Pagoamide A, a Cyclic Depsipeptide Isolated from a Cultured Marine Chlorophyte, Derbesia sp., Using MS/MS-Based Molecular Networking

Article abstract of DOI:10.1021/acs.jnatprod.9b01019

A thiazole-containing cyclic depsipeptide with 11 amino acid residues, named pagoamide A (1), was isolated from laboratory cultures of a marine Chlorophyte, Derbesia sp. This green algal sample was collected from America Samoa, and pagoamide A was isolated using guidance by MS/MS-based molecular networking. Cultures were grown in a light- and temperature-controlled environment and harvested after several months of growth. The planar structure of pagoamide A (1) was characterized by detailed 1D and 2D NMR experiments along with MS and UV analysis. The absolute configurations of its amino acid residues were determined by advanced Marfey's analysis following chemical hydrolysis and hydrazinolysis reactions. Two of the residues in pagoamide A (1), phenylalanine and serine, each occurred twice in the molecule, once in the d- and once in the l-configuration. The biosynthetic origin of pagoamide A (1) was considered in light of other natural products investigations with coenocytic green algae.

Full text of DOI:10.1021/acs.jnatprod.9b01019

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Pagoamide A, a Cyclic Depsipeptide Isolated from a Cultured Marine
Chlorophyte, Derbesia sp., Using MS/MS-Based Molecular
Networking
Yueying Li,†,‡ Hao-Bing Yu,‡,§ Yi Zhang,‡,⊥ Tiago Leao,‡ Evgenia Glukhov,‡ Marsha L. Pierce,∥
Chen Zhang,‡,# Hyunwoo Kim,‡ Huanru Henry Mao,# Fang Fang,‡ Garrison W. Cottrell,#
Thomas F. Murray,∥ Lena Gerwick,‡ Huashi Guan,† and William H. Gerwick*,‡,¶
†Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China
‡Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La
Jolla, California 92093, United States
§Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University,
Shanghai 200433, People’s Republic of China
⊥College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, People’s Republic of China
∥Department of Pharmacology, School of Medicine, Creighton University, Omaha, Nebraska 68178, United States
#Department of Computer Sciences and Engineering, University of California, San Diego, La Jolla, California 92093, United States
¶Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United
States
S
* Supporting Information
ABSTRACT: A thiazole-containing cyclic depsipeptide with
11 amino acid residues, named pagoamide A (1), was isolated
from laboratory cultures of a marine Chlorophyte, Derbesia sp.
This green algal sample was collected from America Samoa,
and pagoamide A was isolated using guidance by MS/MS-
based molecular networking. Cultures were grown in a light-
and temperature-controlled environment and harvested after
several months of growth. The planar structure of pagoamide
A (1) was characterized by detailed 1D and 2D NMR
experiments along with MS and UV analysis. The absolute
configurations of its amino acid residues were determined by
advanced Marfey’s analysis following chemical hydrolysis and
hydrazinolysis reactions. Two of the residues in pagoamide A
(1), phenylalanine and serine, each occurred twice in the
molecule, once in the D- and once in the L-configuration. The biosynthetic origin of pagoamide A (1) was considered in light of
other natural products investigations with coenocytic green algae.
he green algal phylum Chlorophyta is broadly repre-
Tsented in the marine environment and has been a rich
source of novel natural products with 400 published
compounds and with a wide variety of biological activities.
Interestingly, a number of these are of a peptide constitution,
such as the depsipeptide kahalalide F, first reported in 1993
from a Hawaiian Bryopsis species.1 Subsequently a series of
articles clarified stereochemical features of this molecule as well
as reported on a number of related compounds, kahalalides A−
Z2.2−12 Most cyclic kahalalides possess unique structural
features composed of a macrocyclic region that forms from an
ester bond between the C-terminus and the β-hydroxy group
of a threonine residue. Further, they possess a terminal
polyketide moiety or a branched acyclic peptide. A recent
study has shown that the kahalalides are produced by an
intracellular bacterium of the Flavobacteriia family through a
non-ribosomal peptide synthetase (NRPS) pathway.13 These
and related peptide molecules show interesting and highly
diverse biological activities including cytotoxic and antitumor
activity, antimicrobial activity, antileishmanial activity, immu-
nosuppressive properties, and protease inhibitory activity.14−20
Derbesia, a member of the Chlorophyta, is a siphonous
filamentous green alga forming filaments approximately 100
Special Issue: Special Issue in Honor of Jon Clardy
Received: October 15, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b01019
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μm in diameter.21−23 The organism was collected from shallow
coastal waters surrounding America Samoa and successfully
adapted to laboratory culture for the purpose of exploring its
natural products profile.24,25 Previously, only two natural
products have been isolated and reported from this genus,
mebamamides A and B.26 These latter two compounds
possessed a similar macrocyclic arrangement to the kahalalides;
however, neither were cytotoxic, but one (B) did show weak
cellular differentiation-inducing activity.
