Yaku'amides A and B, cytotoxic linear peptides rich in dehydroamino acids from the marine sponge ceratopsion sp.

Article abstract of DOI:10.1021/ja109275z

Two cytotoxic peptides, yaku'amides A (1) and B (2), were isolated from the marine sponge Ceratopsion sp. Their planar structures were elucidated on the basis of spectroscopic data, whereas the absolute configurations were determined by a combination of the Marfey's analysis and dansylation analysis of the total and partial acid hydrolysis products. The growth inhibitory profile of yaku'amide A against a panel of 39 human cancer cell lines was clearly unique and distinguished from other anticancer drugs.

Full text of DOI:10.1021/ja109275z

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Yaku’amides A and B, Cytotoxic Linear Peptides Rich in Dehydroamino Acids
from the Marine Sponge Ceratopsion sp.°
Reiko Ueoka,†,‡ Yuji Ise,§ Susumu Ohtsuka,⊥ Shigeru Okada,† Takao Yamori,¶ and
Shigeki Matsunaga*,†
Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The
UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, Misaki Marine Biological Station, The UniVersity of
Tokyo, Miura, Kanagawa 238-0225, Japan, Takehara Marine Science Station, Hiroshima UniVersity, Takehara,
Hiroshima 725-0024, Japan, and DiVision of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese
Foundation for Cancer Research, Koto-ku, Ariake, Tokyo 135-8550, Japan
Received October 14, 2010; E-mail: assmats@mail.ecc.u-toky.ac.jp
Abstract: Two cytotoxic peptides, yaku’amides A (1) and B (2),
were isolated from the marine sponge Ceratopsion sp. Their
planar structures were elucidated on the basis of spectroscopic
data, whereas the absolute configurations were determined by a
combination of the Marfey’s analysis and dansylation analysis of
the total and partial acid hydrolysis products. The growth inhibitory
Figure 1. 2D-NMR correlations in NTA and CTA.
profile of yaku’amide A against a panel of 39 human cancer cell
lines was clearly unique and distinguished from other anticancer
drugs.
one residue each of Rꢀ-dehydro-Val and ꢀ-hydroxy-Ile, two
residues of ꢀ-hydroxy-Val, and three residues of R,ꢀ-dehydro-Ile
were assigned. Both termini of yaku’amide A (1) were blocked by
unique groups. The partial structure of C-4 to C-9 in the N-terminal
acyl group (NTA) was deduced from the COSY and TOCSY data
(Figure 1). In NTA, the HMBC correlations from H-10/H-11 to
C-1, C-2, and C-3 indicated the presence of a 2,2-dimethyl malonyl
moiety. These two partial structures were connected on the basis
of an HMBC correlation from H-9 to C-3. COSY and TOCSY data
revealed the partial structure of C-1 to C-5 of the C-terminal amine
(CTA). The HMBC correlations from the N-methyl (H3-6 and H3-
7) to C-1 showed the attachment of a dimethylamino group to C-1
in CTA (Figure 1).
Nonribosomal peptides compare well with polyketides in their
potent biological activities, production by prokaryotes or fungi, and
biosynthesis by modular enzymes.1 Marine sponges also contain a
variety of nonribosomal peptides and polyketides.2 In the course
of our search for cytotoxic compounds from marine sponges, we
found that a deep-sea sponge Ceratopsion sp. collected at Yakush-
insone in the East China Sea showed potent cytotoxicity. From the
sponge we have isolated yaku’amides A and B (1 and 2,
respectively), linear peptides with unique N- and C-termini and
rich in dehydroamino acids. Their structures were elucidated by
analysis of spectroscopic data and chemical degradations.
Partial amino acid sequences were deduced by interpretation of
the HMBC data (Figure 2). The HMBC correlation from H-2 of
OHIle to C-1 of NTA indicated the connection between NTA and
OHIle (unit a). The HMBC cross peaks from H-2 of Gly to C-1 of
∆Ile1, from NH of ∆Ile2 to C-1 of Gly, and from NH and H-2 of
Val1 to C-1 of ∆Ile2 indicated the sequence of ∆Ile1-Gly-∆Ile2-
Val1 (unit b). The HMBC correlation from H-2 of Ala to C-1 of
∆Ile3 revealed that ∆Ile3 was attached to Ala (unit c). The
arrangement of Val2-Val3-∆Val-CTA (unit d) was assigned by the
HMBC cross peaks from NH of Val3 to C-1 of Val2, from NH of
∆Val to C-2 of Val3, and from NH of CTA to C-1 of ∆Val. No
sequential data for Ile and two OHVal residues were obtained by
the HMBC data. Then the above partial sequences were integrated
on the basis of the NOESY data (Figure 2). The correlation between
NH of ∆Ile1 and H-2 of OHIle permitted the connection between
unit a and unit b. The cross peaks between NH of Ile and NH,
H-2, H-3, and H3-4 of Val1 and between NH of Ile and NH of
OHVal1 indicated the sequence of Val1-Ile-OHVal1. The connec-
tion of OHVal2 and ∆Ile3 was assigned by the correlation between
NH of ∆Ile3 and NH, H-2, H3-4, and H3-5 of OHVal2, and the
connection of Ala and Val2 was assigned by the correlation between
NH of Val2 and NH, H-2, and H3-3 of Ala. Finally, OHVal1 was
attached to OHVal2 on the basis of the NOESY cross peaks between
methyl singlets of each residue.
The organic layer of the extract of the sponge was subjected to
a modification of the Kupchan’s solvent-solvent partitioning
scheme.3 The CHCl3 fraction was separated by ODS flash chro-
matography, silica gel column chromatography, and reversed-phase
HPLC to give yaku’amides A (1) and B (2).
Yaku’amide A (1) had a molecular formula of C83H145N15O18
which was established by the HR-ESIMS. Analysis of the 1H NMR
spectrum measured in CD3OD in conjunction with the HSQC
spectrum revealed the presence of a large number of aliphatic
methyls, five vinylic methyls, one broad methyl signal, and
R-methines. In DMSO-d6, exchangeable NH signals characteristic
of a peptide were observed and the broad methyl signal was
sharpened. The 13C NMR spectrum showed numerous nonproto-
nated carbons, viz., one ketone, many amide carbonyl carbons, sp2
carbons, and oxygenated quaternary carbons, indicating the presence
of a variety of nonproteinogenic amino acid residues. Analysis of
2D NMR data revealed the presence of one residue each of Gly,
Ala, and Ile and three residues of Val. In addition to these residues,
° Yaku’amides were named after yakushinsone, the collection site.
† Laboratory of Aquatic Natural Products Chemistry, The University of Tokyo.
‡ Present address: Faculty of Life Sciences, Kumamoto University.
§ Misaki Marine Biological Station, The University of Tokyo.
⊥ Hiroshima University.
¶ Japanese Foundation for Cancer Research.
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17692 J. AM. CHEM. SOC. 2010, 132, 17692–17694
10.1021/ja109275z  2010 American Chemical Society

