Medusamide a, a panamanian cyanobacterial depsipeptide with multiple β-amino acids

Article abstract of DOI:10.1021/acs.orglett.5b03110

From a collection of marine cyanobacteria made in the Coiba National Park along the Pacific coast of the Republic of Panama a novel cyclic depsipeptide, given the trivial name medusamide A, has been isolated and fully characterized. Medusamide A contains four contiguous β-amino acid (2R,3R)-3-amino-2-methylhexanoic acid (Amha) residues. This is the first report of multiple Amha residues and contiguous β-amino acid residues within a single cyclic peptide-type natural product. Stereochemical assignment of the Amha residues was completed following the synthesis of reference standards for this β-amino acid and the subsequent derivatization with Marfey's reagent and LC-MS analysis.

Full text of DOI:10.1021/acs.orglett.5b03110

Letter
pubs.acs.org/OrgLett
Medusamide A, a Panamanian Cyanobacterial Depsipeptide with
Multiple β‑Amino Acids
Amanda M. Fenner,† ,†,§,⊥Niclas Engene, Carmenza Spadafora, William H. Gerwick,
,‡,§,⊥
‡,∥
§
‡,⊥
and Marcy J. Balunas*
†Division of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269,
United States
‡
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, United States
§
Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), City of Knowledge, Apartado
0
816-02852, Panama City, Panama
⊥
́
Smithsonian Tropical Research Institute (STRI), Ancon, Apartado 0843-03092, Panama
∥
Department of Biological Sciences, Florida International University, Miami, Florida 33199, United States
*
S Supporting Information
ABSTRACT: From a collection of marine cyanobacteria made in the Coiba National Park
along the Pacific coast of the Republic of Panama a novel cyclic depsipeptide, given the trivial
name medusamide A, has been isolated and fully characterized. Medusamide A contains four
contiguous β-amino acid (2R,3R)-3-amino-2-methylhexanoic acid (Amha) residues. This is the
first report of multiple Amha residues and contiguous β-amino acid residues within a single
cyclic peptide-type natural product. Stereochemical assignment of the Amha residues was
completed following the synthesis of reference standards for this β-amino acid and the
subsequent derivatization with Marfey’s reagent and LC−MS analysis.
1
0
11
12
15
arine cyanobacteria produce a wide variety of secondary
metabolites that are noted for their unique chemical
malevamide B,1 kulokekahilide-1, ulongamides A−F,
3 14
M
guineamide D, lyngbyastatin 3, and carriebowmide.
scaffolds that have a diversity of biomedically relevant
Medusamide A (1) is the first cyanobacterial compound to
have adjacent β-amino acids, a structural motif which suggests a
fundamentally different biosynthetic pathway for their incor-
poration than those previously described.
Medusamide A (1) was isolated from the organic extract of
an encrusting cyanobacterium collected subtidally at four
meters near Coiba Island on the Pacific coast of Panama.
Based on 16S rRNA sequence analysis, the cyanobacterium
does not clade with any known genera of marine cyanobacteria
properties, most notably in the areas of anticancer and anti-
1
−3
inflammation. A prevalent theme in their natural products is
the creation of cyclic lipopeptides with a mixture of proteogenic
as well as unusual amino acids along with polyketide sections.
Understanding the mechanisms by which these organisms
produce and incorporate these unusual structural units is an
area of great scientific interest. Moreover, this understanding
is critical to the manipulation and engineering of these
biosynthetic pathways to create analogues as well as trans-
mission of the genetic machinery to expression hosts to
produce metabolites in large quantities. Thus, discovery of
unique structures from cyanobacteria that do not appear to
result from known biosynthetic pathways provides exciting
avenues for research related to cyanobacterial biosynthesis,
4
−6
(
Figure S5) and is thus likely a new genus. It is most closely
related to members of the genera Trichodesmium, Okeania, and
Oscillatoria. A methanolic fraction of the extract was moderately
inhibitory to cancer cell growth in MCF-7 hormone-dependent
breast cancer cells (73% inhibition at 10 μg/mL). Medusamide
A (1) was isolated from this fraction as a white solid by HPLC
and was found to be inactive in the assays (S19; a second
compound was isolated and determined to be the active
component of the original fraction but was in such small
quantity that structure elucidation was not possible).
