Lipodiscamides A-C, new cytotoxic lipopeptides from discodermia kiiensis

Article abstract of DOI:10.1021/ol501271v

Lipodiscamides A-C, three new lipodepsipeptides, were characterized from the marine sponge Discodermia kiiensis. These structurally rare cyclic lipodepsipeptides were found to possess an unprecedented dilactone macrocycle and, thus, represent a new family of lipopeptides. They are the only lipopeptides bearing 4S-hydroxy-trans-2-enoate, and noncanonical amino acids, l-3-ureidoalanine (Uda), E-dehydronorvaline (Denor), and d-citrulline (Cit). MTT assays against P388 and HeLa cells revealed the moderate cytotoxicity of all three compounds.

Full text of DOI:10.1021/ol501271v

Letter
pubs.acs.org/OrgLett
Lipodiscamides A−C, New Cytotoxic Lipopeptides from Discodermia
kiiensis
Karen Co Tan, Toshiyuki Wakimoto,* and Ikuro Abe*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
S
* Supporting Information
ABSTRACT: Lipodiscamides A−C, three new lipodepsipep-
tides, were characterized from the marine sponge Discodermia
kiiensis. These structurally rare cyclic lipodepsipeptides were
found to possess an unprecedented dilactone macrocycle and,
thus, represent a new family of lipopeptides. They are the only
lipopeptides bearing 4S-hydroxy-trans-2-enoate, and non-
canonical amino acids, L-3-ureidoalanine (Uda), E-dehydro-
norvaline (Denor), and D-citrulline (Cit). MTT assays against
P388 and HeLa cells revealed the moderate cytotoxicity of all
three compounds.
ipopeptides are a class of secondary metabolite hybrids
consisting of a hydrophobic tail attached to a short linear
L
or cyclic peptide. Several bioactive lipopeptides exhibiting
cytotoxic, antimicrobial, immunosuppressant, and surfactant
properties are reported to be produced by various bacteria, such
as Streptomyces, Pseudomonas, and Bacillus,1−4 as well as fungi,
including Fusarium, Aspergillus, and Pochonia.5−7 In contrast,
lipopeptides seem to be a rather rare family among the natural
products derived from the marine sponges, even though
sponges are prolific sources of biologically active metabolites,
such as polyketides, peptides, and terpenoids. In the course of
our search for bioactive secondary metabolites from sponges of
the genus Discodermia, we have reported the isolation of
cytotoxic cyclopeptides, calyxamides from D. calyx8, and
cyclolithistide from D. japonica.9 Furthermore, peptidic
metabolites, such as discodermins and discokiolides, have
been isolated from another species, D. kiiensis.10−12 Herein, we
report a new family of cytotoxic and structurally unique cyclic
lipodepsipeptides characterized from D. kiiensis.
The frozen sponge (1 kg), collected off Shikine-jima Island,
was extracted with EtOH. The EtOH extract was then
partitioned repeatedly between Et2O and H2O, and the organic
extract was repartitioned between n-hexane and 90% aqueous
MeOH. Then, the aqueous MeOH extract was subjected to
flash ODS chromatography, to obtain the 80% aqueous MeOH
fraction. This fraction was purified by reversed-phase HPLC on
a π-NAP column, using an isocratic mode with 50 mM
NH4OAc in 85% MeOH, to afford 4.8, 1.2, and 0.4 mg of 1−3,
respectively (Figure 1).
The major compound, lipodiscamide A, 1, showed an m/z at
966.5289 [M + Na]+ in HR-FABMS, and its molecular formula
was established as C46H73N9O12. A TLC analysis of the
compound revealed that it responds positively to 10% H2SO4
spray, generating a blue spot that turned purple upon continued
heating. When the NMR solvent was changed from CD3OD to
DMSO-d6, five exchangeable protons between 7.5 and 10.5
Figure 1. Structures of lipodiscamides A−C (1−3).
