Discovery and Total Synthesis of Doscadenamide A: A Quorum Sensing Signaling Molecule from a Marine Cyanobacterium

Article abstract of DOI:10.1021/acs.orglett.9b02525

Quorum sensing (QS) plays a critical role in the regulation of bacterial pathogenesis. Doscadenamide A (1a) was isolated from a marine cyanobacterium, its structure elucidated by NMR, and its activity linked to QS induction. The total synthesis of 1a was developed, and the absolute configuration confirmed through comparison of the isolated natural product with synthetic diastereomers. Our preliminary investigation indicated that 1a could activate QS signaling in a LasR-dependent manner.

Full text of DOI:10.1021/acs.orglett.9b02525

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Discovery and Total Synthesis of Doscadenamide A: A Quorum
Sensing Signaling Molecule from a Marine Cyanobacterium
Xiao Liang,†,‡ Susan Matthew,† Qi-Yin Chen,†,‡ Jason C. Kwan,† Valerie J. Paul,§
and Hendrik Luesch*,†,‡
†Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610, United States
‡Center for Natural Products, Drug Discovery and Development (CNPD3), University of Florida, Gainesville, Florida 32610,
United States
§Smithsonian Marine Station, Fort Pierce, Florida 34949, United States
S
* Supporting Information
ABSTRACT: Quorum sensing (QS) plays a critical role in
the regulation of bacterial pathogenesis. Doscadenamide A
(1a) was isolated from a marine cyanobacterium, its structure
elucidated by NMR, and its activity linked to QS induction.
The total synthesis of 1a was developed, and the absolute
configuration confirmed through comparison of the isolated
natural product with synthetic diastereomers. Our preliminary
investigation indicated that 1a could activate QS signaling in a
LasR-dependent manner.
uorum sensing (QS) is an intercellular communication
Q process adopted by a number of bacteria to regulate
diverse physiological activities. This process involves the
production and release of diffusible extracellular signaling
molecules named autoinducers (AIs), which would accumulate
with an increase in bacterial population density.1−3 QS plays a
pivotal role in regulating bacterial pathogenesis. For example,
QS modulates the production of virulence factors such as
pyocyanin and elastase in Pseudomonas aeruginosa during
bacterial growth and infection.4 Thus, QS signaling pathways
are an attractive target for the development of antimicrobial
therapeutic agents. P. aeruginosa is a Gram-negative opportun-
Figure 1. Structures of the endogenous quorum sensing signaling
istic pathogen that can cause serious lung infections in cystic
fibrosis patients5 and microbial keratitis during contact lens
molecules in P. aeruginosa (C4-HSL and 3-oxo-C12-HSL), C8-AHL
produced in a cyanobacterium culture, and natural quorum sensing
wear.6 The AIs that control QS signaling in P. aeruginosa
inhibitors from marine cyanobacteria, lyngbyoic acid and pitinoic acid
A.
include two acylhomoserine lactones with varying alkyl chain
lengths and oxidation states at C-3 [C4-HSL and 3-oxo-C12-
HSL (Figure 1)] and a group of quinolone compounds
homoserine lactone [C8-HSL (Figure 1)] can be produced
in the cyanobacterium culture of Gloeothece PCC6909, and its
[Pseudomonas quinolone signal (PQS)]. They can diffuse freely
across cell membranes and bind intracellularly with corre-
sponding receptor proteins (R proteins).7 These signaling
accumulation corresponds to a characteristic pattern of
autoinduction.13
Marine cyanobacteria have been a valuable source for the
systems form a complex hierarchical quorum sensing network,
where the Las system is considered to be the apex of the
hierarchy.8 Therefore, the LasR receptor is usually considered
discovery of biologically active and structurally unique natural
products, including peptides, polyketides, and peptide−
to be the target for antagonist and agonist development in P.
polyketide hybrids.14 It is noteworthy that marine cyanobac-
teria also produce various AHL-dependent QS inhibitors.15
For instance, lyngbyoic acid (Figure 1), a small cyclopropane-
aeruginosa.8,9 For example, a synthetic non-native AHL, QSI-1,
was demonstrated to be a potent LasR antagonist.10 The
structurally unrelated mimic of AHL, TP-1, is a highly selective
superagonist of the LasR quorum sensing system, while its
derivative TP-5 turned into a moderate QS antagonist.11,12
Moreover, there is evidence showing that N-octanoyl
Received: July 19, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b02525
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Letter
containing fatty acid, was isolated from Lyngbya cf. majuscula
and proven to strongly inhibit the activity of LasR.7 Pitinoic
acid A (Figure 1) was also reported to be a P. aeruginosa
quorum sensing inhibitor.16 In addition to these QS inhibitors,
here we report the isolation, total synthesis, and preliminary
biological investigation of a structurally unprecedented QS
modulator, doscadenamide A [1a (Figure 2)], as a non-HSL
QS agonist in a LasR-dependent manner.