7
−9
chemical ecology, and drug discovery.
Here, we report the isolation and structure elucidation of the
marine cyanobacterial metabolite medusamide A (1), a
depsipeptide containing four contiguous β-amino acid, 3-
amino-2-methylhexanoic acid (Amha) units. The trivial name
reflects the four Amha residues that give it a “snake-hair” like
appearance. Single Amha units have been found in several
cyanobacterial natural products, including the depsipeptides
Received: October 27, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b03110
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. H and 13C NMR Data for 1
1
13C
1H ppm, mult, Hz
COSY
3a, 3b
1
H− C HMBC
13
NOESY
NH-5, 3a, 3b
OLeu
1
2
3
3
4
5
6
7
8
9
1
1
169.9, qC
73.6, CH
40.7, CH2
5.05, dd (3.6, 10.0)
1.77, m
1, 3
a
2, 4
2, 4, 5, 6
NH-5, 4, 5, 6
NH-5
b
1.63, m
24.7, CH
22.9, CH3
21.2, CH3
170.4, qC
68.1, CH
33.9, CH
16.1, CH3
14.1, CH3
174.3, qC
50.9, CH
1.77, m
3a, 3b, 5, 6
2, 3
3, 4
3, 4
2, 3b, 5, 6
4, 5
0.94, m
4
4
0.91, m
4, 3b
Hiva
5.23, m
9, 10
8, 11
8, 11
9, 10
7, 12
8
10
1.53, m
8, 10, 11
8, 9, 11
9, 10
0
1
1.42, d (6.8)
0.94, d (6.5)
7, 8, 9, 11
9, 10
Valine
12
13
4.01, m
NH-1, 14
12, 17
12, 13
14, 15, 16
18, 19
NH-1
7.10, s
13
1
1
1
4
5
6
43.0, CH
12.8, CH3
12.8, CH3
174.6, qC
44.9, CH
51.1, CH
2.80, dq (3.7, 7.0)
1.17, d (7.0)
1.17, d (7.0)
13, 15, 16
13, 15, 16
13, 14
14
14
13, 14
Amha-1
Amha-2
Amha-3
Amha-4
17
1
8
9
2.62, dq (7.3, 7.5)
4.12, m
19, 20
NH-2, 18, 21
19
17, 19
NH-1, 19, 20, 21
1
18, 21, 22, 23, 24
NH-1, NH-2, 18, 20, 21, 22
20, 21, 25, 27
NH-2, 18, 19
NH-2
6.80, s
20
21
22
23
14.0, CH3
35.1, CH2
19.7, CH2
13.7, CH3
174.0, qC
44.5, CH
51.3, CH
1.06, d (6.9)
1.45, m
18
17, 19
19, 22
21, 23
22
19, 22, 23
19, 21, 23
21, 22
18, 19, 22, 23
18, 19, 21, 23
21, 22
1.26, m
0.89 d (3.4)
24
2
5
6
2.52, m
26, 27
NH-3, 25, 28
26
24, 26, 27
31
NH-2, NH-3, 26, 27
NH-3, 25, 27, 28, 30
25, 26, 27, 28, 32, 34
NH-2, NH-3, 25, 26, 30
NH-3, 26, 29, 30
28, 30
2
4.01, m
NH-3
6.09, d (8.2)
1.10, d (7.3)
1.46, m
27
28
29
30
14.7, CH3
34.4, CH2
19.2, CH2
13−14, CH3
174.0, qC
45.9, CH
51.2, CH
25
24, 25, 26
36, 29, 30
26, 28, 30
28, 29
26, 29
28, 30
29
1.28, m
0.9, m
26, 28, 29
31
3
2
3
2.52, m
33, 34
NH-4, 32
33
32, 33, 34
NH-3, NH-4, 33, 34, 35
NH-4, 32, 34, 35
32, 33, 35, 39, 40, 41
NH-3, NH-4, 33, 34, 35
32, 33, 37
3
4.01, m
NH-4
6.65, d (7.5)
1.12, d (7.3)
1.46, m
34
35
36
37
14.8, CH3
31.9, CH2
20.0, CH2
13−14, CH3
174.3, qC
47.2, CH
51.4, CH
32
31, 33
33, 36
35, 37
36
33, 36, 37
33, 35, 37
35, 36