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C O M M U N I C A T I O N S
Figure 2. Structures of yaku’amides and HMBC and NOESY correlations observed in 1.
The geometries of the double bonds in the three ∆Ile residues
were determined on the basis of NOESY data. The correlations
between NH and H-2 of Gly and H3-6 of ∆Ile1 indicated the
Z-configuration in ∆Ile1. The E-configuration in ∆Ile2 was assigned
by the cross peak between H-2 of Gly and H3-6 of ∆Ile2. The cross
peak between NH of Ala and H3-6 of ∆Ile3 indicated the
Z-configuration in ∆Ile3.
and common absolute configurations of component amino acids
suggested that they share the same configurations throughout
the chains.
Yaku’amides A (1) and B (2) exhibited potent cell-growth
inhibitory activity against P388 murine leukemia cells with IC50
values of 14 and 4 ng/mL, respectively. In order to analyze the
mode of action of yaku’amide A (1), we examined its growth-
inhibitory profile against a panel of 39 human cancer cell lines
(JFCR39) that include various human cancers.8 Because yaku’amide
A (1) exhibits clearly a unique profile as compared to any 38
anticancer drugs,8a it is suggested that yaku’amide A (1) has a
unique mode of action in its growth-inhibitory activity.
Yaku’amides are unique in containing many dehydroamino acids
and ꢀ-hydroxyamino acids, both of which are reminiscent of the
thiomycin class of peptides,9 which are synthesized ribosomally
and become mature after a series of post-translational modifica-
tions.10 In contrast, the presence of several D-amino acid residues
in yaku’amides suggests that they are nonribosomal. The N-terminal
and C-terminal blocking groups of yaku’amides are both unprec-
edented as components of natural products.
The absolute configurations of the component amino acids were
determined by the Marfey’s analysis.4 The standard amino acids
were assigned as D-Ala, one L-Val, two D-Val, and D-allo-Ile by
comparing retention times with those of standards. 2S isomers of
OHVal and CTA as well as (2S,3S)- and (2R,3S)-isomers of OHIle
were synthesized as reported in the literature.5 They were derivatized
by L-FDAA and D-FDAA and used as standards for the relevant
isomers and the corresponding enantiomers, respectively.6 The presence
of one L-OHVal, one D-OHVal, (2S,3R)-OHIle, and 2S-CTA was
disclosed by comparing the retention times. In order to establish the
positional assignments of OHVal and Val residues, 1 was subjected
to partial acidic hydrolysis. Yaku’amide A (1) was hydrolyzed with
70% AcOH at 110 °C for 4 days, and the products were separated by
ODS-HPLC to give 3-5, whose sequences were assigned by a
combination of the ESIMS and Marfey’s analysis (Figure 3). The
Marfey’s analysis of 3 and 4 showed that OHVal2 and Val1 were
both in the D-form. The configuration of Val2 was determined to be
L by dansylation of 5 followed by acidic hydrolysis and chiral HPLC
analysis.7 By a process of elimination, the configurations of Val1,
OHVal1, and Val3 were determined as D, L, and D, respectively. The
absolute configuration at C-4 of NTA was left undefined.
Acknowledgment. We thank the officers and crew of T/S
Toyoshio-maru of Hiroshima University for their assistance in
collecting the sponge specimens. We also thank Professor Hidenori
Watanabe and Dr. Naoki Mori for the gift of 3-ethyl-3-methyl
acrylic acid methyl ester.
Supporting Information Available: Experimental procedures,
references, and NMR spectra for yaku’amides A and B (1 and 2) and
noncommercial amino acids synthesized in this study. This material is
available free of charge via the Internet at http://pubs.acs.org.
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