ppm appeared (Figures S1, S3), which were eventually assigned
to α-amino acids, establishing the peptidic nature of 1. The
olefinic protons between 5.3 and 6.6 ppm hinted at the
presence of a polyketide and/or an unsaturated fatty acid
moiety. In brief, the two pairs of diastereotopic methylene
Received: May 4, 2014
Published: June 6, 2014
© 2014 American Chemical Society
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Letter
protons at δH 4.12 and 4.29 (Figure S1) indicated the presence
of two glycine subunits. The two proton signals at δH 4.25 and
4.37 appeared to be the α-protons of two other amino acids,
which were subsequently inferred to be citrulline (Cit) and 3-
ureidoalanine (Uda) by COSY and HMBC correlations (Table
S1). Due to the heavily overlapped signals between 1.4 and 1.7
ppm observed with DMSO-d6, other NMR solvents and
numerous solvent mixtures were tested to achieve better
resolution within this region and to ascertain the number of
methylenes. Eventually, a 2:1 mixture of DMSO-d6/CD3CN
was found to be effective for this purpose (Figures S1−S4) and
also resolved all exchangeable protons (δH 5.31, 2H; δH 5.56,
2H; δH 5.92, 2H) of the two ureido groups. In addition to the
NMR spectral data, the presence of the ureido functionality was
confirmed by its color reaction with 0.06 M dimethylamino-
benzaldehyde under strongly acidic conditions, which gen-
erated a yellow-colored Schiff base.13 The presence of the
dehydronorvaline (Denor) residue was then established by the
COSY correlations from H-37 to H-39 and the appearance of
HMBC correlations of its amide proton with the carbonyl at C-
35 and with the two olefinic carbons C-36 and C-37 using the
2:1 DMSO-d6/CD3CN mixture (Figure S11). Its trans
geometry was deduced by the presence of a NOESY correlation
between the olefinic proton H-37 and its amide proton (Figure
S12). On the other hand, the COSY correlations from H-41 to
H-45 and the HMBC correlations of H-41 and H-42 with the
carbonyl at C-40 confirmed the presence of the 4-hydroxy-5-
The geometry about the diene from C-6 to C-9 was deduced to
be both trans according to the splitting patterns and the JHH
3
coupling constants, which could be observed with CD3OD
(Table S2). On the other hand, the double bond between H-11
and H-12 was inferred to have a cis configuration on the basis of
3
the small JHH coupling constant (10.8 Hz) observed in
DMSO-d6 (Table S3). Finally, the connections between all
subunits were corroborated by the 2D NMR spectral data
(Figure 2). Hence, the planar structure of lipodiscamide A was
determined to be 1.
In contrast, the molecular formula of lipodiscamide B, 2, was
established to be C45H71N9O12 by HR-FABMS, giving an m/z
at 952.5121 [M + Na]+. In terms of its mass, 2 appeared to
have one less CH2 unit than 1. This assumption was clearly
1
supported by its H NMR spectrum in CD3OD (Figure S14),
which was identical to that of 1, except for the integration of its
highest field methyl group CH3-16 (δH 0.90). These protons
were assigned to the terminal methyl group of the fatty acyl
chain and corresponded to three protons in 2. In contrast, the
corresponding region in 1 had an integral intensity of six
protons (CH3-16 and17). Furthermore, its 2D NMR spectral
data were superimposable with those of 1, except for the
appearance of HMQC correlations between the methylene
protons H2-14 (δH 1.31) and C-14 (δC 30.1), and H2-15 (δH
1.32) and C-15 (δC 32.1) (Table S2), providing additional
evidence that 2 differed from 1 only at the terminal portion of
the long chain acyl group. As such, the planar structure of
lipodiscamide B was established as 2.
3
methylhexa-2-enoyl moiety. The large JHH coupling constant
Finally, the molecular formula of lipodiscamide C, 3, was
determined to be C47H75N9O12 by HR-FABMS, yielding an m/
z at 980.5436 [M + Na]+. Based on this, 3 was expected to be
one CH2 unit larger than 1. In CD3OD, the downfield shifts of
H-43 (δH 4.94 in 1 to δH 5.12 in 3) and H-44 (δH 6.53 in 1 to
δH 6.57 in 3) (Figure S21) suggested that the only difference
between the two compounds exists in the 4-hydroxy-2-enoyl
subunit. As evidenced by the COSY and HMBC correlations in
DMSO-d6 (Table S3), where the least overlapping of the
signals from this moiety was found, the 4-hydroxy-5-methylhex-
2-enoyl subunit in 1 is replaced by a 4-hydroxy-5-methylhept-2-
enoate in 3. Aside from this particular difference between 1 and
3, their spectral data were unequivocally indistinguishable from
one another. Therefore, lipodiscamide C was designated as the
planar structure 3.
(15.3 Hz) between H-41 and H-42 led to the assignment of a
trans stereochemistry (Table S1). As for the C19 unsaturated
fatty acyl chain, the C-1 to C-11 and C-12 to C-17 fragments
were established by COSY correlations, and the connection
between these two fragments was confirmed by the HMBC
correlations of H-10, H-13, and H-14 with C-12 (Figure 2).