1.30, m
37
0.9, m
36
38
3
9
0
2.38, dq (7.1, 8.3)
4.12, m
40, 41
NH-5, 39, 42
40
38, 40. 41
42, 43
NH-4, NH-5, 40, 41, 42
NH-4, 39, 41, 42,
2, 3a, 3b, 39, 40, 41, 42
NH-4, 39, 40
43, 44
4
NH-5
6.29, m
41
42
43
44
15.5, CH3
35.7, CH2
19.6, CH2
13−14, CH3
1.09, d (7.3)
1.43, m
39
38, 39
40, 43, 44
44
40, 43
42, 44
43
1.25, m
42, 44
0.85, d (6.8)
42, 44
42, 43
The molecular mass of 1 was measured by HRMS ([M + H]+
m/z = 822.5955), representing a molecular formula of
C H O N with eight degrees of unsaturation. However, the
suggesting five protonated amides. Four of these (δH 6.09−
6.80) were part of overlapping AMX spin systems. In each case,
these amide protons coupled to a four-proton resonance
representing four β protons (δH 4.01−4.12). These latter
resonances were in turn each coupled to a resonance
4
4
79
9
5
1
3
C NMR spectrum of 1 (Table 1; Figure S7) showed only 41
resonances, as a result of overlap of two valine methyl group
resonances and overlap of carbonyl and methyl group
resonances in two of the Amha residues (Amha-2 and -3).
representing four overlapping α protons (δ 2.62−2.38). The
H
four α protons were correlated by HMBC to one of four amide
carbonyls (δC 174.6−174.0) and, taken together with the
additional alkyl substituents described below, afforded four
1
The most downfield region of the H NMR spectrum (Table 1;
Figure S6) contained five broad peaks (δH 6.09−7.10),
B
DOI: 10.1021/acs.orglett.5b03110
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
identical β-amino acids subunits. The fifth amide proton (δH
Connectivity between residues was established using HMBC
and NOESY correlation spectroscopy. HMBC correlations
were observed between the H-8 α-proton of Hiva and the
valine carbonyl (C-12, δ 174.3), the H-13 α-proton of valine
and the Amha-1 carbonyl (C-17, δ 174.6), the H-19 α-proton
of Amha-1 and Amha-2 carbonyl (C-24, δ 174.0), and the H-26
β-proton of Amha-2 and Amha-3 carbonyl (C-31, δ 174.0).
Remaining connectivities were established by NOESY
correlations which included valine NH-1 to Amha-1 H-18
and H-19, Amha-1 NH-2 (δ 6.80) to Amha-2 H-25 and H-27,
Amha-2 NH-3 (δ 6.02) to Amha-3 H-32 (δ 2.52) and H-34 (δ
1.12), Amha-3 NH-4 (δ 6.65) to Amha-4 H-39 (δ 2.38) and H-
41 (δ 1.09), and Amha-4 NH-5 (δ 6.29) to OLeu H-2 (δ 5.05)
and weakly to OLeu H-3a (δ 1.77) and OLeu H-3b (δ 1.63).
Cyclization of the structure provided the final degree of
unsaturation and was corroborated by a weak HMBC
correlation between OLeu H-2 and Hiva C-8.