Figure 3. PGME analysis. Δδ (δS − δR) values for PGME derivatives
of 1a.
corresponding to the α-methyl group, which could be observed
in the COSY spectra of the two PGME derivatives (Figures S8
and S9). To further validate the configuration of 1a and
provide sufficient material for thorough biological investiga-
tion, the total synthesis of 1a was performed.
As depicted in Scheme 1, the retrosynthetic analysis of 1a
relied on the disconnection at the two amide linkages between
Scheme 1. Retrosynthetic Analysis of Doscadenamide A
(1a)
Figure 2. Structure of doscadenamide A (1a).
The cyanobacterium Moorea bouillonii was collected at
Fingers Reef, Guam, and previous investigation of this
cyanobacterium led to the isolation of apratoxin A,17
lyngbyaloside, 2-epi-lyngbyaloside, 18E-lyngbyaloside C, 18Z-
lyngbyaloside C,18 and apratyramide.19 The cyanobacterial
sample was fractionated as described previously,18 and the
isolation was achieved by silica gel column chromatography
and several rounds of reversed-phase HPLC to yield
doscadenamide A (1a) {white solid, [α]20 + 40 (c 0.07,
D
MeOH)}. The HRESIMS spectrum of 1a in the positive mode
exhibited a [M + H]+ peak at m/z 457.3066, which suggested a
molecular formula of C27H40N2O4 with nine degrees of
unsaturation. The structure of 1a was elucidated using a
combination of one-dimensional (1D) and two-dimensional
(2D) NMR techniques. The 1H and 13C NMR spectra of 1a in
CDCl3 (Figures S1 and S2) indicated the presence of several
characteristic signals corresponding to one O-methyl group (δH
3.85 ppm, δC 58.9 ppm), two alkyne groups (δH 1.92−1.94
ppm, δC 68.5, 68.5, 84.6, 84.7 ppm), one α-proton (δH 4.64−
4.66 ppm, δC 59.2 ppm), two α-methyl groups (δH 1.12−1.14
ppm, δC 16.3−18.1 ppm), and several methylene groups (δH
1.30−1.90, 2.06−2.18 ppm, δC 18.4−39.4 ppm). Examination
of the 2D NMR spectra [COSY, TOCSY, HSQC, HMBC, and
NOESY (Figures S3−S7 and Table S1)] in CDCl3 revealed
the structural skeleton of 1a, which features two linear alkyne
amide side chains [Moya1 and Moya2 (Figure 2)] and one
pyrrolinone core [pyLys-OMe (Figure 2)]. To establish the
absolute configuration, a portion of 1a (2 mg) was treated with
ozone at 25 °C for 30 min, followed by oxidative workup and
acid hydrolysis (Scheme S1). The hydrolysate was concen-
trated and partitioned between water and EtOAc. The
resulting aqueous phase was analyzed by chiral HPLC-MS
revealing the presence of L-Lys, establishing the S configuration
of C4 at the pyLys-OMe moiety. The organic residue was
coupled with (S)- and (R)-phenylglycine methyl ester
(PGME)20 (Scheme S2) to analyze the configuration of the
α-methine in the side chain of 1a. The Δδ values (Δδ = δS −
δR) for the PGME derivatives (Figure 3) indicated that the
configurations of the α-methine in both side chains of 1a are
highly likely R and that the overall configuration is 4S,11R,20R.
However, during the investigation, we found a minor
diastereomer signal in addition to the major NMR signal
the pyrrolinone ring and two side chain carboxylic acids, which
in the case of 1a are the same as (R)-2-methyloct-7-ynoic acid
(Moya, 2a). The pyrrolinone ring can be obtained via the
reaction between the doubly protected amino acid Fmoc-L-Lys
(Boc)-OH (5) and Meldrum’s acid (6). As for the synthesis of
2a, the target compound can be achieved in 14 steps using a
method reported in 2005.21 To improve the efficiency and
introduce more flexibility into the production of diverse
carboxylic acids with α-substituted alkyl groups, we developed
an optimized synthetic method (Scheme 2), in which 2a can
be obtained in three steps in 38% overall yield. The
commercially available oct-7-ynoic acid (2c) was activated
using pivaloyl chloride followed by addition of the lithium salt
of the oxazolidinone chiral auxiliary at −78 °C. The resulting
7a was methylated under conventional conditions to yield 8a
as a single diastereomer.22,23 Target compound 2a was
Scheme 2. Optimized Synthesis of Moya (2a)
B
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obtained following alkaline hydrolysis by lithium hydro-
peroxide.24
The total synthesis of 1a was accomplished using synthetic
carboxylic acid 2a (Scheme 3), including generation of the
4S,11S,20R for 1b, 4S,11S,20S for 1c, and 4S,11R,20S for 1d.
