Caldoramide, a Modified Pentapeptide from the Marine Cyanobacterium Caldora penicillata

Article abstract of DOI:10.1021/acs.jnatprod.6b00203

The isolation, structure determination, and biological activities of a new linear pentapeptide, caldoramide (5), from the marine cyanobacterium Caldora penicillata from Florida are described. Caldoramide (5) has structural similarities to belamide A (4), dolastatin 10 (1), and dolastatin 15 (2). We profiled caldoramide against parental HCT116 colorectal cancer cells and isogenic cells lacking oncogenic KRAS or hypoxia-inducible factors 1α (HIF-1α) and 2α (HIF-2α). Caldoramide (5) showed differential cytotoxicity for cells containing both oncogenic KRAS and HIF over the corresponding knockout cells. LCMS dereplication indicated the presence of caldoramide (5) in a subset of C. penicillata samples.

Full text of DOI:10.1021/acs.jnatprod.6b00203

Note
pubs.acs.org/jnp
Caldoramide, a Modified Pentapeptide from the Marine
Cyanobacterium Caldora penicillata
Sarath P. Gunasekera,† Lorelie Imperial,‡,§ Christiana Garst,† Ranjala Ratnayake,‡,§ Long H. Dang,‡,§,⊥
Valerie J. Paul,*,† and Hendrik Luesch*,‡,§
†Smithsonian Marine Station at Ft. Pierce, 701 Seaway Drive, Ft. Pierce, Florida 34949, United States
‡Department of Medicinal Chemistry, §Center for Natural Products, Drug Discovery and Development (CNPD3), and ⊥Department
of Medicine, University of Florida, Gainesville, Florida 32610, United States
S
* Supporting Information
ABSTRACT: The isolation, structure determination, and biological activities
of a new linear pentapeptide, caldoramide (5), from the marine
cyanobacterium Caldora penicillata from Florida are described. Caldoramide
(5) has structural similarities to belamide A (4), dolastatin 10 (1), and
dolastatin 15 (2). We profiled caldoramide against parental HCT116
colorectal cancer cells and isogenic cells lacking oncogenic KRAS or
hypoxia-inducible factors 1α (HIF-1α) and 2α (HIF-2α). Caldoramide (5)
showed differential cytotoxicity for cells containing both oncogenic KRAS and
HIF over the corresponding knockout cells. LCMS dereplication indicated
the presence of caldoramide (5) in a subset of C. penicillata samples.
Here we report caldoramide (5), a new, closely related
structural analogue of belamide A and dolastatin 15 (2), from
the cyanobacterium Caldora penicillata. The taxonomy of this
species has recently been clarified.12 We have also detected via
our dereplication method13 that various geographically
dispersed collections of C. penicillata consistently yielded
known compounds such as dolastatin 10 (1) and/or the
related homologue symplostatin 1 (6); these metabolites can
serve as chemotaxonomic markers for this taxon.12,13 In
addition to caldoramide (5), largazole14,15 and dolastatin 10
(1)15 were isolated from this sample. Cytotoxicity profiling of
caldoramide using three isogenic colorectal cancer cell lines16,17
revealed preferential targeting of oncogenic KRAS and hypoxia-
inducible factor (HIF) pathways.
The wet sample of C. penicillata, a re-collection from the
Florida Keys, was stored frozen and freeze-dried prior to
solvent extraction. The freeze-dried material was extracted first
with EtOAc−MeOH (1:1) and then with EtOH−H2O (1:1) to
give the nonpolar and polar extracts, respectively. The two
extracts were further partitioned to give EtOAc-soluble, n-
BuOH-soluble, and H2O-soluble fractions. The EtOAc-soluble
fraction was subjected to further purification via repeated
chromatography to give a new compound, caldoramide (5), as
well as the known cytotoxic compounds largazole (3) and
dolastatin 10 (1).