7
.10) was correlated by COSY to an α proton (δ 4.01 ppm)
H
that correlated by HMBC to a carbonyl (C-12, δ 174.3), thus
C
indicating that 1 contained a single α-amino acid. Two
1
additional signals occurred in the midfield region of the H
NMR spectrum, corresponding to oxygenated α protons (δH
5
.05, δ 73.6 and δ 5.23, δ 68.1). Compound 1 was thus
C H C
confirmed to be a depsipeptide containing four β-amino acids,
one α-amino acid, and two α-hydroxy acid residues.
Structures of these seven individual residues were deter-
mined using a series of 1D and 2D NMR experiments (Table 1
and SI). These residues were four Amha residues (numbered
Amha-1−4 for clarity), valine (Val), leucic acid (OLeu), and α-
hydroxyisovaleric acid (Hiva). COSY correlations were used to
sequentially connect the resonances of Amha-1: a methyl group
H -20 to a methine H-18 to a second methine H-19. H-19 was
3
coupled to both an amide NH and methylene H -21, which was
2
Collisional-induced fragmentation by LC−MS/MS provided
adjacent to a second methylene H -22 and then terminated
2
additional evidence for the planar structure of 1. Fragmentation
with a methyl group H -23. Taken together with the HMBC
3
+
of the ESI-MS parent ion m/z = 822.31 [C H O N + H]
correlations noted previously from H-18 to an amide carbonyl
44 80
9
5
after initial cleavage between Val and Amha-1, gave the
(C-17), these data defined an Amha residue. Deduction of the
following ions: m/z = 623.26 [OLeu-Amha-Amha-Amha-Amha
other three Amha residues followed a very similar logic with
similar to identical chemical shifts (Table 1 and Figure 1).
+
+
+
=
[
H] ; m/z = 568.24 [Val-Hiva-OLeu-Amha-Amha + H] ; m/z
+
441.13 [Val-Hiva-OLeu-Amha + H] ; and m/z = 314.02
Val-Hiva-OLeu + H] (Figure 2). The peak observed at m/z =
+
Figure 2. Collisionally induced fragments of 1 identified by LC-MS/
MS from ring-opening site between Val and Amha-1.
Figure 1. Planar structure of 1 with supporting 2D NMR correlations.
+
8
04.30 likely corresponds to [M − H O + H] . Ring opening
2
The valine residue was deduced from COSY correlations
between the H-13 methine proton (δH 4.01) and both the
between Amha-1 and Amha-2 produced the acylium ion m/z =
495.10 [OLeu-Amha-Amha-Amha]+ (S14). Additional frag-
mentations following formation of the acylium ion at the initial
ring opening site are included in S14. These fragmentation ions
were annotated with the assistance of mMass prediction
software and align perfectly with the planar structure predicted
from NMR analysis.
remaining NH proton (δ 7.10) and a highly coupled H-14
H
methine proton (δ 2.80). The latter resonance was coupled to
H
both the H-15 and H-16 methyl groups. Leucic acid (OLeu)
was deduced from COSY correlations between the OLeu H-2
methine and the OLeu H-3a (δ 1.77) and H-3b (δ 1.63)
H
H
methylene. Connection to the remainder of this residue was
achieved through HMBC correlations from the OLeu H-3
Stereochemistry was determined using chiral chromatog-
raphy for the hydroxy acids and advanced Marfey’s method for
valine and Amha residues. Co-elution on chiral chromatography
with commercially available standards identified the presence of
L-Hiva and D-OLeu in 1 (see S15). Following the advanced
Marfey’s procedure identified the valine residue in 1 as D-
protons as well as the two methyl groups, OLeu H-5 (δ 0.94)
H
and OLeu H-6, (δ 0.91) to methine OLeu C-4 (δ 24.7). The
H
C
Hiva unit was constructed based on COSY correlations
between methine resonances Hiva H-8 (δH 5.23) and H-9
16
(δH 1.53). The latter resonance was coupled to both methyl
valine. For the Amha residues, standards of the β-amino acid
groups Hiva H-11 (δH 0.94) and H-10 (δH 1.42). A rich
network of HMBC correlations confirmed these seven partial
structures (Figure 1 and Table 1).
