Calothrixamides A and B from the Cultured Cyanobacterium Calothrix sp. UIC 10520

Article abstract of DOI:10.1021/acs.jnatprod.8b00432

Cyanobacteria are a source of chemically diverse metabolites with potential medicinal and biotechnological applications. Rapid identification of compounds is central to expedite the natural product discovery process. Mass spectrometry has been shown to be an important tool for dereplication of complex natural product samples. In addition, chromatographic separation and complementary spectroscopic analysis (e.g., UV) can enhance the confidence of the dereplication process. Here, we applied a droplet-liquid microjunction-surface sampling probe (droplet probe) coupled with UPLC-PDA-HRMS-MS/MS to identify two new natural products in situ from the freshwater strain Calothrix sp. UIC 10520. This allowed us to prioritize this strain for chemical investigation based on the presence of new metabolites very early in our discovery process, saving both time and resources. Subsequently, calothrixamides A (1) and B (2) were isolated from large-scale cultures, and the structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configurations were determined by a combination of chemical degradation reactions, derivatization methods (Mosher's, Marfey's, and phenylglycine methyl ester), and J-based configurational analysis. Calothrixamides showed no cytotoxic activity against the MDA-MB-435, MDA-MB-231, and OVCAR3 cancer cell lines. They represent the first functionalized long-chain fatty acid amides reported from the Calothrix genus and from a freshwater cyanobacterium.

Full text of DOI:10.1021/acs.jnatprod.8b00432

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Calothrixamides A and B from the Cultured Cyanobacterium
Calothrix sp. UIC 10520
Camila M. Crnkovic,†,‡ Aleksej Krunic,† Daniel S. May,† Tyler A. Wilson,§ Diana Kao,⊥
Joanna E. Burdette,† James R. Fuchs,§ Nicholas H. Oberlies,⊥ and Jimmy Orjala*,†
†Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois
60612, United States
‡CAPES Foundation, Ministry of Education of Brazil, Brasília, Federal District 70040-020, Brazil
§Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
United States
⊥Department of Chemistry & Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27402,
United States
S
* Supporting Information
ABSTRACT: Cyanobacteria are a source of chemically diverse
metabolites with potential medicinal and biotechnological
applications. Rapid identification of compounds is central to
expedite the natural product discovery process. Mass spectrom-
etry has been shown to be an important tool for dereplication of
complex natural product samples. In addition, chromatographic
separation and complementary spectroscopic analysis (e.g., UV)
can enhance the confidence of the dereplication process. Here,
we applied a droplet-liquid microjunction-surface sampling
probe (droplet probe) coupled with UPLC-PDA-HRMS-MS/
MS to identify two new natural products in situ from the
freshwater strain Calothrix sp. UIC 10520. This allowed us to
prioritize this strain for chemical investigation based on the
presence of new metabolites very early in our discovery process, saving both time and resources. Subsequently, calothrixamides
A (1) and B (2) were isolated from large-scale cultures, and the structures were elucidated by 1D and 2D NMR spectroscopy
and mass spectrometry. The absolute configurations were determined by a combination of chemical degradation reactions,
derivatization methods (Mosher’s, Marfey’s, and phenylglycine methyl ester), and J-based configurational analysis.
Calothrixamides showed no cytotoxic activity against the MDA-MB-435, MDA-MB-231, and OVCAR3 cancer cell lines.
They represent the first functionalized long-chain fatty acid amides reported from the Calothrix genus and from a freshwater
cyanobacterium.
logical samples represent a chemical mixture of many
compounds in varying concentrations, whose analysis requires
sensitive techniques with high resolving power and dynamic
range. In this context, mass spectrometry (MS) has been
established as an important tool in the natural product
discovery workflow.10−12 For complex mixtures, the resolving
power of MS can be further increased by hyphenation with
chromatographic separation. In addition, high-resolution mass
spectrometry (HRMS) and tandem mass spectrometry (MS/
MS) can be used to identify and dereplicate natural
products.13,14
yanobacteria continue to be a promising source of natural
1,2
Cproducts with great chemical diversity.
Many cyano-
bacterial metabolites have been shown to have important
ecological functions3 or therapeutically relevant activities, such
as antiproliferative, antimicrobial, anti-inflammatory, enzyme
inhibition, and antiviral activities.4,5 Bioassay-guided fractiona-
tion has been used traditionally to discover new bioactive
compounds from various natural sources, including cyanobac-
teria. More recently, omics-based approaches are expanding
the field of natural product discovery.6
Genomic and metagenomic studies constantly reveal the
untapped biosynthetic potential of microorganisms. Genome
mining combined with gene activation in native hosts and
expression in heterologous hosts have been shown to be
powerful strategies for the discovery of natural products.7,8
Complementarily, metabolomics is used to detect the actual
production of metabolites under specific conditions.9 Bio-
