Combining Mass Spectrometric Metabolic Profiling with Genomic Analysis: A Powerful Approach for Discovering Natural Products from Cyanobacteria

Article abstract of DOI:10.1021/acs.jnatprod.5b00301

An innovative approach was developed for the discovery of new natural products by combining mass spectrometric metabolic profiling with genomic analysis and resulted in the discovery of the columbamides, a new class of di- and trichlorinated acyl amides with cannabinomimetic activity. Three species of cultured marine cyanobacteria, Moorea producens 3L, Moorea producens JHB, and Moorea bouillonii PNG, were subjected to genome sequencing and analysis for their recognizable biosynthetic pathways, and this information was then compared with their respective metabolomes as detected by MS profiling. By genome analysis, a presumed regulatory domain was identified upstream of several previously described biosynthetic gene clusters in two of these cyanobacteria, M. producens 3L and M. producens JHB. A similar regulatory domain was identified in the M. bouillonii PNG genome, and a corresponding downstream biosynthetic gene cluster was located and carefully analyzed. Subsequently, MS-based molecular networking identified a series of candidate products, and these were isolated and their structures rigorously established. On the basis of their distinctive acyl amide structure, the most prevalent metabolite was evaluated for cannabinomimetic properties and found to be moderate affinity ligands for CB₁. (Chemical Equation Presented)

Full text of DOI:10.1021/acs.jnatprod.5b00301

Article
pubs.acs.org/jnp
Combining Mass Spectrometric Metabolic Profiling with Genomic
Analysis: A Powerful Approach for Discovering Natural Products
from Cyanobacteria
†
†
†,‡
§
⊥,∥,¶
Karin Kleigrewe, Jehad Almaliti, Isaac Yuheng Tian, Robin B. Kinnel, Anton Korobeynikov,
∇
□
#
◊
Emily A. Monroe, Brendan M. Duggan, Vincenzo Di Marzo, David H. Sherman,
Pieter C. Dorrestein, Lena Gerwick, and William H. Gerwick*
□
†
,†,□
†Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, and □Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California San Diego, San Diego, California, United States
‡
University of California Berkeley, Berkeley, California, United States
§
Hamilton College, Clinton, New York, United States
⊥
∥
Faculty of Mathematics and Mechanics and Center for Algorithmic Biotechnology, Saint Petersburg State University, St.
Petersburg, Russian Federation
¶
Algorithmic Biology Laboratory, Saint Petersburg Academic University, St. Petersburg, Russian Federation
∇
Department of Biology, William Paterson University of New Jersey, Wayne, New Jersey, United States
Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy
#
◊
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States
S Supporting Information
*
ABSTRACT: An innovative approach was developed for the
discovery of new natural products by combining mass
spectrometric metabolic profiling with genomic analysis and
resulted in the discovery of the columbamides, a new class of
di- and trichlorinated acyl amides with cannabinomimetic
activity. Three species of cultured marine cyanobacteria,
Moorea producens 3L, Moorea producens JHB, and Moorea
bouillonii PNG, were subjected to genome sequencing and
analysis for their recognizable biosynthetic pathways, and this
information was then compared with their respective
metabolomes as detected by MS profiling. By genome analysis,
a presumed regulatory domain was identified upstream of several previously described biosynthetic gene clusters in two of these
cyanobacteria, M. producens 3L and M. producens JHB. A similar regulatory domain was identified in the M. bouillonii PNG
genome, and a corresponding downstream biosynthetic gene cluster was located and carefully analyzed. Subsequently, MS-based
molecular networking identified a series of candidate products, and these were isolated and their structures rigorously established.
On the basis of their distinctive acyl amide structure, the most prevalent metabolite was evaluated for cannabinomimetic
properties and found to be moderate affinity ligands for CB1.
5
arine cyanobacteria have emerged as a bountiful source
of structurally diverse and biologically active natural
amenable to informatic-based deductions of structure. With
the mass spectrometric based metabolomics approach,
deductions can be made about the number of compounds
and compound classes present within a natural product extract.
In addition, combining high-resolution mass spectrometry
(HRMS) together with the molecular ion isotopic pattern
M
products, some of which have inspired the development of new
1
pharmaceutical agents. Using the orthologous methods of
genome mining and rapid mass spectrometric dereplication
followed by careful structure elucidation, the discovery process
of new secondary metabolites is becoming increasingly
streamlined and efficient. The genomics approach provides
information about the type of biosynthetic gene cluster present
and, correspondingly, structural predictions about the natural
2
and MS -based fragmentation analyses, it is possible to develop
tentative structural information about unknown compounds.
Therefore, combining genomics and metabolomics makes
possible the linkage of specific compounds to gene clusters
2
−4
products produced.
PKS), nonribosomal peptide synthetases (NRPS), or hybrids
of these two are most commonly encountered and are generally
In cyanobacteria, polyketide synthases
(
Received: April 5, 2015
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00301
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. Moorea Species Studied in This Report, Their Origins and Reported Natural Products, and References to Previously
a
Described Biosynthetic Gene Clusters
NCBI accession
strain
Moorea producens 3L
origin
compounds
cluster known?
13
number
19
20
Curaca
̧
o
barbamide, dechlorobarbamide
yes
HQ696501
NZ_GL890970
HQ696500
AY974560
2
1
11
carmabins A,B
yes
1
2
22
23
curacins A−C, curazole
yes
15
24
Moorea producens JHB JHB22Aug96-1
Jamaica - Hector’s Bay
hectochlorin
yes
14
14
jamaicamides A−C
yes
AY522504
2
5
16
Moorea bouillonii PNG PNG19May05-8 Papua New Guinea - Pigeon Island apratoxins A−C
yes
17
lyngbyabellin A
yes, unpublished
a
9,18
A phylogenetic analysis of these strains was previously published.
and vice versa, and this information can be used to enhance the
discovery and isolation of new natural products.
of chlorination. Owing to their structural relationship to
anandamide and other cannibinomimetic compounds, the two
major compounds columbamides A and B were evaluated for
cannabinoid receptor CB and CB binding efficacy and found
6,7
Herein we describe the discovery of a new class of acyl amide
based on genome comparisons and mass spectrometric
metabolic profiling of three cyanobacterial strains of the
1
2
to be the most potent analogues yet isolated from the marine
8
31
genus Moorea (formerly known as Lyngbya ). This genus is
world.
known to produce many structurally diverse and biologically
active natural products. Moorea producens 3L, collected in
9
RESULTS AND DISCUSSION
■
Curaca
̧
o, produces the tubulin polymerization inhibitor curacin
Identification of Biosynthetic Gene Cluster. The
genome of M. producens 3L was obtained by Sanger and 454
A, the molluscicide barbamide, and the antimalarial compound
carmabin.
1
0−13
11
Moorea producens JHB, obtained from shallow
sequencing, whereas those of M. producens JHB and M.
coastal waters in Jamaica, is known for its production of the
sodium channel blocker jamaicamide and the fungicide
bouillonii PNG were sequenced using Illumina Hiseq; in
addition, for M. bouillonii PNG PACBIO sequencing was also
performed. Assembly of all genomes utilized SPAdes 3.0 and
1
4,15
hectochlorin.
Complementing these, Moorea bouillonii
3
2,33
34
PNG from Papua New Guinea produces the cytotoxic
3.1
with BayesHammer [NN2] read error correction. In
1
6,17
apratoxins A−C and lyngbyabellin A (Table 1).
each case, the source cyanobacteria were contaminated with
heterotrophic bacteria that were associated with the filament
sheath. As a result, a modest complexity metagenomic data set
was obtained, and therefore the sequence data were binned to
obtain the low GC cyanobacterial sequences (43.6% GC)
separate from the higher GC content contaminants. Table 2
Improvements in whole genome sequencing and bioinfor-
matic tools have resulted in a more facile identification of the
biosynthetic gene clusters responsible for the formation of
26
natural products. In particular, the biosynthetic gene clusters
encoding polyketides and nonribosomal peptides are readily
27
detected, and subsequent structure predictions are possible.
