Inhibition of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells by quorum sensing autoinducer-mimics

Article abstract of DOI:10.1039/c2ob26501j

Pseudomonas aeruginosa is a notorious human pathogen associated with a range of life-threatening nosocomial infections. There is an increasing problem of antibiotic resistance in P. aeruginosa, highlighted by the emergence of multi-drug resistant strains.

Full text of DOI:10.1039/c2ob26501j

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PAPER
Inhibition of the production of the Pseudomonas aeruginosa virulence factor
pyocyanin in wild-type cells by quorum sensing autoinducer-mimics†
Bernardas Morkunas,a Warren R. J. D. Galloway,b Megan Wright,b Brett M. Ibbeson,b James T. Hodgkinson,b
Kieron M. G. O’Connell,b Noemi Bartolucci,b Martina Della Valle,b Martin Welcha and David R. Spring*b
Received 31st July 2012, Accepted 10th September 2012
DOI: 10.1039/c2ob26501j
Pseudomonas aeruginosa is a notorious human pathogen associated with a range of life-threatening
nosocomial infections. There is an increasing problem of antibiotic resistance in P. aeruginosa,
highlighted by the emergence of multi-drug resistant strains. Thus the exploration of new strategies for the
treatment of P. aeruginosa infections is clearly warranted. P. aeruginosa is known to produce a range of
virulence factors that enhance its ability to damage the host tissue and cause disease. One of the most
important virulence factors is pyocyanin. P. aeruginosa regulates pyocyanin production using an
intercellular communication mechanism called quorum sensing, which is mediated by small signalling
molecules termed autoinducers. One native autoinducer is N-(3-oxododecanoyl)-L-homoserine lactone
(OdDHL). Herein we report the synthesis of a collection of abiotic OdDHL-mimics. A number of novel
compounds capable of competing with the endogenous OdDHL and consequently, inhibiting the
production of pyocyanin in cultures of wild type P. aeruginosa were identified. We present evidence
suggesting that compounds of this general structural type act as direct antagonists of quorum sensing in
P. aeruginosa and as such may find value as molecular tools for the study and manipulation of this
signalling pathway. A direct quantitative comparison of the pyocyanin suppressive activities of the most
active OdDHL-mimics with some previously-reported inhibitors (based around different general structural
frameworks) of quorum sensing from the literature, was also made.
The ability of pathogenic bacteria such as P. aeruginosa to
cause disease is dependent upon the production of agents termed
Introduction
Pseudomonas aeruginosa is a major opportunistic pathogen.
This Gram-negative bacterium causes a variety of nosocomial
infections and life-threatening diseases in immunocompromised
and debilitated patients1 and is the most predominant cause of
chronic pulmonary infections in cystic fibrosis sufferers.2 Infec-
tions of this bacterium are notoriously difficult to eradicate due
to high levels of intrinsic antibiotic resistance1 and the propen-
sity of P. aeruginosa cells to form antibiotic-resistant biofilms.3
Indeed, P. aeruginosa represents one of the most challenging
pathogenic bacteria to treat1 and multi-drug resistant P. aerugi-
nosa nosocomial infections are increasingly being recognized
worldwide.4 Thus the exploration of innovative new therapeutic
strategies for tackling P. aeruginosa infections is urgently
required.1,2
‘virulence factors’, such as toxins and adhesion molecules, that
actively cause damage to host tissues.5–7 The targeting of viru-
lence factors (e.g. inhibition of their production, delivery or
function) has gained increasing attention as a potential new anti-
bacterial strategy; in principle this would ‘disarm’ pathogens and
allow the host immune system a better chance of clearing the
infection before the bacteria cause too much tissue damage.6
This approach has a number of theoretical advantages over stan-
dard antibiotic treatment.7 For example, many virulence factors
are organism-specific and therefore virulence-targeting drugs
should have a minimum impact upon the host’s commensal
flora.7,8 In addition, provided the targeted virulence factor is not
essential for bacterial survival in vivo then there should be a
weaker selective pressure for the development of resistant
mutants relative to traditional antibiotic treatments.7,8 Recent
years have witnessed considerable interest in the targeting of
virulence factors as a novel therapeutic strategy and there is a
growing body of in vivo data to support this approach.7,8
aDepartment of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, UK. E-mail: spring@ch.cam.ac.uk;
Fax: +44 (0) 1223-336362; Tel: +44 (0) 1223-336498
bDepartment of Biochemistry, University of Cambridge, Cambridge, UK
†Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra for selected compound. Biological screening procedures.
See DOI: 10.1039/c2ob26501j
Amongst the arsenal of virulence factors produced by P. aeru-
ginosa is pyocyanin (1-hydroxy-5-methyl-phenazine), a low
molecular weight redox-active phenazine dye.9 In vitro studies
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Fig. 1 BHL, OdDHL and PQS are natural autoinducers employed by P. aeruginosa in quorum sensing. 1–5 are synthetic OdDHL-based small mol-
ecule inhibitors of LasR-dependent quorum sensing. 1–3 were discovered by our group in a previous study;17 4 and 5 were reported by Blackwell and
co-workers32 and Yoon and co-workers36 respectively.
have shown pyocyanin to have multiple deleterious effects upon
mammalian cells.10 Pyocyanin has been found to be critical for
P. aeruginosa lung infections in mice and it is regarded to play
an important role in colonizing the airways of cystic fibrosis
patients.10,11 Overall there is clear evidence that pyocyanin is
important to the pathogenesis of P. aeruginosa infections.2,11
Unsurprisingly therefore, the targeting of pyocyanin has been
suggested as a therapeutic strategy for treating infections by this
organism.2,8,11 Some of pyocyanin’s toxic effects appear to result
from its ability to redox cycle and place cellular systems under
increased oxidative stress through the production of reactive
oxygen species.9,12 Accordingly several antioxidant therapies
have proved valuable in cystic fibrosis.2,13–15 The inhibition of
pyocyanin production has also been identified as a possible
approach for attenuating the pathogenicity of P. aeruginosa
infections.2,11,16 Indeed, recent years have witnessed consider-
able interest in the discovery of synthetic and non-native natu-
rally occurring compounds which have the ability to inhibit
pyocyanin biosynthesis.17–21
An intercellular signalling process known as quorum sensing
regulates pyocyanin production by P. aeruginosa.22,23 This com-
munication mechanism is mediated by small molecules termed
autoinducers which are synthesized intracellularly by bacterial
cells throughout their growth and are continually released into
the surroundings.24–26 Therefore the extracellular concentration
of the autoinducer (typically) increases in concert with the bac-
terial population cell density; once a threshold concentration is
reached (at which point the population is said to be “quorate”) a
signal transduction cascade is initiated leading to population-
wide changes in gene expression.24,27–32 Quorum sensing
systems can be found in many species of Gram-negative bacteria,
most of which employ N-acylated-L-homoserine lactones
(AHLs) as autoinducers.21,24 The majority of natural AHLs are
based around the same general structural framework, namely a
homoserine lactone ring (the ‘head group’) unsubstituted at the β
and γ positions, which is N-acylated at the α position with an
acyl group (the ‘tail group’).17,21,24 These small molecules are
generated by LuxI-type synthase enzymes and bind to cognate
cytoplasmic LuxR-type receptors to initiate the expression of
genes associated with bacterial group processes.21,24,26,33 Typi-
cally, each bacterial species responds specifically to its own
unique AHL(s), with different LuxI-type synthase and LuxR-
type receptors employed.17,24,32 P. aeruginosa uses two AHL-
based quorum sensing systems. One system employs N-(3-oxo-
dodecanoyl)-L-homoserine lactone (OdDHL) as the autoinducer,
which is generated by LasI with LasR as the cognate receptor
(Fig. 1). The second involves N-butanoyl-L-homoserine lactone
(BHL) as the signalling molecule, which is generated by RhlI
and detected by RhlR. These AHL-based systems are interlinked
with a third system employing a chemically distinct autoinducer
termed the Pseudomonas quinolone signal, PQS. The result is an
intricate hierarchical quorum sensing network.17,24,34,35
Pyocyanin production is regulated by RhlR. Transcription of
the rhlR gene is itself regulated by LasR.22 Thus, lasR mutants
no longer produce pyocyanin, and inhibitors of LasR would be
expected to attenuate pyocyanin biosynthesis.23 Indeed, abiotic
small molecules with this activity profile are known.21,24,37–39
The structure of the natural LasR agonist OdDHL has often
served as a template for the design and synthesis of such
agents.17,24,21,40 For example, we have recently reported the dis-
covery of potent OdDHL-based inhibitors of pyocyanin pro-
duction, in wild-type P. aeruginosa cells in which the native
3-oxo-dodecanoyl tail group is present but the natural homoser-
ine lactone moiety has been substituted with a non-native
aromatic head group (compounds 1–3 Fig. 1).17 These
data clearly suggest that the OdDHL framework can serve as a
promising lead scaffold in developing further novel small
molecule inhibitors of pyocyanin production.
