Nyuzenamides A and B: Bicyclic Peptides with Antifungal and Cytotoxic Activity from a Marine-Derived Streptomyces sp.

Article abstract of DOI:10.1021/acs.orglett.1c00210

Two bicyclic peptides, nyuzenamides A (1) and B (2), were discovered from Streptomyces isolated from suspended matter in deep sea water collected in the Sea of Japan. Their structures were determined through nuclear magnetic resonance and mass spectrometry analyses in combination with X-ray crystallography and the chiral-phase gas chromatography-mass spectrometry method to comprise ten amino acid residues containing four unusual amino acids along with aromatic acyl units. Both compounds displayed antifungal activity against pathogenic fungi and cytotoxicity against P388 murine leukemia cells.

Full text of DOI:10.1021/acs.orglett.1c00210

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Letter
Nyuzenamides A and B: Bicyclic Peptides with Antifungal and
Cytotoxic Activity from a Marine-Derived Streptomyces sp.
Md. Rokon Ul Karim, Yasuko In, Tao Zhou, Enjuro Harunari, Naoya Oku, and Yasuhiro Igarashi*
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ABSTRACT: Two bicyclic peptides, nyuzenamides A (1) and B
(2), were discovered from Streptomyces isolated from suspended
matter in deep sea water collected in the Sea of Japan. Their
structures were determined through nuclear magnetic resonance
and mass spectrometry analyses in combination with X-ray
crystallography and the chiral-phase gas chromatography−mass
spectrometry method to comprise ten amino acid residues
containing four unusual amino acids along with aromatic acyl
units. Both compounds displayed antifungal activity against
pathogenic fungi and cytotoxicity against P388 murine leukemia
cells.
icrobial secondary metabolites have contributed to
Bay, Japan (Figure 1). In this Letter, we describe the isolation,
Mhuman welfare and the scientific community over many structure determination, and bioactivity of 1 and 2.
The producing strain N11-34 was isolated from suspended
matter collected from −311 m depth at the Nyuzen deep sea
water pumping facility, Toyama, Japan.7 The strain was
identified as a member of the genus Streptomyces based on
the 16S rRNA gene sequence analysis. The whole culture broth
of strain N11-34 cultured in A16 liquid medium was extracted
with 1-butanol, and the extract was fractionated by silica gel
chromatography. Careful inspection of the fractions by high-
performance liquid chromatography with a diode-array
detector (HPLC-DAD) indicated the production of unknown
metabolites showing UV spectra characteristic of non-
conjugated benzene rings. UV-guided purification by reverse-
phase HPLC yielded nyuzenamides A (1, 12.3 mg) and B (2,
14.5 mg) from a 3 L culture.
Nyuzenamide A (1) was isolated as a pale yellow,
amorphous powder. The molecular formula was determined
to be C66H81N11O20 on the basis of high-resolution electro-
spray ionization time-of-flight mass spectrometry (HR-
ESITOFMS) that gave a sodium adduct ion [M + Na]+ at
m/z 1370.5554 (Δ + 0.2 mmu). The UV spectrum in
methanol, showing absorption bands at 203 and 273 nm, was
indicative of the presence of benzene ring(s). The IR spectrum
suggested the presence of NH or OH (3291 cm−1) and
years. Among the bacterial genera, Streptomyces is undoubtedly
the most productive genus, accounting for up to 9000
compounds. This figure is three times larger than that of
secondary metabolites from Aspergillus or Penicillum, the most
productive counterparts in eukaryotic microbes.1 Although the
average genome size of Streptomyces is 6 to 8 Mbp, not
exceedingly larger than well-known secondary metabolite
producers in prokaryotes such as myxobacteria (∼13 Mbp)
and cyanobacteria (∼9 Mbp), its average number of
biosynthetic gene clusters for secondary metabolites is quite
large, typically more than 30.2−5 Biosynthetic machineries for
secondary metabolites in Streptomyces are much more diverse
than other prokaryotic organisms, ranging from polyketides
and peptides to isoprenoids, alkaloids, and additional structural
classes derived from other pathways including carbohydrates
and shikimates. Among these, modular type-I polyketide
synthase (PKS) and nonribosomal peptide synthetase
(NRPS) have superior capability of generating complex and
large molecules by virtue of their ability to utilize a range of
small building blocks for chain elongation. In particular, recent
advances in genetic engineering technology are expanding the
possibility of generating new peptide scaffolds, rendering
natural products of the peptide class more attractive targets for
drug lead discovery than ever.6
Received: January 23, 2021
Published: March 4, 2021
In our continuing search for structurally novel secondary
metabolites from marine actinomycetes to obtain new insights
into drug design for antiinfective/anticancer agents, nyuzena-
mides A (1) and B (2), bicyclic peptides possessing several
unusual structural features, were discovered from Streptomyces
isolated from suspended matter in deep sea water of Toyama
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© 2021 American Chemical Society
Org. Lett. 2021, 23, 2109−2113
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aqueous HCl, free amino acids in the hydrolysate of 1 were
derivatized to trifluoroacetic acid (TFA) amide methyl esters
by esterification in hydrochloric methanol followed by
treatment with trifluoroacetic acid anhydride. The derivatized
hydrolysates were analyzed by chiral-phase GC-MS, in
comparison with authentic D- and L-amino acid derivatives,
which established the presence of D-Asx, D-Leu, L-Pro, L-Thr,
and L-Val (Figure S16). The remaining four pairs of amide
protons and α-methines, on the contrary, were all assigned to
be parts of oxygenated unusual α-amino acids, namely,
hydroxyglycine (Hgy), β-hydroxyphenylalanine (Hpa), 4-
hydroxyphenylglycine (Hpg), and α-hydroxytyrosine (Htr).