rganisms from the largely unexplored marine environ-
Oment have proven to be a rich source of secondary
metabolites that are potential therapeutics. Of these, the
benthic marine cyanobacteria have been reported to be a source
of compounds rich in diversity and biological activity.1−3
Cyanobacteria inhabit diverse environments, which could
explain the chemical diversity of the secondary metabolites
that have been isolated from these marine organisms. Among
the compounds that showed interesting biological activities
were the small peptides and depsipeptides collectively named
dolastatins, which were originally isolated from the sea hare
Dolabella auricularia.4 The most potent among the series of
dolastatins, dolastatin 10 (1), has been subsequently isolated
from the cyanobacterium Symploca sp. (now known as Caldora
penicillata) in a yield several magnitudes higher than from the
sea hare.5,6 Specifically, dolastatins 10 (1) and 15 (2) have
displayed extraordinary cytotoxicity to cancer cells.7,8 Their
interesting biological activities have triggered synthetic efforts,
resulting in several drug leads that advanced to clinical trials.9
Inspired by the structure of dolastatin 10, a related cytotoxin
served as the payload for the antibody−drug conjugate
brentuximab vedotin (Adcetris), approved for the treatment
of relapsed Hodgkin lymphoma and anaplastic large-cell
lymphoma.10 Largazole (3), a histone deacetylase (HDAC)
inhibitor, has also been isolated from the cyanobacterium
Symploca (now C. penicillata). Furthermore, a closely related
structure to the dolastatins, belamide A (4), was reported as the
major metabolite of the Panamanian cyanobacterium Symploca
sp. and showed moderate cytotoxicity to the HCT116
colorectal cancer cell line.11
Caldoramide (5) was obtained as an optically active white
powder. HRESI/TOFMS supported the molecular formula of
Received: March 7, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00203
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
indicated the connection between these two amino acids.
HMBC correlations of NH-34 to C-30 (δC 53.6) and C-35 (δC
171.8) and H-36 to C-35 confirmed the connection between
the N,N-dimethylvaline and valine moieties. HMBC correla-
tions between H3-28 (N-Me, δH 3.10) and C-23 (δC 58.3) and
C-29 (δC 172.8) and NOESY correlations shown between H3-
28 and H-23 (δH 5.27) and H3-28 and H-30 (δH 4.81) of valine
linked the valine and N-Me-valine residues. The NOESY
correlations observed between H3-21 (δH 3.21) and H-15 (δH
6.45), H-16 (δH 2.09), and H-23 (δH 5.27) and HMBC
correlations between H3-21 and C-15 (δC 58.7) and C-22 (δC
171.2) connected the N-Me-valine to the N-Me-isoleucine,
which in turn is connected to the remaining C-terminal unit to
satisfy the molecular formula. These data established the planar
structure of caldoramide (5) as N,N-diMe-Val-Val-N-Me-Val-
N-Me-Ile-3-O-Me-4-benzylpyrrolinone.
The absolute configuration was determined by enantiose-
lective HPLC analysis. Determination of the configuration at C-
4 required ozonolysis of 5 prior to hydrolysis with 6 N HCl.
The ozonolysis18 prior to hydrolysis yielded free phenylalanine
in the hydrolysate.11 The retention times of the components of
the hydrolysate were compared to the amino acid standards and
indicated the L-configuration for all amino acids in caldoramide
(5).
