Melonoside A: An ω-Glycosylated Fatty Acid Amide from the Far Eastern Marine Sponge Melonanchora kobjakovae

Article abstract of DOI:10.1021/acs.orglett.6b01678

Melonoside A (1), the first representative of a new class of ω-glycosylated fatty acid amides, was isolated from the Far Eastern marine sponge Melonanchora kobjakovae. The structure of 1, including absolute configuration, was established using detailed analysis of 1D and 2D NMR, CD, and mass spectra as well as chemical transformations. Compound 1 induces autophagy of human cisplatin-resistant germinal tumor cells NCCIT-R.

Full text of DOI:10.1021/acs.orglett.6b01678

Letter
pubs.acs.org/OrgLett
Melonoside A: An ω‑Glycosylated Fatty Acid Amide from the Far
Eastern Marine Sponge Melonanchora kobjakovae
†
,†
†
†
Alla G. Guzii, Tatyana N. Makarieva,* Vladimir A. Denisenko, Pavel S. Dmitrenok,
†
†,‡
‡
†,§
Aleksandra S. Kuzmich, Sergey A. Dyshlovoy, Gunhild von Amsberg, Vladimir B. Krasokhin,
and Valentin A. Stonik†
†G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostoku 690022,
Russia
‡
Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus
Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
*
S Supporting Information
ABSTRACT: Melonoside A (1), the first representative of a new class of ω-glycosylated fatty acid amides, was isolated from the
Far Eastern marine sponge Melonanchora kobjakovae. The structure of 1, including absolute configuration, was established using
detailed analysis of 1D and 2D NMR, CD, and mass spectra as well as chemical transformations. Compound 1 induces autophagy
of human cisplatin-resistant germinal tumor cells NCCIT-R.
arine organisms have been recognized as a rich source of
The molecular formula of 1 was established as C H NO on
43 67 12
−
M
diverse bioactive metabolites with unusual chemical
the basis of NMR and HRESIMS data (m/z 788.4590 [M − H] ,
1
structures. Among them, glycolipids are emerging as an
important class of structurally diverse sponge metabolites that
calcd 788.4591 C H NO ).
43
66
12
In the negative MS/MS spectrum of the parent ion at m/z 788
2
,3
−
frequently have interesting biological activities.
A few
[M − H] , a fragment ion of m/z 612 was observed,
representatives of glycosylated fatty acid amides, isolated from
corresponding to a loss of 176 mass units. The unit was
identified as glucuronic acid after methanolysis of 1 followed by
GLC of the obtained peracetylated methyl glycoside mixture in
comparison to standard samples.
4
,5
marine organisms, have been previously described, including
12,13
6
−11
compounds from sponges,
a marine sediment bacterium,
1
4
and a fungus. The corresponding secondary metabolites exhibit
1
0,11
12
cytotoxic
IL-6 to its receptor.
In continuation of our search for bioactive natural products
and antifungal activities and inhibit the binding of
NMR data (CD OD, Table 1) of 1 supported the presence of a
3
6,7
glucuronic acid unit attached by a β-glycoside bond (anomeric
center δ 105.1; δ 4.27 d, J = 8.0 Hz) to a long hydrocarbon
C
H
15,16
from the Far Eastern marine sponges,
herein, we report the
chain with two disubstituted double bonds (δ 5.30, 5.38; δ
H
C
isolation, structure elucidation, and bioactivity of a glycosylated
fatty acid amide, melonoside A (1), from the marine sponge
Melonanchora kobjakovae (Figure 1). Melonoside A possesses a
new skeleton system differing from other known lipid derivatives
in the structure of the lipid chain (for a list of some previously
reported glycosylated fatty acid amides, see the Supporting
Information (SI)). The compound contains the 2-methoxy-28-
hydroxy fatty acid moiety with two ketone groups, two double
bonds, and glucuronic acid and tyramine termini. Previously,
metabolites from the M. kobjakovae were not studied, except for
1
29.8, 132.5; δ 5.35, 5.36; δ 130.5, 132.0), two ketone groups
H C
(
δ 214.1 and 214.8), flanked by four CH groups (δ 2.43 (6H),
C 2 H
2
.46; δ 43.94, 43.96, 44.0, 44.1), and one OMe group (δ 3.27;
C H
δ 59.1). In addition, a para-disubstituted benzene ring (δ 6.69
C
H
(
2H) and δ 116.9; δ 7.03 (2H) and δ 131.4) connected with a
C H C
pair of mutually coupled methylene groups (δ 2.72 (2H) and
H
3
.41(2H)) revealed a tyramine unit.