Inspired by our previous study,17 we sought to examine the
pyocyanin inhibitory activity of additional OdDHL analogues
containing non-native head groups in order to: (i) identify new
inhibitors of this phenotype and (ii) to gain new insights into the
molecular basis of the activity of compounds of this structural
class. Herein we report the results of this study. A small library
of compounds of this general structural type was synthesized,
a number of which were found to be capable of suppressing the
production of pyocyanin in a wild-type strain of P. aeruginosa.
Such compounds could potentially be exploited in a therapeutic
context for the treatment of human bacterial infections. A com-
parison with the data obtained from our previous study yielded
some new structure–activity information with respect to pyocya-
nin inhibition. Given the strong dependence of pyocyanin pro-
duction upon LasR-mediated quorum sensing (vide supra) we
were also interested in benchmarking the anti-pyocyanin activity
of our OdDHL-mimics against some other compounds pre-
viously reported to have inhibitory activity against LuxR-type
receptors by other researchers. However, a significant issue
encountered when examining the use of small molecules to
modulate phenotypes regulated by quorum sensing pathways is
the lack of standardization between the assays used in different
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reports to assess the biological effects of such agents.21,24,41
Also, it is generally accepted that direct quantitative comparison
of the activities of such compounds obtained from different
studies by different research groups can be misleading and is not
appropriate in many cases.21,24,41,42 Therefore, and to circumvent
these issues, we synthesized several compounds that had pre-
viously been reported by other researchers to have inhibitory
activity against LuxR-type quorum sensing systems. These
included compounds of the same structural class as our library
members (i.e. OdDHL-mimics with non-native head groups). All
compounds were evaluated for their ability to inhibit pyocyanin
production using the same assay.
pyocyanin present was measured and compared with that
obtained when the cells were grown for 13 hours in the absence
of any synthetic AHL mimic (Fig. 2). All compounds were
found to decrease pyocyanin production without affecting
growth. These data could be directly compared with those
obtained for compounds 1–3 from our previous study as an iden-
tical assay protocol was employed (Table 1).
Mode of action considerations. Compounds that affect pyocya-
nin production in P. aeruginosa may be inhibitors of LasR-based
quorum sensing (vide supra). OdDHL, the natural binding
partner of LasR, is very closely related in structure to compounds
1–5, 10–18. Thus it reasonable to propose that the inhibitory
effects of 1–5, 10–18 upon pyocyanin production result from an
inhibition of quorum sensing, with the compounds possibly
acting at the level of the LasR receptor (i.e. they are LasR antag-
onists). Compound 3 has previously been reported to inhibit the
production of elastase, another P. aeruginosa virulence factor
which is regulated by the las branch of the quorum sensing
system (and to a lesser extent, also by the subordinate rhl
branch); a result that is also consistent with direct inhibition of
LasR-based quorum sensing.17 It is worth noting however,
that lasR mutants can still, under certain conditions, express
multiple virulence factors, including pyocyanin, by utilising the
Rhl and PQS systems and there are additional studies which indi-
cate that the quorum sensing hierarchy in this organism is
more complex than the simple las/rhl hierarchy reported
previously.22,46 Thus it is possible that inhibitors of
pyocyanin production in wild-type P. aeruginosa may not affect
the LasR-receptor directly, but instead have an alternative mode
of action.
Results and discussion
OdDHL mimics
Synthesis of OdDHL mimics. A range of OdDHL-mimics
incorporating aromatic head groups differing in both steric and
electronic properties was readily prepared via a sequence which
utilized 2-(2-ethyl-1,3-dioxolan-2-yl)acetic acid (7) as
a
common intermediate (Scheme 1).17,43 EDC-mediated coupling
of 7 with a range of aromatic amines 8 furnished ketone-pro-
tected amide derivatives 9. Acetal deprotection using TFA
yielded the desired final compounds 4, 5 and 10–18. Compounds
4 (Blackwell and co-workers32) and 5 (Yoon and co-workers44)
have previously been reported to be LasR antagonists.
Biological screening of OdDHL-mimics
Assay protocol. Compounds 4, 5 and 10–18 were evaluated for
their effects upon pyocyanin production by the wild type P. aeru-
ginosa strain PAO1. Cells were grown for 13 hours in the pres-
ence of the indicated synthetic AHL mimic (at a concentration of
200 μM) added at the start of the growth period. The amount of
Structure–activity observations. Compound 5, previously
identified as a LasR antagonist by Yoon and co-workers,44 was
found to be less active than six of the novel analogues disclosed
Scheme 1 Synthesis of OdDHL mimics with non-native head groups. The synthesis of 7 from Meldrum’s acid (6) was achieved by following a pre-
viously reported route.43 rt = room temperature. TFA = trifluoroacetic acid.
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Fig. 2 Inhibitory effects of OdDHL-mimics 4, 5 and 10–18 on pyocyanin production in PAO1. Cultures of PAO1 were grown in Luria broth medium
in the presence of OdDHL-mimics (200 μM) with good aeration at 37 °C for 13 hours (initial OD600 of 0.05 t = 0). After growth pyocyanin production
was quantified as previously described.45 No effect on growth was observed for any of the analogues (data not shown). DMSO (−) was added as a
control. The data represents the averages and standard deviations from the results of three independent biological repeats.
Table 1 Summary of the activities of the OdDHL-mimics examined in
our two studies
with this, moving a methoxy group from the meta to ortho pos-
ition (compounds 3 versus 5, respectively) resulted in a consider-
able drop in activity. Assuming these OdDHL-mimics exert their
anti-pyocyanin effects via interaction with the LasR receptor
(vide supra) this may be because ortho groups may inhibit
efficient binding in the LasR pocket, possibly due to steric
factors. Furthermore, the introduction of a second methoxy
group at the ortho position of 3 (to form 15) severely weakened
the pyocyanin antagonistic activity of the ligand. The halide-
functionalized derivatives (2 and 10) had similar inhibitory
activities, suggesting that there is some flexibility in terms of the
steric bulk at the meta position that the LasR binding pocket can
accommodate, again assuming the effects of the compounds are
mediated via binding to the LasR receptor.