Nevertheless, questions still remain whether an identified
biosynthetic gene cluster is functionally expressed and if it is
responsible for the production of a new or a known natural
product. Due to the relative lack of molecular biology
techniques, such as mutagenic gene knockouts and heterolo-
gous expression systems, for cyanobacterial Moorea strains as
well as filamentous marine cyanobacteria in general, other
methods must be used to unequivocally relate a given gene
cluster to a specific natural product. For example, functional
expression of distinctive biosynthetic enzymes from these
clusters and characterization of their specificity and chemical
reactivity has been used in several cases to confirm the
connection between gene cluster and compound (e.g.,
Table 2. QUAST (Quality Assessment Tool for Genome
Assemblies) Results for the Three Moorea Strains
3
L
JHB 22Aug96-1
PNG 19May05-8
no. of contigs
largest contig
total length
N50
161
960
579
593 782
8 478 612
123 305
19
144 691
9 501 724
20 017
145
135 461
8 576 967
31 790
77
L50
GC (%)
43.68
43.67
43.65
depicts the quality of the three draft genomes using the Quality
35
Assessment Tool (QUAST) for Genome Assemblies. The
published genome from M. producens 3L had 161 contigs, while
the DNA sequences for M. producens JHB and M. bouillonii
PNG were more fragmented, with 960 and 579 contigs,
respectively. The approximate sizes of the Moorea genomes
appear to be between 8 and 9.5 MB (Table 2).
1
4,28,29
barbamide, curacin A, jamaicamide A
). Another con-
ceivable approach is to identify similar or nearly identical
biosynthetic genes between different cyanobacterial genomes
and to compare this information with that generated from
parallel metabolomic studies. In the current study, the latter
approach was taken in that all of the NRPS and PKS
biosynthetic gene clusters of the Moorea strains listed above
were identified in their respective genomic data sets, and this
information was then juxtaposed with mass spectrometric
Because the focus of this study was to detect biosynthetic
gene clusters and their respective encoded compounds, we
performed a manual assembly to close gaps (unpublished data)
in cases where putative biosynthetic gene clusters were split
between two contigs. Biosynthetic gene clusters were identified
in the three draft genomes using common bioinformatics tools,
30
profiles observed with the Molecular Networks algorithm to
identify a family of functionally expressed novel metabolites.
Subsequently, these metabolites were isolated in high purity
from laboratory cultures, and their structures rigorously
determined as a series of acyl amides with unique positions
2
7,36,37
including AntiSMASH and NaPDos.
The genome of M.
producens 3L contains six PKS, NRPS, or hybrid biosynthetic
pathways that include the curacin A, barbamide, and carmabin
B
DOI: 10.1021/acs.jnatprod.5b00301
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Figure 1. MultiGeneBlast result for the columbamide, jamaicamide A, and curacin A biosynthetic gene clusters. At the 3′ end of each pathway is an
open reading frame for a putative regulatory serine histidine kinase (*).
pathways. The M. producens JHB strain has eight biosynthetic
pathways in addition to those encoding for jamaicamide and
hectochlorin production. In the genome of M. bouillonii PNG,
seven PKS/NRPS-based biosynthetic pathways are present,
including those for apratoxin and lyngbyabellin biosynthesis.
From LC-MS results as well as previous reports, M. producens
depicts the molecular network obtained from combining the
data sets generated from analysis of the extracts of M. producens
3L, M. producens JHB, and M. bouillonii PNG. The color of the
node reflects the strain [M. producens 3L (red), M. producens
JHB (yellow), and M. bouillonii PNG (green)]. The size of each
individual node reflects the abundance of the compound based
2
3L and M. producens JHB produce large amounts of the hybrid
on the number of MS -spectra scans present. The square-
PKS-NRPS metabolites curacin A and jamaicamide A,
shaped nodes indicate matches to previously characterized
compounds. Each node is labeled with its respective parent
mass.
1
2,14
respectively.
The biosynthetic gene clusters for both of
these natural products are known, and intriguingly, they each
are located adjacent to a regulatory serine histidine kinase
The major compounds produced by M. producens 3L are the
3
8
12
13
21
gene. This regulatory kinase is highly homologous between
the two Moorea strains (96.1% identity based on protein
sequence), suggesting the hypothesis that it is specifically
associated with highly expressed natural product gene clusters.
We therefore reasoned that searching for the gene encoding
this regulatory enzyme within the M. bouillonii PNG genome
sequence might identify new natural product biosynthetic gene
clusters. Such a BLAST search was conducted using the entirety
of the kinase protein sequence from M. producens 3L
curacins, barbamide, and carmabins, whereas those of
24
Moorea producens JHB are the hectochlorins and jamaica-
mides. The metabolic profile of M. bouillonii PNG reflects the
14
17
16
biosynthesis of lyngbyabellin A and the apratoxins. In
addition, two clusters (marked with black squares) were
particularly interesting because the isotopic pattern of the
parent masses indicated di- and trichlorinated species. In
addition, the relatively large size of the nodes reflected the fact
that these molecules were abundant in M. bouillonii PNG.
Moreover, these features of size and chlorination were generally
in accord with the prediction of the new biosynthetic gene
cluster in M. bouillonii PNG. Consequently, the biosynthetic
gene cluster was analyzed in greater detail, the encoded
compounds were isolated, and their chemical structures were
elucidated by NMR, resulting in the discovery of columbamides
A (1), B (2), and C (3).
(
WP_008191774.1) and revealed one highly homologous
sequence in the M. bouillonii PNG genome. Investigation of
the gene neighborhood for this kinase revealed a new and
undescribed biosynthetic gene cluster with several unique
39
features, as described below. To evaluate the potential
expression of novel metabolites by this gene cluster, the
metabolic profile of each strain was analyzed by mass
3
0,40
spectrometric molecular networking
and then utilized in a
Analysis of the Col Biosynthetic Gene Cluster. Table 3
and Figure 3 summarize the deduced function of each gene and
depict its involvement in the putative biosynthetic pathway. On
the basis of the subsequent structure elucidation of the
columbamides, the gene cluster is designated as “col”. Natural
product biosynthetic gene clusters are often found associated
with transposases; in this case, two were identified at the
presumed 5′-end boundary of the gene cluster (Orf1,
comparative metabolomics approach to complement the
genomic analysis.
Metabolic Profiling of Moorea Strains. Cultured biomass
of M. producens 3L, M. producens JHB, and M. bouillonii PNG
was harvested and chemically extracted, and the metabolic
30
profile of each was evaluated using molecular networking.
This mass spectrometry based tool uses fragmentation patterns
to determine the relative similarity of different metabolites.
Molecules with similar fragmentation patterns cluster together
1
4,24,42
Orf2).
These genetically mobile elements can play a
role in the horizontal acquisition and distribution of natural
4
3
and are visualized as a series of nodes (=molecules) and edges
product gene clusters between different strains. The initial
biosynthetic enzyme of the cluster, ColA, encodes for a putative
acyl-CoA synthetase. Cyanobacterial biosynthetic gene clusters
often contain acyl-ACP synthetase starting units (jamaicamide
2
(
=similarities) in Cytoscape. In addition, by comparing MS -
fragmentation patterns against the natural product MS/MS
library, the dereplication process is accelerated, and novel
compound classes can be easily identified. In the current case,
the extracts were analyzed using both ion trap (ITMS) and
QTof mass spectrometers; this allowed production of a
robustly annotated network by utilizing chemical standards
1
4,24
JamA, hectochlorin HctA),
and commonly hexanoic acid is
loaded onto an acyl carrier protein. In this case, BlastP analysis
revealed a 63% identity between colA and puwC of the
lipopeptide puwainaphycin biosynthetic gene cluster and a 57%
identity between colA and the fatty acyl ACP ligase of the
41
generated from our in-house pure compound library as well
as accurate formula assignments from the HRMS data. Figure 2
18,44
olefin-synthase (OLS) pathway.