Substructures a−c were established by COSY, HSQC, and
HMBC experiments (Figure 2). Substructure a contains a
1
13
17
tyramine unit attached to the C-1 by an amide bond. H− C
fatty acid composition.
correlation between H -1′ (δ 3.41) and C-1 (δ 175.8)
2
H
C
The EtOH extract of M. kobjakovae (dry weight 5.3 g) was
concentrated and partitioned between aqueous EtOH and n-
hexane. The EtOH-soluble materials were separated by ODS
flash chromatography, further by Sephadex LH-20 and reversed-
phase HPLC to give a difficult to separate mixture of closely
related compounds and individual 1, being one of the main
constituents of the fraction, as a colorless amorphous solid (6.3
confirmed the attachment of the tyramine residue to C-1. HMBC
cross-peak OCH /C-2 confirmed the placement of the O-methyl
ether unit at C-2. Location of the double bond at C-5/C-6 was
assigned through COSY experiments and HMBC correlations.
3
Received: June 10, 2016
mg, 0.11% of dry weight; [α] −37 (c 0.18, EtOH).
D
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b01678
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Figure 1. Structure of melonoside A (1).
1
13
Table 1. H (700 MHz) and C (175 MHz) NMR Data of Melonoside A (1)
a
a
position
δC
δH mult (J in Hz)
COSY
HMBC
1, 2′, 3′
1′, 4′, 8′
position
δC
δH mult (J in Hz)
COSY
12, 14
HMBC
c
1
2
3
4
5
6
′
′
′
42.3
3.41, t (7.3)
2.72, m
2′
1′
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28a
28b
1″
2″
3″
4″
5″
25.3
30.6
30.6
25.1
44.0
1.52, m
1.27, m
1.27, m
1.52, m
2.43, m
11, 12, 14
12, 13, 15
14, 16, 17
15, 17, 18
15, 16, 18
36.3
13
131.6
131.4
116.9
157.6
16
c
c
′, 8′
′, 7′
′
7.03, d (8.5)
6.69, d (8.5)
5′, 7′
4′, 8′
2′, 5′, 6′, 7′
3′, 4′, 6′, 8′
15, 17
16
214.1
44.1
d
NH
7.89
1′
2.46, t (7.3)
2.27, m
20
18, 20, 21
18, 19, 21, 22
19, 20
1
175.8
83.5
59.1
34.7
23.3
19, 21
20, 22
21, 23
22, 24
23
2
3.54, dd (4.4, 7.5)
3.27, s
3a, 3b
1, 3, 4, −OCH3
2
129.8
132.5
5.30, m
OCH3
5.38, m
23, 24
c
3
3
4
4
5
6
7
8
9
1
1
1
a
1.60, m
4a, 4b
4a, 4b
3a, 3b, 4b, 5
2, 3, 5, 6
4a, 4b
7
1, 2, 4, 5
1, 4, 5
28.7
2.04, m
21, 22, 24
22
c
b
a
1.66, m
30.8
1.34, m
24.3
2.03, m
2, 3, 5, 6
30.6
27.6
31.4
71.6
1.27, m
b
2.11, m
130.5
132.0
28.5
3a, 3b, 4a, 5
5.35, m
1.38, m
27
27
3, 4
1.61, m
26, 28a, 28b
27, 28b
27, 28a
2″
1″, 3″
2″, 4″
3″, 5″
4″
25, 27, 28
26, 27, 1″
26, 27, 1″
28, 3″
1″, 3″
5.36, m
4, 7
3.52, dt (6.9, 9.3)
3.91, dt (6.9, 9.3)
4.27, d (8.0)
3.21, t (8.0)
3.38, m
2.03, m
6, 8
8, 9
c
30.9
1.32, m
7, 9
6, 9, 10
7, 8, 10
7, 11
105.1
75.5
78.4
74.2
75.6
c
25.2
1.54, m
8, 10
9
c
0
1
2
43.96
2.43, m
4″
214.8
3.47, t (8.7)
3.65, m
c
43.94
2.43, m
13
11, 13, 14
b
6″
nd
a
13
b
c
d
C NMR assignments supported by HSQC and HMBC data. Not detected. Signals may be interchangeable. Exchanged with a deuterium oxide.
