Synthesis and biological activity of methylated derivatives of the Pseudomonas metabolites HHQ, HQNO and PQS

Article abstract of DOI:10.3762/bjoc.15.18

Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine–metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively.

Full text of DOI:10.3762/bjoc.15.18

Synthesis and biological activity of methylated derivatives of
the Pseudomonas metabolites HHQ, HQNO and PQS
Sven Thierbach1, Max Wienhold2, Susanne Fetzner*1 and Ulrich Hennecke*2,3
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 187–193.
1Institute for Molecular Microbiology and Biotechnology, University of
doi:10.3762/bjoc.15.18
Münster, Corrensstr. 3, 48149 Münster, Germany, 2Organic
Chemistry Institute, University of Münster, Corrensstr. 40, 48149
Münster, Germany and 3Organic Chemistry Research Group,
Departments of Chemistry and Bioengineering Sciences, Vrije
Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium
Received: 29 September 2018
Accepted: 02 January 2019
Published: 21 January 2019
Associate Editor: J. S. Dickschat
Email:
Susanne Fetzner* - fetzner@uni-muenster.de; Ulrich Hennecke* -
ulrich.hennecke@vub.be
© 2019 Thierbach et al.; licensee Beilstein-Institut.
License and terms: see end of document.
*
Corresponding author
Keywords:
antibiotic acitivity; methylation; Pseudomonas aeruginosa; quinolones;
quorum sensing
Abstract
Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the
Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa,
have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones,
methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation
and iodine–metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activi-
ty comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa
especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated
derivatives of HQNO and HHQ, respectively.
Introduction
2
-Alkyl-4(1H)-quinolones (AQs) have been identified as natural tunistic pathogen Pseudomonas aeruginosa, AQ derivatives
products produced by higher plants of the Rutaceae family as with heptyl or nonyl side chains are prevalent [3,7-9]. 2-Heptyl-
well as by some microorganisms including Alteromonas, Burk- 3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal,
holderia and Pseudomonas species [1-9]. Plant-derived AQs PQS) and its biosynthetic precursor 2-heptyl-4(1H)-quinolone
occur with alkyl chains of different lengths, branches and unsat- (HHQ, 1) are important signaling molecules involved in
uration and can be O- or N-methylated [1-3]. In the oppor- quorum sensing and as such play an important role in virulence
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Beilstein J. Org. Chem. 2019, 15, 187–193.
regulation [3,10-12]. Another metabolite from the AQ biosyn-
thesis pathway of P. aeruginosa is 2-heptyl-1-hydroxy-4(1H)-
quinolone (generally referred to as 2-heptyl-4-hydroxyquino-
line N-oxide, HQNO), which is not active in quorum sensing,
but interferes with the respiratory electron transport via inhibi-
tion of the cytochrome bc1 complex and moreover inhibits
pyrimidine biosynthesis [9,12-14]. Therefore, HQNO is toxic to
many organisms. In dual-species co-cultures of P. aeruginosa
and Staphylococcus aureus, which model interactions relevant
to patients with cystic fibrosis, HQNO is a major factor for
killing of S. aureus [15]. While HQNO seems to be generally
toxic to Gram-positive bacteria, we could show that some
bacteria are able to transform HQNO into less toxic metabolites
[
16]. For example, Mycobacterium abscessus, which like
P. aeruginosa and S. aureus can occur in the lung of cystic
fibrosis patients [17-19], is able to methylate HQNO to give
2
-heptyl-1-methoxy-4(1H)-quinolone (HMOQ). HMOQ is a
Scheme 1: Methylation of HHQ (1).
significantly less efficient inhibitor of the respiratory chain
cytochromes in Mycobacteria than HQNO and therefore the
methylation of HQNO can be seen as detoxification strategy was methylated using MeI and K2CO3 [23]. This procedure
[
16].
provided a mixture of O- and N-methylated products with a
slight preference for OMe-HHQ (3). OMe-HHQ (3) could be
Considering the natural occurrence of N- as well as O-methyl- easily separated from the N-methylation product by column
ated AQs in plants and the use of methylation of HQNO in chromatography and could be isolated in 32% yield. Surprising-
Mycobacteria as detoxification reaction, we decided to investi- ly, the N-methylated product under these conditions was not
gate the effect of methylation of AQs on their biological proper- NMe-HHQ (2), but instead a second methylation in the benzylic
ties more systematically. To this end, we prepared selectively position had occurred to give N-methyl-2-(1-methylheptyl)-
methylated derivatives of the most important heptyl-AQs in- 4(1H)-quinolone (4), which could be isolated in 14% yield.
cluding HHQ, HQNO and PQS derivatives. These synthetic
compounds were tested for their antimicrobial properties For the synthesis of methylated HQNO derivatives, HHQ (1)
against S. aureus and for their quorum sensing activity.