The OLS pathway is
C
DOI: 10.1021/acs.jnatprod.5b00301
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Journal of Natural Products
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Figure 2. Molecular network (A) derived from mass spectrometric analysis of extracts of M. producens 3L (red), M. producens JHB (yellow), and M.
bouillonii PNG (green). In the nodes, squares indicate consensus MS/MS spectra to compounds in a MS/MS library of known molecules. The
2
respective compound name of an identified molecule is given next to the square node. The node size is representative of the numbers of MS spectra
2
obtained for that specific m/z. The thickness of the edges between nodes indicates the degree of similarity between their respective MS spectra.
+
+
Multiple clusters per compound derives from the fact that [M + H] and [M + Na] parent ions sometimes fragment differently. The black
rectangles indicate the clusters of interest and are enlarged in B.
responsible for hydrocarbon production in cyanobacteria and
have a variety of unique genes that encode for proteins involved
in the halogenation of natural products. Examples include HctB
in hectochlorin biosynthesis, BarB1 and BarB2 in barbamide
biosynthesis, and the cryptic halogenase CurA in curacin A
18
utilizes long-chain fatty acids as initial substrates. On the basis
of subsequent structure elucidation of the compounds formed
by this biosynthetic gene cluster, the colA gene loads
dodecanoic acid onto the acyl carrier protein ColC.
45−47
biosynthesis.
For ColD and ColE, the BlastP search
The next gene in the cluster, colB, encodes for a putative
HNH endonuclease [77% identity to HNH-endonuclease of M.
producens 3L (WP_008186580.1)]. A functional role for ColB
is not apparent at this time. The next two genes, colD and colE,
most likely encode for a new type of halogenase, although the
initial BLAST hits for these were for oxygenases. Cyanobacteria
yielded weak hits to p-aminobenzoate N-oxygenases AurF
(ColD: 27% identity, 47% similarity, ColE: 30% identity, 44%
similarity) and to the protein CylC (ColD: 48% identity, 66%
similarity, ColE: 47% identity, 62% similarity), which is
potentially a cryptic halogenase involved in the carbon−carbon
4
8,49
bond activation of cylindrocyclophane biosynthesis.
The
D
DOI: 10.1021/acs.jnatprod.5b00301
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Table 3. Deduced Functions of the Open Reading Frames in the Col Gene Cluster
amino
identity/
similarity
protein
acids
proposed function
sequence similarity (protein, origin)
sucrose synthase, Moorea producens
accession no.
ORF7
ORF6
ORF5
413
91
97%, 98%
66%, 80%
28%, 40%
WP_008191839.1
WP_015134379.1
EGI82761.1
transposase, Leptolyngbya sp. PCC 7376
51
glycyl-tRNA synthetase subunit alpha, Streptococcus
pneumoniae GA17570
ORF4
ORF3
ORF2
ORF1
ColA
384
34
sucrose synthase, Moorea producens
98%, 98%
WP_008191839.1
37
transposase, Dolichospermum circinale
61%, 75%
74%,85%
63%/79%
57%/71%
77%, 87%
31%/50%
WP_028082114.1
ADO19008.1
33
transposase, Nostoc flagelliforme str. Sunitezuoqi
PuwC, Cylindrospermum alatosporum CCALA 988
fatty acyl ACP ligase, Moorea bouillonii PNG5-198
HNH endonuclease, Moorea producens
583
dodecanoyl-ACP synthetase
AIW82280.1
AHH34187.1
WP_008186580.1
EUA89990.1
ColB
ColC
432
119
unknown
ACP
short-chain dehydrogenase family protein, Mycobacterium
ulcerans str. Harvey
ColD
463
halogenase
CylC, Cylindrospermum licheniforme UTEX B 2014
48%/66%
27%/47%
AFV96137.1
ESA35271.1
p-aminobenzoate N-oxygenase, Leptolyngbya sp. Heron
Island J
ColE
ColF
471
halogenase
CylC, Cylindrospermum licheniforme UTEX B 2014
p-aminobenzoate N-oxygenase AurF, Oscillatoria acuminata
CrpB, Nostoc sp. ATCC 53789
47%/62%
30%, 44%
53%, 69%
AFV96137.1
WP_015146466.1
ABM21570.1
3712
KS-AT-(DH)-KR-ACP-KS-AT-DH-
ER-KR-ACP
ColG
2227
35
C-A(Ser)-OMT-NMT-PCP-R
unknown
NcpB, Nostoc sp. ATCC 53789
51%, 66%
54%, 36%
16%, 32%
73%, 83%
91%, 91%
81%, 93%
97%,96%
96%, 97%
84%, 84%
94%, 96%
80%, 88%
AAO23334.1
ColH
hypothetical protein, Alicyclobacillus pohliae
Acyltransferase, Mycobacterium tuberculosis
choloylglycine hydrolase-like protein, Lyngbya majuscula
hypothetical protein, Lyngbya majuscula
hypothetical protein, Gloeocapsa sp. PCC 73106
hypothetical protein, Moorea producens
tRNA 2-selenouridine synthase, Moorea producens
hypothetical protein, Moorea producens
guanylate cyclase, Moorea producens
WP_018133539.1
WP_031725540.1
AAS98791.1
ColI
459
723
41
acyltransferase
ORF1′
ORF2′
ORF3′
ORF4′
ORF5′
ORF6′
ORF7′
ORF8′
AAS98793.1
33
WP_006530731.1
WP_008191272.1
WP_008191777.1
WP_008180717.1
WP_008191774.1
WP_008189692.1
49
355
31
1169
220
regulatory enzyme
putative exosortase, PEP-CTERM interaction domain protein,
Moorea producens
ORF9′
269
putative peptidoglycan-binding domain-containing protein,
Moorea producens
97%, 98%
WP_008189690.1
ORF10′
ORF11′
424
428
zinc protease, Desulfotomaculum alkaliphilum
peptidase M16, Moorea producens
26%, 47%
97%, 98%
WP_031514442.1
WP_008189686.1
Figure 3. Proposed biosynthetic pathway of the columbamides in M. bouillonii PNG. AS: acyl synthetase, ACP: acyl carrier protein, Hal: halogenase,
KS: β-ketoacyl-ACP synthase; AT: acyl transferase, DH: β-hydroxy-acyl-ACP dehydratase, KR: β-ketoacyl-ACP reductase, ER: enoyl reductase, C:
condensation domain, A: adenylation domain, MT: methyltransferase, PCP: peptidyl carrier protein, R: reductase, AcylT: acyltransferase. Using
multiple bioinformatic methods, the first DH domain in ColF appears to be absent and is therefore colored in light gray. As discussed in the text, the
DH domain in the second PKS module of ColF is believed to provide this biochemical property.
E
DOI: 10.1021/acs.jnatprod.5b00301
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1
13
1
Table 4. H and C NMR Spectroscopic Data for Columbamides A (1), B (2), and C (3) in CDCl at 600 MHz for H and 150
3
MHz for 13
C
a
columbamide A (1)
columbamide B (2)
columbamide C (3)
no.