In the current study, we discovered a new cyclic nontoxic
depsipeptide from cultures of a marine Derbesia sp. Isolation of
this novel compound was facilitated and guided by using MS/
MS-based molecular networking as enabled through the Global
Natural Product Social (GNPS) platform (https://gnps.ucsd.
edu)27,28 as well as a calcium influx/oscillation assay. This
analysis revealed a small cluster composed of two related
compounds at m/z 1112 and 1114 that had no matches in the
GNPS database. Subsequently, purification of the m/z 1112
metabolite afforded a novel cyclic depsipeptide named here as
pagoamide A (1). The planar structure of compound 1 was
determined by MS, UV, and detailed 1D and 2D NMR
analyses. Absolute configurations of the amino acid moieties
were determined by advanced Marfey’s analysis following
chemical hydrolysis or hydrazinolysis reactions as well as DFT
calculations in combination with ROESY results. While the
extract and pagoamide-containing fractions showed inhibition
activity in the calcium oscillation assay, pure pagoamide A was
inactive, indicating that the bioactive compounds were other
components in these fractions. The extract, pagoamide A-
containing fractions, and pure pagoamide A were also
evaluated for H-460 human lung cancer cell cytotoxicity;
however, all of these materials were inactive at the maximum
concentrations tested (10 μg/mL for impure materials, 30 μM
for pure pagoamide A).
Figure 1. Photomicrograph of ASF14JUL14-1A Derbesia sp. from
culture (bar = 50 μm).
salts and afforded both aqueous and organic extracts. The
organic extract was separated using silica gel vacuum liquid
chromatography (VLC) to obtain nine fractions. These were
subjected to untargeted LC-MS/MS analysis as well as
evaluation in a calcium influx/oscillation assay.32−34 The
biological assay indicated that VLC fractions H and I potently
suppressed the amplitude of spontaneous calcium oscillations
in neocortical neurons, suggesting the presence of neuro-
modulatory secondary metabolites. By detailed comparison of
the LC-MS/MS and molecular networking outcomes with the
biological assay results, a small cluster of compounds was
targeted for isolation with parent masses of m/z 1112 and
1114. The MS/MS fragmentations suggested these compounds
were peptides, and ensuing reversed-phase silica gel column
chromatography followed by HPLC was successful in the
isolation of 3.2 mg of pure pagoamide A (1). Unfortunately,
upon isolation of pure pagoamide A, it was found to be inactive
in the calcium oscillation assay, and thus other trace
components of these fractions must constitute the active
materials.
Pagoamide A (1) was obtained as a white powder, and the
molecular formula was determined by HRESIMS as
C51H67N11O12S2 from observation of the sodium adduct ion
peak at m/z 1112.4292. Of the 24 degrees of unsaturation
inherent in this molecular formula, nine were revealed as
amide-type carbonyls from 13C NMR resonances at δC 168−
1
174. The H NMR, 13C NMR, and MS/MS fragmentations
exhibited characteristic profiles for a peptide-type structure
(Table 1, Figures 2 and 3). The 1H NMR spectrum contained
two signals at δH 8.10 (H-39) and 8.31 (H-44) with associated
13C NMR resonances at δC 124.2 and 124.4, and these could
be assigned to the methine groups of two substituted thiazole
rings; this was confirmed by diagnostic HMBC correlations
and accounted for six additional degrees of unsaturation
(Table 1).35−37 One Gly-Cys thiazole ring was confirmed by
Marfey’s analyses both with or without ozonolysis. Two phenyl
rings, accounting for 8 more degrees of unsaturation, were
revealed by 10 protons appearing at δH 7.00−7.30. The
remaining unaccounted degree of unsaturation was thus
deduced to be due to an overall macrocyclic ring structure.
An intense singlet at δH 2.15 (H3-50, H3-51) integrated for
six protons and suggested the presence of two N-methyl
groups in pagoamide A. These methyl protons and an α-
methine carbon at δC 71.9 (C-46) were correlated in the
HMBC spectrum. This α-methine proton was coupled to a β-
methine proton at δH 2.26 (H-47), which in turn was coupled
to two γ-methyl groups at δH 0.75 and 0.99, respectively (H3-
48, H3-49), thus constructing a terminating residue as N,N-
dimethyl valine. The correlations between eight amide NH
RESULTS AND DISCUSSION
■
A culture sample of a Derbesia sp. green macroalga (Figure 1)
was obtained in 12−18 m water depth by scuba diving in
Fagatele Bay, America Samoa, and codified as ASF-14JUL14-
1A after separation and purification from co-collected
cyanobacteria under an experimental environment. Purification
of the living collection was achieved using standard methods in
microbiology, and cultures were maintained under a light- and
temperature-controlled environment in Erlenmeyer flasks with
salt water BG11 media.29−31 Morphologically, this cultured
material was consistent with a Derbesia species (Figure 1);
unfortunately, DNA sequencing of the metagenomic DNA