Of the OdDHL-mimics examined in this study, 4 and 10 are the
only examples which do not contain a heteroatom on the aro-
matic head group which would be expected to be capable of
forming hydrogen bonding interactions. If one supposes that
these two OdDHL-mimics act as competitive LasR antagonists,
then the relatively high inhibitory activities of these compounds
corroborates recent observations by our own group (and that of
Blackwell and co-workers) which question the proposed critical
nature of a hydrogen bonding interaction between the head
group of AHLs or AHL-mimics and the LasR receptor for strong
binding.32
Overall, from the available data it is difficult to draw any firm
SAR conclusions regarding the effects of the aromatic ring head
group on the ability of members of the OdDHL-mimic com-
pound class to inhibit pyocyanin production. There appears to be
a subtle interplay between the structural and electronic properties
of the aromatic moiety governing compound activity, which is
which is consistent with previous observations by the Blackwell
and co-workers.32 Consequently the rational modification of
compounds of this sort in order to improve properties (e.g.
activity) is challenging. This is a well-known problem associated
with AHL-based compounds.24,41
Compound
% Inhibitiona
1
2
3
4
67 2b
73 4b
93 2b
80 4b
5
45
70
38
49
62
47 17
12
39 21
6
4
6
4
4
10
11
12
13
14
15
16
17
18
8
64
68
5
3
a % Inhibition of pyocyanin production as determined from the data
illustrated in Fig. 2. b Data obtained from previous study.17 Compound
concentration of 200 μM in all cases.
in this report and also less active than compounds 1–3 identified
in our previous study.17 The most active of the AHL-mimic
screened in the current study was 4, a compound that had pre-
viously been reported by the Blackwell group to be a potent
LasR antagonist.32 The activity of 4 compares favourably with
the three strongest inhibitors of pyocyanin production that we
identified previously,17 with only 3 being noticeably more active
(Table 1). 3 bears an electron-donating meta-methoxy group; the
aryl ring would therefore be expected to be more electron rich
than that of 4. Compound 12 also contains an electron donating
substituent in the meta-position; however, this derivative was
found to be considerably less active than 3 (and indeed, slightly
less active than 13 which has an electron withdrawing meta-nitro
group). These data suggest that steric, as well as electronic
factors, play an important role in determining activity. Consistent
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Comparative assessment of OdDHL-mimics and some
structurally distinct literature inhibitors of LuxR-type proteins
Synthesis of some literature inhibitors of LuxR-type proteins.
Of the OdDHL-mimics with non-native head groups examined,
the two most potent inhibitors of pyocyanin production (and by
inference LasR-mediated quorum sensing) were found to be
3 and 4. We were interested in comparing the activity of these
compounds with some examples of literature inhibitors of LuxR-
type quorum sensing which were based around different general
structural frameworks in an attempt to gain further insights into
the molecular features required for inhibition of pyocyanin pro-
duction. Specifically we focused upon: (i) 19–20, AHL-mimics
with non-native tail groups reported by Geske et al.,47 (ii)
21–22, AHL mimics in which both the natural homoserine
lactone head group and acyl chain had been replaced with non-
native structural moieties (identified by Teasdale et al.48) and
(iii) PD-12, an example of a quorum sensing inhibitor with a
non-AHL core scaffold identified by Müh et al.49 (Fig. 3).
Scheme 2 Synthesis of AHL-mimics 19 and 20 with non-native tail
groups. rt = room temperature.
19 and 20 were readily accessed via EDC-mediated coupling
of (S)-(−)-(α)-amino-γ-butyrolactone hydrobromide (23) with
indole-3-butyric acid and 4-bromophenyl acetic acid respectively
(Scheme 2). In a similar fashion, amide bond formation between
2-phenylethylamine (24) and isobutyric acid furnished 21
whereas the use of isovaleric acid generated target compound 22
(Scheme 3). PD-12 was synthesized via a three-step sequence
from ethyl cyanoacetate (25) (Scheme 4).50,51 Reaction
with sodium azide furnished tetrazole 26. Alkylation with
Scheme 3 Synthesis of AHL-mimics 21 and 22. rt
temperature.
= room
Scheme 4 Synthesis of PD-12. rt = room temperature.
1-bromododecane yielded 27 and subsequent ester hydrolysis
furnished the desired target compound.
Biological screening. Compounds 19–22 and PD-12 were
evaluated for their effects upon pyocyanin production by the
wild type P. aeruginosa strain PAO1 using the method described
above at a compound concentration of 200 μM. However, all
these compounds were found to have very similar levels of
inhibitory activities at this concentration, therefore experiments
were repeated at a lower ligand concentration (50 μM) to
increase resolution. (Fig. 4 and Table 2). The OdDHL-mimics 3
and 4 were found to be significantly stronger inhibitors of pyo-
cyanin synthesis. This was not necessarily unexpected in the
case of compounds 21–22, which had not previously been tested
Fig. 3 OdDHL-mimics 3 and 4 together with the structures of known
literature quorum sensing modulators examined in this report. 19–20 are
potent antagonists against LasR (Geske et al.47), 21–22 have been
reported to inhibit quorum sensing-controlled phenotypes in Gram nega-
tive bacteria, presumably by competing with native AHLs for binding to
the LuxR-type receptors48 and PD-12 is an inhibitor of LasR-dependent
gene expression which is also believed to interact directly with the
native AHL-binding site on LasR.49
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pyocyanin production by targeting the LasR receptor (vide
supra) these data corroborate our earlier study, and observations
from other researchers, which suggest that the natural OdDHL
acyl tail group is important for the LasR modulatory activity of
compounds which are based around the AHL scaf-
fold.17,21,24,32,52 The large difference in activity between the two
OdDHL-mimics 3–4 and PD-12 was surprising. Though PD-12
it is based upon a structural framework that is clearly distinct
from that of AHLs, it does retain the natural 3-oxo-dodecanoyl
tail group of OdDHL. PD-12 has been identified as a potent
LasR antagonist in a bacterial reporter strain.49 PD-12 has also
previously been reported to inhibit pyocyanin production by
approximately 40% in a wild-type PAO1 strain (derived from an
alternative source to that used in our study) at a compound con-
centration of 10 μM (in an assay that also employed slightly
different conditions to those used in our study).49 This provides
a clear illustration of how misleading it can be to directly
compare, in a quantitative fashion, the activities of compounds
affecting quorum-sensing regulated phenotypes obtained from
different studies by different research groups (even in studies
investigating the modulation of the same protein).21,24,42
Probing the binding of OdDHL-mimcs to LasR. Bottomley
et al.53 and Schuster et al.54 have reported that the LasR protein
is soluble and stable only if it is expressed in the presence of its
cognate ligand, OdDHL.41 Presumably correct binding of
OdDHL to LasR causes the protein to adopt a soluble confor-
mation.41 This observation provided a convenient (albeit crude)
assay to determine whether our most active antagonist (com-
pound 3) might bind directly to LasR and thereby inhibit pyo-
cyanin production. The expression of full length LasR yields
only insoluble inclusion bodies, even in the presence of the
native ligand OdDHL.41,53,54 However, when the DNA-binding
domain is removed (leaving just the ligand-binding domain and
dimerization determinants) the protein was fully soluble when
expressed in trans in Escherichia coli in the presence of OdDHL
(Fig. 5). In the absence of OdDHL, the LasR-LBD exclusively
partitioned into insoluble inclusion bodies (Fig. 5). In the pres-
ence of compound 3 the protein was roughly equally distributed
between the soluble and insoluble fractions, indicating that this
compound probably binds directly to the LasR-LBD and elicits a
conformational change similar to that caused by OdDHL
Bottomley et al. previously showed that weak binding of
non-cognate AHLs to LasR elicits a similar result.53 This lends
credence to the hypothesis that the inhibitory effect of 3 (and
presumably, also the other OdDHL-mimics examined,) upon
pyocyanin production results – at least in part – from modulation
of LasR-based quorum sensing as a consequence of direct
binding to the LasR receptor. Compounds of this general form
may therefore represent antagonists of quorum sensing in
P. aeruginosa.
Fig. 4 Inhibitory effects of various compounds on pyocyanin pro-
duction in PAO1. Cultures of PAO1 were grown in Luria broth medium
in the presence of compounds (50 μM) with good aeration at 37 °C for
13 hours (initial OD600 of 0.05 t = 0). After growth pyocyanin pro-
duction was quantified as previously described.45 No effect on growth
was observed for any of the analogues (data not shown). DMSO (−) was
added as a control. The data represents the averages and standard devi-
ations from the results of three independent biological repeats.