Dereplication is a vital step for modern natural product
discovery platforms, and applying it as early as possible can
greatly expedite the discovery process.15 For instance,
Received: May 29, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00432
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a
Table 1. NMR Spectroscopic Data of Calothrixamides A (1) and B (2), CDCl3
calothrixamide A (1)
calothrixamide B (2)
δH, mult (J in Hz) COSY
position
δC, mult
δH, mult (J in Hz)
COSY
HMBC
δC, mult
HMBC
1
2
3
170.1, C
128.9, C
170.1, C
128.9, C
129.8, CH 7.26, ddq (11.8, 1.3, 4, 25
0.9)
122.1, CH 6.18, ddd (11.8,
10.9, 0.9)
1, 2, 4, 5, 25
2, 5, 6
129.8 CH 7.26, ddq (11.8, 1.3, 4, 25
0.9)
122.1, CH 6.18, ddd (11.8,
10.9, 0.9)
1, 2, 4, 5, 25
2, 5, 6, 25
4
3, 5
3, 5
5
6
7
145.6, CH 5.54, t (10.6)
4, 6
5, 7, 24
6
1, 3, 6, 7, 24
4, 5, 7, 8
145.6, CH 5.54, t (10.6)
32.7, CH 2.73, m
37.4, CH2 1.22, m
1.34, m
4, 6
5, 7, 24
6
3, 6, 7, 24
4, 5, 7, 8
32.7, CH
2.73, m
37.4, CH2 1.22, m
1.34, m
8
9
10
11
12
13
27.5, CH2 1.22, m
29.8, CH2 1.24, m
29.6, CH2 1.24, m
29.6, CH2 1.24, m
29.7, CH2 1.24, m
25.8, CH2 1.25, m
1.29, m
27.5, CH2 1.22, m
29.8, CH2 1.24, m
29.6, CH2 1.24, m
29.6, CH2 1.24, m
29.7, CH2 1.24, m
25.8, CH2 1.25, m
1.29, m
14
15
31.6, CH2 1.46, m
1.51, m
15
13, 15
31.6, CH2 1.46, m
1.51, m
78.6, CH 4.66, dt (8.7, 4.3)
15
13,15
78.5, CH
4.66, dt (8.7, 4.3)
14, 16
13, 16, 17, 23,
−OCONH2
14, 16
13, 16, 17, 23,
−OCONH2
16
17
36.5, CH
32.8, CH2 1.11, m
1.38, m
1.61, m
15, 17, 23
16
17, 23
15, 16, 18, 23
36.5, CH 1.61, m
32.9, CH2 1.10, m
1.37, m
15, 17, 23
16
15, 17, 18, 23
15, 16, 18, 23
18
19
20
26.8, CH2 1.35, m
1.37, m
29.3, CH2 1.37, m
1.40, m
33.9, CH2 2.03, m
2.04, m
27.3, CH2 1.26, m
1.32, m
29.7, CH2 1.24, m
21
18, 19, 21,
22
32.0, CH2 1.25, m
21
22
139.2, CH 5.80, ddt (17.1, 10.2, 20, 22
6.7)
19, 20
22.8, CH2 1.29, m
22
21
20, 22
20, 21
114.4,
CH2
4.93, ddt (10.2, 2.4, 20, 21
1.2)
20, 21
14.3, CH3 0.87, t (7.1)
4.99, ddt (17.1, 2.1,
1.6)
23
24
25
1’
14.5, CH3 0.87, d (6.8)
21.3, CH3 0.98, d (6.6)
12.9, CH3 1.95, d (1.3)
16
6
3
15, 16, 17
5, 6, 7
1, 2, 5
2′, 3′
14.5, CH3 0.86, d (6.8)
21.3, CH3 0.98, d (6.6)
12.9, CH3 1.95, d (1.3)
48.3, CH 4.16, m
16
6
3
15, 16, 17
5, 6, 7
1, 2
48.3, CH
4.16, m
2′, 3′,
2′, 3′,
2′, 3′
−NH−
−NH−
2’
67.6, CH2 3.58, ddd (10.9, 6.2, 1′, −OH
1′, 3′
67.6, CH2 3.58, ddd (10.9, 6.2, 1′, −OH
1′, 3′
4.9)
4.8)
3.72, ddd (10.9, 6.0,
3.5)
3.72, ddd (10.9, 5.8,
3.5)
3′
−OH
17.3, CH3 1.24, d (6.9)
3.17, dd (5.9, 4.9)
1′
2′
1′
1′, 2′
1, 1′
17.3, CH3 1.24, d (6.9)
3.18, dd (5.7, 4.8)
1′
2’
1’
1′, 2′
1, 1′
−NH−
−OCONH2 157.4, C
5.90, d (7.0)
4.61, br
5.91, d (7.0)
4.61, br
157.4, C
a
Frequency of 900 MHz for H and 226 MHz for 13C.
1
dereplication of microbial samples in situ can significantly
reduce the time spent in sample preparation, with the
additional benefit of providing information about the spatial
distribution of metabolites. Studies have investigated the use of
ambient ionization MS techniques, such as desorption
electrospray ionization (DESI) and direct analysis in real
time (DART), for the dereplication of microbial natural
products.16,17 With a broad use in metabolite imaging, ambient
ionization MS techniques display two major disadvantages for
dereplication: absence of chromatographic separation and lack
of mutually supportive spectroscopic data, e.g., UV. Chroma-
tography allows for the determination of retention time and
identification of isomers, while additional spectroscopic data
increase the confidence of the dereplication process. The
droplet-liquid microjunction-surface sampling probe (droplet
probe) combines the advantages of in situ analysis with the
capabilities of chromatography hyphenated to analytical
techniques.18 In this study, we used a droplet probe coupled
to a UPLC-UV-HRMS-MS/MS system to dereplicate
cyanobacterial compounds in situ.
B
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Previously used to analyze human tissue,19 plant samples,20
and fungal cultures,18,21−25 the droplet probe has been shown
to be a robust tool for analyzing chemistry in situ, and it has the
advantages of requiring little, if any, sample preparation and
can be applied to irregular surfaces. In short, it consists of a
sampling needle that is positioned millimeters above the
surface to be sampled. It dispenses a droplet of solvent that
forms a microjunction with the sample surface, performing a
microextraction, which is directed to the analytical system.
Using this technique, two new compounds, calothrixamides A
(1) and B (2), were identified in the cyanobacterial strain
Calothrix sp. UIC 10520.
molecular formula. Investigation of the 1H NMR and
multiplicity-edited HSQC spectra (Table 1) revealed six
olefinic protons (methine δH 7.26, 6.18, 5.80, and 5.54;
terminal methylene δH 4.99 and 4.93), four heteroatom-
bearing protons (methine δH 4.66 and 4.16; methylene δH 3.72
and 3.58), and two aliphatic methine protons (δH 2.73 and
1.61). It also confirmed 12 aliphatic methylene groups and four
methyl groups (δH 1.95, 1.24, 0.98, and 0.87), the most
deshielded one consistent with a vinyl methyl group. Together,
these data accounted for three out of the five degrees of
unsaturation. Four protons attached to heteroatoms were also
observed (1H δH 5.90, 2H δH 4.61, and 1H δH 3.17). Finally,
two carbonyl groups (δC 170.1 and 157.4) accounted for the
two missing degrees of unsaturation.