δC, type
173.5, C
δH (J [Hz])
HMBC
1, 3, 4
δC
δH (J [Hz])
HMBC
δC
174.2
δH (J [Hz])
HMBC
1, 3, 4
1
2
3
4
5
6
7
8
9
173.1
33.9
28.0
129.3
131.1
32.3
28.6
25.9
38.5
34.2, CH2
28.6, CH2
129.3, CH
131.1, CH
32.5, CH2
29.1, CH2
26.2, CH2
38.5, CH2
2.40, m
2.40, m
2.32, m
5.45, m
5.45, m
1.99, m
1.33, m
1.42, m
1.71, m
1, 3, 4
4, 5
6
34.2
28.0
2.41, m
2.33, m
5.46, m
5.46, m
1.99, m
1.34, m
1.39, m
2.32, m
2, 4, 5
2, 4, 5
5.45, m
3, 6, 4, 5
3, 4, 5, 6
4, 5, 7, 8
5, 6, 8, 9
7, 9, 10
7, 8, 10
129.0
131.0
32.0
3, 4, 5, 6
3, 4, 5, 6
4, 5, 7, 8
5, 6, 9
5.45, m
6
1.99, m
4, 5, 7
6, 8
9
1.35, m*
1.35, m*
1.70, h (8.9,
28.7
26.0
6, 7, 9, 10
7, 8, 10, 11
8, 10, 11
38.3
1.70, tp (10.5, 5.5,
4.3)
7
.7)
3.88, ddd (12.8, 8, 9, 11, 12
.1, 4.9)
1.70, h (8.9,
.7)
1.53, td (9.1,
.0)
10
11
12
13
14
15
64.3, CH
38.5, CH2
26.5, CH2
28.2, CH2
26.9, CH2
32.6, CH2
45.2, CH
63.9
38.5
25.9
28.6
25.8
43.5
73.5
3.88, ddd (12.8, 8, 9, 11,
8.1, 5.0)
64.1
38.3
26.0
28.6
26.4
32.4
45.0
3.88, tt (8.2, 4.9)
8, 9, 11, 12
8
12
9, 10, 12
1.71, dtd (12.1,
8.8, 8.1, 4.1)
9, 10, 12
1.70, tp (10.5, 5.5,
4.3)
8, 9, 10,
12, 13
7
10, 11, 13,
14
1.43, m
1.33, m
1.55, m
2.2, m
11
1.54, ddt (23.2, 13.1, 10, 11, 14
6.1)
5
1.35, m*
11, 12, 13,
12, 14
1.33, m
11, 12, 14,
15
1
4, 15
1.45, dq (13.5,
12, 13, 15,
16
12, 13,
15, 16
1.45, dq (13.0, 6.6,
5.9)
12, 13, 15,
16
6
.7)
1.78, p (6.9)
13, 14, 16
13, 14,
1.78, p (6.9)
13, 14, 16
1
6
1
1
1
6
7
8
3.53, t (6.7)
14, 15
5.75, t (6.0)
14
3.54, t (6.7)
14, 15
1, 18
31.9 (27.6), CH3 2.95, s (2.82, s) 1, 18 1, 18
31.6 (27.3)
51.9 (55.1)
2.96, s (2.82, s) 1, 18
33.8 (27.4) 3.02, s (2.84, s)
52.1 (55.3), CH
4.90, tt (6.8,
.8) (4.27, m)
1, 17, 19,
20
4.90, tt (5.5, 5.5)
(4.27, m)
57.8 (57.8) 4.38, tt (5.2,5.2)
(4.13, tt (6.3, 6.3)
1, 17, 19,
20
6
19
71.1, CH2
3.58, dd (10.3,
.9)
18, 20, 23
70.9 70.8 70.7 3.58, dd (10.3,
6.8)
18, 20,
23
70.9
3.57, dd (10.3, 4.9) 18, 20, 19
6
3
.48, dd (10.4,
.3)
3.49, dd (10.3,
5.1)
3.67, dd (10.2, 7.4)
5
20
62.2, CH2
4.26, dd (11.5,
.9)
1, 18, 19,
21
61.9
4.27, dd (11.5,
7.9)
18, 19,
21
62.3
3.79, d (3.9) 3.76, m 18, 19
7
4
.20, dd (11.9,
.8)
4.20, dd (11.8,
4.9)
4
2
2
1
2
170.9, C
170.6
20.7
21.0, CH3
2.04, s
.05, s
3.32, s
.34, s
21
19
2.04, s
2.05, s
3.32, s
3.34, s
21
19
2
23
59.1, CH3
59.0
59.1 (59.1) 3.34, s (3.35, s)
19
3
a
*
represents overlapping signals; chemical shift values in parentheses are those resulting from minor conformer.
former gene, aurF, is involved in the biosynthesis of aureothin;
however, the exact functioning and mechanism of the encoded
enzyme are controversial. The Hertweck group identified AurF
as a dimanganese enzyme, while the Zhao group reported AurF
as a nonheme di-iron monoxygenase. Finally, the Bollinger
group recently reported that it catalyzes a four-electron
The next gene in the cluster, colF, matches a bimodular PKS
motif. The protein sequence shows 53% similarity to CrpB,
responsible for cryptophycin biosynthesis in Nostoc sp. ATCC
53
53789. In this biosynthetic sequence, the acyl ACP is
extended by two rounds of ketide extension by the two PKS
modules found in ColF. Bioinformatics analysis in concert with
the elucidated structure indicated the presence of the following
domain structure: KS-AT-(DH)-KR-ACP-KS-AT-DH-ER-KR-
ACP. On the basis of the structure of the isolated compound,
the first polyketide module should contain a dehydratase
domain. However, using common bioinformatics tools such as
Delta BlastP, AntiSMASH, and 2metDB, a dehydratase domain
50−52
oxidation within a di-iron cluster.
The similarity in protein
structure of ColD and ColE to AurF suggests that the
halogenation mechanism at an unactivated center on an alkyl
chain might be similar to the oxidation of aminoarenes to
nitroarenes. Consistent with this deduction, the cylC gene
product is likely involved in the carbon−carbon bond activation
of cylindrocyclophane, possibly through a cryptic chlorination
2
7,54,55
was not detectable.
Interestingly, the curacin A pathway
4
8,49
event.
Ultimately, through isolation of the encoded
also has expected DH domains in each of the CurG, CurH,
CurI, CurJ, and CurK proteins, but only three functional DH
compounds 1−3, it was deduced that one putative halogenase
introduces either one or two chlorine atoms at the terminal end
of the alkyl chain, whereas the other introduces a chlorine atom
at the ω-7 position of the fatty acid chain. Clearly, halogenation
of these positions involves a highly unusual C−H bond
activation and clearly warrants future investigation.
2
3,56
domains were evident.
It is believed that these functional
DH domains provide the required biochemical activity for
those modules in which the domain is absent; however, to date,
the mechanism behind this presumed multiple use of DH
domains remains unknown.
F
DOI: 10.1021/acs.jnatprod.5b00301
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Journal of Natural Products
Article
Figure 4. Selected 2D NMR data for columbamide A.
Next, the NRPS module (ColG) extends the acyl ACP by an
amino acid. On the basis of the Stachelhaus predictions, the
amino acid residues in the substrate-binding pocket of the A-
domain are consistent with those encoding for the amino acid
A (1), C H Cl NO for columbamide B (2), and
2
3
40
3
4
C H Cl NO for columbamide C (3).
21
39
2
3
The planar structures of each of the three analogues were
deduced from 1D and 2D NMR data (Supporting Information,
Figures S1−S9, S11−S21). The major metabolite, columba-
mide A (1), possessed an N-methyl amide as revealed from a
three-proton singlet (distributed between δ 2.95 and δ 2.82
57,58
serine (Supporting Information Table S1).
The absence of
an epimerase domain suggests that L-serine is incorporated into
the molecule; this configuration was confirmed through
subsequent hydrolysis and Marfey’s analysis (Figure S10).
Using the Delta BlastP function of the NCBI, two
methyltransferase domains were identified, and subsequent
structure elucidation confirmed the presence of one O-methyl
and one N-methyl group. The sequences of the ColG
methyltransferases were compared to known O- and N-
methyltransferases (see Supporting Information for multiple
sequence alignments, Tables S2 and S3). The N-methyltrans-
ferase domain of ColG shares an 80% identity to the N-
methyltransferase domain of BarG. By contrast, the O-
methyltransferase domain of ColG does not share a high
sequence homology to any known O-methyltransferase,
perhaps due to its unique positioning within an NRPS module.