Table 2 Summary of the comparative assessment
Compound
% Inhibitiona
3
4
74
77
22
13
30
25
9
3
4
5
7
2
4
11
19
20
21
22
PD-12
a % Inhibition of pyocyanin production as determined from the data
illustrated in Fig. 2. Compound concentration of 50 μM in all cases.
against P. aeruginosa. In addition, our earlier study suggested
that the incorporation of both non-native head and tail regions
into an AHL-mimic (as is the case in 21–22) is generally associ-
ated with decreased inhibitory activity (relative to compounds
which retain the native acyl chain). The data for 19–20 implies
that non-native tail groups are not well tolerated with respect to
pyocyanin inhibition by AHL-mimics, though additional ana-
logues which retain the natural homoserine lactone head group
but which incorporate other abiotic tail moieties need to be
examined in order to explore this trend further. Overall the data
for the AHL-mimics 3–4 and 19–22 suggests that the natural
3-oxo-dodecanoyl tail group of OdDHL is important for the
inhibition of pyocyanin production by compounds which mimic
the structure of AHLs. Assuming that these compounds affect
Conclusions
We have reported the design, synthesis and biological evaluation
of a collection of abiotic compounds based around the structure
of the natural P. aeruginosa signalling molecule OdDHL in
which the native homoserine lactone head group has been
replaced with a non-native aromatic moiety. The majority of
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sensing in P. aeruginosa (note again that the las system is gener-
ally regarded as standing at the apex of the quorum sensing hier-
archy in P. aeruginosa). The possibility that the OdDHL-mimics
examined may inhibit quorum sensing in this organism has some
interesting implications. In addition to pyocyanin production,
quorum sensing also regulates additional elements associated
with P. aeruginosa pathogenicity.8,24,55–57 These include biofilm
formation (in many growth conditions)3 and the production of
the virulence factors elastase, exotoxin A and alkaline protease
(specifically regulated by the las system).58 In addition, the rhl
system regulates the virulence factor hydrogen cyanide and is
also required for optimal production of alkaline protease and
elastase.57,58 Thus there is tremendous interest in disrupting
AHL-mediated quorum sensing in P. aeruginosa;21 it is possible
that, as putative quorum sensing inhibitors, the OdDHL-mimics
studied in this report may have broad-spectrum inhibitory
activity against several aspects of P. aeruginosa virulence. Fur-
thermore such compounds could have value as molecular tools
to study and manipulate quorum sensing pathways in P. aerugi-
nosa. Potentially these compounds could affect the quorum
sensing systems of other species of Gram-negative bacteria and
may therefore may prove to be of value in a wide range of other
applications where the inhibition of quorum sensing is desir-
able.24,49,59 However, there is evidence that the length of the
AHL acyl chain, even in non-lactone derivatives, can give high
levels of receptor selectivity.32 Thus the OdDHL-mimics may
only have a significant impact upon quorum sensing in P. aerugi-
nosa. This could be advantageous in the context of possible
therapeutic applications of such agents; in contrast to broad-
spectrum quorum sensing inhibitors, narrow-spectrum inhibitors
of this sort would be expected to interfere less with the commen-
sal (and beneficial) microbial flora of the host.60 Investigations
exploring both species selectivity of the OdDHL-mimics and
their ability to affect other quorum sensing-regulated phenotypes
in P. aeruginosas are on going and results will be reported in
due course.
Fig. 5
A Coomassie blue-stained 12% SDS-polyacrylamide gel
showing the partitioning of LasR-LBD in the insoluble pellet (P) and
soluble (S) fractions of E. coli cell lysates obtained from cultures grown
in the presence or absence of OdDHL and compound 3. The far left lane
is a molecular weight ladder. From bottom to top of the molecular
weight ladder: the bands below the red rectangle are molecular weights
of 7 kDa and 17 kDA; the bands above the rectangle are 23 kDa and
30 kDa.
these OdDHL-mimics, including several structurally novel
derivatives, were found to inhibit the production of pyocyanin, a
molecule that enhances the virulence of P. aeruginosa, in a wild-
type strain of the organism. The fact that this class of compounds
effectively compete with endogenously-produced OdDHL to
antagonize pyocyanin production means that they have the
potential to be exploited in a therapeutic context for the develop-
ment of novel anti-pseudomonal agents. Of more general signifi-
cance it has been argued that the identification of such small
molecule modulators is needed in order to better evaluate the
therapeutic potential of targeting virulence factors.8
Overall it proved difficult to delineate SARs for pyocyanin
inhibition by this class of compounds. There appears to be a
subtle interplay between the structural and electronic properties
of the aromatic head group governing compound activity, which
is difficult to rationalize. Thus the deliberate modification of
compounds of this sort in order to improve properties (e.g.
activity) would be expected to be challenging, which may hinder
their suitability as lead-compounds for further rational
development.
The OdDHL-based derivatives 1–5, 10–18 are closely related
in structure to the natural autoinducer OdDHL, which is known
to interact with the LasR receptor, and pyocyanin production is
regarded as being regulated by LasR-based quorum sensing.
These observations are consistent with the notion that 1–5,
10–18 reduce the level of pyocyanin production by disrupting
OdDHL-dependent activation of LasR. Furthermore we have pre-
sented direct experimental evidence that OdDHL-mimic 3, one
of the most potent inhibitors of pyocyanin production explored
in our two studies, is capable of causing LasR to adopt a soluble
conformation. This result is indicative of direct binding of 3 to
LasR and provides further support that 3 (and, by inference,
possibly the other OdDHL-mimics examined) is an antagonist of
the LasR receptor and an inhibitor of LasR-based quorum
Of the OdDHL-mimics with non-native aromatic head groups
examined, the two most potent inhibitors of pyocyanin pro-
duction in wild-type P. aeruginosa cells (strain PAO1) were
found to be 3, discovered by our research group17 and 4, initially
reported to be a LasR antagonist by the Blackwell group.32
A
comparison of the anti-pyocyanin activities of these compounds
with some known literature inhibitors of LuxR-type quorum
sensing, which were based around different general structural
frameworks, was then made. Importantly all compounds were
synthesized in house and biologically evaluated using the same
assay system to allow a direct quantitative comparison of activi-
ties. In general the data suggested that the natural 3-oxo-dodeca-
noyl tail group of OdDHL is important for the inhibition of
pyocyanin production by compounds which mimic the structure
of AHLs. This adds to the growing body of evidence that this
structural feature is important for the LasR modulatory activity
of compounds that are based around the AHL scaffold.17,21,24,52
Compounds 3 and 4 were found to be significantly stronger
inhibitors of pyocyanin production than the other compounds
examined. Significantly this included the compound PD-12, a
molecule based around a non-AHL core structure, which had
previously been reported to be a potent inhibitor of this pheno-
type in a wild-type strain of this organism.49 In our hands, the
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potency of PD-12 in inhibiting pyocyanin production was sig-
nificantly lower than that reported in the literature. This provides
a clear illustration of the importance of adopting standardized
assay systems to enable truly comparative analyses to be made.
In this context the establishment of a small ‘test’ compound
library of known quorum sensing inhibitors may be valuable.
Such a library could be made available to all quorum sensing
researchers that publish new inhibitors, thus allowing the activi-
ties of new agents to be directly benchmarked against know
inhibitors.