Hydroxylation at the α- or β-positions of Hgy, Hpa, and Htr
was evident from sequences of COSY correlations from amide
protons to hydroxy protons (δH 5.73, 6.21, and 7.15), whereas
the presence of an aromatic ring in Hpa, Hpg, and Htr was
supported by HMBC correlations from ortho-aromatic protons
to benzylic carbons (Figure 2). Oxygenation of the aromatic
rings in Hpg and Htr was inferred by deshielded chemical
shifts of carbons at the para and meta positions (δH 147.5,
147.8, and 160.3). Because both of the proton pairs
symmetrically positioned on the phenoxy ring of Hpg (H4/
H8 and H5/H7) were magnetically inequivalent, the ring
rotation appeared to be severely restricted, which implied a
connection of the phenolic oxygen to some other part of the
molecule.
In addition to these amino acid residues, another
component, a methyl- and hydroxy-substituted 2-(isochro-
man-1-yl)acetyl moiety (Ica), was assembled from the
remaining three spin-coupled fragments and two nonproto-
nated aromatic carbons (δC 138.4, Ica-C4; 136.7, Ica-C9). A
bundle of four aromatic protons (δH 7.12, 5.71, 6.92, and 7.29)
had a doublet−triplet−triplet−doublet coupling pattern and
correlated with both of these nonprotonated carbons in the
HMBC spectrum, showing a typical signature of a 1,2-
disubstituted benzene ring. The two substituents on this ring
were an oxymethine (Ica-3) paired with a deshielded
methylene (δH 2.64, 3.22/δC 41.5, Ica-2) and a hydroxy-
substituted methine (Ica-10) linking to a methyl-substituted
oxymethine (Ica-11), as supported by HMBC correlations
from protons at both ends of the aromatic fragments to these
methine carbons. Another HMBC correlation from Ica-H11 to
Ica-C3 confirmed an ether bridge between Ica-C3 and Ica-C11,
thus completing an isochroman core. Furthermore, the
deshielded methylene and neighboring oxymethine protons
showed HMBC correlations to a carbonyl carbon (δC 175.9,
Ica-C1), which indicated a connection of the Ica unit to a
hydroxy or amino functionality in the peptide chain.
Figure 1. Structures of nyuzenamides A (1) and B (2).
carbonyl (1644 cm−1) functionalities. Consistent with these
analytical data, an initial inspection of 1D and 2D nuclear
magnetic resonance (NMR) data revealed the presence of
multiple aromatic protons/carbons (δH 6.40−7.39; δC 116.0−
131.6), 12 carbonyl carbons (δC 168.4−175.9), and 9 amide
protons (δH 7.51−9.10), respectively, connecting to α-
methines/methylenes (δH 3.21−5.77; δC 42.7−71.4), suggest-
ing the peptidic nature of this molecule (Table S1).
Analysis of correlation spectroscopy (COSY), heteronuclear
single quantum coherence (HSQC), and heteronuclear
multiple bond correlation (HMBC) spectral data allowed the
assignment of six common amino acids, Asx, Gly, Leu, Pro,
Thr/allo-Thr, and Val (Figure 2), which accounted for five out
of the nine signatures of α-amino acids. In parallel with the
NMR analysis, chiral gas chromatography−mass spectrometry
(GC-MS) analysis was conducted to confirm the amino acid
composition and their absolute configurations. After heating in
The partial alignments of the amino acid residues were
established by HMBC correlations from amide protons to the
adjacent amide carbons, revealing Pro-Leu-Hgy-Htr-Asx and
Thr-Hpg-Val-Gly-Hpa sequences. From the Thr β-proton was
observed an HMBC correlation to a carboxyl carbon of Asx,
which connected two peptide fragments via an ester linkage,
whereas a correlation from the Thr α-proton to the Ica
carbonyl carbon verified the acylation of the Thr amino group
by the Ica unit.