Interpretation of the COSY spectrum allowed the assembly
of four fragments that were connected based on HMBC
correlations (Figure 1). C-1′, C-2′, and C-3′ (fragment 1)
RESULTS AND DISCUSSION
■
Strain UIC 10520 was identified to be a Calothrix sp. by
morphological characterization and phylogenetic analyses
using a partial 16S rDNA gene sequence (see Experimental
Section and Supporting Information S1 and S2). The strain
was grown on solid Z medium and was analyzed by droplet
probe UPLC-UV-HRMS-MS/MS. This revealed the presence
of two compounds of m/z values of 515.3817 and 517.3975
([M + Na]+), consistent with the formulas C29H52N2O4 and
C29H54N2O4, respectively. For both compounds, losses of
−CH3NO2 and −C3H9NO were detected in the MS2 spectra
(Supporting Information S3). Preliminary dereplication of
these chemistry data, all of which were acquired in situ, failed
to match this information with any known cyanobacterial
compound, inspiring us to proceed with the isolation and
characterization of these new chemical entities. Traditionally,
we spend 6−8 weeks growing 8 L cultures, which are then
lyophilized, extracted, and fractionated before we select strains
for natural products chemistry investigations.26 Using the in
situ analysis afforded by the droplet probe, we can detect the
presence of new compounds much earlier in the process and
prioritize these for evaluation.1,27 For this study we used 4-
week-old agar plates. However, it is worth noting that droplet
probe analysis of cyanobacteria can be performed after 10 to
15 days or as soon as there is microscopic growth on agar
plates.
Figure 1. Key 2D NMR correlations of calothrixamide A (1).
composed an alaninol moiety, with COSY correlations
extending to the exchangeable protons at δH 3.17 (−OH)
and 5.90 (−NH−). HMBC correlations indicated that the
−NH− group was linked to C-1 (δC 170.1), and C-1 was in
turn connected to the methyl diene fragment by a correlation
between C-1 and H3-25. H3-25 also showed an HBMC
correlation to C-2 and a COSY correlation to C-3. The
fragment from C-3 to C-7 (fragment 2) was constructed based
on the interpretation of COSY correlations. The 2E,4Z
configuration of the conjugated diene was assigned based on
NOE correlations between H-25 and H-4, H-4 and H-5, and
H-3 and H-6. This assignment was further supported by
3
proton coupling constants (3JH‑3,H‑4 = 11.8 Hz, JH‑4,H‑5 = 10.9
Hz). This fragment was extended based on an HMBC
correlation between H-6 and C-8. Further assignment of the
aliphatic chain was hampered by overlapping signals in the
COSY, TOCSY, and HMBC spectra. To overcome this, we
performed an 1H−13C HSQC-TOCSY experiment, which
revealed a correlation between H-6 and H-9.
Cell material from an 18 L culture of Calothrix sp. UIC
10520 was harvested, lyophilized, and extracted with CH2Cl2/
MeOH, 1:1. The concentrated extract was fractionated using
Diaion HP-20SS and a stepwise gradient of isopropyl alcohol
(IPA) and H2O, followed by automatic fractionation by RP-
HPLC. The HPLC fractions containing the target compounds
At the aliphatic terminus, fragment 4 (C-22 to C-20) was
assembled based on COSY correlations and further extended
to C-19 and C-18 based on HMBC. A key HMBC was
observed between H-17 and C-18, allowing the connection of
C-18 to fragment 3 (C-14 to C-17 and C-23). The chemical
shift of C-15 (δC 78.5) indicated a neighboring oxygen atom.
In addition, an HBMC between H-15 and the carbon signal at
δC 157.4 was observed. This moiety was finally assigned as a
carbamate ester based on the characteristic chemical shift of
the carbonyl at δC 157.4 ppm. The carbamate ester also
accounted for the broad proton signal at δH 4.61 integrating for
two protons and with no correlations in the HSQC spectrum.
The assignment of the broad proton signal as OCONH2 was
further supported by analysis of the base hydrolysis product of
1 and comparison with the intact compound in pyridine-d5
(see Experimental Section). Fragment 3 was extended to C-13
1
were analyzed by H NMR, which further supported that the
compounds were new. Compound purification by semi-
preparative RP-HPLC yielded calothrixamides A (1) and B
(2).
Calothrixamide A (1) was obtained as a pale orange oil.
HRMS data in both positive and negative modes (m/z
515.3817 [M + Na]+ and m/z 491.3857 [M − H]−) were
consistent with the formula C29H52N2O4, implying 5 degrees of
unsaturation. The DEPTQ spectrum contained three non-
protonated carbons, eight methine, 14 methylene, and four
methyl carbons, accounting for all 29 carbons suggested by the
C
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based on HMBC and to C-12 based on an HSQC-TOCSY
correlation between H-15 and H-12. Finally, the two missing
methylene groups (C-10 and C11) were assigned to the
aliphatic chain between fragments 2 and 3. This structure is
supported by the MS/MS data acquired in situ and used for
preliminary dereplication. The observed losses of −CH3NO2
and −C3H9NO are consistent with the fragmentation of the
carbamate ester and the alaninol groups, respectively.
Calothrixamide B (2) was also obtained as a pale orange oil.
Its molecular formula was deduced to be C29H54N2O4 (m/z
517.3981 [M + Na]+ and m/z 493.4015 [M − H]−), with one
fewer degree of unsaturation than 1. Again, losses consistent
with carbamate ester and alaninol features were observed in the
1
Figure 2. Δδ (δS − δR) values (ppm) for (a) (S)- and (R)-PGME
MS/MS spectrum of 2. Investigation of the DEPTQ and H
derivatives 3a and 3b and (b) (S)- and (R)-MTPA derivatives 5a and
5b.