Differential cytotoxicity profiling of isogenic knockouts of
cancer genes can be used to determine target pathway
specificity. Oncogenic KRAS and activated HIF pathways
have been linked to angiogenesis, cell growth, survival, and
metastasis.16,17,19 To test if caldoramide’s mechanism of action
involves these pathways, we used the parental HCT116
colorectal cancer cell line that contains one oncogenic KRAS
allele and one wild-type KRAS allele, HCT116 cells without the
oncogenic KRAS allele (HCT 116WT KRAS), and HCT116 cells
depleted of HIF-1α and HIF-2α (HCT116HIF‑1α−/−HIF‑2α−/−).16
In a recently reported gene expression profiling of isogenic HIF
and KRAS knockout cell lines, we showed that there is a
significant overlap between global gene expression affected by
oncogenic KRAS with genes affected by both HIF-1α and HIF-
2α.17 This signature overlap indicates that HIF transcriptional
activity is regulated by oncogenic KRAS. Caldoramide (5)
showed preferential antiproliferative activity against cells
containing oncogenic KRAS or HIF transcription factors, viz.,
parental HCT 116 over HCT116HIF‑1α−/−HIF‑2α−/− and over
HCT116WT KRAS (Figure 1). The potency of caldoramide in
HCT116HIF‑1α−/−HIF‑2α−/− was slightly decreased (IC50 shifts
from 3.9 μM to 5.2 μM) and maximum efficacy reduced to 67%
compared with parental HCT116 cells. The IC50 shift in
HCT116WT KRAS was more pronounced (3.9 μM to 8.6 μM),
while efficacy was unaffected.
The cytotoxicity profiles of both dolastatin 10 (1) and
largazole (3) have been previously determined using the same
cell lines. Knockout cells were much less susceptible to
dolastatin 10 with >50-fold shifts in IC50 values compared
with parental cells with concomitant reduced efficacy (Table
2).17 The HDAC inhibitor largazole also showed
differential cytotoxicity for parental HCT116 over
HCT116HIF‑1α−/−HIF‑2α−/−, but this difference in cytotoxicity is
only seen with HCT116HIF‑1α−/−HIF‑2α−/− and not in the cells
lacking oncogenic KRAS (Table 2).17 Caldoramide (5),
although structurally related, is less potent than dolastatin
1017 across the three cell lines investigated. Thus, all three
compounds have varying activity profiles against the HCT116
isogenic cell lines that were tested, suggesting that the dose−
C37H60N5O6. Following the interpretation of DQF COSY,
edited HSQC, and HMBC experiments (Table 1), the H and
1
13C NMR signals were assignable to one OMe group, N,N-
dimethylvaline (C-35 to C-42), valine (C-29 to N-34), N-Me-
valine (C-22 to C-28), N-Me-isoleucine (C-14 to C-21), a
putative phenylalanine (C-4 to C-12), and a conjugated enol
ether (C-1 to C-3). The remarkable chemical shifts recorded
for a methine carbon (C-2, δC 94.8; δH 4.80) adjacent to a
nonprotonated carbon (C-3, δC 178.1) suggested oxygenation
at the latter position. Further, HMBC correlations between H-2
(δH 4.80) and C-1 (δC 169.1) and C-4 (δC 59.7); H-4 (δH 4.83)
and C-2 (δC 94.8); and H2-6 (δH 3.30, 3.08) and C-3 (δC
178.1) were observed, and these data established a substituted
pyrrolinone ring (C-1 to C-5) similar to that reported for
belamide A (4).11 The HMBC correlation connected the OMe
(δH 3.72) to the C-3 (δC 178.1) position in the pyrrolinone
ring. Additional HMBC correlations from the H2-6 methylene
protons (δH 3.30, 3.08) to C-3 (δC 178.1), C-7 (δC 134.7), and
C-8/12 (δC 129.6) connected the benzyl moiety to the C-4
position of the pyrrolinone ring. These data supported a 3-
methoxy-4-benzylpyrrolinone moiety present at the C-
terminus, and therefore, it was apparent that the N,N-
dimethylvaline residue formed the N-terminus. Sequencing of
the remaining three amino acid residues was accomplished by
analyzing the HMBC and NOESY data (Table 1). NOESY
correlations of H-34 (N−H, δH 6.92) to H-36 (δH 2.44) and
H3-41/42 (N,N-dimethyl, δH 2.23) of the N,N-dimethylvaline
and to H-30 (δH 4.81) and H-31 (δH 1.96) of the valine moiety
B
DOI: 10.1021/acs.jnatprod.6b00203
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) for Caldoramide (5)
a
b
c
unit
C/H no.
δC mult.