H
H
61
due to two rotamers around the CO−N bond ) that showed
an HMBC correlation to the amide carbonyl carbon at δ
C
173.5. This carbon was connected by HMBC to a distinctive
spin system composed of two methylenes (δ 4.26/4.20, δH
H
3.58/3.48) that bordered either side of a deshielded methine
(δH 4.90/4.27). An O-methyl group, appearing as a three-
proton singlet at δH 3.32/3.34, was attached to the more
shielded of these two methylene groups, according to HMBC
data. An acetoxy group was attached to the second methylene
group (δ 4.26/4.20), as revealed by HMBC correlations from
H
both a three-proton singlet at δ 2.04/2.05 and the deshielded
H
methylene protons to an acetoxy carbonyl at δ 170.9. Location
C
of this functionalized serinol fragment distal to the amide
carbonyl was confirmed by HMBC correlations from H-18 to
59
This is the first report for such an arrangement. Lastly, the
peptidyl carrier protein (PCP)-bound product is released by a
reductase-releasing mechanism, presumably as a primary
alcohol. The reductase domain shows a 48% identity and
C-1 and from H -17 to both C-1 and C-18 (Table 4, Figure 4).
3
A 1,2-disubstituted alkene was evident from the two nearly
identical alkene protons at δH 5.45 connected by HSQC to
carbons with chemical shifts of δC 129.3 and 131.1. These
protons showed COSY correlations to allylic methylene
protons at δH 2.32 and 1.99. The former of these allylic
66% similarity to the LtxA release domain of the lyngbyatoxin
biosynthetic gene cluster, a terminating domain that has been
demonstrated to catalyze an NADPH-dependent reductive
60
cleavage to form a primary alcohol. ColH encodes for a small
hypothetical protein (35 amino acids), whose function is not
clear at this time. In a final postmodular assembly step, ColI, a
predicted acyltransferase, is thought to acetylate the primary
alcohol to form columbamides A (1) and B (2).
protons was located adjacent to the C-2 protons at δ 2.40 by
H
HMBC, thereby placing the olefin at C-4/C-5. The E-geometry
1
13
of the double bond was indicated by characteristic H and
C
NMR chemical shifts for isolated olefins in alkyl chains (e.g.,
62
methyl elaidate and methyl oleate). This was confirmed by
3
Structure Elucidation of Columbamides. Cultured M.
measurement of the J coupling constant (14 Hz) between
HH
13
bouillonii PNG biomass was repetitively extracted with CH Cl /
the two olefinic protons from the C satellites observed in the
HSQC spectrum (Figure S22).
2
2
63
MeOH (2:1, v:v) and fractionated using normal-phase vacuum
liquid chromatography (NP VLC). A fraction containing a
relatively abundant series of lipid metabolites containing
multiple halogen atoms was further purified using a reversed-
phase C18 column followed by preparative HPLC purification.
The structure elucidation of columbamide A was accomplished
using an integrated approach that made use of traditional
analytical data as well as bioinformatics from the gene cluster.
For example, the molecular formula was determined consider-
ing the HRMS data, the isotopic pattern of the parent ion, and
information deduced from the biosynthetic pathway. According
to the latter, the formula should contain halogen atoms, one
nitrogen atom, four oxygen atoms, two methyl groups, and a
fatty acid chain. Using this information, the three molecular
formulas were determined as C H Cl NO for columbamide
One chlorine atom was located attached to a terminal
methylene group at δH 3.53 (δC 45.2, C-16) and, by its
appearance as a triplet, indicated an adjacent second methylene
group at δ 1.78 (δ 32.6, C-15). The latter methylene showed
H
C
a COSY correlation to yet another methylene group at δ 1.45
H
(δC 26.9, C-14). An H2BC NMR experiment, providing
correlations between protons and carbons specifically separated
by two bonds, defined the methylene at δ 1.45 to be adjacent
H
to a methylene carbon at δ 28.2 with associated protons at δ
C
H
64
1.35 (C-13). H2BC data further placed this δ 28.2 carbon
C
adjacent to a methylene at δ 1.53 (δ 26.5, C-12). By COSY,
H
C
this methylene was adjacent to yet another CH group at δH
2
1.70 (δ 38.5, C-11). Finally, the latter resonance was located
C
by COSY and HMBC next to the second site of chlorination
2
3
41
2
4
G
DOI: 10.1021/acs.jnatprod.5b00301
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Journal of Natural Products
C-10) with a characteristic methine resonance at δ 3.88 (δC
Article
6
6
(
grenadamide, and semiplenamides A, B, and G. All of these
compounds were shown to have binding activity with the
cannabinoid receptors CB and CB . Comparing K values,
H
6
4.3).
Confirmation that the second chlorine atom resided at the ω-
position was obtained by locating this functional group at the
1
2
i
7
columbamide B (K = 0.41 μM) is slightly more active for CB
i
1
C-10 position relative to the carboxylate of a hexadecenoic acid
derivative. Despite overlap in both the proton and carbon
dimensions between the methylenes to either side of C-10, an
HMBC from the α-chloro methine proton to a carbon
resonance at δc 26.2 allowed connectivity to the upstream
portion of the carbon chain. This connectivity of C-10 to C-9
to C-8 could also be established from two sequential H2BC
correlations (Figure 4 and Table 4). H2BC correlations from
H -8 (δ 1.35) to C-7 (δ 29.1) were reinforced by HMBC
than the previously reported marine-derived CB1 ligand
mooreamide A (K = 0.47 μM). However, the columbamides
i
31
are not as CB receptor selective as was mooreamide A.
1
These two columbamides are the highest affinity ligands for
31
CB and CB ever identified from marine natural products.
1
2
CONCLUSION
■
Employing a combination of orthologous techniques, such as
genome mining and mass spectrometric networking, led to the
isolation and characterization of the structurally unique acyl
amides columbamides A−C (1−3). The Global Natural
Products Social Networking is an efficient tool for dereplicating
known compounds as well as identifying new compound
2
H
C
correlations from H -9 to C-7. By COSY as well as H2BC, the
2
C-7 methylene could be placed adjacent to the somewhat
deshielded allylic methylene at δH 1.99 (H -6). As this
2
methylene was already assigned in relationship to the
carboxylate terminus, an (E)-10,16-dichlorohexadec-4-enoate
residue was defined.
The only difference between columbamides A (1) and B (2)
was the presence of an additional chlorine atom. The location
41
classes. Using the Cytoscape platform, nodes are labeled by
parent mass, colored by strain, shaped by library hits, and sized
by number of spectra. Armed with this information, large
numbers of metabolites can be easily visualized and assessed,
and interesting new targets for isolation can be rapidly detected.
In addition, the pie chart node application allows identification
of which compounds are shared between Moorea strains and
of this addition was indicated by the triplet proton at δ 5.75
H
with its associated carbon shift of δ 73.5 ppm, indicative of a
C
terminal gem-dichloro functionality. Indeed, these bands
replaced those of the terminal chloro-methylene group in
columbamide A (1) [see Supporting Information for stacked
68
which compounds are unique to each strain. In the current
study, we found that the majority of secondary metabolites are
strain specific. Further, we came to focus on the identification
of several unknown products of M. bouillonii PNG. Through
molecular networking the columbamides were detected, and
this information was integrated with genomic information.
However, the fragmentation pattern of columbamide C, due to
the lack of the acetyl group, was different from the
fragmentation pattern of columbamides A and B, and therefore,
columbamide C was grouped into a different cluster (Figure 2).
These new natural products are interesting for the concise
integration of structural units and the uniqueness of the
positions of chlorination. In this regard, this pathway, along
1
H and 2D NMR spectra (Figures S1, S11−17) and Table 4].
In addition, the adjacent methylene group (C-15) was more
deshielded (δH 2.2 and δ 43.5). The other portions of the
C
molecule were identical to columbamide A.
In a similar fashion, the structure of columbamide C was
easily deduced as being the same as columbamide A but lacking
the acetoxy group at C-20 [see Supporting Information for
1
stacked H and 2D NMR spectra (Figures S1, S18−21) and
Table 4]. The chemical shifts of the hexadecenoic acid portion
of columbamide C were identical to columbamide A. The lack
of the acetoxy group at C-20 resulted in a more shielded shift of
48,49
the hydroxy-substituted methylene group at δ 3.79/3.76 (C-
H
with others such as that for cylindrocyclophane,
appears to
2
0) and the adjacent α-proton (C-18) at δ 4.38/4.13.