failed to capture a phylogenetically informative sequence. After
a number of scale-up experiments, the culture was harvested
and extracted using CH2Cl2/MeOH (2:1). The crude extract
was washed with distilled H2O to remove polysaccharides and
B
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a
1
Table 1. H and 13C NMR Data for Pagoamide A (1) in DMSO-d6
residue
position δC, type
δH, mult. (J, Hz)
HMBC
residue
position δC, type
δH, mult. (J, Hz)
HMBC
phenylalanine-1
1
2
3
4
5, 9
6, 8
7
NH
10
11
170.4, C
52.4, CH
36.1, CH2
137.9, C
128.1, CH
129.2, CH
126.2, CH
NH
8.53, s
30
4.85, m
3.18, m
1
threonine
serine-2
30
31
32
33
NH
34
35
36
169.1, C
54.1, CH
2, 4, 5−9
4.85, m
71.3, CH 5.65, qd (6.6, 3.2)
1
7.19, m
7.19, m
7.13, m
3, 4, 6−8
3−5, 7, 9
5−9
16.0, CH3
1.11, d (6.6)
6.86, d (9.5)
31, 32
34
169.9, C
54.5, CH
7.74, d (9.35)
10
4.87, m
34, 37
valine
170.0, C
62.4, CH2 3.75, dd (11.0, 4.3); 34, 35
3.93, dd (11.1,
58.7, CH 3.87, dd (7.9, 4.6) 10, 12, 13,
4.1)
14, 15
NH
37
38
39
40
41
NH
42
43
44
45
46
8.07, d (8.0)
37
12
13
14
NH
15
16
17
18
29.3, CH
19.1, CH3
16.4, CH3
1.84, m
13, 14
Gly-Cys thiazole
159.7, C
148.8, C
124.2, CH
170.3, C
40.8, CH2
0.59, d (6.9)
0.27, d (6.9)
7.38, d (7.9)
11, 12, 14
11, 12, 13
15
8.10, s
37, 38, 40
phenylalanine-2
172.5, C
53.0, CH
34.1, CH2
138.1, C
4.74, d (6.4)
9,26, t (6.2)
40, 42
42
4.58, m
3.16, m
15, 18
16
N,N-diMe-Val-
Cys thiazole
161.1, C
148.5, C
124.4, CH
168.4, C
71.9, CH
19, 23 128.0, CH
7.19, m
7.19, m
17, 18,
20−22
8.31, s
42, 43, 45
20, 22 129.1, CH
17−19, 21,
23
3.51, m
45, 47, 48,
49, 50,
51
21
NH
24
25
26
NH
27
28
126.1, CH
7.13, m
8.21, d (8.9)
19−23
24
47
48
49
50
51
29.5, CH
19.4, CH3
20.1, CH3
41.3, CH3
41.3, CH3
2.26, m
0.75, d (6.5)
0.99, d (6.5)
2.15, s
45, 46
alanine
173.5, C
50.1, CH
16.7, CH3
46, 47, 49
46, 47, 48
46, 51
4.03, quint (6.8)
1.15 d (7.3)
9.12, d (5.7)
24, 26
24, 25
25, 26, 27
2.15, s
45, 50
serine-1
171.4, C
57.4, CH
60.7, CH2
a
Data recorded at 500 MHz (1H NMR) and 125 MHz (13C NMR).
4.14, t (7.0)
3.57, m
27, 29
27, 28
29
Figure 2. Select correlations used to determine the planar structure of
pagoamide A (1). Bolded bonds represent proton-based spin systems
deduced by COSY correlations. Arrows represent correlations
observed in the HMBC spectrum.
Figure 3. Selected MS/MS fragments of pagoamide A (1).
protons (Table 1) and their corresponding α-methine protons
were determined by the COSY and HMBC experiments, and
these spin systems could be extended to the amino acid side
chains in each case. This analysis revealed that in addition to
the two cysteine-derived thiazoles and one N,N-dimethyl
valine, pagoamide A possessed two serines, two phenylalanines,
one valine, one alanine, one glycine, and one threonine. The
sequence of residues in the peptide was determined by HMBC
correlations from the amide NH protons to the carbonyl
equivalents in the adjacent residue or, in some cases, from β-
protons to carbonyls (Table 1, Figure 2), resulting in the series
N,N-dimethyl-valine-cysteine thiazole, glycine-cysteine thia-
zole, serine, threonine, serine, alanine, phenylalanine, valine,
and phenylalanine. Finally, the phenylalanine carboxyl
terminus could be connected as an ester to the β-hydroxy
group of threonine through observation of a key HMBC
correlation. Thus, the planar structure of pagoamide A was
revealed as a new cyclic despipeptide formed from 11 amino
acid moieties (Figure 2).
C
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Figure 4. (A) Conformationally optimized stereostructures of the two possible absolute configurations of pagoamide A (1): (A1) Serine-1 as D and
serine-2 as L, (B1) serine-1 as L and serine-2 as D. Protons were omitted for clarity. (B) Conformationally optimized stereostructures of two possible
absolute configurations for pagoamide A (1) were discriminated using selected ROESY correlations (e.g., between Ser-2 and Phe-1 residues and
between Thr and Val residues). The Gly-Cys thiazole, N,N-diMe-Val-Cys thiazole, and unrelated protons were omitted for clarity. (C)
Conformationally optimized stereostructures of two possible absolute configurations of pagoamide A (1) were discriminated using additional
selected ROESY correlations (e.g., between Ser-1 and NH’s of Ala and the Phe-2). The side chains and unrelated protons were omitted for clarity.
The configurations of the amino acid moieties in pagoamide
A (1) were investigated using advanced Marfey’s analysis and
involved ozonolysis, hydrolysis, chemical derivatization with D-
FDAA, and LC-MS comparison against available stand-
ards.37−40 The configuration of the N,N-dimethyl valine
residue was determined as L by comparing the hydrolyzed
product with synthetic D/L N,N-dimethyl valine standards by
chiral-phase HPLC [(D)-penicillamine stationary phase]. The
remainder of the hydrolyzed products was subjected to
chemical derivatization with D-FDAA and comparison with
the D-DAA derivatives of the authentic amino acid standards.