δH (J in Hz)
COSY
HMBC
1, 4
3, 7
NOESY
Bn-O-Me-pyrrolinone
1
169.1, C
94.8, CH
178.1, C
59.7, CH
2
4.80, s
13
6
3
4
4.83, dd (4.8, 3.4)
6
N-5
6
35.6, CH2
3.30, dd (13.7, 4.8)
3.08, dd (13.7, 3.4)
4
4
3, 4, 7, 8/12
3, 4, 7, 8/12
4, 8/12
4, 8/12
7
134.7, C
8/12
9/11
10
129.6, CH
128.3, CH
127.0, CH
58.4, CH3
172.2, C
7.00, dd (7.5, 1.3)
7.20, m
9/11
6, 10
7
4, 11
8/12, 10
9/11
7.14, m
8/12
3
13
3.72, s
2, 8/12
N-Me-Ile
14
15
58.7, CH
34.3, CH
25.1, CH2
6.45, d (10.9)
2.09, m
16
14, 17, 19, 21
15, 17, 18
17, 19
16
15, 17, 19
16, 18
16, 18
17
17, 18, 19
15, 16, 18
15, 16, 18
16, 17
17
1.26, m
15, 16, 18, 19
15, 16, 18, 19
16, 17
1.10, m
18
10.6, CH3
14.5, CH3
0.84, t (7.5)
0.89, d (6.8)
19
16
15, 16, 17
15, 16
N-20
21
31.8, CH3
171.2, C
3.21, s
15, 22
15, 16, 23
N-Me-Val
22
23
58.3, CH
27.2, CH
19.6, CH3
18.3, CH3
5.27, d (10.3)
2.38, m
24
22, 24, 25, 28
22, 23, 25
23, 24, 26
23, 24, 25
21, 25, 26, 28
23, 25, 26, 28
23, 24, 28
24
23, 25, 26
25
0.89, d (6.9)
0.75, d (6.9)
24
24
26
23, 24, 28,
N-27
28
30.5, CH3
172.8, C
3.10, s
23, 29
23, 24, 30, 31
Val
29
30
53.6, CH
31.0, CH
17.8, CH3
19.9, CH3
4.81, dd (8.9, 3.4)
1.96, m
31, 34
30, 32, 33
31
29, 31, 32, 33
29, 30, 32, 33
30, 31, 33
30, 31, 32
30, 35
28, 31, 32, 34
30, 32, 33, 34
30, 31, 34
31
32
0.93, d (6.9)
0.98, d (6.9)
6.92, d (8.9)
33
31
30, 31
NH-34
35
30
30, 31, 36, 41/42
N,N-diMe-Val
171.8, C
36
76.6, CH
27.7, CH
20.2, CH3
17.7, CH3
2.44, d (6.2)
2.08, m
37
35, 38, 39, 41
35, 36, 38, 39
36, 37, 39
34, 37, 38, 41/42
36, 38, 39, 41/42
36, 37, 41/42
37
36, 38, 39
38
0.99, d (6.9)
0.92, d (6.9)
37
37
39
36, 37, 38
36, 37, 41/42
40-N
41
43.0, CH3
43.0, CH3
2.23, s
36
34, 36, 38, 39
34, 36, 38, 39
42
2.23, s
36
a
b
2/3
COSY correlations are from proton(s) stated to the indicated proton(s). HMBC correlations, optimized for
J
= 8 Hz, are from proton(s)
CH
c
stated to the indicated carbon. NOESY correlations are from proton(s) stated to the indicated proton(s).
response analyses of these cell lines might be used as a platform
to group antiproliferative compounds by mechanism.
Extracts, both nonpolar and polar, for the different
collections of cyanobacterial samples were eluted with MeOH
through C18 solid phase extraction columns to remove
interference from the polar compounds for HPLC-MS analysis.
Detection of known compounds has provided a guide into
prioritization of extracts at the early stage for further
purification. The sensitive, reliable, and fast HPLC tandem
mass spectrometry fragmentation (HPLC-MS/MS) was used
to screen several extracts that were collected from various sites
for the presence of known compounds. We monitored the
parent ion and the most stable fragment ion to detect the
presence of each compound. Using this technique, we have
simultaneously scanned for the presence of dolastatin 10 (1),
Figure 1. Effects of caldoramide (5) on cell viability of isogenic
HCT116 cell lines.