H
possess a novel type of halogenase distinct from the 2-
oxoglutarate-dependent radical halogenases we and others
previously characterized as a part of the barbamide biosynthetic
Next, the stereocenter of the dimethylated and acetylated
serinol residue was established through Marfey’s analysis. D-
and L-N-methyl-O-methylserinol standards were synthesized
28,46
pathway.
These new metabolites are biologically significant
65
according to standard protocols. The two standards were
derivatized with Marfey’s reagent (L-FDAA) and compared to
the derivatized hydrolysate of columbamide A. From the
matching retention times, it was clear that this residue derived
from L-serine, in accordance with the analysis of the
biosynthetic pathway (Figure S10).
as a result of their potent inhibition of the CB and CB
1
2
receptors. Comparison of the genomes from M. producens JHB
and 3L led to the identification of a highly homologous serine
histidine kinase sensor closely associated with major secondary
metabolite biosynthetic gene clusters (jamaicamide A in JHB
and curacin A in 3L, Figure 1). The concept of a “genome hot
spot” for secondary metabolite production is quite intriguing, as
an aid in both informatically locating new biosynthetic gene
clusters and potentially understanding their regulation.
However, whether the locus of the biosynthetic gene cluster
itself plays a crucial role or if the guanylate cyclase with its
serine histidine kinase domain plays an important function in
regulating secondary metabolite production remains unan-
swered.
Biological Properties of Columbamides. The relative
structural homology between the columbamides and the
endogenous cannabinoid ligands, anandamide and 2-arachido-
noyl glycerol, led us to evaluate the two most plentiful
columbamides (1, 2) for binding properties to the cannabinoid
receptors CB and CB . Columbamides A and B were found to
1
2
be potent ligands for the CB and CB receptor [for CB
1
2
1
(
±standard error): columbamide A: K = 0.59 ± 0.08 μM,
i
columbamide B: K = 0.41 ± 0.06 μM; for CB : columbamide
A: K = 1.03 ± 0.12 μM, columbamide B: K = 0.86 ± 0.09 μM],
i
2
i
i
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a Jasco P-2000 polarimeter. IR spectra were measured
on a Thermo Electron Corporation Nicolet IR 100 FT-IR. H and C
■
with columbamide B being slightly more active than
columbamide A. Both compounds showed almost 2-fold
greater affinity for CB compared to CB . Previous studies
1
13
1
2
have reported the isolation of other cyanobacterial-derived acyl
NMR spectra were recorded on a 600 MHz Bruker Avance III
spectrometer with a 1.7 mm Bruker TXI cyroprobe. Spectra were
31
66,67
amides, such as mooreamide A, serinolamides A and B,
H
DOI: 10.1021/acs.jnatprod.5b00301
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Journal of Natural Products
Article
referenced to residual CDCl solvent signals with resonances at δH/C
similarity. Hits to library inputs were marked as squares. The size of
the node is representative of the number of MS spectra present for
3
2
7
.26/77.1. Semipreparative HPLC was performed on a Waters 515
pump system equipped with a Waters 996 PDA detector.
each parent mass. To visualize which compounds are shared between
Cyanobacteria. Moorea bouillonii PNG05-198 was collected in
May 2005 by scuba in 3−10 m water depth, where it was found
growing tangled around the coral Stylophora pistillata off Pigeon
Island, Papua New Guinea. M. producens 3L was originally collected
the three Moorea strains, the pie-chart-creating tool (nodeCharts
6
8
plugin for Cytoscape) was used. Pie slices are proportional to the
number of MS2 spectra for each parent mass and are therefore a proxy
for its relative quantity. To simplify the network, nodes created by
solvent background were removed from the network.
Isolation of Genomic DNA. Genomic DNA was isolated from live
cultures of M. producens JHB and M. bouillonii PNG using a standard
phenol/chloroform/isoamyl alcohol (PCI) extraction protocol. In
brief, two grams (wet weight) of cultured biomass was rinsed with
fresh SW BG-11 media, flash frozen in liquid nitrogen, and ground to a
fine powder using a prechilled mortar and pestle. The powder was
resuspended in 10 volumes of lysis buffer (10 mM Tris pH 8, 0.1 M
EDTA pH 8, 0.5% w/v SDS, and 20 μg/mL RNase) and incubated at
near CARMABI Research Station in Curac a̧ o, Netherlands Antilles.
Moorea producens JHB 22Aug96-1 was collected in Hector’s Bay,
Jamaica. Live cultures have been maintained in SW BG-11 media
under laboratory conditions.
Metabolic Profiling. Each strain was grown in SW BG-11 media.
After harvest, the cells were freeze-dried and extracted with (2:1, v/v)
CH Cl /MeOH. The extraction procedure was repeated until the
2
2
supernatant was colorless. After removal of the solvent, the extracts
were dissolved in CH CN. To remove compounds that would not
3
elute from reversed-phase column material, each extract was purified
using a disposable 100 mg reversed-phase C18 catridge (Bond Elut-C18
3
7 °C for 30 min. Proteinase K was added (100 μg/mL final
concentration), and samples were incubated at 50 °C for 1 h. After
cooling the samples to room temperature (rt), one volume of
equilibrated phenol (Life Technologies) was added and samples were
mixed for 10 min. Phases were separated by centrifugation at 3300 rcf
for 10 min, and the aqueous phase was removed to a new tube. The
phenol extraction was repeated twice followed by a chloroform/
isoamyl alcohol (24:1) (Life Technologies) extraction. DNA was
precipitated and purified by ethanol precipitation. Genomic DNA was
quantified using a NanoDrop (Thermo Scientific) and qualified by gel
electrophoresis. Genomic DNA was sequenced at the genomics core
facility at the University of Michigan using Illumina HiSeq and at the
University of California San Diego genomics facility using PACBIO
OH, 100 mg, 1 mL, Agilent Technologies). The column was
conditioned with CH CN, and then an aliquot of extract was added
3
to the column and eluted with 4 mL of CH CN. The solvent was
3
removed, and the residue dissolved in CH CN to give a 10 mg/mL
3
solution. The samples were analyzed via HPLC coupled to a Thermo
Finnigan LCQ Advantage Max mass spectrometer or an Agilent 6530
Accurate Mass Q-TOF MS system. The Thermo Finnigan mass
spectrometer was attached to a Thermo Finnigan Surveyor
Autosampler-Plus, a LC-Pump-Plus, and a PDA-Plus system. ESI
conditions were set to 325 °C capillary temperature, 5 kV source
voltage, and 69 psi sheath gas flow rate. Four scan events were set up:
positive total ion count of m/z 100−2000, followed by three data-
(
M. bouillonii PNG). Assembly was done using the SPAdes Genome
2
dependent MS scans of the first, second, and third most intense ions
32
Assember 3.0 followed by Opera to produce cyanobacterial scaffolds.
Further binning was performed to obtain cyanobacterial-specific
scaffolds.
from the first scan event. The collision energy was 35%, the minimum
5
intensity 10 counts, the isolation width m/z 2, the dynamic exclusion
count 5 with a repeat duration of 1 min, an exclusion list of 25, and an
exclusion duration of 1.5 min.
The Agilent 6530 Accurate Mass Q-TOF MS system was attached
to a 1290 Infinity Binary LC system (Agilent Technologies). ESI
conditions were set to 300 °C capillary temperature, 3.5 kV VCap, and
Bioinformatics Analysis. Biosynthetic gene clusters were
identified using the genome mining software programs AntiSMASH,
27,37,54
NaPDoS, and met2db.
NRPS adenylation domain substrate
71
predictions were made using NRPSpredictor2. Annotations were
refined using Delta BlastP to identify conserved domains.
55
1
0 L/min gas flow. The scan events were set up as follows: positive
2
Phylogenetic analysis was performed by comparing the N- and O-
methyltransferase domains using the Web-based tool at www.
phylogeny.fr. Sequences of methyltransferases were obtained from
total ion count of m/z 100−1700, followed by data-dependent MS
scans. The collision energy was ramped with a 0.5 slope and a 30
offset; three precursors per cycle were fragmented with a MS abs
threshold of 200, isolation width of m/z 1.3, and an active exclusion
count of 5 with a repeat duration of 0.5 min.