The valine and threonine residues were of L-configuration,
whereas the alanine was D-configured. However, the two serine
and two phenylalanine residues each comprised one D- and one
L-configured residue, a result that provided four possibilities for
the absolute configuration of compound 1. Subsequently, mild
base hydrolysis of pagoamide A provided the acyclic peptide
with phenylalanine-1 as the carboxy terminus.6 A hydrazinol-
ysis reaction was employed to selectively release this terminal
phenylalanine residue,11 which was then subjected to Marfey’s
analysis to reveal its L configuration.38,39 This result necessarily
defined the phenylalanine-2 residue to be D-configured, which
leaves two possible absolute configurations for compound 1.
D
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1
Figure 5. Nine most similar natural products to pagoamide A (1) using the SMART 2.0 tool based on H−13C HSQC spectra (similarity
coefficient above 0.8 out of 1).45
Conformational analysis using Discovery Studio 2.5 gave
eight conformers in total [5 for structure A and 3 for structure
B (Figure S24)]. Reoptimization was carried out using density
functional theory (DFT) calculations in the Gaussian 09
program for the eight conformers. In the calculated lowest
energy conformers, conformer A1 and conformer B1 showed
Boltzmann distributions with Gibbs free energies of more than
99.99%. (Figure 4A) The other six conformers were A2
(8.505e-15%), A3 (3.621e-17%), A4 (2.737e-20%), A5
(5.391e-24%), B2 (0.004%), and B3 (1.300e-13%), each
with very minor Boltzmann distributions (Figure S24). It was
observed that the side chain in A1 extends out from the
macrocycle versus partially folding back over the macrocycle as
in B1 (Figure 4A). Discrimination between these two
possibilities (A1 and B1, Figure 4B,C) was achieved using
ROESY correlations. The ROESY correlations observed
between the α-proton of Ser-2 (H-35) and β-protons of Ser-
2 (H-36) and NH of the Phe-1 residue and the correlations
between the α-proton of Val (H-11), NH of the Val, and NH
of the Thr were consistent with the stable conformer wherein
serine-1 is L and serine-2 is D (B1, Figure 4B); conversely,
these long-range correlations were inconsistent with the major
conformer, where serine-1 is D and serine-2 in the side chain is
L (A1, Figure 4B). Consistent with this result, ROESY
correlations were observed between H-28 of Ser-1 and the
NH of both the Ala and the Phe-2 residues; this was only
consistent with structure B1, wherein Ser-1 is of L-
configuration (B1, Figure 4C).
Pagoamide A (1) possesses two thiazole ring moieties that
are predicted to derive from the cyclization between cysteine
and N,N-dimethyl valine in one case and with glycine in a
second instance. It was interesting that only the terminal valine
moiety was N-methylated, as N-methylation is frequently
observed in marine-derived cyclic and acyclic peptides.41,42
Additionally, three out of 11 amino acids were of D-
configuration. These types of structural modifications are
somewhat more consistent with a biogenesis through the
ribosomally synthesized and post-translationally modified
peptide (RiPPs) pathway rather than a nonribosomal peptide
synthetase route.43,44 Partial support for this comes from
preliminary genome sequence data for this Derbesia sp.
(Illumina HiSeq 400 assembled with metaSPAdes 3.12).
AntiSmash analysis of the biosynthetic gene clusters in this
eukaryotic alga failed to identify any NPRS pathways that
E
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could potentially encode for pagoamide A (1) biosynthesis.
However, as noted above, the somewhat related kahalalides
were recently shown to be produced via NRPS metabolism by
an endosymbiotic bacterium in another coenocytic green alga,
a Bryopsis sp.13
through microbial culture or genetic methods, other
production strategies are possible. For example, the cyclic
peptide kahalalide F, originally isolated from a coenocytic
Bryopsis sp. green alga as well as from a nudibranch that feeds
on the alga Elysia rufescens, was shown to derive from an NRPS
pathway found within a bacterium living within the cells of the
alga.13 This severely genome-reduced bacterium was deduced
to be of the class Flavobacteriia and given the name Candidatus
Endobryopsis kahalalidefaciens. Given the related structures of
pagoamide A and kahalalide F in the sense that both are cyclic
peptides comprised of D and L amino acids (kahalalides H, J, P,
and Q possess both D- and L-phenylalanine residues), it may
point to a general trend of intracellular bacterial symbionts
with significant natural product biosynthetic capacity asso-
ciated with coenocytic algae. These as well as questions
concerning other bioactive constituents of this cultured
Derbesia sp. (e.g., those causing modulation of calcium
oscillations) are the focus of ongoing investigations.