was converted to HQNO (5) using a literature procedure [24],
which was slightly modified to provide HQNO in 69% yield
over three steps without purification by column chromatogra-
phy (see Supporting Information File 1 for details). Selective
Results and Discussion
Synthesis of methylated heptyl-AQs
The synthesis of methylated AQ derivatives started from HHQ methylation of one of the O-atoms of HQNO was attempted
1), which is conveniently available via Conrad–Limpach reac- using various methods. Treatment of HQNO (5) in MeOH/Et2O
(
tion in large quantities [20,21]. Upon treatment of HHQ (1) with TMS-DA resulted in selective methylation of the N–O
with n-butyllithium and dimethyl sulfate mainly N-methylation oxygen atom to provide 1-methoxy-2-heptyl-4(1H)-quinolone
was observed accompanied by a small amount of the N,O- (HMOQ (6), Scheme 2) [16]. The methylation was highly selec-
dimethylquinolonium ion. Pure NMe-HHQ (2) was obtained in tive and no methylation at the 4-position oxygen atom was ob-
moderate yield of 51% by column chromatography (Scheme 1). served, however, conversion and, therefore, yields remained
low even with a large excess of reagent. Addition of HBF4 did
Selective O-methylation of an AQ has been reported using not improve the yield, but led to side products, which proved to
diazomethane [1]. To avoid explosive diazomethane, methyla- be difficult to remove.
tion of HHQ with trimethylsilyldiazomethane (TMS-DA) in
methanol was carried out, which indeed provided selectively Several attempts were made to obtain the 4-O-methylated
OMe-HHQ (3). However, even using a large excess of TMS- HQNO by direct methylation of HQNO (5), however, treat-
DA low conversion and yields of less than 20% OMe-HHQ (3) ment of HQNO with strong bases such as NaH followed by a
were observed. Addition of HBF4 did improve the reactivity methylating agent (MeI) did not provide methylated HQNO de-
[
22], but instead of the desired 3 dimethylation to give the N,O- rivatives, but complex mixtures containing decomposition prod-
dimethylquinolonium ion was observed. Alternatively, HHQ ucts including HHQ (1) and methylated HHQ derivatives. The
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Beilstein J. Org. Chem. 2019, 15, 187–193.
Scheme 2: Synthesis of methylated HQNO derivatives.
N–O bond appeared to be unstable under these alkylation condi-
tions. Therefore, the synthesis of the 4-methoxy derivative was
carried out starting from OMe-HHQ (3). Upon treatment of 3
with mCPBA the compound was cleanly oxidized to 4OMe-
HQNO (7, Scheme 2). Purification by column chromatography
provided the highly polar compound 7 in acceptable yield
(
45%).
Finally, the synthesis of methylated PQS derivatives was
carried out. Initially, the direct conversion of OMe-HHQ (3)
into 4OMe-PQS (11) was attempted. However, the standard
Scheme 3: Synthesis of methylated PQS derivatives.
methodology for the conversion of HHQ into PQS such as amounts of oxidative side products including N-methylisatin
formylation followed by oxidation failed when applied to OMe- were obtained, which also impeded the isolation of the desired
HHQ (3). Alternatively, ortho-metalation next to the methoxy product 12. Nevertheless, enough material could be obtained to
group of OMe-HHQ (3) followed by borylation/oxidation was enable biological investigations on this compound.
investigated. Several trials using different lithium bases failed
and only small amounts of oxidation products in the benzylic Growth inhibition of S. aureus
position could be observed. Instead of direct ortho-metalation, a To get a better understanding of the influence of methylation on
strategy based on halogen–lithium exchange proved to be more the antibiotic properties of AQ compounds, their effect on the
suitable. To this end, HHQ (1) was converted into 3I-HHQ (8) growth of S. aureus Newman was investigated (Figure 1). To be
following a literature procedure [21]. 3I-HHQ (8) was then able to compare the growth in the presence of the only weakly
methylated using the MeI/K2CO3 conditions to give a mixture antibiotic HHQ derivatives, growth experiments were con-
of OMe-3I-HHQ (9, 21%) and NMe-3I-HHQ (10, 24%, ducted at a concentration of 100 µM. Planktonic cultures of
Scheme 3). Interestingly, in the case of 3I-HHQ (8) the ratio of P. aeruginosa in LB medium were reported to contain up to
N-methylation versus O-methylation was almost 1:1 and no 50 µM PQS and up to 70 µM HQNO [25]. As observed previ-
further methylation of 10 in the benzylic position was observed. ously, HQNO (5) inhibited the growth of S. aureus significant-
With the methylated compounds 9 and 10 in hand, the halogen- ly, while HHQ (1) had only a moderate effect. Interestingly, the
lithium exchange was attempted. Treatment of 9 with n-butyl- methylated HQNO derivatives 6 and 7 were either equally po-
lithium at −78 °C followed by the addition of B(OMe)3 and tent as HQNO or, in the case of 7, even led to a stronger growth
finally in situ oxidation using sodium perborate provided the reduction. While most of HHQ and PQS derivatives showed no
desired 4OMe-PQS derivative 11 in moderate yield (35%, or only very weak effects on the growth of S. aureus, NMe-PQS
Scheme 3). The same procedure could be applied to NMe-3I- (12) caused a similar growth inhibition as HQNO. However, the
HHQ (10), however, the resulting NMe-PQS derivative 12 was AQs did not significantly affect the formation of static biofilms
only isolated in low yield (14%). Presumably, this compound is of S. aureus under the conditions tested (see Figure S1, Sup-
not too stable during the final oxidation step as significant porting Information File 1).