NMR spectra indicated that both compounds were closely
related. The most striking difference was the lack of resonances
assigned as the terminal olefin in 1 (δC 139.2 and 114.4, δH
5.80, 4.99, and 4.93). Instead, spectroscopic data of 2 showed
aliphatic resonances consistent with a methylene and terminal
methyl groups (δC 22.8 and 14.3, δH 1.29 and 0.87). The
planar structure of 2 was elucidated by the analysis of COSY,
HSQC, HMBC, and HSQC-TOCSY spectra. Coupling
constants indicated the 2E,4Z configuration of the conjugated
diene (3JH‑3,H‑4 = 11.8 Hz, 3JH‑4,H‑5 = 10.9 Hz). Comparison of
the electronic circular dichroism (ECD) spectra of 1 and 2
suggested the same absolute configuration of both compounds
surrounding the conjugated diene (Supporting Information
S17). Comparable coupling constants and carbon chemical
shifts supported the same absolute configuration of both
compounds at C-15 and C-16.
of the six possible relative conformations for C-15 and C-16.
2,3
Additional
J
coupling constants and strong NOE
C,H
correlations between H-23/H-15 and H-15/H-16 supported
the assignment of 15R* and 16S* (Figure 3).
The absolute configuration of the alaninol moiety was
determined to be S by Marfey’s analysis of the acid hydrolysate
of 1 and comparison to authentic standards (Supporting
Information S18). To determine the absolute configuration of
the stereogenic center at C-6, we subjected 2 to ozonolysis and
oxidative workup followed by derivatization with (S)- or (R)-
phenylglycine methyl ester (PGME).28 Reactions were
performed on calothrixamide B (2) due to the absence of
the terminal alkene, which would create a second carboxylic
acid, potentially complicating the analysis. The PGME amides
3a [(S)-PGME derivative] and 3b [(R)-PGME derivative]
1
were isolated by HPLC and analyzed by H NMR. NMR
revealed the HPLC isolates to be enantiomerically enriched
mixtures, containing a 3:1 ratio of the expected product and its
respective enantiomer. This was due to the partial racemization
of PGME under the reaction conditions (Supporting
Information S19). The Δδ values obtained from the analysis
of 1H NMR spectra of 3a and 3b revealed the 6R configuration
(Figure 2a). The absolute configuration of C-15 was elucidated
by Mosher’s ester analysis. First, 1 was subjected to base
hydrolysis, yielding (2E,4Z)-15-hydroxy-2,6,16-trimethyldoco-
sa-2,4,21-trienoic acid (4). Next, 4 was esterified with (R)- and
(S)-MTPA-Cl at the C-15 hydroxy group to give the (S)- and
(R)-MTPA esters 5a and 5b, respectively. Δδ values extracted
from the comparison of 1H NMR spectra of 5a and 5b
indicated the configuration of C-15 to be 15R (Figure 2b).
Finally, the relative configuration between C-15 and C-16 was
Figure 3. Newman projections for C-16/C-15. All possible
conformations are presented along with predicted size of JC,H
coupling constants. Double arrows indicate NOE correlations.
Conformation highlighted by the box is consistent with experimental
values.
3
Calothrixamides as pure compounds or as a 1:1 mixture of 1
and 2 were found to be inactive against MDA-MB-435 human
melanoma, MDA-MB-231 breast cancer, and OVCAR3
ovarian epithelial cancer cell lines at 10 μM.
Long-chain fatty acid amides have been reported from
marine cyanobacteria. They include the malyngamides,30
pitiamides,31,32 grenadamides,33,34 jamaicamides,35 semiplena-
mides,36 besarhanamides,37 credneramides,38 kimbeamides,39
smenamides,40 parguerene,41 mooreamide A,42 columba-
established by J-based conformational analysis supported by
NOE correlations.29 The
J
coupling constants were
2,3
C,H
obtained from a phase-sensitive HMBC spectrum. The large
coupling constants between C-14 and H-16 (3JC‑14,H‑16 = 7.3
Hz) and C-17 and H-15 (3JC‑17,H‑15 = 6.6 Hz) indicated both
pairs to be in anti conformations. This allowed for only one out
D
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previously.18 Sampling spots were manually selected, and a 3 μL
droplet of 70:30 MeOH/H2O was applied as the extraction solvent.
This solvent system retains the droplet formation and integrity during
the in situ analysis and is compatible with LC-MS systems.18 The
droplet was allocated to the surface and drawn back into the sampling
syringe 5 times per spot before injection into the UPLC-HRMS
system. Additional UPLC-UV-HRMS experiments were performed at
UIC Research Resources Center on a Shimadzu LCMS-IT-TOF set
as follows: C18 column (2.1 × 50 mm × 1.7 μm), 0.5 mL/min mobile
phase flow rate, gradient of 50−100% B for 7 min with 1 min wash
and 2 min re-equilibration, PDA acquiring 190−450 nm at 4.17 Hz
sampling rate, HRMS acquisition in positive and negative modes from
150 to 3000 m/z.
Biological Material. Calothrix sp. UIC 10520 was obtained from
a sample collected in July 2015 at Dixon Page Park, Illinois (N
41°50′42″, W 89°29′54″). The unialgal strain UIC 10520 was
isolated by micropipet isolation techniques.54 For the in situ
metabolomic analysis, UIC 10520 was grown for 4 weeks in 60 ×
15 mm plates containing solid Z medium. To allow for the isolation of
1 and 2, strain was cultured for 6 weeks in 18 L of liquid Z medium55
(four 2.8 L Fernbach flasks, each filled with 2 L of medium, and one
13 L flask containing 10 L of medium). Cultures were kept at 22 °C,
under constant sterile aeration, and illuminated by fluorescent lights at
2.6 klx following an 18/6 h light/dark cycle. Cells were harvested by
centrifugation and lyophilized.
mides,43 laucysteinamide A,44 and others. These compounds
have been associated with biological activities, such as
insecticidal, cytotoxic, calcium and sodium channel modu-
lation, and cannabinoid receptor binding. The cytotoxic
nocuolin A, found in freshwater cyanobacterial species,
contains structural and biosynthetic features that resemble
the fatty acid amides.45 However, it was suggested to form a
new class of secondary metabolites, due to the presence of a
unique oxadiazine ring. Thus, the here-reported calothrix-
amides are one of the first functionalized long-chain fatty acid
amides isolated from freshwater cyanobacteria.