To explore this aspect in somewhat greater depth, we
applied our recently described Small Molecule Automatic
Recognition Technology (SMART) program, which is a
machine learning tool that recognizes structurally related
compounds on the basis of their HSQC spectra.45 The
pagoamide A HSQC image was evaluated in SMART 2.0 that
had been trained with 25,434 marine and terrestrial natural
products and provided close hits for echinomycin A,
bistratamide F, didmolamide A, bistratamide E, ulongamide
A, patellamide E, banyascyclamide A, xentrivalpeptide A, and
guineamide B (Figure 5). Ulongamide A, banyascyclamide A,
and guineamide B are metabolites of free living marine
cyanobacteria, whereas bistratamides E and F, didmolamide A,
and patellamide E are produced by cyanobacteria living within
an ascidian.37,46−50 Echinomycin A and xentrivalpeptide A are
metabolites reported from actinomycetes and proteobacteria,
respectively.51,52 The dominant feature being recognized by
the SMART 2.0 algorithm appears to be that the molecule is a
cyclic peptide with thiazole/oxazole and aromatic ring
structures. This analysis provides some additional support for
the hypothesis that pagoamide A (1) is the result of
prokaryotic, possibly symbiotic, metabolism within the context
of a eukaryotic host. Nevertheless, additional investigations are
needed to confirm the origin of pagoamide A (1) in this
cultured Derbesia sp.
In conclusion, in this work, we report the isolation and
structure determination of the cyclic depsipeptide pagoamide
A (1) from a cultured Derbesia sp. marine Chlorophyte. This
finding expands the scope of secondary metabolites to be
described from this underexplored genus, previously only
investigated for its lipids, carotenoids, and, in one case, two
structurally unique cyclic peptides, mebamamides A and B.26
Detailed cultivation experiments were performed to obtain
monocultures suitable for long-term scale-up experiments, and
this enabled the accumulation of sufficient biomass for
chemical investigations. MS/MS-based molecular networking
facilitated the efficient isolation of pagoamide A through its
detection in the various repeat cultures and subfractions
produced during the chromatographic process. This was
complemented by calcium influx/oscillation and cytotoxicity
assays that helped to guide the isolation and purification of this
new metabolite. By integrating multiple analytical methods and
synthetic reaction procedures, the complete stereostructure of
pagoamide A (1) was fully elucidated.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured in MeOH with a JASCO P-2000 polarimeter operated at
25 °C. Ultraviolet and visible (UV−vis) spectra were recorded using a
Beckman Coulter DU 800 spectrophotometer. All NMR data except
13C NMR spectra were obtained from a JEOL ECZ 500 MHz
spectrometer with a 3 mm detection probe. The 13C NMR spectrum
was recorded with a Varian VX 500 MHz spectrometer with a Varian
XSens 2 channel (1H and 13C) NMR cold probe optimized for
directly observing 13C NMR. The NMR solvent was DMSO-d6
purchased from Cambridge Isotope Laboratories, Inc. with 99.8%
deuterium and using the solvent peaks for the internal standard (δH
2.50 and δC 39.1). LCMS data were obtained with a Thermo Finnigan
LCQ Advantage Max mass spectrometer coupled to a Thermo
Finnigan Surveyor HPLC system with a Kinetex C18 100 × 4.6 mm ×
5 μm analytical column (Phenomenex) (monitoring 200−600 nm and
m/z 200−1600 in positive ion mode). HRESIMS data were recorded
using an Agilent 6530 Accurate-Mass QTOF mass spectrometer in
positive ion mode. Semipreparative HPLC purification was performed
on a Thermo Dionex UltiMate 3000 HPLC system with a YMC-
Triart C18 (250 mm × 10 mm x 5 μm) column. Analytical HPLC was
performed on a Thermo Dionex UltiMate 3000 HPLC system with a
Phenomenex Chirex 3126 D-penicillamine column (250 mm × 4.6
mm, 5 μm). HPLC-grade and LC-MS-grade CH3CN were purchased
from Thermo Fisher Scientific. HPLC-grade H2O was obtained by
filtration using a Milli-Q Direct water purification system (Millipore).
Optical density (OD) values were measured on a ThermoElectron
Multiskan Ascent plate reader.
Organism Collection, Identification, and Cultivation. The
sample was collected from 12 to 18 m water depth in Fagatele Bay,
American Samoa (S 14°21′47.714″, W 170°45′43.902″ on July 14,
2014). Cultures of a Symploca sp. marine cyanobacterium were
initially collected; however, the cyanobacterium died, leaving a fine
siphonous green unialga alive in the culture flask, codified as
ASF14JUL14-1A. Based on its microscopic morphology, this sample
was identified as a Derbesia sp.21,22,53,54 No attempt was made to
produce axenic cultures of this cultured Chlorophyte; however, there
was no visible evidence of microbial growth. The cultures were
initially grown in 12 × 250 mL autoclaved flasks with SWBG11 media
under 27.0−27.6 °C and 16 h/8 h light/dark cycle at 400−650 l×
condition with semicontinuous dilution.24,25,29 After 2 weeks of
growth, the cultures were transferred into autoclaved 2 L flasks for
another one month until they fully occupied the culture volume. The
culture was harvested by filtration from the SWBG11 culture media
and lyophilized overnight before grinding into a fine powder.
Relatively large and complex natural products such as
pagoamide A impose significant challenges in structure
elucidation due to NMR signal degeneracy and a multitude
of stereogenic centers that require assignment. These
difficulties are compounded when dealing with relatively
small amounts of the natural product material. And while
laboratory cultivation can in principle overcome the small
quantity issue, it poses its own unique problems, as oftentimes
the cultured organism grows slowly requiring months for scale-
up.