methanol at −70 °C. This reaction yielded 3, which was analyzed−
by HRESIMS. The peak of the deprotonated molecule [M − H]
was observed at m/z 353.1456 (calcd for 3 (C H O ),
1
4
26 10
3
53.1453), which permitted determination of the position of one
double bond at C-21.
The exact location of the ketone group and double bond
positions was achieved by the following chemical trans-
formations (Figure 3). Compound 1 was transformed into
11,18-diaminomelonoside A (2) by reductive amination with
sodium cyanoborohydride in ammonium acetate solution. Then,
Figure 2. Partial structures of 1 with selected COSY, HMBC, and
NOESY correlations.
Substructure b consists of one keto group separated from the
disubstituted double bond by two CH groups.
2
The Z-geometry of double bonds in both substructures was
inferred from the carbon chemical shifts of the adjacent allylic
carbons, which have chemical shifts at lower values than 29
1
8,19
ppm
and correlation between H-20 and H-23 in the NOESY
spectra (Table 1).
Substructure c contains a glucuronic acid residue attached by a
β-O-glycosidic bond to C-28. Characteristic NMR chemical
shifts of the anomeric H-1″ (δH 4.27) and C-28 (δ 71.6),
C
together with HMBC correlations between the two signals,
confirmed the location of glucuronic acid at the end position.
However, the assembly of these substructures was difficult due to
several NMR overlapping methylene groups.
Initial attempts to determine the position of ketone groups in 1
by either MS analysis of Baeyer−Villiger oxidation products or
reductive ozonolysis of 1 and 2 were unsuccessful. Unambiguous
data were obtained when 1 was subjected to ozonolysis in
Figure 3. Derivatization and ozonolysis of 1.
B
DOI: 10.1021/acs.orglett.6b01678
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
amino groups in 2 were acetylated with 20% Ac O in MeOH in
expression of autophagy-related proteins LC3B-II and
SQSTM1/p62 at a concentration of 10 μM (SI).
2
the presence of catalytic amounts of DMAP. N,N′-Diacetamide
of melonoside A (4) was subjected to ozonolysis followed by
reductive workup and acetylation. These reactions yielded 5,
which was confirmed by HRESIMS and GC−MS (Figure 4).
In summary, 1 represents a new class of glycolipid derivatives
with an unprecedented C43 skeleton consisting of glucuronic
acid, multifunctionalized fatty acids, and tyramine. The most
unique structural feature of 1 is the presence of (2S,5Z,21Z)-28-
hydroxy-2-methoxy-11,18-dioxooctacosa-5,21-dienoic acid,
which was never described before from natural sources. The
novel structure of 1, combined with its ability to induce
autophagy, should make it a useful chemical biology tool.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Figure 4. EIMS fragmentations (70 eV) of 5.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b01678.
The HRESIMS of 5 exhibited a peak of the protonated molecule
Experimental details, copies of 1D and 2D NMR, CD
spectra of 1, and bioassay results (PDF)
+
at 457.3278 [M + H] (calcd for C H N O , 457.3272),
2
4
45
2
6
confirming the location of the double bonds at C-5 and C-21.