To determine the absolute stereochemistry at the α-carbons
in Cit and 3-Uda, acid hydrolysis was performed by heating
each lipopeptide in 6 M HCl at 110 °C for 24 h. This process
converted the ureido amino acids to ornithine (Orn) and 2,3-
diaminopropionic acid (Dpr),14 respectively, which were then
derivatized to the corresponding N-trifluoroacetyl/methyl
esters and analyzed by chiral-phase GC-MS. Standard amino
acids were derivatized and analyzed in the same manner.
Consequently, the results showed that Orn, and therefore Cit,
was in the D-form in all three compounds. However, 3-Uda
underwent racemization through a 5-dihydrouracil intermedi-
ate,13 as expected under the acidic reaction conditions, yielding
both the L- and D-products in a 3:2 ratio. To resolve this
problem, mild acid hydrolysis was performed with a shorter
reaction time of 6 h. This condition circumvented the acid-
induced racemization, and L-Dpr was solely detected (Figure
S27). As a result, all three compounds were determined to
possess L-3-Uda.
Next, to elucidate the absolute configurations at C-3 and C-5,
Figure 2. Key COSY and HMBC correlations of 1.
all four diastereomeric standards of 6′ were synthetically
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Letter
prepared from optically pure 4 (Scheme 1).15−17 In the case of
(S)-4, a mixture of (2S,4R)- and (2S,4S)-5 was obtained. These
Scheme 2. Synthesis of the Stereoisomers of 8 and 10
Scheme 1. Synthesis of the Stereoisomers of 6′
Scheme 3. Degradation Schemes of 1−3 To Determine the
Absolute Configurations at C-3, C-5, C-43 (and C-44)
diastereomers were separated by silica gel chromatography, and
their absolute configurations were determined by reaction with
2-methoxyphenylacetic acid (MPA) (Figure S28). On the other
hand, (R)-4 produced a mixture of the allylated lactones
(2R,4S)- and (2R,4R)-5′, which were also separated by silica gel
chromatography. Their relative configurations were then
determined by NOESY analyses (Figures S29−30). Afterward,
5/5′ were subjected to ozonolysis, oxidative workup, and
methyl esterification. These reactions yielded all possible
stereoisomers of 6′, which were analyzed by chiral-phase GC-
MS (Figure S31). In addition, because the syn-isomers were
only partially lactonized, the acyclic dimethylester products
(2S,4S)-6 and (2R,4R)-6 were also detected together with their
respective lactones. The lipopeptides were subjected to
ozonolysis at −78 °C, followed by oxidative workup, acid
hydrolysis, and methyl esterification (Scheme 3). The
derivatized natural products were subsequently analyzed by
chiral-phase GC-MS and gave a peak that coeluted with
(2R,4S)-6′ (Figure S32). Hence, the 3S,5R-configuration was
assigned to all three compounds.
Finally, to establish the absolute stereochemistry at C-43 in
1−2, standards were prepared by the methyl esterification of
commercially available, optically pure 2-hydroxyisovaleric acid
(hiv) 7. In addition, in the cases of C-43 and C-44 in 3, the
isoleucine stereoisomers 9 were diazotized and hydrolyzed to
afford the corresponding 2-hydroxy-3-methylpentanoic acids
(hmpa),18 which were converted to the respective methyl esters
10 (Scheme 2). Meanwhile, the three lipopeptides were
individually subjected to ozonolysis in a 1:2 mixture of 2.5 M
methanolic NaOH and CH2Cl2 at −78 °C, thus cleaving the
Δ41,42 olefin to a methyl ester (Scheme 3).19 We decided to
resort to such conditions because conventional ozonolysis, as
well as OsO4-catalyzed cleavage with Oxone,20 failed to break
this double bond. Subsequently, methanolysis was performed
by heating the reaction mixture at 50 °C for 1 h. These
reactions conveniently converted the 4-hydroxy-2E-enoyl
substituents in 1 and 2 to the hiv methyl ester 8, and that in
3 to the hmpa methyl ester 10. The ozonolyzed methanolysates
were then analyzed by GC-MS, and the retention times were
compared with the appropriate synthetic standard. Based on
the chiral-phase GC-MS results, 1 and 2 possess an S-
configuration at C-43, since the methyl 2-hydroxyisovalerate
detected in their ozonolyzed methanolysates coeluted with
synthetic (S)-8 (Figure S33). In the case of 3, a syn-
stereochemistry was initially assigned to the C-43/C-44 bond
using nonchiral GC-MS conditions (Figure S34). On the other
hand, under the chiral conditions employed, the derivatized 3
lacked a peak corresponding to 10. Thus, the heating at 50 °C
was continued overnight, and the subsequent chiral-phase GC-
MS analysis revealed the presence of (2R,3S)- and (2S,3S)-10
(Figure S35). This led to the conclusion that C-44 should have
an S-stereochemistry. As for the C-43 position, it also possesses
an S-configuration on the basis of the following: (1) nonchiral
GC-MS analysis established a syn-stereochemistry; (2) 1 and 2
both have an S-stereochemistry at this position, making it more
biogenetically plausible to have a 43S-stereochemistry, as well.