However, in some cases, associated or intracellular bacterial
symbionts have been found to be responsible for the
production of bioactive compounds associated with macro-
organisms (e.g., algae, sponges, tunicates), and thus either
DNA Extraction and Sequencing. A DNA sample was obtained
from live cultures of Derbesia sp. by carefully grinding freshly
harvested filaments using a mortar and pestle under liquid nitrogen.
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DNA was extracted using the QIAGEN bacterial genomic DNA
extraction kit using the standard kit protocol. The library was
prepared with the miniaturized version of the KAPA HyperPlus kit,
followed by quantification using the PicoGreen ds DNA assay. The
metagenomic DNA was sequenced with Illumina HiSeq 4000 and
subsequently assembled with metaSPAdes 3.12.
product was dissolved in a 7:1 mixture of EtOH/H2O and evaporated
to dryness in vacuo. A sample of L-Val was subjected to the same
reaction conditions. The identities of both products were confirmed
56,57
1
by comparing their H NMR features with literature values.
N,N-Dimethyl-L-valine: white powder; 1H NMR (500 MHz,
DMSO-d6 TMS) δ 2.65 (d, J = 9.4 Hz, 1H), 2.28 (s, 6H), 1.90
(m, 1H), 0.92 (d, J = 6.7 Hz, 2H), 0.83 (d, J = 6.5 Hz, 2H).
N,N-Dimethyl-D-valine: white powder; 1H NMR (500 MHz,
DMSO-d6 TMS) δ 2.64 (d, J = 9.4 Hz, 1H), 2.28 (s, 6H), 1.90
(m, 1H), 0.92 (d, J = 6.8 Hz, 2H), 0.83 (d, J = 7.0 Hz, 2H).
Chemical Degradation of Pagoamide A (1) and Absolute
Configuration Analysis. A 0.1 mg sample of pagoamide A (1) was
dissolved in 1 mL of CH2Cl2 and then treated with ozone for 10 min
at rt.37,40 Next, the product was subjected to acid hydrolysis in a
sealed vial at 110 °C with 200 μL of 6 M HCl for 16 h. The reaction
mixture was cooled to rt and evaporated to dryness under a stream of
N2. A 20 μL aliquot of this hydrolysis product as well as the synthetic
N,N-dimethylvaline standards were injected onto an analytical HPLC
system using a Phenomenex Chirex 3126 (D)-penicillamine column
(250 mm × 4.6 mm with 5 μm particles and 110 Å pore size) eluting
with 2 M CuSO4 for 15 min at a flow rate of 2 mL/min. The
remainder of the hydrolysis product and both D- and L-amino acid
standards (serine, threonine, alanine, phenylalanine, and valine) were
treated with 100 μL of 1 M NaHCO3 and 200 μL of D-FDAA acetone
solution (1.0%) at 40 °C for 1 h and then neutralized with 100 μL of
1 M HCl to obtain the D-DAA derivatives. The D-DAA derivatives
were then evaporated to dryness under N2, and the products dissolved
in 1 mL of MeOH and filtered over C18 SPE cartridges before LCMS
analysis. A 10 μL aliquot was taken and injected into an analytical
LCMS to compare against the derivatized authentic amino acid
standards.38,39 Retention times for the D-DAA-derivatized amino acid
standards were as follows: L-Ser (2.99 min), D-Ser (2.97 min), L-Thr
(4.07 min), D-Thr (3.24 min), L-allo-Thr (3.67 min), D-allo-Thr (3.37
min), L-Ala (6.12 min), D-Ala (4.71 min), L-Phe (13.54 min), D-Phe
(12.37 min), L-Val (12.13 min), and D-Val (9.52 min).
Extraction and Separation. The scale-up cultivation of the
Derbesia sp. was repeated eight times, and for each, a separate
extraction and isolation procedure was performed. The freeze-dried
and powdered biomass was exhaustively extracted in 2:1 CH2Cl2/
MeOH (six times). The extracts (#2245EX) were dried under
vacuum and then washed with DI H2O (3×) to remove residual salt
(#2245AQ), affording 1.6 g of organic portion (#2245CR). The
organic portion was subjected to VLC separation over grade H silica
gel. The column was eluted to provide nine subfractions: 2245CR_A
(100% hexanes, 64.5 mg), 2245CR_B (10% EtOAc/hexanes, 107.6
mg), 2245CR_C (20% EtOAc/hexanes, 316.4 mg), 2245CR_D (40%
EtOAc/hexanes, 185.1 mg), 2245CR_E (60% EtOAc/hexanes, 32.4
mg), 2245CR_F (80% EtOAc/hexanes, 45.2 mg), 2245CR_G (100%
EtOAc, 13.7 mg), 2245CR_H (25% MeOH/EtOAc, 507.5 mg), and
2245CR_I (100% MeOH, 211.3 mg). Fractions 2245CR_H and
2245CR_I were combined and then applied to a 5000 mg/20 mL
Gracepure C18 max SPE cartridge to produce seven subfractions:
2245CR_HI_1(100% H2O, 263.2 mg), 2245CR_HI_2 (20%
CH3CN/H2O, 20.6 mg), 2245CR_HI_3 (40% CH3CN/H2O, 4.9
mg), 2245CR_HI_4 (60% CH3CN/H2O, 82.6 mg), 2245CR_HI_5
(80% CH3CN/H2O, 76.7 mg), 2245CR_HI_6 (100% CH3CN, 56.2
mg), and 2245CR_HI_7 (2:1 CH2Cl2/MeOH, 187.6 mg).