C
DOI: 10.1021/acs.jnatprod.6b00203
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Table 2. Comparison of Cytotoxicity Profiles of the Three Coproduced Metabolites against Isogenic HCT116 Cells
IC50
efficacy relative to parental HCT116, %
oncogenic KRAS ko
oncogenic KRAS ko
compound
parental HIF-1α−/−2α−/−
(HCT116WT KRAS
)
parental HIF-1α−/−2α−/−
(HCT116WT KRAS
100
)
caldoramide (5)
dolastatin 10 (1)
3.9 μM
5.2 μM
65 nM
18 nM
8.6 μM
24 nM
3.7 nM
100
100
100
67
74
70
a
0.48 nM
6.41 nM
83
67
a
largazole (3)
a
Values taken from ref 17.
1
MHz for H and 150.9 MHz for 13C. The edited-HSQC experiment
symplostatin 1 (6), largazole (3), and caldoramide (5) in an
array of extracts and partitions from various collections of
cyanobacterial samples (Figure 2). Most samples contained the
was optimized for JCH = 140 Hz, and the HMBC spectrum was
optimized for
2/3
1
J
= 8 Hz. H NMR chemical shifts (referenced to
CH
residual CHCl3 observed at δH 7.25) were assigned using a
combination of data from 2D DQF COSY and multiplicity-edited
HSQC experiments. Similarly, 13C NMR chemical shifts (referenced to
CDCl3 observed at δC 77.0) were assigned on the basis of multiplicity-
edited HSQC experiments. The HRMS data were obtained using an
Agilent 6210 LC-TOF mass spectrometer equipped with an APCI/ESI
multimode ion source detector at the Mass Spectrometer Facility at
the University of California, Riverside, California. Silica gel 60 (EMD
Chemicals, Inc. 230−400 mesh) was used for column chromatog-
raphy. All solvents used were of HPLC grade (Fisher Scientific).
Collection, Extraction, and Isolation. The sample of cyano-
bacterial assemblage of Caldora penicillata for this study was collected
from Big Pine Ledges (24.55302 N × 81.38028 W), a reef site near Big
Pine Key, Florida, on March 30, 2015. This sample was a re-collection
of FK 12-18, collected at the same location and well characterized by
both microscopic and molecular methods in our recent paper that
described this species,12 and was consistent with the prior collection
morphologically and chemically.12,13 A voucher specimen of the re-
collection (FK 15-1) is maintained at the Smithsonian Marine Station,
Fort Pierce, FL. The freeze-dried material (96.6 g) was first extracted
with EtOAc−MeOH (1:1) followed by H2O−EtOH (1:1). The
combined lipophilic and polar extract was partitioned between EtOAc
and H2O, and the aqueous portion subsequently partitioned between
n-BuOH and H2O. Concentration of these extracts furnished 0.775 g
(0.8%) of an EtOAc-soluble fraction and 2.275 g (2.3%) of a BuOH-
soluble fraction. The EtOAc-soluble fraction (0.760 g) was purified on
a column of SiO2 (25 g) using a step gradient system of hexanes−30%
EtOAc, EtOAc, EtOAc−10% MeOH, and MeOH to give four
subfractions (1a−1d). Subfraction 1b (0.106 g), eluting with EtOAc,
was further chromatographed on a column of C18 silica (7 g) using a
step gradient system of MeOH−10% H2O, MeOH, and EtOAc to give
three subfractions. The first subfraction (0.068 g), eluting with
MeOH−10% H2O, was rechromatographed on a Si column (6 g)
using a step gradient system of hexanes−30% EtOAc, hexanes−60%
EtOAc, EtOAc, and MeOH to give another four subfractions. The last
two subfractions were combined (0.053 g) and pushed through a SiO2
Sep-Pak with EtOAc followed by MeOH. Fraction 1 (0.019 g), eluting
with EtOAc, was further purified by reversed-phase HPLC (semi-
preparative 250 × 10 mm, 5 μm, RP-18, flow 3.0 mL/min) using
MeOH−8.5% H2O to give the known compound largazole (3) (tR =
8.8 min, 2.0 mg yield, 0.002% dry wt) and caldoramide (5) (tR = 9.4
min, 5.9 mg yield, 0.006% dry wt). Subfraction 1d (0.194 g), eluting
with MeOH, was separated on a column of C18 (12 g) using a step
gradient system of MeOH−H2O followed by MeOH to give four
subfractions. Subfraction 2 (6.5 mg), eluting with MeOH−10% H2O,
was further purified by reversed-phase HPLC (semipreparative 250 ×
10 mm, 5 μm, RP-18, flow 3.0 mL/min) using MeOH−23% H2O to
give the known compound dolastatin 10 (1) (1.3 mg yield, tR = 24
min, 0.001% dry wt). The two known compounds largazole14 and
dolastatin 1015 were identified by LCMS data, and their structures
Figure 2. Distribution of the three structural classes of compounds
isolated from marine cyanobacteria from Florida.
