Nazumazoles A-C, cyclic pentapeptides dimerized through a disulfide bond from the marine sponge theonella swinhoei

Article abstract of DOI:10.1021/acs.orglett.5b01020

A mixture of nazumazoles A-C (1-3) was purified from the extract of the marine sponge Theonella swinhoei. The mixture was eluted as an extraordinarily broad peak in the reversed-phase HPLC. The structures of nazumazoles were determined by interpretation o

Full text of DOI:10.1021/acs.orglett.5b01020

Letter
pubs.acs.org/OrgLett
Nazumazoles A−C, Cyclic Pentapeptides Dimerized through a
Disulfide Bond from the Marine Sponge Theonella swinhoei
Kazuya Fukuhara, Kentaro Takada,* Shigeru Okada, 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
S
* Supporting Information
ABSTRACT: A mixture of nazumazoles A−C (1−3) was purified from the
extract of the marine sponge Theonella swinhoei. The mixture was eluted as
an extraordinarily broad peak in the reversed-phase HPLC. The structures of
nazumazoles were determined by interpretation of the NMR data and
chemical degradations. Nazumazoles contain one residue each of alanine-
derived oxazole and α-keto-β-amino acid residue. Nazumazoles exhibited
cytotoxicity against P388 cells.
arine sponges of the genus Theonella have been shown to
structure elucidation, and biological activity of the constituents of
Mbe a rich source of structurally unique and biologically the peak, nazumazoles A−C (1−3).
active secondary metabolites.1,2 From T. swinhoei with a yellow
interior (T. swinhoei Y) collected at Hachijo Island we have
isolated more than 40 bioactive polyketides and modified
peptides such as onnamides,3 polytheonamides,4 aurantosides,5
and orbiculamides.6 Recently, a symbiotic filamentous bacte-
rium, Candidatus Entotheonella sp., was identified as the sole
producer of a variety of natural products from this sponge.7 We
have analyzed the extracts of marine invertebrates by LCMS and
found that the extract of T. swinhoei Y contained unidentified
metabolites, among which was an extraordinarily broad peak in
ODS-HPLC (Figure 1, Figure S1, Supporting Information). We
The MeOH extract of the sponge was partitioned between
H2O and Et2O, and the organic layer was further partitioned
between 90% MeOH and n-hexane. The H2O and 90% MeOH
fractions were combined and fractionated by ODS flash chro-
matography, gel permeation chromatography, gel permeation
HPLC, and ODS-HPLC to afford nazumazoles A−C (1−3) as
an inseparable mixture.
The HRESIMS of the mixture of nazumazoles A−C showed
the molecular formulas of nazumazoles A, B, and C to be
C50H68N14O16S2, C51H70N14O16S2, and C52H72N14O16S2, respec-
tively. Interestingly, 1−3 gave different molecular ion peaks in
the ESIMS when the solvent of the sample and carrier liquid was
altered from MeCN to MeOH or EtOH. For example, the
protonated ion peaks of nazumazole C (3) were observed at m/z
1213, 1277, and 1305, respectively, when using MeCN, MeOH,
and EtOH, indicating that two molecules of MeOH or EtOH
were incorporated into the molecular ion, reminiscent of the
FABMS data of cyclotheonamide A, in which alcohol adducts
were formed at the α-keto-β-amino acid residue.9
1H and 13C NMR data (Table 1) suggested the peptidic nature
of the molecules with several amide 1H signals and carbonyl 13C
signals. Interpretation of the COSY, HSQC, and TOCSY spectra
revealed the presence of Cys, 2,3-diaminopropionic acid (Dpr)
and 4-methylproline (MePro) residues. The α-amino group of
the Dpr residue was formylated as indicated by the HMBC
correlations between H-14 (δ 4.76) and the formyl 13C signals
(δ 160.7) and between the formyl 1H (δ 7.99) and C-14 (δ 50.5)
Figure 1. LCMS charts of the crude fractionof T. swinhoei Y: (a) UV
chromatogram (integration of 210−400 nm) with assignments of some
known compounds3,5,6,8 and (b) MS chromatogram of a sum of the
responses of the ions at m/z 1185, 1199, and 1213.