Accordingly, 2245CR_HI_2 and 2245CR_HI_3 were combined
and further purified by semipreparative RP-HPLC using a YMC-
Triart C18 (250 mm × 10 mm × 5 μm) column eluted with mixtures
of H2O with 0.1% formic acid and CH3CN with 0.1 formic acid using
the following gradient: 10% CH3CN/H2O for 5 min, 10% CH3CN/
H2O to 99% CH3CN/H2O in 15 min, 99% CH3CN/H2O for 5 min,
99% CH3CN/H2O back to 10% CH3CN/H2O in 1 min, and then
maintained in 10% CH3CN/H2O for 4 min at a flow rate of 2 mL/
min. The aqueous portion (2245AQ) was subjected to semi-
preparative RP-HPLC using the same conditions to accumulate
small additional amounts of pagoamide A (1), eluting at 15.2 min and
yielding 3.2 mg in total from all fractions.
Another portion of pagoamide A (0.1 mg) was treated with the
same acid hydrolysis and derivatization protocol described above, but
without the prior ozonolysis step. The retention time of the D-DAA-
derivatized Gly-Cys moiety was 5.99 min and showed an m/z 410
(Gly-Cys Thiazole) (Supporting Information S23).
A third portion of pagoamide A (0.1 mg) was hydrolyzed in a
sealed vial with 0.1 N NaOH in aqueous MeOH (50 μL) at 37 °C for
90 min. The partially hydrolyzed product was added to 1 mg of
Amberlite GC50 under argon, followed by 40 μL of distilled
hydrazine. The reaction mixture was heated at 80 °C for 5 days.
Molecular Networking. The organic portion (2245CR) and
fractions 2245CR_A−I were analyzed by LCMS/MS. Samples were
dissolved in MeOH at 1 mg/mL and then filtered through a
Gracepure C18 SPE cartridge before LCMS analysis. A 10 μL aliquot
of each sample was injected and eluted with a gradient of H2O
containing 0.1% formic acid and CH3CN containing 0.1% formic acid
with a gradient method as follows: 30% CH3CN/H2O for 5 min, 30%
CH3CN/H2O to 99% in 17 min, held at 99% CH3CN/H2O for 3
min, then 99% CH3CN/H2O to 30% CH3CN/H2O in 1 min, and
finally held at 30% CH3CN/H2O for 4 min with the flow rate of 0.7
mL/min. Mass spectra were recorded in positive ESI mode (m/z
200−1600) and with an automated full dependent MS/MS scan
enabled. All chromatograms were converted digitally to .mzXML files
using MSConvert software (www.proteowizard.sourceforge.net). The
organic extract (#2245CR) and the fractions (#2245CR_A−I) were
submitted to GNPS molecular networking for dereplication.27,28,55
The node and table file from GNPS were inputted into Cytoscape
software to generate a molecular networking. The cluster containing
the sodiated pagoamide A ion at m/z 1112 was used to direct further
purification.
After cooling to rt, the reaction mixture and both the D and
L
authentic phenylalanine amino acid standards were treated with 100
μL of 1 M NaHCO3 and 200 μL of D-FDAA acetone solution (1.0%)
at 40 °C for 1 h and then neutralized with 100 μL of 1 M HCl to
obtain the D-DAA derivatives. A 20 μL aliquot of this product and the
phenylalanine standards were injected into an analytical HPLC
coupled with a YMC-Triart C18 column (250 mm × 10.0 mm, 5
μm), and their retention times compared as follows: L-Phe (20.04
min), D-Phe (19.56 min).6,11,38,39
Conformer and Stereoisomer Calculations. The conforma-
tional analyses were carried out in the Discovery Studio 2.5 software
package. The results showed the eight lowest energy conformers for
two proposed structures (Figure S24) whose relative energies were
within 10.0 kcal/mol. Subsequently, the conformers were reoptimized
using DFT at the B3LYP/6-31+G(d,p) level in the gas phase by the
GAUSSIAN 09 program. The B3LYP/6-31+G(d,p) harmonic
vibrational frequencies were also calculated to confirm their stability.
Biological Evaluation. Animal care and handling complied with
protocols approved by the Creighton University Institutional Animal
Care and Use Committee and employed measures to minimize pain
and discomfort (0801.3). Swiss-Webster gravid dams were euthanized
by CO2 asphyxiation, and embryonic day 16 mice were removed
under sterile conditions. Cerebrocortices were dissected, stripped of
meninges, minced, and incubated with trypsin for 25 min at 37 °C.
Cells were dissociated by trituration in isolation buffer containing
Pagoamide A (1): white powder; [α]25D +5.5 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 215 (4.37), 220 (4.39), 234 (4.59); 1H and 13
C
NMR data, Table 1; HRESIMS m/z 1112.4292 [M + Na]+ (calcd for
C51H67N11O12S2Na+, 1112.4304).