Calothrixamides also add a new chemical class to the short
list of compounds reported from the Calothrix genus. Calothrix
natural products include the calothrixins (alkaloids),46
calophycin (peptide),47 daklakapeptin (peptide),48 brominated
phenols,49 and sesquiterpenes.50 Distributed worldwide,
Calothrix strains can be found in freshwater and marine
environments, from temperate to tropical regions.51 Despite
genomic studies showing the potential of Calothrix for natural
product production,52,53 little is known about the chemical
diversity of the genus.
In summary, in situ dereplication by droplet probe UPLC-
UV-HRMS-MS/MS guided the discovery of calothrixamides A
(1) and B (2) from strain Calothrix sp. UIC 10520. In the
natural products setting, this technique has been used to
analyze both plant and fungal cultures;18,20−25 however, this is
the first report on cyanobacterial cultures. We were encouraged
by our ability to screen through scores of cultures with this
technique, thereby selecting those most likely to biosynthesize
new chemical entities for time- and resource-consuming scale
up and chemical analysis. Calothrixamides are the first
functionalized long-chain fatty acid amides reported from the
Calothrix genus and from freshwater cyanobacteria. In our
hand, no cytotoxicity was detected for the calothrixamides
against a set of cancer cells. Ecological roles and biological
activities at other targets are yet to be determined for the
calothrixamides.
Morphological Identification. Cultured UIC 10520 was
analyzed under a Zeiss Axiostar Plus light microscope equipped
with a Moticam 5 camera (Supporting Information S1). Observed
morphological features were as follows: heteropolar filaments with
basal subspherical heterocyte and narrowed toward apex; uniseriate
trichomes slightly constricted at cross-walls; barrel-shaped cells
isodiametric at the base and elongated toward the end; no akinetes.
Morphological identification of UIC 10520 was based on the
taxonomic systems by Komarek et al.56,57
DNA Extraction, 16S Amplification, and Sequencing.
Cyanobacterial cells from a liquid culture were concentrated by
centrifugation, separated from the media, and resuspended in lysis
buffer (2.5 mL, 10 mM Tris, 0.1 M EDTA, 0.5% w/v SDS) and
lysozyme solution (1 mg/mL). The mixture was incubated for 30 min
in a water bath at 37 °C. Proteinase K was added to the mixture to a
final concentration of 100 μg/mL, and the mixture was incubated for
1 h in a water bath at 50 °C. The mixture was centrifuged to pellet the
cells, and the supernatant was removed. DNA was then extracted from
the cell pellet using the Nucleospin soil kit (Macherey-Nagel).
Cyanobacteria-specific primers 109F and 1509R were used to amplify
part of the 16S rRNA gene by PCR. The PCR reaction mixture
consisted of 2 μL of DNA (50 ng/μL), 10 μL of Taq buffer, 2 μL of
each primer (10 μM), 1 μL of dNTPs (10 μM), 0.5 μL of Taq DNA
polymerase, and 32.5 μL of nuclease-free H2O. PCR was executed in
an Applied Biosystems 2720 thermal cycler with a program of 2 min
denaturation at 95 °C, followed by 35 amplification cycles of 30 s at
95 °C, 30 s at 49 °C, and 2 min at 72 °C, with a final 5 min extension
at 72 °C. PCR products were purified using a GeneJet PCR
purification kit (Thermo). Sanger sequencing was performed on the
purified PCR product using 109F, 359F, and 1509R primers. The
partial 16S rRNA sequence of the UIC 10520 is available in GenBank
under the accession number MG984605.
Extraction and Isolation. Lyophilized cell mass of UIC 10520
was extracted three times with CH2Cl2/MeOH, 1:1. Remaining cell
material was filtered, and the extraction solution was concentrated in
vacuo to yield 809 mg of dry extract. Extract was fractionated by
vacuum liquid chromatography using a Diaion HP-20SS column and a
step gradient of IPA/H2O (0:100, 20:80, 40:60, 70:30, 90:10, and
100:0). LC-MS analysis identified 1 and 2 in the fractions eluting at
70:30 (F4), 90:10 (F5), and 100:0 (F6) IPA/H2O. A 1:1 mixture of
F5/F6 was subjected to reversed-phase semipreparative HPLC using a
Phenomenex Onyx Monolithic C18 column (100 × 4.6 mm), 4 mL/
min flow rate, and automatic fraction collection every 30 s. The 16
min method included a gradient of 70−90% MeOH in H2O over 10
min, a 3 min wash with 100% MeOH, and re-equilibration for 3 min.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a PerkinElmer 241 polarimeter. UV spectra were
acquired on a Varian Cary 5000 spectrophotometer. ECD spectra
were obtained using a JASCO J-710 CD spectrometer. IR spectra
were measured on a Thermo-Nicolet 6700 with a Smart iTR
accessory. 1D and 2D NMR spectra were acquired on a Bruker 800