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Beilstein J. Org. Chem. 2019, 15, 187–193.
Figure 2: Inhibition of cellular O2 consumption rate (cOCR) of
S. aureus Newman. Cell suspensions (OD600 nm of 0.5) were incubat-
ed with 100 µM of the indicated AQs (1–12), PQS (P) or an equal
amount of DMSO as the control (C). Black and grey bars show the
mean cOCR of three biological replicates. Error bars represent the
standard error. One-way ANOVA was used for statistical analysis, and
significant differences compared to the control are indicated
(* P < 0.05, ** P < 0.01, *** P < 0.001).
Quorum sensing activity
The compounds HHQ, PQS and HQNO are strongly related to
quorum sensing and virulence in P. aeruginosa. HHQ and PQS
act as signal molecules and, by interacting with the transcrip-
tional regulator PqsR, induce the expression of the AQ biosyn-
thetic genes. Such positive feedback or “auto-induction” loops
are considered a hallmark of quorum sensing systems. There-
fore, in order to detect potential effects of the methylated AQ
derivatives on the AQ-based quorum sensing, we analyzed
their influence on the production of PQS and HQNO by
P. aeruginosa PAO1 (Figure 3).
For most compounds tested, PQS and HQNO contents of the
cultures were unaffected compared to the DMSO control, indi-
cating neither quorum sensing nor antiquorum sensing activity.
However, besides the well-known quorum sensing signal mole-
cule HHQ (1), also the two methylated PQS derivatives 11 and
Figure 1: Overview of AQ compounds (A), and effect of AQs on the
growth of S. aureus Newman (B). 24-Well plates were inoculated at an
optical density (OD600 nm) of 0.1 and supplemented with 100 µM of the
indicated AQs (1–12), PQS (P) or an equal amount of DMSO as the
control (C). Black and grey bars show the mean optical density after
4
hours of cultivation at 37 °C of 3 biological replicates. Error bars
1
2 and the HQNO derivative 6 elicited PQS and HQNO produc-
represent the standard error. One-way ANOVA was used for statistical
analysis, and significant differences compared to the control are indi-
cated (* P < 0.05, ** P < 0.01, *** P < 0.001).
tion by P. aeruginosa PAO1, suggesting quorum sensing activi-
ty. However, the significant activity of the HQNO derivative 6
is unexpected, as HQNO is not a signal. We assume that the
HQNO is known to inhibit complex III of the respiratory chain weak N–O bond is cleaved under the assay conditions and HHQ
and therefore leads to a measurable reduction in O2 consump- is generated, which could be the cause of the observed effects.
tion by cells of S. aureus [16]. The investigation of the Experiments with a luciferase-based bioreporter strain [26] con-
O2-consumption rates of S. aureus in response to AQs revealed firmed that compounds 11 and 12 act via addressing the tran-
that among the compounds tested, HQNO (5) had the strongest scriptional regulator PqsR of the AQ-based quorum sensing
effect (Figure 2). The 4-O-methylated HHQ and HQNO deriva- system, while compound 6 did not elicit a significant response
tives 3 and 7, but not the respective PQS derivative 11, also in- (see Figure S2, Supporting Information File 1, for details). The
hibited O2 consumption. In contrast, only weak to moderate data on the PQS derivatives 11 and 12 suggest that the presence
inhibition was observed with derivatives methylated either at of a substituent at one of the heteroatoms does not necessarily
the nitrogen atom (2, 4 and 12) or at the N–O oxygen atom (6). preclude quorum sensing activity, and that formation of a 1H-4-
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Beilstein J. Org. Chem. 2019, 15, 187–193.
Growth inhibition experiments against S. aureus showed that
HQNO derivatives (5, 6 and 7) were generally more efficient
than HHQ (1) and its derivatives (2–4 and 8). Interestingly,
while PQS and 4OMe-PQS did not have any activity,
NMe-PQS (12) was about as active as the HQNO derivatives.