KI in 3 M aq. AcOH). Infrared spectra were recorded neat or as
a solution in the designated solvent on a Perkin-Elmer Spectrum
One spectrometer with internal referencing. Selected absorption
maxima (νmax) are reported in wavenumbers (cm−1). The
abbreviation “br” indicates a broad peak. Melting points were
obtained using a Büchi® melting point apparatus (model B-545)
and are uncorrected. Proton magnetic resonance spectra were
recorded using an internal deuterium lock at ambient probe
temperatures (unless otherwise stated) on the following instru-
ments: Bruker DPX-400 (400 MHz), Bruker Avance 400 QNP
(400 MHz) Bruker Avance 500 BB ATM (500 MHz) and Bruker
Avance 500 Cryo Ultrashield (500 MHz). Chemical shifts (δH)
are quoted in ppm, to the nearest 0.01 ppm, and are referenced to
the residual non-deuterated solvent peak. Coupling constants (J)
are reported in Hertz to the nearest 0.5 Hz. Data are reported as
follows: chemical shift, integration, multiplicity [br, broad; s,
singlet; d, doublet; t, triplet; q, quartet; quint, quintet; sextet;
sept, septet; m, multiplet; or as a combination of these (e.g. dd,
dt, etc.)], coupling constant(s) and assignment. Proton assign-
ments were determined either on the basis of unambiguous
chemical shift or coupling pattern, by patterns observed in 2D
experiments (1H–1H COSY, HMBC and HMQC) or by analogy
to fully interpreted spectra for related compounds. Carbon mag-
netic resonance spectra were recorded by broadband proton spin
decoupling at ambient probe temperatures (unless otherwise
stated) using an internal deuterium lock on the following instru-
ments: Bruker DPX-400 (100 MHz), Bruker Avance 400 QNP
(100 MHz) and Bruker Avance 500 BB ATM (125 MHz) and
Bruker Avance 500 Cryo Ultrashield (125 MHz). Chemical
shifts (δC) are quoted in ppm, to the nearest 0.1 ppm, and are
referenced to the residual non-deuterated solvent peak. Where
appropriate, coupling constants are reported in Hertz to the
nearest 0.5 Hz and data are reported as for proton magnetic res-
onance spectra without integration. Assignments were supported
by DEPT editing and determined either on the basis of unam-
biguous chemical shift or coupling pattern, by patterns observed
in 2D experiments (HMBC and HMQC) or by analogy to fully
interpreted spectra for related compounds. LCMS spectra were
recorded on an HP/Agilent LCMS APCI 120-1000 full gradient
machine. The ionisation technique used was electron ionisation
(EI). High resolution mass spectroscopy measurements were
recorded in-house using a Waters LCT Premier Mass Spec-
trometer or a Micromass Quadrapole-Time of Flight (Q-ToF)
spectrometer. Mass values are reported within the error limits of
5 ppm mass units. The ionisation technique used was electro-
spray ionization (ESI).
It is worth noting that small molecules that modulate AHL-
based quorum sensing are typically discovered through culture-
based screening assays that employ bacterial reporter strains
(so-called biosensor strains) where the expression of an easily
assayable phenotypic output is under the control of a LuxR-
(type)-regulated promoter.17,21,60 Functional AHL synthases are
not present in such strains; thus transcription of the reporter gene
is, in principle, entirely dependent upon the addition of the
exogenous AHL (or a functional equivalent).17,21,60 Antagonist
screening trials using biosensor strains involve the addition of
the cognate AHL at a fixed concentration (usually, just enough to
stimulate expression of the reporter gene); antagonists are ident-
ified by their ability to compete with the native autoinducer and
reduce reporter gene expression.17,21,60 The advantage of such a
system is that it is well-defined17,60 However, it does not accu-
rately mimic the situation in wild-type cells, where the endogen-
ous AHL is continually produced and can therefore more
effectively outcompete any added antagonist.17,21,60 In this study
we therefore chose to work with wild-type cells and screen for
the inhibition of a quorum sensing-dependent phenotype (pyo-
cyanin production). Studies such as this, which use wild-type
bacteria, may be of more direct relevance to native situations
than those employing biosensor strains, and may therefore better
facilitate the discovery of small molecule modulators of quorum
sensing with useful real-world applications.60
Experimental
General information
Reactions were performed using oven-dried glassware under an
atmosphere of nitrogen with anhydrous, freshly distilled solvents
unless otherwise stated. Dichloromethane, ethyl acetate, metha-
nol, n-hexane, acetonitrile and toluene were distilled from
calcium hydride. Diethyl ether was distilled over a mixture of
lithium aluminium hydride and calcium hydride. Petroleum ether
was distilled before use and refers to the fraction between
30–40 °C. All other reagents were used as obtained from com-
mercial sources. Room temperature refers to ambient tempera-
ture. Yields refer to chromatographically and spectroscopically
pure compounds unless otherwise stated. All flash chromato-
graphy was carried out using slurry-packed Merck 9325 Keisel-
gel 60 silica gel. Were possible, reactions were monitored by
thin layer chromatography (TLC) performed on commercially
prepared glass plates pre-coated with Merck silica gel 60 F254
or aluminium oxide 60 F254. Visualisation was by the quench-
ing of UV fluorescence (vmax = 254 nm) or by staining with
ceric ammonium molybdate, potassium permanganate or Dra-
gendorff ’s reagent (0.08% w/v bismuth subnitrate and 2% w/v
General procedure 1: synthesis of β-keto amides from 2-(2-
nonyl-1,3-dioxolan-2-yl)acetic acid (7). EDC (1.2 equiv.),
DMAP (1.7 equiv.) and the appropriate aromatic amine (1.3
equiv.) were added to a solution of 2-(2-nonyl-1,3-dioxolan-2-
yl)acetic acid (7, 1.0 equiv.) in CH2Cl2 (∼7 mL per mmol 7) at
room temperature. The reaction mixture was stirred at room
temperature until TLC analysis indicated complete consumption
of the starting acid (∼18 hours). Aqueous HCl (10% v/v solu-
tion, ∼7 mL per mmol 7) was added to the reaction mixture
with stirring. The aqueous and organic layers were separated and
the aqueous layer was extracted with CH2Cl2 (×3). The com-
bined organic extracts were dried (MgSO4) and the solvent
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N-(3-Bromophenyl)3-oxododecanammide (10). Prepared by
removed under reduced pressure to yield the ketone-protected
amide (the crude product material could be purified by column
chromatography if desired or used directly in the next step). TFA
(∼10 equiv.) and H2O (∼0.2 mL per mmol ketone-protected
amide substrate) were added dropwise to the ketone-protected
amide with stirring at room temperature. The reaction mixture
was stirred at room temperature until TLC analysis indicated
complete consumption of starting material (∼18 hours). Sat.
aqueous NaHCO3 (∼15 mL per mmol substrate) was added
dropwise at room temperature with stirring. CH2Cl2 was added
and the aqueous and organic layers separated. The aqueous layer
was extracted with CH2Cl2 (×3). The combined organic extracts
were dried (MgSO4) and the solvent removed under reduced
pressure. The crude product material was purified by column
chromatography.