Therefore, the complete structures were concluded to be those
depicted in Figure 1.
Lipodiscamides A−C are structurally unique cyclic lip-
opentapeptides with the 3-hydroxyl group of the fatty acyl
chain attached to the C-terminus of the amino acid chain by an
ester linkage forming a macrocylic lactone. They are the first
characterized D-Cit-bearing lipopeptides.14,21−23 In addition,
there is only one example of a secondary metabolite bearing a
3-Uda residue and only one report of the occurrence of Denor,
which was found in a thiopeptide antibiotic.24−26 The α,β-
dehydroamino acids are known to be components of bioactive
peptides, and their geometrical configurations significantly
affect their bioactivity. Although the Z-isomer is thermodynami-
cally more stable,27,28 lipodiscamides have the unusual E-Denor
residue. Furthermore, the presence of the 4-hydroxy-2E-enoyl
unit is unprecedented. The same subunit, but with the cis
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geometry, exists in antitumor substances isolated from a
Pseudomonas sp.29 Interestingly, in lipodiscamides, this 4-
hydroxyl group is linked to the fatty acyl chain by a second
ester bond within the macrocycle. As such, lipodiscamides serve
as the first examples of a new family of dilactone lipopeptides.
In terms of bioactivity, lipodiscamides A−C showed
moderate cytotoxicity against P388 murine leukemia cells,
with IC50 values of 23, 20, and 31 μM, and moderate to weak
cytotoxicity against HeLa cells, with IC50 values of 18, 26, and
46 μM, respectively. However, they did not exhibit
antimicrobial activity against Bacillus cereus, methicillin-sensitive
Staphylococcus aureus, Escherichia coli, and Candida albicans.
This unexpected lack of antimicrobial activity might be due to
the insufficient overall positive charge contributed by the
carbamoylated functionalities of these molecules.
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ASSOCIATED CONTENT
* Supporting Information
■
S
(25) Debono, M.; Michael Molloy, R.; Occolowitz, J.; Paschal, J.;
Hunt, A.; Michel, K.; Martin, J. J. Org. Chem. 1992, 57, 5200−5208.
(26) Favret, M.; Paschal, J.; Elzey, T.; Boeck, L. J. Antibiot. 1992, 45,
1499−1511.
Description of experimental procedures and spectroscopic data
of 1−3. This material is available free of charge via the Internet
at http://pubs.acs.org.
(27) Yasuno, Y.; Hamada, M.; Yamada, T.; Shinada, T.; Ohfune, Y.
Eur. J. Org. Chem. 2013, 1884−1888.
AUTHOR INFORMATION
Corresponding Authors
■
(28) Ward, D.; Vasquez, A.; Pedras, M. J. Org. Chem. 1999, 64,
1657−1666.
*E-mail: wakimoto@mol.f.u-tokyo.ac.jp.
*E-mail: abei@mol.f.u-tokyo.ac.jp.
Notes
(29) Nakajima, H.; Takase, S.; Terano, H.; Tanaka, H. J. Antibiot.
1997, 50, 96−99.
NOTE ADDED AFTER ASAP PUBLICATION
The authors declare no competing financial interest.
■
The abstract graphic has been updated and the R2 substituents
in compounds 1 and 2 in Figure 1 have been corrected. The
revised version was re-posted on June 20, 2014.
ACKNOWLEDGMENTS
■
This work was partly supported by the Nagase Science
Technology Foundation, the Astellas Foundation for Research
on Metabolic Disorders, the CREST program from Japan
Science and Technology Agency, and Grants-in-Aid from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy (MEXT), Japan.
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