From a comparison of the NMR spectra of all four
diastereomers with those of the isolated natural product
doscadenamide A (Figure 4 and Figures S10 and S11), the four
1
diastereomers displayed different H NMR signals at δ 5.42−
Scheme 3. Total Synthesis of Doscadenamide A (1a)
5.55 ppm, δ 3.11−3.28 ppm, δ 2.04−2.21 ppm, δ 1.90−1.95
1
ppm, δ 1.32−1.56 ppm, and δ 1.09−1.22 ppm. Only the H
and 13C NMR spectra of synthetic 1a matched those of the
natural product. Meanwhile, 1a and 1b exhibited virtually the
same 13C NMR spectrum as well as the natural product, while
1c and 1d displayed 13C NMR spectra different from those of
the isolated doscadenamide A (Figure S11). In addition, the
optical rotation value of 1a {[α]20 +54.3 (c 0.07, MeOH)}
D
further confirmed that the absolute configuration of synthetic
diastereomer 1a is consistent with the isolated doscadenamide
A, verifying the proposed configuration of doscadenamide A.
Doscadenamide A (1a) and QS signaling molecule 3-oxo-
C12-HSL [C12 (Figure 1)] share structural similarities
because they both feature a five-membered ring core and
long alkyl side chains. In addition, 1a superficially resembles
tetramic acid derivative compounds, and C12 has been
previously shown to undergo rearrangement to form a tetramic
acid derivative.27 Thus, we proposed 1a may exhibit QS
modulatory activities. Our preliminary screening results
indicated that 1a can activate the 3-oxo-C12-HSL-responsive
reporter plasmid pSB1075,28 a plasmid encoding LasR and
containing a light-producing luxCDABE cassette expressed in
E. coli (Figure 5A). However, 1a was not able to activate the
related reporter pTIM5319,7 which is identical to pSB1075 but
lacks the AHL-binding site LasR (Figure 5B), which suggests
that 1a activates QS via the AHL-binding site. To validate the
activity of 1a, we tested its effect on wild-type P. aeruginosa
using activating doses (10, 100, and 1000 μM) based on
reporter assay results. Because 10 μM already caused almost
maximal induction of the QS pigment pyocyanin production
(Figure S15), we selected 10 μM for subsequent comparative
biological investigations. After further optimization of the assay
condition using a more concentrated cell density, we included
the other three diastereomers. As shown in Figure 5C, after
treatment with 1a−1d, pyocyanin levels were elevated in
response to all diastereomers after 6 h. Our preliminary results
indicated that the C11 and C20 configurations are not crucial
for QS activation by the doscadenamide scaffold but may play
a slight modulatory role. However, the assay is sensitive to cell
density, timing, and other factors. In-depth SAR studies are
ongoing to understand the molecular basis. The observed
agonistic activities may provide a starting point for the
development of potential superagonists with new structural
skeletons. Those superagonists are expected to artificially
regulate virulence factor production such as pyocyanin and
activate QS at lower bacterial cell populations and, thus, to
stimulate the host immune system to clear the infection when
fewer bacterial cells are present.3
pyrrolinone core and two coupling processes to assemble the
structure. Pyrrolidine-2,4-dione 9a was prepared through
condensation of 5 with 6 in the presence of EDCI and
DMAP, followed by thermolysis.25 This intermediate was used
in the next step without purification. Conversion of 9a into its
O-methylated derivative 4 was achieved by treatment with
trimethylsilyldiazomethane. Subsequently, the N-Fmoc pro-
tecting group in 4 was removed using piperidine to yield the
secondary amide of tetramic acid 3. The first coupling was
accomplished by condensation of the anion derived from
deprotonation of 3 by nBuLi and active ester 10a derived from
activation of 2a by pentafluorophenol to yield 11a.26 On the
basis of the 1H NMR spectrum of 11a, there was <5% impurity
1
(Figure S12). In addition, from comparison of the H NMR
spectra of 11a and its epimer at C20, 11b, we found that this
impurity may be introduced due to minor epimerization at
C20 (Figure S13). After removal of the Boc protecting group
with TFA, intermediate 12a was condensed with 2a using
typical coupling conditions to afford target compound 1a in six
1
steps in 30% overall yield from 3. The H NMR spectrum of
In summary, we have isolated a new quorum sensing
modulating agent, doscadenamide A (1a), from a marine
cyanobacterium. Its total synthesis has been accomplished
through development of an efficient and flexible scheme.