successfully purified the peak, which exhibited cytotoxicity
Received: April 9, 2015
against P388 murine leukemia cells. We describe the isolation,
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b01020
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. 1H and 13C NMR Data for the Mixture of
Nazumazoles A (1)−C (3) in DMSO-d6
a
a
residue
Knv
position
δC , type
δH (J, Hz)
b
1
2
3
4
163.5, C
197.2, C
54.6, CH
31.4, CH2
4.82, m
1.94, m
1.56, m
1.48, m
1.40, m
0.90, m
8.59, d (6.0)
5
19.1, CH2
13.3, CH3
6
3-NH
Kle
1
2
3
4
163.5, C
197.3, C
53.3, CH
38.0, CH2
4.87, m
1.76, m
1.46, m
1.78, m
0.90, m
0.93, m
8.56, m
signals (Figure 2). There were 1H NMR spin systems attributable
to leucine and norvaline (Nva) residues, whose nitrogen
substituted methine 1H signals were correlated to a 13C signal at
δ 197.3 (197.2), suggesting the presence of α-ketoleucine (Kle)
and α-ketonorvaline (Knv) residues.10 The presence of a
disubstituted oxazole ring was deduced from the HMBC
5
24.8, CH
21.0, CH3
22.9, CH3
6
5-Me
3-NH
1
data, in which a singlet aromatic H signal (δ 8.54) directly
MePro
7
8
9
172.4, C
59.5, CH
37.1, CH2
coupled to a 13C signal at δ 142.4 by 213 Hz was correlated to 13C
signals at δ 134.5 and δ163.5.9 Further HMBC correlations from
the H-23 (δ 5.01) and H-24 (δ 1.54) signals to the C-22
(δ 163.4) signal suggested that the oxazole ring was a part of an
alanine-derived oxazole (AlaOx). At this moment, two amide
carbonyl 13C (δ 159.6 and 163.5) signals were left unassigned,
one of which was in the α-keto-β-amino acid residue and the
other attached to the AlaOx residue as inferred from a process of
elimination.
4.44, m
2.42, m
1.29, m
2.21, m
3.90, m
2.94, m
0.99, d(6.4)
10
11
33.2, CH
53.9, CH2
12
13
14
15
15.8, CH3
167.0, C
Dpr
50.5, CH
38.7, CH3
4.76, m
3.68, m
3.45, m
7.98, m
7.48, m
7.99, s
The amino acid sequences of 1−3 were determined by HMBC
and NOESY data (Figure 2). HMBC correlations between
3-NH/C-7, H-11α/C-13, H-14/C-13, H-15β/C-16, H-18β/
C-16, and H-17/C-19 suggested the sequence of -Knv (Kle)-
MePro-isoDpr-Cys-. The amide 1H signal of the Cys residue was
correlated to a 13C signal at δ 159.6, and the nitrogen-substituted
14-NH
15-NH
CHO
16
160.7, CH
170.6, C
Cys
methine 1H signal of the AlaOx residue was correlated to a 13
C
17
52.7, CH
39.1, CH2
4.58, m
3.14, m
2.71, m
7.81, m
signal at δ 163.5 in the HMBC spectrum. Therefore, the Cys
residue was shown to be connected to the AlaOx residue via
a carbonyl carbon and the AlaOx residue was linked to the
Knv/Kle residue.
18
17-NH
19
AlaOx
159.6, C
134.5, C
Because the sizes of the resulting pentapeptide fragments were
about half of the molecular weights of nazumazoles A−C, we
considered that these peptides were either cyclic decapeptides or
cyclic pentapeptides dimerized through a disulfide bond. In order
to clarify this issue, the mixture of 1−3 was reduced consecutively
with NaBH4 and DTT and subjected to alkylation with
4-vinylpyridine to afford a mixture of 4a and 4b and that of 5a
and 5b, whose HRESIMS suggested that they were reduced
cyclic pentapeptides with an S-pyridylethyl group.11 In 4a and 4b,
the α-carbon of Knv residue was reduced to a secondary alcohol
(rKnv residue), whereas in 5a and 5b the pertinent residue was
replaced by the reduced Kle (rKle) residue. Even though the
structures of 4a, 4b, 5a, and 5b were confirmed by the 2D NMR
data, each pair of diastereomers was inseparable by HPLC.
Therefore, 1−3 were cyclic pentapeptides dimerized through a
disulfide bond.
20
21
142.4, CH
8.54, s
b
22
163.5,
C
23
43.7, CH
15.7, CH3
5.01, m
24
1.54, d(6.9)
8.95, m
23-NH
a
Recorded at 150 MHz for 13C and 600 MHz for 1H at 40 °C.
Chemical shifts of C-1 and C-22 overlapped each other.
b
We then examined the mode of dimerization by analyzing the
NaBH4 reduction products of the mixture of nazumazoles A−C,
which gave three sharp HPLC peaks (Figure S3, Supporting
Information, 6−8). The ESIMS and 2D NMR data demon-
strated that the smallest congener (peak A, Figure S3) contained
Figure 2. Key COSY, TOCSY, HMBC, and NOESY correlations in 1−3.
B
DOI: 10.1021/acs.orglett.5b01020
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(4) Hamada, T.; Matsunaga, S.; Yano, G.; Fusetani, N. J. Am. Chem. Soc.
2005, 127, 110−118.
two rKnv residues, the second congener (peak B, Figure S3)
contained one each of rKnv and rKle residue, and the largest
congener (peak C, Figure S3) contained two rKle residues.12
(5) Matsunaga, S.; Fusetani, N.; Kato, Y.; Hirota, H. J. Am. Chem. Soc.
1991, 113, 9690−9692.