metabolites dolastatin 10 (1) and/or the structurally related
symplostatin 1 (6), as well as largazole (3). Furthermore, we
found that in the extracts tested, caldoramide (5) did not elute
alone, but has been consistently coproduced with dolastatin 10
(1) or symplostatin 1 (6). This characteristic has been shown
by largazole (3), which was reported to be coproduced with the
same metabolites.13 Lastly, from the extracts tested, caldor-
amide (5) was detected in four samples, all of which belong to
C. penicillata. This suggests that the presence of caldoramide
(5) is genus or species specific.
We have described the isolation of a modified peptide,
caldoramide (5), from the marine cyanobacterium C. penicillata.
Our tandem MS/MS method has revealed that this compound,
like largazole, is coproduced with dolastatin 10 (1) and/or its
homologue symplostatin 1 (6). While only moderately
cytotoxic, similar to belamide A (4), we have shown that
caldoramide (5) is able to inhibit the growth of cells that have
both oncogenic KRAS and HIF-1α and HIF-2α transcription
factors (parental HCT 116) to a greater extent than cells
lacking those genes (HCT116HIF‑1α−/−HIF‑2α−/− and
HCT116WT KRAS). A related yet not identical selectivity profile
has been determined for dolastatin 10 (1) and largazole (3).17
Consequently, all three coproduced compounds derived from
the same extract appear to act as indirect HIF inhibitors and
exert different profiles, suggesting complementary mechanisms.
1
1
EXPERIMENTAL SECTION
were confirmed by comparing H NMR spectra with the H NMR
■
spectra published in the literature.
General Experimental Procedures. The optical rotations were
recorded on a Rudolph Research Analytical Autopol III automatic
polarimeter. UV spectrophotometric data was acquired on a Shimadzu
PharmaSpec UV−visible spectrophotometer. NMR data were
collected on a JEOL ECA-600 spectrometer operating at 600.17
Caldoramide (5): colorless, amorphous powder; [α]25 +11.1 (c
D
1
0.36, MeOH); UV (MeOH) λmax (log ε) 240 (4.19) nm; H and 13C
NMR data, Table 1; HRESI/TOFMS m/z 670.4527 [M + H]+ (calcd
for C37H60N5O6, 670.4538).