general procedure
1
using 3-bromoaniline (0.11 mL,
1.01 mmol). The crude ketone-protected amide was purified by
column chromatography (SiO2, 7 : 3 petroleum ether : EtOAc) to
yield the ketone-protected amide as a red oil (0.154 g, 48%)
which was used in the next step of the reaction. The crude
material obtained after treatment with TFA was purified by
column chromatography (SiO2, 5 : 5 petroleum ether : Et2O) to
yield the title compound as a pale pink solid (0.094 g, 69%). Rf
0.4 (5 : 5 petroleum ether : Et2O); νmax (neat)/cm−1 3283, 3253,
2916 (NH), 1715 (CvO ketone), 1660 (CvO amide), 1587,
1547, 1479; δH (500 MHz; CDCl3) 9.34 (1H, s, NH), 7.81 (1H,
t, J = 2.0 Hz, NHCCHBr), 7.43 (1H, dt, J = 8.0 Hz, 1.5 Hz, aryl
CH), 7.23 (1H, dt, J = 8.0 Hz, 1.5 Hz, aryl CH), 7.16 (1H, t, J =
8.0 Hz, aryl CH), 3.55 (2H, s, COCH2CO), 2.56 (2H, t, J =
7.5 Hz, COCH2(CH2)7CH3), 1.60 (2H, t, J = 7.5 Hz,
CH2CH2CO), 1.36–1.16 (12H, br m, 6 × CH2), 0.87 (3H, t, J =
7.0 Hz, CH3); δC (125 MHz; CDCl3) 207.9 (CvO ketone),
163.7 (CvO amide), 138.8 (NHC), 130.2 (CH), 127.4 (CH),
123.0 (CH), 122.6 (C), 118.5 (CH), 48.7 (COCH2CO), 44.2
(CH2), 31.8 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 28.9
(CH2), 23.3 (CH2), 22.6 (CH2), 14.1 (CH3); HRMS (ESI+) m/z
found [M + H]+ 368.1233, C18H27NO2Br79+ requires 368.1220;
m.p. 55–58 °C (5 : 5 petroleum ether : Et2O).
3-Oxo-N-phenyldodecanamide (4). Prepared by general pro-
cedure 1 using aniline (0.92 g, 9.8 mmol). The crude ketone-
protected amide was used directly without further purification.
The crude material obtained after treatment with TFA was
purified by column chromatography (SiO2, 8 : 2 petroleum ether :
EtOAc) to yield the title compound as a white solid (44%
overall). Rf 0.3 (8 : 2 petroleum ether : EtOAc); δH (400 MHz;
CDCl3) 9.16 (1H, s, NH) 7.56–7.53 (2H, m, aryl H), 7.35–7.31
(2H, m, aryl H), 7.14–7.10 (1H, m, aryl H), 3.56 (2H, s,
COCH2CO), 2.58 (2H, t, J = 7.5 Hz, COCH2(CH2)7CH3),
1.66–1.58 (2H, m, CH2), 1.32–1.26 (12H, m, 6 × CH2),
0.89–0.86 (3H, m, CH3); δC (100 MHz; CDCl3) 208.0 (CvO
ketone) 163.2 (CvO amide), 137.5 (C), 128.9 (CH), 124.5
(CH), 120.1 (CH), 48.8 (COCH2CO2), 44.3 (COCH2-
(CH2)7CH3), 31.8 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2),
29.0 (CH2), 23.4 (CH2), 22.6 (CH2), 14.1 (CH3); νmax (neat)/
cm−1 2918 (NH), 1729 (CvO ketone), 1709 (CvO amide);
3-Oxo-N-((3-trifluoromethoxy)phenyl)dodecanamide (11).
Prepared by general procedure 1 using 3-(trifluoromethoxy)
aniline (0.93 g, 5.88 mmol). The crude ketone-protected amide
was used directly without further purification. The crude material
obtained after treatment with TFA was purified by column
chromatography (SiO2, 8 : 2 petroleum ether : EtOAc) to yield
the title compound (0.1113 g, 37% overall). Rf 0.63 (8 : 2
petroleum ether : EtOAc); νmax (neat)/cm−1 2912 (N–H), 1713
(CvO ketone), 1665 (CvO amide); δH (500 MHz; CDCl3) 9.40
(1H, s, NH), 7.59 (1H, s, aryl CH), 7.40 (1H, m, aryl CH), 7.31
(1H, t, J = 8.0 Hz, aryl CH), 7.00–6.94 (1H, m, aryl CH), 3.56
+
HRMS (ESI+) m/z found [M + H]+ 290.2119, C18H28NO2
requires 290.2120; m.p. 80–85 °C (8 : 2 petroleum ether :
EtOAc). This data is consistent with that previously reported.32
(2H, s, COCH2CO), 2.56 (2H, t,
J
=
7.5 Hz,
COCH2(CH2)7CH3), 1.31–1.23 (14H, m, 7 × CH2), 0.86 (3H, t,
J = 6.5 Hz, CH3); δC (100 MHz; CDCl3) 208.1 (CvO ketone),
163.5 (CvO amide), 149.5 (C), 138.8 (C), 129.9 (CH),
118.0 (CH), 116.5 (CH), 112.8 (CH), 48.3 (CH2), 44.3
(CH2), 31.8 (CH2), 29.3 (CH2), 29.3 (CH2), 29.2 (CH2), 28.5
(CH2), 23.3 (CH2), 22.6 (CH2), 14.0 (CH3); δF (376 MHz;
CDCl3) −57.99 (CF3); m.p. 50–55 °C (8 : 2 petroleum
ether : EtOAc).
N-(2-Methoxyphenyl)-3-oxododecanamide (5). Prepared by
general procedure 1 using o-anisidine (0.12 mL, 1.05 mmol).
The crude ketone-protected amide was used directly without
further purification. The crude material obtained after treatment
with TFA was purified by column chromatography (SiO2, 7 : 3
petroleum ether : EtOAc) to yield the title compound as a pale
brown solid (36% overall). Rf 0.8 (8 : 2 petroleum ether :
EtOAc); δH (400 MHz; CDCl3) 9.30 (1H, s, NH), 8.32 (1H, dd,
J = 8.0 Hz, 2.0 Hz, aryl H), 7.05 (1H, td, J = 8.0 Hz, 2.0 Hz,
aryl H), 6.96 (1H, dd, J = 8.0, 1.5 Hz, aryl H), 6.90–6.87 (1H,
m, aryl H), 3.92 (3H, s, OCH3), 3.57 (2H, s, COCH2CO), 2.58
(2H, t, J = 7.5 Hz, COCH2(CH2)7CH3), 1.65–1.59 (2H, m,
CH2), 1.31–1.22 (12H, m, 6 × CH2), 0.89–0.86 (3H, m, CH3);
δC (100 MHz; CDCl3) 207.0 (CvO ketone) 163.3 (CvO
amide), 148.3 (COCH3), 127.4 (C), 124.0 (CH), 120.9 (CH),
120.1 (CH), 110.0 (CH), 55.8 (COCH3), 50.0 (COCH2CO2),
44.1 (COCH2(CH2)7CH3), 35.4 (CH2), 31.8 (CH2), 29.4 (CH2),
29.3 (CH2), 29.0 (CH2), 23.4 (CH2), 22.6 (CH2), 14.1 (CH3);
νmax (neat)/cm−1 2921 (NH), 1711 (CvO ketone), 1669 (CvO
amide); HRMS (ESI+) m/z found [M + Na]+ 342.2059,
C19H29NO3Na+ requires 342.2040; m.p. 36–38 °C (7 : 3 pet-
roleum ether : EtOAc).
N-(3-Ethoxyphenyl)-3-oxododecanamide (12). Prepared by
general procedure 1 using m-phenetidine (0.14 mL, 1.07 mmol).