MHz spectrometer or on a Bruker AVII 900 MHz spectrometer, the
latter equipped with a 5 mm TCI cryoprobe. NMR chemical shifts
were referenced to residual solvent peaks (CDCl3 δH 7.26 and δC
77.16; Pyr-d5 δH 8.74 and δC 150.35). The majority of UPLC-UV-
HRMS-MS/MS experiments were performed at UNCG using a
Waters Acquity UPLC system (WatersCorp.) equipped with a
photodiode array detector (PDA) and coupled to a Thermo
QExactive Plus MS (Thermo-Fisher). UPLC was performed at 0.3
mL/min through a C18 (2.1 × 50 mm × 1.7 μm) column equilibrated
at 40 °C. Mobile phases consisted of H2O (A) and CH3CN (B), both
acidified with 0.1% formic acid (FA). A gradient program was set
from 15% to 100% B for 8 min, held at 100% B for 1.5 min, and re-
equilibrated to initial conditions. The PDA acquisition window was
set from 200 to 500 nm and sampling rate at 5 points/s. HRMS
spectra were acquired in positive and negative modes at a resolution
of 70 000, scanning range from 20 to 2000 m/z, spray voltage of 3.7
kV, nitrogen sheath gas set to 25 arb, and capillary temperature of 350
°C. Automatic data-dependent fragmentation with a collision energy
of 35 eV was applied to obtain the MS/MS spectra. A droplet probe
system was used for in situ analyses, as detailed in this journal
E
DOI: 10.1021/acs.jnatprod.8b00432
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Journal of Natural Products
Article
Fractions eluting between 4 and 6 min were shown to contain 1 and
2. Dereplication based on H NMR, HRMS, MS/MS, and database/
329.2933) was concentrated to dryness and separated into two equal
portions for derivatization with PGME. Each portion was mixed with
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HBTU) (1 mg, 2.6 μmol), 1-hydroxybenzotriazole (HOBt)
(0.5 mg, 3.7 μmol), (S)- or (R)-PGME (0.5 mg, 2.5 μmol),
dimethylformamide (DMF) (25 μL), triethylamine (25 μL), and one
drop of CH2Cl2 to ensure solubility. Reaction mixtures were stirred
for 5 h at rt. After 5 h, 1 mL of EtOAc was added to the mixtures,
forming organic layers, which were each washed with a saturated
aqueous solution of NH4Cl (3 × 1.0 mL). The organic layers were
concentrated and purified by HPLC to yield enantioenriched mixtures
of (S)-PGME (3a) or (R)-PGME (3b) amide derivatives of 11-
(carbamoyloxy)-2,12-dimethyloctadecanoic acid [Agilent Microsorb
Dynamax C18 column (250 mm × 10 mm), 4 mL/min flow rate,
gradient of H2O (A) and CH3CN (B) set from 60% to 85% B over 20
min, from 85% to 100% B for 10 min, and held at 100% for 10 min
before re-equilibration].
1
literature search identified no hits, indicating these compounds to be
potentially new. Compound isolation was performed by reversed-
phase semipreparative HPLC using an Agilent C18 column (250 ×
10.0 mm), 4 mL/min flow rate, and a gradient of H2O (A) and
CH3CN (B) with the following program: 60% B for 5 min, 60−85% B
for 25 min, 85−100% B for 10 min, 100% B for 10 min, 100−60% B
for 5 min, 60% B for 10 min. Compounds 1 (6.5 mg) and 2 (4.5 mg)
eluted at 29.5 and 34 min, respectively.
Calothrixamide A (1): pale orange oil; [α]25D +39 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 213 (2.97), 294 (3.01) nm; ECD
(CH3CN) λmax (Δε) 211.0 (−0.44), 261.5 (+1.33); IR (neat) νmax
3351 (br), 2925, 2854, 1709, 1642, 1593, 1525, 1456, 1392, 1322,
1
1046, 992, 909, 781, 747 cm−1; H and 13C NMR (CDCl3) Table 1;
1H NMR (Pyr-d5, 800 MHz) δ 8.10 (1H, d, J = 7.6 Hz, NH), 7.68
(1H, d, J = 11.8 Hz, H-3), 7.39 (2H, br, OCONH2), 6.50 (1H, m,
OH), 6.37 (1H, ddd, J = 11.8, 10.8, 0.9 Hz, H-4), 5.85 (1H, ddt, J =
17.0, 10.3, 6.7 Hz, H-21), 5.51 (1H, t, J = 10.6 Hz, H-5), 5.14 (1H, dt,
J = 8.5, 4.1 Hz, H-15), 5.07 (1H, m, H-22a), 5.01 (1H, m, H-22b),
4.67 (1H, m, H-1’), 4.00 (1H, dt, J = 10.8, 4.9 Hz, H-2’a), 3.92 (1H,
dt, J = 10.8, 5.4 Hz, H-2’b), 2.65 (1H, m, H-6), 2.14 (3H, s, H-25),
2.03 (2H, m, H-20), 1.77 (1H, m, H-16), 1.74 (1H, m, H-14a), 1.61
(1H, overlapped, H-14b), 1.60 (1H, overlapped, H-17b), 1.53 (1H,
m, H-13b), 1.47 (1H, m, H-13a), 1.42 (3H, d, J = 6.8 Hz, H-3′), 1.37
(1H, overlapped, H-19b), 1.34 (1H, overlapped, H-19a), 1.25 (1H,
overlapped, H-17a), 1.41−1.12 (14H, H-7, H-8, H-9, H-10, H-11, H-
12, H-18), 1.01 (3H, d, J = 6.8 Hz, H-23), 0.85 (3H, d, J = 6.6 Hz, H-
24); HRESIMS m/z 515.3817 [M + Na]+ (calcd for C29H52N2O4Na,
515.3825).
1
3a: H NMR (CDCl3, 800 MHz) δ 7.36−7.31 (PGME’s aromatic
protons), 6.51 (1H, d, J = 7.0), 5.60 (1H, d, J = 7.2), 4.68 (1H, m),
4.53 (2H, br), 3.73 (3H, s), 2.26 (1H, m), 1.63 (1H, m), 1.61 (1H,
m), 1.53 (overlapped), 1.46 (1H, m), 1.37 (1H, m), 1.35−1.18
(overlapped), 1.15 (3H, d, J = 6.9), 0.88 (3H, t, J = 7.0), 0.87 (3H, d,
J = 6.8); HRESIMS m/z 541.3650 [M + Na]+ (calcd for
C30H50N2O5Na, 541.3617).