7
2
SBSPKS. The nucleotide sequence of the columbamide pathway
genes has been submitted to GenBank under the accession number
KP715425.
The same gradient was used on both machines. The separation was
performed using a Phenomenex Kinetex C-18 100 Å 100 × 4.6 mm
column with a 700 μL/min flow and a linear gradient from 5% A (A:
CH CN, B: 0.1% HCOOH in H O) for 3 min to 100% A in 45 min,
Structure Elucidation. About 5.5 g (wet weight) of M. bouillonii
PNG was repetitively extracted with CH Cl /MeOH (2:1, v/v),
2
2
yielding 389 mg of extract. Subsequently the extract was fractionated
by silica gel vacuum liquid chromatography (VLC) using a stepwise
gradient solvent system of increasing polarity beginning from 100%
hexanes to 100% EtOAc to 100% MeOH, yielding nine subfractions.
The fraction eluting with 40% EtOAc and 60% hexanes (fraction D,
3
2
which was maintained for 10 min. Afterward the column was
equilibrated back to starting conditions.
The data were converted to mzXML format, a text-based format for
mass spectrometry data by using MSConvert, part of the
69
3
.8 mg) was separated further using a 100 mg reversed-phase C18
ProteoWizard package, and uploaded to the Global Natural Products
Social Molecular Networking Web site. The mass spectrometry data
are available through MassIVE Public GNPS Data sets
solid-phase extraction cartridge (Bond Elut-C18 OH, 100 mg, 1 mL,
Agilent Technologies) and a stepwise gradient solvent system of 25%,
5
0%, 75%, and 100% CH CN/H O, respectively. The 75% eluting
(
MSV000079016, gnps.ucsd.edu). These data were analyzed using
3 2
41
fraction was further purified using reversed-phase HPLC: Phenomenex
Kinetex C-18 100 Å 100 × 10 mm column with a 4 mL/min flow and
the molecular networking workflow. The input data were searched
against annotated reference spectra of the MS library within GNPS.
The GNPS MS library contains ITMS and QTof reference spectra of
2
2
a linear gradient from 75% A (A: CH CN, B: H O) for 3 min to 95%
3 2
4
1
previously isolated compounds of the Gerwick laboratory.
A in 24 min. Afterward, the column was equilibrated back to starting
conditions. The yield of columbamide A was 2 mg, columbamide B 1
Computationally, the algorithms compare MS2 spectra by their
similarity and assign similarity scores. The basic underlying algorithm
mg, and columbamide C 0.5 mg.
3
0,40,41,68
23
was published in detail elsewhere.
For the network presented
Columbamide A (1): [α]
D
−11.3 (c 0.27, CHCl ); IR (neat) νmax
3
−1
1
13
in this paper, the parent mass peak tolerance was set to 2 Da and the
ion tolerance for mass fragments was set to 0.5 Da. The minimum
cluster size was set to 1, and the cosine score was set to 0.7. For
visualization, the created molecular networks were imported into the
2927, 2856, 1621, 1452, 1124, 1089, 1046 cm ; H and C NMR,
Table 4 and Supporting Information; HRESIMS m/z 466.2478 [M +
+
H] (calcd for C23
H
42Cl
NO
, 466.2485); MS/MS (CID 32.3%; [M +
2
4
+
H] ) m/z (%) 370 (100), 398 (30), 334 (27), 102 (14), 362 (6).
7
0
23
program Cytoscape 2.8.3. Each node was labeled with their
respective parent mass. The edges between nodes indicated the level
of similarity between nodes, with thicker lines indicating higher
Columbamide B (2): [α] 8.7 (c 0.77, CHCl ); IR (neat) νmax
2928, 2853, 1627, 1467 cm ; H and C NMR, Table 4 and
Supporting Information; HRESIMS m/z 500.2087 [M + H] (calcd
D 3
−1
1
13
+
I
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Journal of Natural Products
Article
+
for C H Cl NO , 500.2096); MS/MS (CID 32.5%; [M + H] ) m/z
(
Synthesis of tert-Butyl 1-Hydroxy-3-methoxypropan-2-yl-
carbamate (6). To a stirred solution of ester 5 (75 mg, 0.32 mmol) in
23
41
3
4
%) 404 (100), 368 (99), 432 (35), 396 (30), 102 (17), 162 (7).
Columbamide C (3): [α]23 −27.5 (c 0.2, CHCl ); IR (neat) ν
MeOH at 0 °C was added NaBH in several portions. The reaction
D
3
max
4
−1
1
13
3
401, 2931, 2859, 1627, 1465, 1124 cm ; H and C NMR, Table 4
mixture was stirred at rt overnight, treated with water, and extracted
with ethyl acetate. The combined organic layer was washed with brine,
dried over anhydrous Na SO , and evaporated. The residue was
+
and Supporting Information; HRESIMS m/z 424.2377 [M + H]
calcd for C H Cl NO , 424.2380); MS/MS (CID 32.1%; [M +
(
21
40
2
3
2
4
+
H] ) m/z (%) 356 (100), 320 (46), 370 (17), 120 (4).
purified by flash chromatography on silica gel in ethyl acetate/hexanes
1
Marfey’s Analysis of Columbamide A and Synthesis of
Standards. To a solution of columbamide A (1) (0.4 mg) in MeOH
100 μL) was added 9 N HCl (200 μL), and the reaction stirred in a
microwave reactor at 140 °C. After 20 min the reaction was quenched
by 200 μL of saturated sodium bicarbonate solution, followed by
addition of 45 μL of L-FDAA (0.5% solution in acetone). The solution
was heated at 40 °C for 60 min, then quenched by addition of 150 μL
of 2 N HCl. The mixture was diluted with 200 μL of CH CN, and 30
μL of the solution was analyzed by using the HPLC-ITMS described
(
5
(
3
7
10−30%) to afford 6 as a colorless oil (48 mg, 74%). H NMR (6,
00 MHz, CDCl ) δ 5.26 (br s, 1H), 3.77−3.73 (m, 2H), 3.68−3.64
m, 1H), 3.56 (dd, J = 9.4, 3.8 Hz, 1H), 3.50 (dd, J = 9.5, 4.5 Hz, 1H),
.37 (s, 3H), 1.45 (s, 9H); C NMR (125 MHz, CDCl ) δ 156.3,
9.8, 72.9, 63.5, 59.3, 51.6, 28.5; LRESIMS m/z 206.34 [M + H]
3
(
13
3
+
(
calcd for C H NO 206.14).
9 20 4
Synthesis of tert-Butyl 1-(tert-Butyldimethylsilyloxy)-3-me-
thoxypropan-2-yl(methyl)carbamate (7). To a solution of alcohol
(34 mg, 0.17 mmol) in CH Cl (2 mL) at 0 °C were added tert-
3
6
2
2
above.
butyldimethylsilyl chloride (26 mg, 0.17 mmol) and imidazole (13 mg,
0.19 mmol). The reaction was stirred overnight at rt and then
quenched by addition of water. The organic layer was separated,
washed with 2 N HCl, dried over anhydrous Na SO , and evaporated.
The standard alcohols were synthesized according to the literature
procedure. Esterification of O-methylserine (4) followed by Boc
protection gave product 5 in 82% yield over two steps. Reduction of
ester 5 with NaBH gave alcohol 6, which was protected with TBDMS
to give compound 7. It was deprotected using TFA, and the crude
product was reacted with L-FDAA Marfey’s reagent, in the presence of
65
2
4
4
The crude product was used in the next step without additional
purification.