The thus-assembled peptolide sequence contained one
additional oxygen atom and one fewer H3N molecule
compared with the molecular formula, with six open termini
of Pro-N, Htr-6-O, Htr-7-O, Asx-C4, Hpg-6-O, and Hpa-C1
remaining to be connected. Possible connectivities among
these termini were partially given by a rotating frame
Figure 2. Key 2D NMR correlations for nyuzenamide A (1).
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Overhauser effect spectroscopy (ROESY) spectrum (Figure
S7). First, an amide linkage between Pro-N and Hpa-C1 was
suggested by ROESY correlations Pro-H5/Hpa-H2 and Pro-
H5/Hpa-H3 (Figure 2). In addition, ROESY correlations Hpg-
H5/Htr-H5 and Hpg-H7/Htr-H5 implied an ether bridge
between Hpg and Htr, which eliminated the excessive oxygen,
as previously assumed. An exchangeable proton at δH 9.63,
showing no COSY or HMBC correlation, was accordingly
attributable to a phenolic proton at Htr-7-O, and the rest of
the atoms, NH2, were likely the components of a carboxamide
blockade at the Asx-C4 terminus. No further information was
available in the NMR data to validate these speculations. To
our delight, however, during the storage of the NMR sample in
a refrigerator, crystalline particles were formed, which were
recovered and recrystallized from a mixture of MeOH−
CH2Cl2 to provide colorless needles. The single-crystal X-ray
analysis elucidated the gross structure of 1, in which Pro and
Hpa were connected through an amide bond, and Hpg and Htr
were connected through an ether bridge at Hpg-C6 and Htr-
C6, whereas a carboxamide group was present on Asx to form
Asn (CCDC accession no. 2026824, Figure 3). The Flack
eventually were assembled into a 2-(2-hydroxy-1-methoxy-
propyl)phenylpropenoyl group (Ppa), lacked the oxymethine
and methylene units present in 1 and instead added methoxy
(δH 2.69, s/δC 56.0) and disubstituted E-olefin (δH 6.84, d, J =
15.7 Hz; 8.03, d, J = 15.6 Hz) groups, suggesting the alteration
of the substituents on the benzene ring (Figure 4). In fact, both
Figure 4. Key 2D NMR correlations for nyuzenamide B (2).
of the double-bond protons exhibited HMBC correlations to a
carbonyl carbon at δC 168.8 (Ppa-C1) and a nonprotonated
aromatic carbon at δC 135.4 (Ppa-C4), establishing a 2-
propenoyl unit conjugated with a phenyl ring. The β-proton
(Ppa-H3) of the acyl unit further correlated with another two
aromatic carbons, one protonated (δC 127.1, Ppa-C5) and the
other nonprotonated (δC 138.7, Ppa-C9), which, together with
a four proton sequence traced in the COSY spectrum,
supported a 1,2-disubstituted benzene structure. Finally, on
the basis of the COSY and HMBC correlations, a 2-hydroxy-1-
methoxypropyl group was placed at Ppa-C9 as the other
substituent on the ring to complete the modified cinnamoyl
unit as well as the gross structure of 2. The absolute
configurations of the common amino acids were identical to
those in 1, as determined by chiral-phase GC-MS analysis, and
the same conclusion might be applicable to the rest of the
amino acids in consideration of the global resemblance of the
NMR spectra and biogenetic identity. The two chiral centers in
the Ppa group were tentatively assigned to be 10R and 11S
based on the interconvertible relationship of Ica and Ppa
through β-elimination or Michael addition.
Nyuzenamides A (1) and B (2) were inactive against Gram-
positive and -negative bacteria and a yeast but selectively
inhibited the growth of filamentous fungi (Table S3).
Compound 1 was more potent than 2 against plant and
human pathogens, Glomerella cingulata NBRC5907 and
Trichophyton rubrum NBRC5467, with a minimum inhibitory
concentration (MIC) of 3.1 and 6.3 μg/mL, respectively. The
MIC values of 2 against these fungi were 25 μg/mL. In
addition, 1 and 2 exhibited cytotoxicity against P388 murine
leukemia cells with IC50 of 4.9 and 6.2 μM (6.6 and 8.4 μg/
mL, respectively).
Figure 3. X-ray crystal structure of nyuzenamide A (1).
parameter (0.01) was small enough to verify the results from
the chiral-phase GC-MS analysis and assign the remaining
absolute configurations for the unusual amino acids and a
nonpeptidic unit to be (S) for Hpg, (2S, 3S) for Hpa, (S) for
Hgy, (2R, 3S) for Htr, and (3S, 10R, 11S) for Ica (Figure 1).
Nyuzenamide B (2) was also obtained as a pale yellow
amorphous powder. Its molecular formula C67H83N11O20,
determined by HR-ESITOFMS that displayed a sodium
adduct ion [M + Na]+ at m/z 1384.5706, was 14 amu larger
than 1, corresponding to the increment of a CH2 fragment.