1
3b: H NMR (CDCl3, 800 MHz) δ 7.36−7.31 (PGME’s aromatic
protons), 6.39 (1H, d, J = 7.0), 5.58 (1H, d, J = 7.2), 4.68 (1H, m),
4.50 (2H, br), 3.73 (3H, s), 2.26 (1H, m), 1.64 (1H, m), 1.61 (1H,
m), 1.52 (overlapped), 1.47 (1H, m), 1.37 (1H, m), 1.35−1.18
(overlapped), 1.12 (3H, d, J = 6.9), 0.88 (3H, t, J = 7.0), 0.87 (3H, d,
J = 6.8); HRESIMS m/z 541.3647 [M + Na]+ (calcd for
C30H50N2O5Na, 541.3617)
Calothrixamide B (2): pale orange oil; [α]25D +50 (c 0.17, MeOH);
UV (MeOH) λmax (log ε) 210 (2.74), 297 (2.79) nm; ECD
(CH3CN) λmax (Δε) 208.5 (−0.52), 268.0 (+1.57); IR (neat) νmax
3340 (br), 2924, 2854, 1708, 1646, 1593, 1526, 1456, 1392, 1320,
1046, 990, 780, 746 cm−1; 1H and 13C NMR (CDCl3) Table 1;
HRESIMS m/z 517.3975 [M + Na]+ (calcd for C29H54N2O4Na,
517.3981).
Base Hydrolysis and Mosher’s Ester Analysis. Approximately
1 mg of 1 was dissolved in 0.5 mL of MeOH and reacted with 0.5 mL
of 0.8 N NaOH in a high-pressure tube at 110 °C for 18 h. The
hydrolysate was dried, resuspended in H2O, and neutralized with HCl
to pH 7. The reaction mixture was extracted with EtOAc three times.
The combined organic layers were dried to afford (2E,4Z)-15-
hydroxy-2,6,16-trimethyldocosa-2,4,21-trienoic acid (4).
Acid Hydrolysis and Marfey’s Analysis. Approximately 0.3 mg
of 1 was hydrolyzed with 1 mL of 6 N HCl in a high-pressure tube
kept at 110 °C for 18 h. The hydrolysate was sequentially dried and
resuspended with 0.5 mL of H2O twice to remove the residual HCl.
The hydrolysate of 1 and (S)-alaninol and (R)-alaninol standards
were derivatized with Advanced Marfey’s reagent (FDLA) according
to the following protocol. Approximately 0.2 mg of the hydrolysate or
standards was dissolved in 110 μL of acetone, 50 μL of H2O, and 20
μL of 1 N NaHCO3. Next, 20 μL of L-FDLA solution (10 mg/mL in
acetone) was added, and the mixtures were stirred for 1 h at 40 °C.
Once at room temperature (rt), reactions were quenched with 20 μL
of 1 N HCl. Reaction mixtures were dried under vacuum and
resuspended in CH3CN for LC-MS analysis. LC-MS analyses were
carried out using a Phenomenex Kinetex C18 column (50 × 2.1 mm,
1.7 μm) and a gradient of H2O (A) and CH3CN (B) both with 0.1%
formic at 0.5 mL/min flow rate. A gradient program was set from 25%
to 65% B for 9 min. Extracted ion chromatograms for m/z 370.17
revealed the following retention times: 3.4 min for (S)-alaninol
derivative, 4.2 min for (R)-alaninol derivative, 3.4 min for the
derivatized hydrolysate of 1 (Supporting Information S18). The
absolute configuration of the alaninol moiety was assigned as S.
Ozonolysis and PGME Derivatization. A solution of 2 (3.5 mg,
0.007 mmol) in MeOH (140 μL) was cooled to −78 °C, and ozone
was bubbled through the solution until a pale blue color persisted.
The reaction vessel was opened to the atmosphere, and the mixture
was slowly warmed to rt. The resulting mixture was concentrated in
vacuo. Formic acid (95% in H2O, 70 μL) and H2O2 (35% in H2O, 70
μL) were added to the resulting residue and stirred for 17 h at rt. The
reaction mixture was concentrated in vacuo. Toluene (3 × 2 mL) was
then successively added to the mixture and removed under vacuum to
facilitate the removal of any volatile aqueous reagents. The resulting
residue containing 11-(carbamoyloxy)-2,12-dimethyloctadecanoic
acid (HRESIMS m/z 394.2920 [M + Na]+, calcd for C21H41NO4Na
1
4: H NMR (Pyr-d5, 800 MHz) δ 8.13 (1H, d, J = 12.0 Hz), 6.52
(1H, dd, J = 12.0, 11.0 Hz), 5.87 (1H, ddt, J = 16.8, 10.3, 6.8 Hz),
5.48 (1H, t, J = 10.4 Hz), 5.08 (1H, m), 5.02 (1H, m), 3.79 (1H, dt, J
= 8.3, 3.7 Hz), 2.82 (1H, m), 2.38 (3H, s), 2.06 (2H, m), 1.75 (4H,
m), 1.66 (1H, m), 1.61 (1H, m), 1.52 (1H, m), 1.44 (1H, m), 1.43−
1.18 (overlapped), 1.12 (3H, d, J = 6.8 Hz), 0.95 (3H, d, J = 6.6 Hz);
HRESIMS m/z 415.3196 [M + Na]+ (calcd for C25H44O3Na,
415.3188).