Synthesis of N,N-Dimethylvaline Standards. D-Valine (1.00 g,
8.541 mmol) was dissolved in DI H2O (45 mL), and then
formaldehyde (37% w/w, 1.4 mL, 17.1 mmol) and 5% Pd−C (1.0
g) were added. The reaction mixture was hydrogenated at rt for 5
days under H2 gas and then refluxed for 30 min. The mixture was
filtered through a bed of Celite, and the filtrate dried in vacuo. The
G
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soybean trypsin inhibitor and DNase, centrifuged, and resuspended in
Eagle’s minimal essential medium with Earle’s salt (MEM) and
supplemented with 1 mM L-glutamine, 10% fetal bovine serum, 10%
horse serum, 100 IU/mL penicillin, and 0.10 mg/mL streptomycin
(pH 7.4). Cells were plated onto poly-L-lysine-coated 96-well (9 mm),
clear-bottomed, black-well culture plates (MidSci, St. Louis, MO,
USA) at a density of 1.5 × 105 cells/well. Cells were then incubated at
37 °C in a 5% CO2 and 95% humidity atmosphere. Cytosine
arabinoside (ARA-C; 10 μM) was added to the culture medium 24 h
after plating to prevent proliferation of non-neuronal cells. From day 4
in vitro, culture media was changed every other day using a serum-free
growth medium containing Neurobasal Medium supplemented with
B-27, 100 IU/mL penicillin, 0.10 mg/mL streptomycin, and 0.2 mM
L-glutamine. Experiments were performed on cerebrocortical cultures
at 12 days in vitro.
MS/MS, ESIMS, HRESIMS, 1D/2D NMR spectra of
compound 1; LCMS chromatograms of Marfey’s
analysis hydrolysis and derivatization products of 1
with and without ozonolysis, along with derivatized
amino acid standards; Marfey’s analysis hydrolysis,
hydrazinolysis, and derivatization products of 1, along
with derivatized amino acid standards; MS2-based
molecular networking figure for the crude and VLC
fractions, bioassay screening result (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*Tel: (858)-534-0578. E-mail: wgerwick@ucsd.edu.
Cerebrocortical neuron cultures were used for [Ca2+] measure-
ments at day 12 in vitro (DIV12). Briefly, the growth medium was
removed and replaced with dye-loaded medium (100 μL per well)
containing 4 μM fluo-3 AM and 0.04% pluronic acid in Locke’s buffer
(8.6 mM HEPES, 5.6 mM KCl, 154 mM NaCl, 5.6 mM glucose, 1.0
mM MgCl2, 2.3 mM CaCl2, 0.1 μM glycine, pH 7.4). After 1 h of
incubation in dye-loaded medium, the neurons were washed 4× with
fresh Locke’s buffer (180 μL per well) using an automated microplate
washer (Bio-Tek Instruments Inc.) and transferred into a FLIPR II
(Molecular Devices, Sunnyvale, CA, USA). Cells were excited at 488
nm, and Ca2+-bound fluo-3 emission was recorded at 515−575 nm at
1.2 s intervals for calcium influx and 0.5 s intervals for calcium
oscillations. For calcium influx, baseline fluorescence was recorded for
1 min, 20 μL of 10× concentrations of compounds was added to wells
at a rate of 20 μL/s, and the fluorescence was monitored for an
additional 5 min. For calcium oscillations, baseline fluorescence was
recorded for 2 min, 20 μL of 10× concentrations of compounds was
added to wells at a rate of 20 μL/s, and the fluorescence was
monitored for an additional 2 min. Graphpad Prism 7 software was
used to analyze time and concentration response relationships.
Calcium oscillation data are a plot of the raw Fluo3 fluorescence reads
over time. With the calcium influx graph, Fluo3 fluorescence was
expressed as (Fmax − F0), where Fmax is the maximum and Fo is the
baseline line fluorescence measured in each well.32−34
ORCID
Marsha L. Pierce: 0000-0002-7265-0104
Chen Zhang: 0000-0003-4805-0392
Lena Gerwick: 0000-0001-6108-9000
William H. Gerwick: 0000-0003-1403-4458
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by NIH grant GM107550 (to
L.G. and W.H.G.). We acknowledge the Donald C. and
Elizabeth M. Dickinson Foundation for providing the JEOL
NMR spectrometer. We thank A. Mrse and B. Duggan for
facilitating acquisition of NMR data and Y. Su for acquiring
HRESIMS data at the University of California San Diego. We
appreciate G. Li for providing conformer and stereochemical
calculations at the Ocean University of China. Y.L. acknowl-
edges financial support provided by a fellowship from the
Ocean University of China, PR China.
The extract 2245CR as well as all nine VLC fractions 2245CR_A−I
were evaluated for cytotoxicity using NCI-H460 human large cell lung
carcinoma cells for 48 h at two concentrations (10 and 1 μg/mL)
using the MTT staining method for cytotoxicity.58 Pagoamide A was
screened in this assay system at 10 concentrations prepared by half-
logarithmic dilutions starting at 30 μM, run in duplicate. Doxorubicin
and 1% DMSO were used as positive and negative controls,
respectively. The optical density of the stained lysate was measured
at 570 and 630 nm.
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