To a solution of tert-butyl 1-(tert-butyldimethylsilyloxy)-3-methox-
ypropan-2-ylcarbamate in THF (1 mL) was added NaH (60%
dispersion in mineral oil, 22 mg, 0.47 mmol) at 0 °C. After stirring at
the same temperature for 30 min, a solution of MeI (30 μL, 0.59
mmol) in DMF (30 μL) was added. The reaction mixture was warmed
̈
excess Hunig’s base, to prepare the standards. The O-methyl-L-serine
gave the R-enantiomer of alcohol 8, while the O-methyl-D-serine
resulted in the S-enantiomer of compound 8. The Marfey’s derivatives
L-FDAA of the hydrolysate and standards were analyzed with HPLC-
ITMS by using a Phenomenex Luna 5 μm C18 column (4.6 × 250
to rt and stirred overnight. It was quenched with H O, and the organic
mm). The HPLC conditions started with 10% CH CN and 90%
HCOOH (0.1%) in H O followed by a gradual increase to 50%
CH CN and 50% HCOOH in H O (0.1%) over 100 min at a flow rate
of 0.4 mL/min with monitoring the total ion count from 100 to 2000
m/z. The retention time for the L-FDAA derivative of the S-alcohol
was 72.4 min, while it was 64.3 min for the R-alcohol. The retention
time of the L-FDAA derivative of the hydrolysate product gave a peak
with a retention time of 64.7 min, corresponding to the R-alcohol from
the L-serine.
2
3
layer was separated. The aqueous layer was extracted with EtOAc, and
2
the combined organic layer was dried over anhydrous Na SO and
2
4
3
2
concentrated. The residue was purified by flash chromatography on
2
2
silica gel in CH
1.0, CHCl ) for the S-enantiomer of 7 (derivative from O-methyl-L-
serine). [α] +10.7 (c 1.0, CHCl ) for the R-enantiomer of 7
3
Cl
to give product 7 (48 mg, 85%). [α]
−12.6 (c
2
2
D
3
22
D
1
(derivative from O-methyl-D-serine). H NMR (7, 500 MHz, CDCl )
3
δ 4.24 (br s, 1H), 3.75−3.66 (m, 2H), 3.61−3.57 (m, 1H), 3.53−3.47
(
6
5
m, 2H), 3.33 (s, 3H), 2.84 (s, 3H), 1.46 (s, 9H), 0.89 (s, 9H), 0.15 (s,
H); 13C NMR (125 MHz, CDCl ) δ 156.3, 79.7, 71.0, 62.1, 59.0,
3
+
7.2, 31.6, 28.8, 26.2, −5.3; LRESIMS m/z 334.18 [M + H] (calcd for
C H NO Si 334.24).
16
36
4
CB1 and CB2 Receptor Binding Assays. CB1 and CB2
displacement assays were performed according to a previously
7
3
reported protocol. Membranes from HEK-293 cells stably trans-
fected with human recombinant CB receptor (B = 2.9 pmol/mg
1
max
protein) or human recombinant CB receptor (B = 6.0 pmol/mg
2
max
3
protein) were incubated with the high-affinity ligand [ H]-CP-55,940
0.4 nM/K = 0.11 nM and 0.53 nM/K = 0.19 nM for CB and CB ,
(
d
d
1
2
respectively). The radioactive ligand was displaced with 10 μM WIN
55212-2 as the heterologous competitor to determine nonspecific
Synthesis of Methyl 2-(tert-Butoxycarbonylamino)-3-
methoxypropanoate (5). To a solution of D- or L-O-methyl serine
(
0.15 g, 1.26 mmol) in MeOH (3 mL) at 0 °C was added thionyl
binding (K values 8.8 and 0.9 nM for CB and CB , respectively). The
i
1
2
chloride (1 mL) dropwise. The reaction mixture was warmed to room
temperature and stirred overnight. The solvent was evaporated to give
a yellow solid, which was used without further purification.
compounds were tested according to the protocol described by the
manufacturer (PerkinElmer). Displacement curves were obtained by
3
incubating columbamides A and B with [ H]-CP-55,940 for 90 min at
A mixture of O-methylserine methyl ester hydrochloride salt in
30 °C. K values were calculated by applying the Cheng-Prusoff
i
CH Cl (7 mL) at 0 °C was added to Hu
̈
nig’s base (0.66 mL, 3.78
equation to the IC50 values obtained from GraphPad for the
displacement of the bound radioligand by increasing concentrations
of the test compounds. Data are means of at least three replicates.
2
2
mmol) and Boc-anhydride (275 mg, 1.26 mmol). The reaction
mixture was warmed to rt and stirred overnight, after which it was
diluted with CH Cl (10 mL) and washed with 1 N HCl and brine.
2
2
The combined organic layer was dried over anhydrous Na SO and
2
4
ASSOCIATED CONTENT
evaporated. It was purified by flash chromatography on silica gel in
CH Cl /hexanes (75−100%) to give methyl 2-(tert-butoxycarbonyl)-
■
2
2
* Supporting Information
S
1
3
-methoxypropanoate (5) as a yellow oil (0.23 g, 82%). H NMR (500
Stachelhaus prediction, multiple sequence alignments of
methyltransferases, NMR data, and Marfey’s analysis chromato-
grams. The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/
acs.jnatprod.5b00301.
MHz, CDCl ) δ 5.37 (d, J = 8.5 Hz, 1H), 4.41 (dd, J = 8.2, 3.8 Hz,
3
1
3
1
H), 3.83−3.77 (m, 1H), 3.76 (s, 3H), 3.59 (dd, J = 9.5, 3.5 Hz, 1H),
.35 (s, 3H), 1.45 (s, 9H); 13C NMR (125 MHz, CDCl ) δ 171.4,
3
55.7, 80.1, 72.7, 59.4, 54.0, 52.6, 28.4; LRESIMS m/z 234.17 [M +
+
H] (calcd for C H NO 234.13).
10
20
5
J
DOI: 10.1021/acs.jnatprod.5b00301
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
23) Chang, Z.; Sitachitta, N.; Rossi, J. V.; Roberts, M. A.; Flatt, P.
AUTHOR INFORMATION
Corresponding Author
Tel: +1 (858) 534-0578. Fax: +1 (858) 534-0576. E-mail:
wgerwick@ucsd.edu.
■
*
M.; Jia, J.; Sherman, D. H.; Gerwick, W. H. J. Nat. Prod. 2004, 67,
356−67.
24) Ramaswamy, A. V.; Sorrels, C. M.; Gerwick, W. H. J. Nat. Prod.
1
(
2
007, 70, 1977−86.
Notes
(25) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett, T.
The authors declare no competing financial interest.
H. J. Am. Chem. Soc. 2001, 123, 5418−23.
(
26) Calteau, A.; Fewer, D. P.; Latifi, A.; Coursin, T.; Laurent, T.;
ACKNOWLEDGMENTS
K.K. was supported by a fellowship within the Postdoc-
Programme of the German Academic Exchange Service
DAAD); R.K. was supported by the Hamilton College; A.K.
was supported in part by the Russian Science Foundation
grant 14-50-069). This work was supported from NIH-R01
Jokela, J.; Kerfeld, C. A.; Sivonen, K.; Piel, J.; Gugger, M. BMC
Genomics 2014, 15, 977.
■
(27) Blin, K.; Medema, M. H.; Kazempour, D.; Fischbach, M. A.;
Breitling, R.; Takano, E.; Weber, T. Nucleic Acids Res. 2013, 41,
W204−W212.
(
(28) Flatt, P. M.; O’Connell, S. J.; McPhail, K. L.; Zeller, G.; Willis,
(
C. L.; Sherman, D. H.; Gerwick, W. H. J. Nat. Prod. 2006, 69, 938−44.
(29) Gu, L.; Wang, B.; Kulkarni, A.; Gehret, J. J.; Lloyd, K. R.;
Gerwick, L.; Gerwick, W. H.; Wipf, P.; Hakansson, K.; Smith, J. L.;
Sherman, D. H. J. Am. Chem. Soc. 2009, 131, 16033−5.
GM107550 and NIH CA108874.
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(
(
(
1
(
̈
(
(
1
(
(
(
(
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rtz, P.; Nyberg, N.
(
(
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DOI: 10.1021/acs.jnatprod.5b00301
J. Nat. Prod. XXXX, XXX, XXX−XXX