Specifically, we developed an efficient method for synthesizing
α-alkylated carboxylic acids. The quorum sensing activating
activity of 1a was verified in the Gram-negative bacterium P.
aeruginosa. Doscadenamide A (1a) could serve as a new
template for the development of QS superagonists with new
1a indicated <5% impurity and that synthetic 1a had a
diastereomeric purity that was higher than that of the natural
product doscadenamide A (see the methyl region in Figure 4).
To further validate the configuration of the stereocenters in
the side chain, the three other diastereomers [1b−1d (Figure
4)] of 1a were also synthesized using the described synthetic
method. Similarly, there is <5% impurity shown in the H
NMR spectra of 1b−1d. Specifically, the absolute config-
urations of the three stereocenters are 4S,11R,20R for 1a,
1
C
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Figure 4. Comparison of selected regional 1H NMR spectra of the isolated natural product doscadenamide A and synthetic diastereomers 1a−1d
(maroon, olive, green, navy, and purple bottom to top, respectively) in CDCl3 (600 MHz) at 27 °C, with structures of synthetic diastereomers 1a−
1d shown on the left.
Figure 5. Preliminary determination of the activating activity of doscadenamide A (1a) in (A) pSB1075, the lasR-luxCDABE reporter constructs
expressed in E. coli, and (B) related reporter pTIM5319, which lacks a functional AHL-binding domain. The bacterial cultures were treated with 1a
in a dose−response manner and solvent control at 37 °C for 6 h before luminescence was measured. Results are expressed as fold activation
compared to the solvent control. Effect of doscadenamide A (1a) and its diastereomers (1b−1d) as well as positive control 3-oxo-C12-HSL (C12)
at 10 μM on the (C) production of pyocyanin in wild-type P. aeruginosa after being shaken for 6 h at 37 °C. Data are presented as means the
standard deviation (**P < 0.01; ****P < 0.0001) compared to the solvent control using one-way ANOVA (n = 3).
skeletons to explore the potential of these activators as
therapeutics or chemical tools.
Experimental Procedures, Table S1, Figures S1−47,
including NMR spectra and assay data, and Supple-
mentary References (PDF)
ASSOCIATED CONTENT
■
AUTHOR INFORMATION
■
S
* Supporting Information
Corresponding Author
*E-mail: luesch@cop.ufl.edu.
ORCID
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b02525.
Valerie J. Paul: 0000-0002-4691-1569
D
DOI: 10.1021/acs.orglett.9b02525
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Letter
(20) Toki, M.; Ooi, T.; Kusumi, T. J. Nat. Prod. 1999, 62 (11),
1504−1509.
Hendrik Luesch: 0000-0002-4091-7492
Author Contributions
(21) Chen, H.; Feng, Y.; Xu, Z.; Ye, T. Tetrahedron 2005, 61 (47),
11132−11140.
X.L.: total synthesis, biological experiments with P. aeruginosa,
experimental design, and manuscript writing. S.M.: isolation
and structure elucidation. Q.-Y.C.: supervision of the total
synthesis experimental design. J.C.K.: biological evaluation
using reporter assays. V.J.P.: sample collection and identi-
fication and manuscript editing. H.L.: project supervision,
experimental design, and manuscript editing. All authors have
given approval to the final version of the manuscript.
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Notes
The authors declare the following competing financial
interest(s): The University of Florida has filed a patent
application related to the subject of the manuscript.
(27) Kaufmann, G. F.; Sartorio, R.; Lee, S. H.; Rogers, C. J.; Meijler,
M. M.; Moss, J. A.; Clapham, B.; Brogan, A. P.; Dickerson, T. J.;
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ACKNOWLEDGMENTS
■
The authors acknowledge financial support from the National
Institutes of Health, National Cancer Institute Grant
R01CA172310 and National Institute of General Medical
Sciences Grant P41GM086210, and the Debbie and Sylvia
DeSantis Chair professorship (H.L.). The authors thank Dr.
Max Teplitski (formerly Soil and Water Sciences Department,
University of Florida Institute of Food and Agricultural
Sciences, Gainesville, FL) for providing reporter strains and
support with the reporter assays.
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