The crude ketone-protected amide was used directly without
further purification. The crude material obtained after treatment
with TFA was purified by column chromatography (SiO2, gradi-
ent 8 : 2 petroleum ether : EtOAc to 7 : 3 petroleum ether :
EtOAc) to yield the title compound (0.032 g, 12% overall). Rf
0.6 (7 : 3 petroleum ether : EtOAc); νmax (neat)/cm−1 3299
(C–H), 2916 (N–H), 1718 (CvO ketone), 1661 (CvO amide),
1606, 1535, 1496; δH (500 MHz; CDCl3) 9.15 (1H, s, NH),
7.26–7.25 (1H, m, aryl CH), 7.20 (1H, t, J = 8.0 Hz, aryl CH),
7.03 (1H, ddd, J = 8.0 Hz, 2.0 Hz, 1.0 Hz, aryl CH), 6.69–6.61
(1H, m, aryl CH), 4.02 (2H, q, J = 7.0 Hz, OCH2CH3), 3.55
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(2H, s, COCH2CO), 2.57 (2H, t,
J
=
7.5 Hz,
(SiO2, 8 : 2 petroleum ether : EtOAc) to yield the title compound
(0.0109 g, 44% overall). Rf 0.28 (8 : 2 petroleum ether : EtOAc);
νmax (neat)/cm−1 2914 (N–H), 1703 (CvO ketone), 1677 (CvO
amide); δH (500 MHz; CDCl3) 9.29 (1H, s, NH), 8.02 (1H, d,
J = 3.0 Hz, aryl CH), 6.75 (1H, dd, J = 9.0 Hz and 3.0 Hz, aryl
CH), 6.54 (1H, dd, J = 9.0 Hz and 3.0 Hz, aryl CH), 3.83 (3H,
s, OCH3), 3.73 (3H, s, OCH3), 3.52 (2H, s, COCH2CO), 2.56
(2H, t, J = 7.5 Hz, COCH2(CH2)7CH3), 1.31–1.28 (14H, m, 7 ×
CH2), 0.84 (3H, t, J = 7.0 Hz, CH3); δC (100 MHz; CDCl3)
206.8 (CvO ketone), 163.5 (CvO amide), 153.7 (C), 142.6
(C), 128.1 (C), 110.9 (CH), 108.6 (CH), 106.4 (CH), 56.4
(CH3), 55.7 (CH3), 50.0 (CH2), 44.0 (CH2), 31.8 (CH2), 29.3
(CH2), 29.3 (CH2), 29.2 (CH2), 28.9 (CH2), 23.3 (CH2), 22.7
(CH2), 14.1 (CH3); HRMS (ESI+) m/z found [M + H]+
COCH2(CH2)7CH3), 1.66–1.58 (2H, m, CH2CH2CO), 1.40 (3H,
t, J = 7.0 Hz, OCH2CH3), 1.33–1.19 (12H, br m, 6 × CH2), 0.88
(3H, t, J = 7.0 Hz, CH3); δC (125 MHz; CDCl3) 208.0 (CvO
ketone), 163.4 (CvO amide), 159.5 (C), 138.7 (C), 129.6 (CH),
112.1 (CH), 111.0 (CH), 106.3 (CH), 63.5 (CH2), 48.8 (CH2),
44.1 (CH2), 31.8 (CH2), 29.35 (CH2), 29.35 (CH2), 29.2 (CH2),
29.0 (CH2), 23.3 (CH2), 22.6 (CH2), 14.8 (CH3), 14.1 (CH3);
HRMS (ESI+) m/z found [M + Na]+ 356.2217, C20H31NO3Na+
requires 356.2196; m.p. 41–44 °C (7 : 3 petroleum
ether : EtOAc).
N-(3-Nitrophenyl)-3-oxododecanamide (13). Prepared by
general procedure 1 using 3-nitroaniline (0.18 g, 1.37 mmol).
The crude ketone-protected amide was used directly without
further purification. The crude material obtained after treatment
with TFA was purified by column chromatography (SiO2, 6 : 4
petroleum ether : EtOAc) to yield the title compound (0.0512 g,
18% overall). Rf 0.77 (6 : 4 petroleum ether : EtOAc); νmax
(neat)/cm−1 2919 (N–H), 1716 (CvO ketone), 1678 (CvO
amide); δH (500 MHz; CDCl3) 9.65 (1H, s, NH), 8.44 (1H, s,
aryl CH), 7.92 (1H, d, J = 8.5 Hz, aryl CH), 7.89 (1H, d, J =
8.0 Hz aryl CH), 7.49–7.44 (1H, m, aryl CH), 3.60 (2H, s,
COCH2CO), 2.57 (2H, t, J = 7.5 Hz, COCH2(CH2)7CH3),
1.31–1.18 (14H, m, 7 × CH2), 0.85 (3H, t, J = 7.0 Hz, CH3); δC
(100 MHz; CDCl3) 207.8 (CvO ketone), 164.1 (CvO amide),
148.5 (C), 138.6 (C), 129.7 (CH), 125.6 (CH), 119.0 (CH),
114.8 (CH), 48.4 (CH2), 44.1 (CH2), 31.7 (CH2), 29.2 (CH2),
29.2 (CH2), 29.1 (CH2), 28.9 (CH2), 23.2 (CH2), 22.6 (CH2),
14.0 (CH3); HRMS (ESI+) m/z found [M + H]+ 335.1996,
C18H27N2O4+ requires 335.1971; m.p. 68–93 °C (6 : 4 petroleum
ether : EtOAc).
+
350.2327, C20H32NO4 requires 350.2331; m.p. 48–55 °C (8 : 2
petroleum ether : EtOAc).
N-(3,4-Dimethoxyphenyl)-3-oxododecanamide (16). Prepared
by general procedure 1 using 3,4-dimethoxyaniline (0.16 g,
1.08 mmol). The crude ketone-protected amide was used directly
without further purification. The crude material obtained after
treatment with TFA was purified by column chromatography
(SiO2, 8 : 2 petroleum ether : EtOAc) to yield the title compound
(0.173 g, 56% overall). Rf 0.83 (8 : 2 Petroleum Ether : EtOAc);
νmax (neat)/cm−1 2914 (N–H), 1703 (CvO ketone), 1677 (CvO
amide); δH (500 MHz; CDCl3) 9.09 (1H, s, NH), 7.23 (1H, d,
J = 2.5 Hz, aryl CH), 6.95 (1H, dd, J = 8.5 Hz and 2.5 Hz, aryl
CH), 6.77 (1H, d, J = 8.5 Hz aryl CH), 3.82 (3H, s, OCH3), 3.81
(3H, s, OCH3), 3.50 (2H, s, COCH2CO), 2.53 (2H, t, J = 7.5
Hz, COCH2(CH2)7CH3), 1.31–1.18 (14H, m, 7 × CH2), 0.84
(3H, t, J = 7.0 Hz, CH3); δC (100 MHz; CDCl3) 207.9 (CvO
ketone), 163.5 (CvO amide,) 148.9 (C), 145.9 (C), 131.2 (C),
112.1 (CH), 111.2 (CH), 104.9 (CH), 56.0 (CH3), 55.8 (CH3),
48.9 (CH2), 44.1 (CH2), 31.8 (CH2), 29.3 (CH2), 29.3 (CH2),
29.2 (CH2), 28.9 (CH2), 23.3 (CH2), 22.7 (CH2), 14.0 (CH3);
3-Oxo-N-(3-phenoxyphenyl)oxododecanamide (14). Prepared
by general procedure 1 using 3-phenoxyaniline (0.22 g,
1.23 mmol). The crude ketone-protected amide was used directly
without further purification. The crude material obtained after
treatment with TFA was purified by column chromatography
(SiO2, 8 : 2 petroleum ether : EtOAc) to yield the title compound
(0.1086 g, 41% overall). Rf 0.36 (8 : 2 petroleum ether : EtOAc);
νmax (neat)/cm−1 2916 (N–H), 1717 (CvO ketone), 1660 (CvO
amide); δH (500 MHz; CDCl3) 9.25 (1H, s, NH), 7.34–7.25 (3H,
m, aryl CH), 7.25–7.20 (2H, m, aryl CH), 7.12–7.06 (1H, m,
aryl CH), 7.01–6.97 (2H, m, aryl CH), 6.75–6.71 (1H, m, aryl
CH), 3.49 (2H, s, COCH2CO), 2.52 (2H, t, J = 7.5 Hz,
COCH2(CH2)7CH3), 1.26–1.24 (14H, m, 7 × CH2), 0.87 (3H, t,
J = 7.0 Hz, CH3); δC (100 MHz; CDCl3) 207.4 (CvO ketone),
163.7 (CvO amide), 157.7 (C), 156.7 (C), 138.8 (C), 129.8
(CH), 129.6 (CH), 123.3 (CH), 118.9 (CH), 114.6 (CH), 114.5
(CH), 110.5 (CH), 49.0 (CH2), 43.8 (CH2), 31.7 (CH2), 29.2
(CH2), 29.2 (CH2), 29.1 (CH2), 28.8 (CH2), 23.2 (CH2), 22.5
(CH2), 14.0 (CH3); HRMS (ESI+) m/z found [M + H]+
+
HRMS (ESI+) m/z found [M + H]+ 350.2338, C20H32NO4
requires 350.2331; m.p. 69–74 °C (8 : 2 petroleum
ether : EtOAc).