For the Mosher’s ester reaction, two equal portions of 4 were
separated in oven-dried 4 mL vials and dried under vacuum. To each
vial were added four activated molecular sieves, a stir bar, and 200 μL
of pyridine-d5. Vials were stirred for 10 min at rt, after which 1.8 μL of
R- or S-MTPA-Cl was added. The reaction mixtures were stirred for
16 h at rt to allow formation of the S-MTPA ester of 4 (5a) and R-
1
MTPA ester of 4 (5b), respectively. Direct H NMR analysis of the
reaction mixtures was hindered due to overlapping reagent and
impurity signals, and derivatives were isolated by RP-HPLC [Agilent
Microsorb Dynamax C18 column (250 mm × 10 mm), 4 mL/min
flow rate, gradient of H2O (A) and CH3CN acidified with 0.1% FA
(B) from 80% to 100% B over 20 min, 100% B for 15 min, followed
by re-equilibration].
1
5a: partial H NMR (Pyr-d5, 800 MHz) δ 8.07 (1H, d, J = 10.6
Hz), 6.47 (1H, m), 5.87 (1H, ddt, J = 16.8, 10.7, 6.9), 5.64 (1H, m),
5.31 (1H, m), 5.10 (1H, m), 5.04 (1H, m), 2.84 (1H, m), 2.23 (3H,
br), 2.05 (1H, m), 1.79 (1H, m), 1.70 (1H, m), 1.57 (1H, m), 1.00
(3H, d, J = 6.6), 0.96 (3H, d, J = 6.8); HRESIMS m/z 631.3563 [M +
Na]+ (calcd for C35H51F3O5Na, 631.3586).
1
5b: partial H NMR (Pyr-d5, 800 MHz) δ 8.07 (1H, d, J = 10.5
Hz), 6.47 (1H, m), 5.87 (1H, ddt, J = 16.8, 10.7, 6.9), 5.65 (1H, m),
5.31 (1H, m), 5.10 (1H, m), 5.04 (1H, m), 2.84 (1H, m), 2.23 (3H,
br), 2.03 (1H, m), 1.78 (1H, m), 1.75 (1H, m), 1.64 (1H, m), 0.99
F
DOI: 10.1021/acs.jnatprod.8b00432
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Journal of Natural Products
Article
(3H, d, J = 6.5), 0.90 (3H, d, J = 6.8); HRESIMS m/z 631.3552 [M +
Na]+ (calcd for C35H51F3O5Na, 631.3586).
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Rep. 2017, 34, 6−24.
Antiproliferative Assay. Cytotoxicity assays against MDA-MB-
435 human melanoma cancer cells, MDA-MB-231 human breast
cancer cells, and OVCAR3 human ovarian cancer cells were
performed following established protocols.58 Taxol was used as
positive control (IC50 values of 3.1, 2.6, and 7.5 nM against MDA-
MB-435, MDA-MB-231, and OVCAR3, respectively).
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00432.
■
̂
(15) Gaudencio, S. P.; Pereira, F. Nat. Prod. Rep. 2015, 32, 779−
S
810.
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Rondeau, D. Phytochem. Anal. 2016, 27, 354−363.
(17) Watrous, J.; Roach, P.; Alexandrov, T.; Heath, B. S.; Yang, J. Y.;
Kersten, R. D.; van der Voort, M.; Pogliano, K.; Gross, H.;
Raaijmakers, J. M.; Moore, B. S.; Laskin, J.; Bandeira, N.;
Dorrestein, P. C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, E1743−
E1752.
Photomicrograph and phylogenetic analysis of Calothrix
sp. UIC 10520, results from the droplet probe UPLC-
UV-HRMS-MS/MS analysis of Calothrix sp. UIC 10520,
1D and 2D NMR and ECD spectra of 1 and 2, results of
Marfey’s analysis (PDF)
(18) Sica, V. P.; Raja, H. a.; El-Elimat, T.; Kertesz, V.; Van Berkel, G.
J.; Pearce, C. J.; Oberlies, N. H. J. Nat. Prod. 2015, 78, 1926−1936.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1 312 996 5583. E-mail: orjala@uic.edu (J. Orjala).
■
(20) Sica, V. P.; El-Elimat, T.; Oberlies, N. H. Anal. Methods 2016,
8, 6143−6149.
ORCID
́
(21) Rivera-Chavez, J.; Raja, H. A.; Graf, T. N.; Gallagher, J. M.;
James R. Fuchs: 0000-0002-8743-6389
Jimmy Orjala: 0000-0002-4420-2048
Notes
Metri, P.; Xue, D.; Pearce, C. J.; Oberlies, N. H. RSC Adv. 2017, 7,
45733−45741.
(22) Paguigan, N. D.; Raja, H. A.; Day, C. S.; Oberlies, N. H.
Phytochemistry 2016, 126, 59−65.
The authors declare no competing financial interest.
́
(23) Sica, V. P.; Rees, E. R.; Raja, H. A.; Rivera-Chavez, J.; Burdette,
J. E.; Pearce, C. J.; Oberlies, N. H. Phytochemistry 2017, 143, 45−53.
(24) Sica, V. P.; Rees, E. R.; Tchegnon, E.; Bardsley, R. H.; Raja, H.
A.; Oberlies, N. H. Front. Microbiol. 2016, 7, 1−14.
(25) Sica, V. P.; Figueroa, M.; Raja, H. A.; El-Elimat, T.; Darveaux,
B. A.; Pearce, C. J.; Oberlies, N. H. J. Ind. Microbiol. Biotechnol. 2016,
43, 1149−1157.
ACKNOWLEDGMENTS
■
We thank R. Ahadi and S. Romanowski for assisting with
cyanobacterial strain isolation and culture. This research was
supported by NCI/NIH PO1 CA125066, CAPES/Science
without Borders BEX 13055-13-5, and The Office of the
Director, National Institutes of Health (OD) National Center
for Complementary and Integrative Health (NCCIH)
T32AT007533. D.K. was supported by F31 AT009264 from
the NCCIH. We thank Drs. V. Kertesz and G. J. Van Berkel
(Oak Ridge National Laboratory) for inspiration and guidance
with the droplet probe.
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In Bioactive Compounds from Natural Sources, Second ed.; CRC Press,
2011; pp 37−64.
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DOI: 10.1021/acs.jnatprod.8b00432
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