The 4-O-methylated derivatives of HQNO (7) and HHQ (3) but
not the PQS derivative 11 were shown to reduce O2 consump-
tion similar to HQNO, possibly indicating inhibition of the
respiratory chain as proven for HQNO. However, the reduction
in O2 consumption is less pronounced for HMOQ (6) and
NMe-PQS (12). Therefore, it is possible that growth inhibition
is following a different mechanism than for HQNO (5). The in-
vestigation of AQ levels in cultures of P. aeruginosa revealed
quorum sensing activity for the two PQS derivatives 11 and 12,
suggesting once more that AQ-based quorum sensing in
P. aeruginosa tolerates significant structural diversity on the
signaling molecule [27-29].
Figure 3: Impact on AQ quorum sensing by the newly synthesized AQ
derivatives. Cultures of P. aeruginosa PAO1 were supplemented with
Experimental
100 µM of AQs (1–12) or an equal amount of DMSO as the control (C),
Detailed synthetic procedures for all new compounds including
copies of their NMR spectra can be found in Supporting Infor-
mation File 1. HHQ and HQNO were prepared by adjusted and
optimized literature procedures (see Supporting Information
File 1 for details) [20,21]. 3-Iodo-2-heptyl-4(1H)-quinolone (3I-
HHQ) was prepared according to a literature procedure [21].
and the concentrations of PQS (grey bars) and HQNO (black bars), as
readout of the signal-induced upregulation of AQ biosynthesis, were
measured at an early (after 4 hours; left) and a late stage (after
24 hours; right) of quorum sensing. Because P. aeruginosa is able to
convert HHQ (1) to PQS, the PQS data measured after addition of 1 to
the cultures represent a combination of auto-induction of AQ synthesis
by HHQ, and biotransformation of HHQ to PQS. However, HHQ is not
a biosynthetic precursor of HQNO. Means and standard errors of five
biological replicates are shown. One-way ANOVA was used for statis-
tical analysis, and significant differences compared to the control (C)
are indicated (* P < 0.05, ** P < 0.01).
Bioassays
The growth of S. aureus strain Newman in the presence of AQs
was followed by measuring the optical density of cell suspen-
oxo-tautomeric form (which is impeded by the O-methylation in sions in 24-well microtiter plates. To this end, freshly grown
1) is not a prerequisite for quorum sensing activity [27]. cells were diluted in LB medium to an OD600 nm of 0.2. Each
1
Furthermore, in agreement with previous results, our results well contained 1 mL of cell suspension including 100 µM of the
show the importance of the 3-hydroxy group, which turns non- AQ derivatives or an equal amount of DMSO. The plates were
active compounds 2 and 3 into PQS agonists, irrespective of sealed with optically clear and gas permeable membranes, incu-
4
-O-methylation (11) or N-methylation (12) [27-29].
bated at 37 °C while shaking and the OD600 nm was measured at
certain time points. Experiments were done in biological tripli-
cates (n = 3).
Conclusion
In this study we describe the synthesis of selectively methyl-
ated AQs and the influence of methylation on the bioactivity. The influence of AQs on the cellular consumption of O2 was
While the methylation of HHQ is straightforward, selective studied as previously described [16]. Briefly, exponentially
preparation of methylated HQNO and PQS derivatives required growing cells of S. aureus Newman were diluted in LB to an
optimized reaction conditions and multistep syntheses. The OD600 nm of 0.5. After adding 100 µM AQs and incubating for
strategy to “oxidize” the 3-position of AQs via iodination, lithi- 10 min, 500 µL cell suspension was transferred to the chamber
um–halogen exchange and electrophilic trapping can offer a of a Clark-type oxygen electrode and the O2 consumption was
flexible approach to new 3-substituted AQs starting from an measured for 2–3 min. Experiments were done in biological
easily available synthetic intermediate. While we decided to triplicates (n = 3).
oxidize the newly formed boronic acid ester in situ, its isolation
should be possible and enable further modification, for exam- The effect of AQs on the quorum sensing activity in
ple by ipso-substitution with heteroatom electrophiles. The ap- P. aeruginosa was studied by using AQ biosynthesis as measur-
plication of the boronic acid in Suzuki–Miyaura coupling able output. The induction of AQ biosynthesis by methylated
should also be possible [30,31].
AQs was followed by measuring PQS and HQNO concentra-
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Additional biological data, detailed synthetic procedures
and copies of 1H and 13C NMR spectra of all compounds.
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Acknowledgements
Financial support by the DFG (grant FE 383/25-1 to S. F., and
Heisenberg grants HE 6020/3-1 and HE 6020/4-1 to U. H.) is
gratefully acknowledged. A kind donation of analytical equip-
ment from BASF to U. H. is gratefully acknowledged.
ORCID® iDs
Susanne Fetzner - https://orcid.org/0000-0002-7228-1871
Ulrich Hennecke - https://orcid.org/0000-0002-4904-6793
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