, W.; Portus, J. H.; Robinson, B. L. The chemotherapy of
rodent malaria, XXII. The value of drug-resistant strains of P. berghei
in screening for blood schizontocidal activity. Ann. Trop. Med.
Parasitol. 1975, 69, 155−171.
(16) A few compounds which have similar structures to kozupeptins
were reported; see: (a) Fujie, K.; Sato, T.; Nishikawa, M.; Hatanaka,
́
H.; Hashimoto, M. JP 09/221497, August 26, 1997. (b) Ortiz-Lopez,
́
́
F. J.; Monteiro, M. C.; Gonzalez-Menendez, V. G.; Tormo, J. R.;
Genilloud, O.; Bills, G. F.; Vicente, F.; Zhang, C.; Roemer, T.; Singh,
S. B.; Reyes, F. Cyclic Colisporifungin and Linear Cavinafungins,
Antifungal Lipopeptides Isolated from Colispora cavincola. J. Nat.
Prod. 2015, 78, 468−475.
E
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