D
DOI: 10.1021/acs.jnatprod.6b00203
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Ozonolysis, Acid Hydrolysis, and Enantioselective HPLC
Analysis. Compound 5 (1.0 mg) was dissolved in 1.5 mL of CH2Cl2
and ozonized at −78 °C. The solvent was evaporated, and the product
was suspended in 6 N HCl (0.5 mL) and heated at 110 °C for 12 h in
a sealed tube. The hydrolysate was concentrated to dryness. The
residue was reconstituted in 0.2 mL of H2O and analyzed by
enantioselective HPLC, comparing the retention times with those of
authentic standards [Phenomenex Chirex (D) penicillamine, 4.6 × 250
mm, 5 μm; solvent 2.0 mM CuSO4 and mixtures of 2.0 mM CuSO4−
CH3CN (95:5 or 92:8); detection at 254 nm]. Using 2.0 mM CuSO4
with a flow rate of 0.8 mL/min, the retention times (tR min) for
authentic standards were N,N-diMe-L-Val (11.5) and N,N-diMe-D-Val
(14.4), and with a flow rate of 1.0 mL/min, the retention times (tR
min) for authentic standards were N-Me-L-Val (19.5) and N-Me-D-Val
(31.6). Using 2.0 mM CuSO4−CH3CN (95:5) with a flow rate of 1.0
mL/min, the retention times (tR min) for authentic standards were L-
Val (14.4) and D-Val (19.4) and with a flow rate of 0.8 mL N-Me-L-
allo-Ile (25.3), N-Me-D-allo-Ile (37.1), and N-Me-L-Ile (29.1). N-Me-D-
Ile was not available for this analysis. N-Me-D-Ile and N-Me-D-allo-Ile
are reported to have close retention times.18 No peak appeared close
to the retention time of 37 min in the hydrolysate. Using 2.0 mM
CuSO4−CH3CN (92:8) with a flow rate of 1.0 mL/min, the retention
times (tR min) for authentic standards were L-Phe (80.3) and D-Phe
(84.5). The absolute configurations of the amino acid moieties in the
hydrolysate were confirmed by comparing the retention times in their
respective chromatography traces as N,N-diMe-L-Val (11.5), N-Me-L-
Val (19.5), L-Val (14.4), N-Me-L-Ile (29.1), and L-Phe (80.3).
HPLC-MS Profiling. Cyanobacteria samples that were previously
fractionated using 100% MeOH through a C18 solid phase extraction
column were reconstituted in MeOH to 1 mg/mL concentration.
Samples for analysis were diluted to 10 000 ng/mL in MeCN from the
methanolic stock solutions. For each sample, 10 μL was injected and
analysis was done using the following conditions: (1) HPLC: 0.1%
HCOOH in MeCN−0.1% HCOOH in H2O [20−100% MeCN for 25
min followed by 100% MeCN for 5 min, flow rate 0.25 mL/min, on a
Hypersil GOLD aQ C18 polar end-capped LC column]; (2) MS and
MS/MS: Thermo Fisher LCQ-Deca system with ESIMS in positive
ion mode and CID 35%. Detection of largazole (3), symplostatin 1
(6), and dolastatin 10 (1) using MRM was previously reported.13
Cell Viability Assay. Human colorectal cancer cell lines HCT116,
HCT116HIF‑1α−/−HIF‑2α−/−, and HCT 116WT KRAS were cultured in
Dulbecco’s modified Eagle medium supplemented with 10% fetal
bovine serum and maintained in 5% CO2 at 37 °C. The cells were
seeded in a 96-well plate (10 000 cells/well) and treated with different
concentrations of 5 after 24 h. The cell viability was measured 48 h
following treatment with MTT dye using the manufacturer’s protocol
(Promega).
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health, NCI grant R01CA172310. Financial support from the
Smithsonian Competitive Grants Program for Science
supported collection and an internship for C.G. We thank
the Harbor Branch Oceanographic Institute at Florida Atlantic
University spectroscopy facility for 600 MHz NMR spectrom-
eter time and optical rotation measurements. The high-
resolution mass spectrometric analysis was performed by the
UCR mass spectrometer facility, Department of Chemistry,
University of California at Riverside. We are grateful to E.
Bartels and the staff at the Mote Tropical Research Laboratory
for assistance with collections. E. Bartels also kindly provided
the photograph for the TOC graphic. This is contribution
number 1036 from the Smithsonian Marine Station at Fort
Pierce.
REFERENCES
■
(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.;
Prinsep, M. R.; Northcote, P. T.; Prinsep, M. R. Nat. Prod. Rep. 2014,
31, 160−258.