were synthesized in enantiomeric excess as previously
reported. The hydrogenation step was incomplete, but fully
hydrogenated product was identified via LC−MS. The mixture
14
C
DOI: 10.1021/acs.orglett.5b03110
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
of hydrogenated and nonhydrogenated enantiomers was
reacted with either D-FDLA or L-FDLA, following the advanced
Marfey’s procedure, and the resulting Amha diastereomers were
resolved into four distinct peaks by LC−MS (S18). Hydrolyzed
Present Address
A.M.F.) Department of Chemistry, University of Hawai’i at
Manoa, Honolulu, HI 96822.
(
Notes
1
was reacted with L-FDLA, and the conjugated Amha coeluted
The authors declare no competing financial interest.
as a single peak with the major peak of the L-FDLA Amha
standard, indicating 1 contains only (2R,3R)-Amha residues
ACKNOWLEDGMENTS
■
(S18). In addition, we used advanced Marfey’s technique to
We acknowledge funding from the Fogarty International
Center (FIC) International Research Scientist Development
Award (IRSDA) to M.J.B. (K01 TW008002) and FIC
International Cooperative Biodiversity Group (ICBG) (U01
TW006634) to W.H.G.
synthesize D/L-FDAA Amha conjugates, providing further
evidence of the Amha stereochemistry. Hydrolyzed 1
conjugated with L-FDAA coeluted with the major peak of the
L-FDAA Amha, confirming the presence of only (2R,3R)-Amha
in medusamide A (1).
One of the more intriguing aspects of medusamide A’s
structure is the presence of four identical and adjacent Amha
residues. To the best of our knowledge, multiple adjacent β-
amino acids have not been previously observed in any
cyanobacterial or other marine natural product. Moreover,
only a very few marine natural products possess multiple β-
amino acids (dihydrocyclotheonamide A, cyclocinamide A,
REFERENCES
■
(
(
1) Gerwick, W. H.; Moore, B. S. Chem. Biol. 2012, 19, 85−98.
2) Fattorusso, E.; Taglialatela-Scafati, O. Mar. Drugs 2009, 7, 130−
1
52.
(
1
3) Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem. Rev. 2009,
09, 3012−3043.
(4) Daly, J. W. J. Nat. Prod. 2004, 67, 1211−1215.
17,18
theonellamides F, B, C, and onchidin),
and none have
(5) Jones, A. C.; Monroe, E. A.; Eisman, E. B.; Gerwick, L.; Sherman,
D. H.; Gerwick, W. H. Nat. Prod. Rep. 2010, 27, 1048−1065.
6) Mahmud, T.; Flatt, P. M.; Wu, X. J. Nat. Prod. 2007, 70, 1384−
391.
7) Blunt, J. W.; Copp, B. R.; Hu, W.-P.; Munro, M. H. G.;
Northcote, P. T.; Prinsep, M. R. Nat. Prod. Rep. 2007, 24, 31−86.
8) Choi, J. Y.; Plummer, M. S.; Starr, J.; Desbonnet, C. R.; Soutter,
contiguous β-amino acids. This suggests involvement of a
unique biosynthetic pathway to account for such an unusual
structural motif as in medusamide A (1).
The known biosynthetic pathways for cyanobacterial
metabolites containing β-amino acids, such as microcystin
(
1
(
(
[
contains a 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-
decadienoic acid (Adda) residue] and nostophycin [contains a
-amino-2,5-dihydroxy-8-phenyloctanoic acid (Ahoa) residue],
H.; Chang, J.; Miller, J. R.; Dillman, K.; Miller, A. a; Roush, W. R. J.
Med. Chem. 2012, 55, 852−870.