The position of carbonyl groups at C-11 and C-18 was
determined on the basis of mass spectral fragmentation of 5 by
EIMS (Figure 4).
AUTHOR INFORMATION
Corresponding Author
■
Determination of the absolute configuration of the sugar unit
was carried out after acid hydrolysis of 1 with 2 M TFA followed
by preparation of acetylated (R)-octyl glycosides. GC analysis of
these compounds and comparison with authentic samples
revealed the D-configuration of the glucuronic acid.
*E-mail: makarieva@piboc.dvo.ru.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was a part of the project (Grant No. 20140513)
funded by the Ministry of Oceans and Fisheries, Korea.
■
Absolute configuration of the C-2 asymmetric center in 1 was
assigned by analysis of the CD spectra of the aglycone
melonoside A (6), model compounds 7a, 7b, 8a, and 8b, and
literature data for CD spectra of 2-methoxy fatty acids (Figure
1
8,20,21
DEDICATION
In memory of Vladimir B. Krasokhin.
5).
CD data for (R)-2-methoxypropionic acid (8a) and
■
§
REFERENCES
■
(
1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep,
M. R. Nat. Prod. Rep. 2015, 32, 116−211 and earlier reviews.
2) Fattorusso, E.; Mangoni, A. Marine Glycolipids. In Progress in the
(
Chemistry of Organic Natural Products; Hertz, W., Kirby, G. W., Moore,
R. E., Steiglich, W., Tamm, Ch., Eds.; Springer, 1997; pp 215−301.
(
3) Fedorov, S. N.; Makarieva, T. N.; Guzii, A. G.; Shubina, L. K.;
Figure 5. Structures of tyramine derivative 6 and model compounds 7a,
b, 8a, and 8b.
Kwak, J. Y.; Stonik, V. A. Lipids 2009, 44, 777−785.
4) Kalinin, V. I.; Ivanchina, N. V.; Krasokhin, V. B.; Makarieva, T. N.;
Stonik, V. A. Mar. Drugs 2012, 10, 1671−1710.
7
(
(
5) Dembitsky, V. M. Lipids 2005, 40, 641−660.
20,21
(R)-2-methoxy fatty acid methyl esters
demonstated a strong
(6) Sata, N.; Asai, N.; Matsunaga, S.; Fusetani, N. Tetrahedron 1994,
5
(
0, 1105−1110.
negative Cotton effect at 210−212 nm and a weaker positive one
at 240 nm. We attempted to hydrolyze the amide bond in 6 and
to isolate a fatty acid moiety, but these efforts were unsuccessful.
Model compounds 7b and 8b with tyramine residue were
synthesized (SI). Tyramine derivatives (7b, 8b) have opposite
Cotton effects when compared with acids (7a, 8a). These data
showed that the sign of a Cotton effect is independent of the
length of the hydrocarbon chain in 2-methoxy acids but is highly
sensitive to amidation by tyramine.
7) Goobes, R.; Rudi, A.; Kashman, Y. Tetrahedron 1996, 52, 7921−
7
(
928.
8) Warabi, K.; Zimmerman, W. T.; Shen, J.; Gauthier, A.; Robertson,
M.; Finlay, B. B.; van Soest, R.; Andersen, R. J. Can. J. Chem. 2004, 82,
02−112.
9) Peddie, V.; Takada, K.; Okuda, S.; Ise, Y.; Morii, Y.; Yamawaki, N.;
1
(
Takatani, T.; Arakawa, O.; Okada, S.; Matsunaga, S. J. Nat. Prod. 2015,
78, 2808−2813.
(10) Ohta, S.; Ohta, E.; Ikegami, S. J. Org. Chem. 1997, 62, 6452−6453.