Comprehensive analysis of 2D NMR spectra of 2 revealed that
the ten amino acid residues, including four unusual amino
acids, were totally identical to those in 1, indicating a structural
difference in the N-acyl substituent on Thr. The amino acid
sequence was also proven to be the same by a series of HMBC
correlations (Table S2).
Although HMBC correlations to support connectivities
between Pro and Hpa and Hpg and Htr were lacking, the same
set of ROESY correlations observed for 1 was also present,
again supporting the amide linkage between Pro and Hpa and
the ether linkage between Hpg and Htr (Figure S13). The
remaining molecular parts for the nonpeptidic moiety, which
In summary, the chemical investigation of a marine-derived
Streptomyces strain N11-34 led to the discovery of two novel
bicyclic peptides, nyuzenamides A (1) and B (2). The four
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unusual amino acids in 1 and 2 are quite rare in nature. Hgy is
known to be a component of spergualin, an antitumor
antibiotic from Bacillus,8 and dolyemycins, cyclic peptides
from Streptomyces.9 Hpa is a component of cyclic peptides
from Streptomyces, dolyemycins, RP-1776,10 ohmyungsamy-
cins,11 atratumycin,12 and atrovimycin.13 Htr is found in
vancomycins from Amycolatopsis14 and ralstonins from
Ralstonia solanacearum.15 Hpg is also a building block of
vancomycins. The diphenyl ether bridge between aromatic
amino acids is seen in some cyclic peptides such as
vancomycins and seongsanamides,16 but the linkage pattern
between Htr and Hpg is unreported to date. The nonpeptidic
units in 1 and 2, oxygenated Ica and Ppa moieties, are also rare
in nature. Similar structural parts, namely, the ortho-
substituted cinnamates, are reported as an acyl decoration
unit in RP-1776, atratumycin, atrovimycin, cinnapeptin,17 and
eudistamides,18 all of which are the monocyclic depsipeptides
from Streptomyces. The aromatic acyl portions in 1 and 2 are
likely derived from the PKS pathway.12,13,17,18 Starting from a
propenylphenylpropenoate, dioxidation at the propenyl double
bond (e.g., epoxidation/hydrolysis cascade) would give rise to
the diol. Michael addition of the hydroxy group could lead to
cyclic ether formation, thus yielding the Ica moiety. Further
methylation of one hydroxy group could afford the Ppa moiety
(Figure 5). Although many bicyclic peptides such as
AUTHOR INFORMATION
■
Corresponding Author
Yasuhiro Igarashi − Biotechnology Research Center and
Department of Biotechnology, Toyama Prefectural University,
Imizu, Toyama 939-0398, Japan; orcid.org/0000-0001-
5114-1389; Phone: +81-766-56-7500; Email: yas@pu-
toyama.ac.jp; Fax: +81-766-56-2498
Authors
Md. Rokon Ul Karim − Biotechnology Research Center and
Department of Biotechnology, Toyama Prefectural University,
Imizu, Toyama 939-0398, Japan; orcid.org/0000-0002-
1891-8894
Yasuko In − Department of Physical Chemistry, Osaka
University of Pharmaceutical Sciences, Takatsuki, Osaka
569-1041, Japan
Tao Zhou − Biotechnology Research Center and Department
of Biotechnology, Toyama Prefectural University, Imizu,
Toyama 939-0398, Japan; orcid.org/0000-0002-6359-
2598
Enjuro Harunari − Biotechnology Research Center and
Department of Biotechnology, Toyama Prefectural University,
Imizu, Toyama 939-0398, Japan; orcid.org/0000-0001-
8726-0865
Naoya Oku − Biotechnology Research Center and Department
of Biotechnology, Toyama Prefectural University, Imizu,
Toyama 939-0398, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c00210
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. Takayuki Kumagai at the Federation of Fisheries
Cooperative Association of Nyuzen, Toyama, Japan for
providing spent filters at the deep sea water intake pit. We
also thank Prof. N. Itoh and Dr. H. Toda at Toyama
Prefectural University for allowing and instructing us on the
use of the Shimadzu GC-MS system. P388 cells were obtained
from JCRB Cell Bank under accession code JCRB0017 (lot
06252002). This research was supported by JSPS KAKENHI
grant no. JP16K07719 to Y.I.
Figure 5. Plausible biosynthetic relationship between Ica and Ppa
moieties.
seongsamides and salinamides19 are known from nature,
overall, 1 and 2 have no structural resemblance to any of the
known natural peptides. The discovery of 1 and 2 expands the
structural diversity of microbial peptides and provides new
scaffolds for the development of new antifungal antibiotics.
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ASSOCIATED CONTENT
* Supporting Information
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The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c00210.
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CCDC 2026824 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
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Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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