3
(9) Chang, Z.; Flatt, P.; Gerwick, W. H.; Nguyen, V.-A.; Willis, C. L.;
Sherman, D. H. Gene 2002, 296, 235−247.
involve the formation of β-amino acids from the pathway-
initiating polyketide structural unit. Specifically, a β-ketocarbox-
ylate is transaminated by a PLP-dependent aminotransferase to
form the β-amino function. Following a series of NRPS-
mediated incorporations of amino acids, the β-amino group
nucleophile is employed in the macrocyclization event and
(10) Horgen, F. D.; Yoshida, W. Y.; Scheuer, P. J. J. Nat. Prod. 2000,
6
(
3, 461−467.
11) Kimura, J.; Takada, Y.; Inayoshi, T.; Nakao, Y.; Goetz, G.;
Yoshida, W. Y.; Scheuer, P. J. J. Org. Chem. 2002, 67, 1760−1767.
12) Luesch, H.; Williams, P. G.; Yoshida, W. Y.; Moore, R. E.; Paul,
(
V. J. J. Nat. Prod. 2002, 65, 996−1000.
(13) Tan, L. T.; Sitachitta, N.; Gerwick, W. H. J. Nat. Prod. 2003, 66,
764−771.
1
9−21
completes the biosynthetic pathway.
However, the
microcystin/nostophycin pathway for forming β-amino acids
is inconsistent with the structure of 1, as this would require
multiple separate starting points or an iterative use of an NRPS
(14) Williams, P. G.; Moore, R. E.; Paul, V. J. J. Nat. Prod. 2003, 66,
1
356−1363.
22
23,24
(15) Gunasekera, S. P.; Ritson-Williams, R.; Paul, V. J. J. Nat. Prod.
module (as found in fungi and bacteria
), and implies
2
(
008, 71, 2060−2063.
16) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada, K.
Anal. Chem. 1997, 69, 3346−3352.
17) Clark, W. D.; Corbett, T.; Valeriote, F.; Crews, P. J. Am. Chem.
unprecedented reactions to interconnect these residues. Hence,
it appears more likely that multiple Amha units are formed
separately from the assembly line, and then incorporated as
activated amino acids through an entirely NRPS-type
mechanism, such as observed for the incorporation of β-
(
Soc. 1997, 119, 9285−9286.
(18) Romanova, N. N.; Tallo, T. G.; Rybalko, I. I.; Zyk, N. V.;
Shvyadas, V. K. Chem. Heterocycl. Compd. 2011, 47, 395−417.
25
alanine into jamaicamide A. More research is needed to clarify
the novel biosynthetic assembly illustrated by medusamide A
(
̈ ̈
19) Tillett, D.; Dittmann, E.; Erhard, M.; von Dohren, H.; Borner,
T.; Neilan, B. A. Chem. Biol. 2000, 7, 753−764.
(1).
(20) Fewer, D. P.; Osterholm, J.; Rouhiainen, L.; Jokela, J.; Wahlsten,
M.; Sivonen, K. Appl. Environ. Microbiol. 2011, 77, 8034−8040.
ASSOCIATED CONTENT
Supporting Information
(21) Kudo, F.; Miyanaga, A.; Eguchi, T. Nat. Prod. Rep. 2014, 31,
■
1
056−1073.
22) Boettger, D.; Hertweck, C. ChemBioChem 2013, 14, 28−42.
(23) Blodgett, J. A.; Oh, D. C.; Cao, S.; Currie, C. R.; Kolter, R.;
Clardy, J. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 11692−11697.
24) Gaitatzis, N.; Silakowski, B.; Kunze, B.; Nordsiek, G.; Blocker,
H.; Hofle, G.; Mu
*
S
(
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b03110.
(
̈
̈
̈
ller, R. J. Biol. Chem. 2002, 277, 13082−13090.
Full experimental details and spectral data (PDF)
(25) Edwards, D. J.; Marquez, B. L.; Nogle, L. M.; McPhail, K.;
Goeger, D. E.; Roberts, M. A.; Gerwick, W. H. Chem. Biol. 2004, 11,
8
17−833.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: (860) 486-3051. E-mail: marcy.balunas@uconn.edu.
D
DOI: 10.1021/acs.orglett.5b03110
Org. Lett. XXXX, XXX, XXX−XXX