(6) Fusetani, N.; Sugawara, T.; Matsunaga, S.; Hirota, H. J. Am. Chem.
Soc. 1991, 113, 7811−7812.
(7) Wilson, M. C.; Mori, T.; Ruckert, C.; Uria, A. R.; Helf, M. J.;
Takada, K.; Gernert, C.; Steffens, U. A. E.; Heycke, N.; Schmitt, S.;
Rinke, C.; Helfrich, E. J. N.; Brachmann, A. O.; Gurgui, C.; Wakimoto,
T.; Kracht, M.; Crusemann, M.; Hentschel, U.; Abe, I.; Matsunaga, S.;
Kalinowski, J.; Takeyama, H.; Piel, J. Nature 2014, 506, 58−62.
(8) (a) Kobayashi, J.; Sato, M.; Ishibashi, M.; Shigemori, H.;
Nakamura, T.; Ohizumi, Y. J. Chem. Soc., Perkin. Trans 1 1991, 10,
2609−2611. (b) Itagaki, F.; Shigemori, H.; Ishibashi, M.; Nakamura, T.;
Sasaki, T.; Kobayashi, J. J. Org. Chem. 1992, 57, 5540−5542.
(c) Kobayashi, J.; Itagaki, F.; Shigemori, I.; Takao, T.; Shimonishi, Y.
Tetrahedron 1995, 51, 2525−2532.
(9) Fusetani, N.; Matsunaga, S.; Matsumoto, H.; Takebayashi, Y. J. Am.
Chem. Soc. 1990, 112, 7053−7054.
(10) The presence of Kle and Knv residues was confirmed by the
Marfey’s analysis of the hydrolysate, which gave peaks for of DAA-
derivatized rKnv (m/z 400) and DAA-derivatized rKle (m/z 414) (cf.
the experimental section in the Supporting Information). This was
confirmed by the HMBC data of 4a, 4b, 5a, and 5b.
(11) The reduction product of the mixture of 1−3 with DTT gave a
broad HPLC peak in ODS-HPLC [m/z 594 and 608 (M + H)+], and the
two constituents were not separable.
(12) Each peak in Figure S2 (Supporting Information) contains four
diastereomers which were not separable by ODS-HPLC. Each peak gave
two sets of NMR signals depending on the configuration of the newly
generated secondary alcohol.
The absolute configurations of the amino acid residues in
1−3 were determined by Marfey’s method.13 Ala, Cys, Dpr, and
MePro were liberated by standard acid hydrolysis. Knv and Kle
residues were analyzed as Nva and Leu residues, respectively, by
oxidation with H2O2. Two diastereomers of 4-methylproline
were synthesized from trans-4-hydroxy-L-proline as described in
the literature and used as standards.14 Marfey’s analysis with
detection by ESIMS revealed the presence of L-Ala, D-Cys, L-Dpr,
cis-4-methyl-L-proline, L-Leu, and L-Nva (Figures S30−S32,
Supporting Information).
The mixture of nazumazoles A (1)−C (3) exhibited
cytotoxicity against P388 murine leukemia cells with an IC50
value of 0.83 μM. The cytotoxicity was diminished when the
ketone in 1−3 was reduced, whereas the cytotoxicity was lost by
further reduction and alkylation of the thiol group (Table S8,
Supporting Information).
(13) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
(14) Murphy, A. C.; Mitova, M. I.; Blunt, J. W.; Munro, M. H. G. J. Nat.
Prod. 2008, 71, 806−809.
(15) Ueda, M. Chem. Lett. 2012, 41, 658−666.
ASSOCIATED CONTENT
■
S
* Supporting Information
Description of experimental procedure and spectroscopic data of
1−8. The Supporting Information is available free of charge on
the ACS Publications website at DOI: 10.1021/acs.orglett.5b01020.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: atakada@mail.ecc.u-tokyo.ac.jp.
*E-mail: assmats@mail.ecc.u-tokyo.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partly supported by a Grant-in-Aid for Scientific
Research on Innovative Areas “Chemical Biology of Natural
Products”15 and JSPS KAKENHI Grant Nos. 25252037,
25712024, and 25660163 from The Ministry of Education,
Culture, Sports, Science and Technology, Japan.
REFERENCES
■
(1) Bewley, C. A.; Faulkner, D. J. Angew. Chem., Int. Ed. 1988, 37,
2162−2178.
(2) Winder, P. L.; Pomponi, S. A.; Wright, A. E. Mar. Drugs. 2011, 9,
2643−2682.
(3) (a) Sakemi, S.; Ichiba, T.; Kohmoto, S.; Saucy, T.; Higa, T. J. Am.
Chem. Soc. 1988, 1 (10), 4851−4853. (b) Matsunaga, S.; Fusetani, N.;
Nakao, Y. Tetrahedron 1992, 48, 8369−8376.
C
DOI: 10.1021/acs.orglett.5b01020
Org. Lett. XXXX, XXX, XXX−XXX