(11) Fujita, M.; Nakao, Y.; Matsunaga, S.; Seiki, M.; Itoh, Y.; van Soest,
R. W. M.; Fusetani, N. Tetrahedron 2001, 57, 1229−1234.
The CD spectrum of 6 displayed a negative Cotton effect at
λmax = 200 nm (Δε = −2.53) and 220 nm (Δε = −2.13), similar
to the CD spectrum of 7b, indicating the S-configuration of C-2
in 1 (SI). The proposed configuration is opposite in comparison
to that reported for naturally occurring 2-methoxy fatty acids,
(
12) Tareq, F. S.; Kim, J. H.; Lee, M. A.; Lee, H. S.; Lee, Y. J.; Lee, J. S.;
Shin, H. J. Org. Lett. 2012, 14, 1464−1467.
13) Tareq, F. S.; Lee, H. S.; Lee, Y. J.; Lee, J. S.; Shin, H. J. Lipids 2015,
0, 513−519.
14) Wright, A. D.; Osterhage, C.; Konig, G. M. Org. Biomol. Chem.
003, 1, 507−510.
15) Guzii, A. G.; Makarieva, T. N.; Denisenko, V. A.; Dmitrenok, P. S.;
(
5
(
2
18,20−22
which are known to possess the R-configuration.
So far,
2
S-hydroxylated fatty acids have been detected only in some
23−26
bacterial lipopolysaccharides.
(
Melonoside A (1) induces autophagy of human cisplatin-
Kuzmich, A. S.; Dyshlovoy, S. A.; Krasokhin, V. B.; Stonik, V. A. Org.
resistant germinal tumor cells NCCIT-R, decreasing the
Lett. 2010, 12, 4292−4295.
C
DOI: 10.1021/acs.orglett.6b01678
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(
16) Makarieva, T. N.; Ogurtsova, E. K.; Denisenko, V. A.; Dmitrenok,
P. S.; Tabakmakher, K. M.; Guzii, A. G.; Pislyagin, E. A.; Es’kov, A. A.;
Kozhemyako, V. B.; Aminin, D. L.; Wang, Y.-M.; Stonik, V. A. Org. Lett.
2
(
2
(
014, 16, 4292−4295.
17) Rodkina, S. A.; Imbs, A. B.; Krasokhin, V. B. Russ. J. Mar. Biol.
008, 34, 384−390.
18) Carballeira, N. M.; Montano, N.; Amador, L. A.; Rodriguez, A. D.;
Golovko, M. Y.; Golovko, S. A.; Reguera, R. M.; Alvarez-Velilla, R.;
Balana-Fouce, R. Lipids 2016, 51, 245−256.
(
19) Mudianta, I. W.; Skinner-Adams, T.; Andrews, K. T.; Davis, R. A.;
Hadi, T. A.; Hayes, P. Y.; Garson, M. J. J. Nat. Prod. 2012, 75, 2132−
143.
20) Ayanoglu, E.; Popov, S.; Kornprobst, J. M.; Aboud-Bichara, A.;
Djerassi, C. Lipids 1983, 18, 830−836.
21) Ayanoglu, E.; Kornprobst, J. M.; Aboud-Bichara, A.; Djerassi, C.
2
(
(
Tetrahedron Lett. 1983, 24, 1111−1114.
(
(
22) Carballeira, N. M. Prog. Lipid Res. 2002, 41, 437−456.
23) Gibbons, H. S.; Lin, S.; Cotter, R. J.; Raetz, C. R. H. J. Biol. Chem.
2
(
000, 275, 32940−32949.
24) Hancock, I. C.; Humphreys, G. O.; Meadow, P. M. Biochim.
Biophys. Acta, Lipids Lipid Metab. 1970, 202, 389−391.
(
(
25) Rietschel, E. T. Eur. J. Biochem. 1976, 64, 423−428.
26) Bryn, K.; Rietschel, E. T. Eur. J. Biochem. 1978, 86, 311−315.
D
DOI: 10.1021/acs.orglett.6b01678
Org. Lett. XXXX, XXX, XXX−XXX