(2) Tan, L. T. Phytochemistry 2007, 68, 954−979.
(3) Van Wagoner, R.; Drummond, A. K.; Wright, J. L. C. Adv. Appl.
Microbiol. 2007, 61, 89−217.
(4) Yamada, K.; Kigoshi, H. Bull. Chem. Soc. Jpn. 1997, 70, 1479−
1489.
(5) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Fujii, Y.; Kizu, H.; Boyd,
M.; Boettner, F. E.; Doubek, D. L.; Schmidt, J. M.; Chapuis, J. C.
Tetrahedron 1993, 49, 9151−9170.
(6) Luesch, H.; Moore, R. E.; Paul, V. J.; Mooberry, S. L.; Corbett, T.
H. J. Nat. Prod. 2001, 64, 907−910.
(7) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A.; Boettner,
F. E.; Kizu, H.; Schmidt, J. M.; Baczynskyj, L.; Tomer, K. B.; Bontems,
R. J. J. Am. Chem. Soc. 1987, 109, 6883−6885.
(8) Pettit, G. R.; Kamano, Y.; Dufresne, C.; Cerny, R. L.; Herald, C.
L.; Schmidt, J. M. J. Org. Chem. 1989, 54, 6005−6006.
(9) Flahive, E.; Srirangam, J. In Anticancer Agents from Natural
Products; Cragg, G. M.; Kingston, D. G. I.; Newman, D. J., Eds.; CRC
Press, Taylor and Francis Group: Boca Raton, FL, 2005; Chapter 11,
pp 191−213.
(10) Senter, P. D.; Sievers, E. L. Nat. Biotechnol. 2012, 30, 631−637.
(11) Simmons, T. L.; McPhail, K. L.; Ortega-Barría, E.; Mooberry, S.
L.; Gerwick, W. H. Tetrahedron Lett. 2006, 47, 3387−3390.
(12) Engene, N.; Tronholm, A.; Salvador-Reyes, L. A.; Luesch, H.;
Paul, V. J. J. Phycol. 2015, 51, 670−681.
(13) Salvador-Reyes, L. A.; Engene, N.; Paul, V. J.; Luesch, H. J. Nat.
Prod. 2015, 78, 486−492.
ASSOCIATED CONTENT
■
(14) Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008, 130,
1806−1807.
S
* Supporting Information
(15) Salvador-Reyes, L. A.; Luesch, H. Nat. Prod. Rep. 2015, 32,
478−503.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00203.
(16) Burkitt, K.; Chun, S. Y.; Dang, D. T.; Dang, L. H. Mol. Cancer
Ther. 2009, 8, 1148−1156.
1H, 13C, COSY, HMBC, 2D NOESY, and HSQC NMR
(17) Bousquet, M. S.; Ma, J. J.; Ratnayake, R.; Havre, P. A.; Yao, J.;
Dang, N. H.; Paul, V. J.; Carney, T. J.; Dang, L. H.; Luesch, H. ACS
Chem. Biol. 2016, 11, 1322−1331.
spectra of caldoramide (5) in CDCl3 (PDF)
(18) Taori, K.; Liu, Y.; Paul, V. J.; Luesch, H. ChemBioChem 2009,
10, 1634−1639.
AUTHOR INFORMATION
■
(19) Talks, K. L.; Turley, H.; Gatter, K. C.; Maxwell, P. H.; Pugh, C.
W.; Ratcliffe, P. J.; Harris, A. L. Am. J. Pathol. 2000, 157, 411−421.
Corresponding Authors
*Tel (V. J. Paul): (772) 462-0982. Fax: (772) 461-8154. E-
mail: paul@si.edu.
*Tel (H. Luesch): (352) 273-7738. Fax: (352) 273-7741. E-
mail: luesch@cop.ufl.edu.
Notes
The authors declare no competing financial interest.
E
DOI: 10.1021/acs.jnatprod.6b00203
J. Nat. Prod. XXXX, XXX, XXX−XXX