N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-oxododecanamide
(17). Prepared by general procedure 1 using 6-amino-1,4-benzo-
dioxan (0.13 mL, 1.06 mmol). The crude ketone-protected
amide was used directly without further purification. The crude
material obtained after treatment with TFA was purified by
column chromatography (SiO2, 7 : 3 petroleum ether : EtOAc) to
yield the title compound (0.07 g, 26% overall). Rf 0.7 (7 : 3
petroleum ether : EtOAc); νmax (neat)/cm−1 3298 (C–H), 2918
(N–H), 1711 (CvO ketone), 1655 (CvO amide), 1547, 1507,
1466, 1417; δH (500 MHz; CDCl3) 9.00 (1H, s, NH), 7.18 (1H,
d, J = 2.5 Hz, aryl CH), 6.91 (1H, dd, J = 9.0 Hz, 2.5 Hz, aryl
CH), 6.79 (1H, d, J = 9.0 Hz, aryl CH), 4.28–4.17 (4H, m,
OCH2CH2O), 3.52 (2H, s, COCH2CO), 2.55 (2H, t, J = 7.5 Hz,
COCH2(CH2)7CH3), 1.60 (2H, quint, J = 7.5 Hz, CH2CH2CO),
1.36–1.14 (12H, br m, 6 × CH2), 0.87 (3H, t, J = 7.0 Hz, CH3);
δC (125 MHz; CDCl3) 207.9 (CvO ketone), 163.3 (CvO
amide), 143.4 (C), 140.5 (C), 131.2 (C), 117.1 (CH), 113.7
(CH), 109.9 (CH), 64.4 (CH2), 64.2 (CH2), 48.8 (CH2), 44.1
(CH2), 31.8 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.0
(CH2), 23.3 (CH2), 22.6 (CH2), 14.1 (CH3); HRMS (ESI+) m/z
+
382.2375, C24H32NO3 requires 382.2382; m.p. 40–45 °C (8 : 2
petroleum ether : EtOAc).
N-(2,5-Dimethoxyphenyl)-3-oxododecanamide (15). Prepared
by general procedure 1 using 2,5-dimethoxyaniline (0.16 g,
1.06 mmol). The crude ketone-protected amide was used directly
without further purification. The crude material obtained after
treatment with TFA was purified by column chromatography
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found [M + Na]+ 370.2000, C20H29NO4Na+ requires 370.1994;
m.p. 53–55 °C (7 : 3 petroleum ether : EtOAc).
0.6 mol equiv.) were added to a stirred solution of (S)-(−)-(α)-
amino-γ-butyrolactone hydrobromide (23, 169 mg, 0.93 mmol)
and 4-bromophenyl acetic acid (200 mg, 0.93 mmol) in anhy-
drous CH2Cl2 (10 mL). The reaction mixture was stirred at room
temperature for 18 hours and then diluted with CH2Cl2, washed
with HCl (10% v/v solution), dried (MgSO4), and the solvent
removed under reduced pressure. The resulting yellow-white
solid was purified by column chromatography (SiO2, 5 : 1
EtOAc : CH2Cl2) to yield the title compound as a white solid
(233 mg, 84%). Rf 0.26 (5 : 1 EtOAc : CH2Cl2); νmax (neat)/
cm−1 3291 (NH), 1778 (CvO ester), 1649 (CvO amide), 1542
(CvC aromatic); δH (500 MHz; CDCl3) 7.49–7.46 (2H, m, aryl
CH), 7.17–7.14 (2H, m, aryl CH), 6.09 (1H, d, J = 6.5 Hz, NH),
4.56–4.46 (1H, m, NHCH), 4.44 (1H, td, J = 9.0 Hz and 1.0 Hz,
CHH′OC(vO)), 4.26 (1H, ddd, J = 11.5 Hz, 9.5 Hz and 6.0 Hz,
CHH′OC(vO)), 3.56 (2H, s, C(vO)CH2), 2.81–2.75 (1H, m,
CHH′CHNH), 2.17–2.04 (1H, m, CHH′CHNH2); δC (100 MHz;
CDCl3) 175.0 (CvO ester), 170.8 (CvO amide), 133.0 (C),
132.2 (CH), 1331.0 (CH), 121.6 (C), 66.0 (CH2), 49.4 (CH),
42.5 (CH2), 30.3 (CH2); LCMS (ES+) (MeCN) 299 (M + H)+;
m.p. 154–156 °C (5 : 1 EtOAc : CH2Cl2). This data is in accord-
ance with that previously reported.47
N-(Benzo[d][1,3]dioxol-5-yl)-3-oxododecanamide
(18). Pre-
pared by general procedure 1 using 3,4-(methylenedioxy)aniline
(0.145 g, 1.06 mmol). The crude ketone-protected amide was
used directly without further purification. The crude material
obtained after treatment with TFA was purified by column
chromatography (SiO2, 7 : 3 petroleum ether : EtOAc) to yield
the title compound as a light brown solid (0.0346 g, 13%
overall). Rf 0.6 (7 : 3 petroleum ether : EtOAc); νmax (neat)/cm−1
3294 (CH), 2916 (N–H), 1710 (CvO ketone), 1654 (CvO
amide), 1507, 1495, 1414; δH (500 MHz; CDCl3) 9.09 (1H, s,
NH), 7.24 (1H, d, J = 2.0 Hz, aryl CH), 6.84 (1H, dd, J =
8.5 Hz, 2.0 Hz, aryl CH), 6.74 (1H, d, J = 8.5 Hz, aryl CH),
5.94 (2H, s, OCH2O), 3.53 (2H, s, COCH2CO), 2.56 (2H, t, J =
7.5 Hz, COCH2(CH2)7CH3), 1.65–1.59 (2H, m, CH2CH2CO),
1.32–1.21 (12H, br m, 6 × CH2), 0.87 (3H, t, J = 7.0 Hz, CH3);
δC (125 MHz; CDCl3) 208.0 (CvO ketone), 163.3 (CvO
amide), 147.7 (C), 144.3 (C), 131.8 (C), 113.7 (CH), 108.0
(CH), 102.9 (CH), 101.2 (CH2), 48.6 (CH2), 44.2 (CH2), 31.8
(CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.0 (CH2), 23.3
(CH2), 22.6 (CH2), 14.1 (CH3); HRMS (ESI+) m/z found
[M + H]+ 334.2044, C19H28NO4 requires 334.2013; m.p.
+
N-Phenethylisobutyramide (21). A solution of isobutyric acid
(0.21 mL, 2.26 mmol), EDC (0.525 g, 2.73 mmol), DMAP
(0.03 g, 0.24 mmol) and 2-phenylethylamine (24, 0.29 mL,
2.3 mmol) in anhydrous CH2Cl2 (6 mL) was stirred at room
temperature under nitrogen for 12 hours. The solution was con-
centrated under high vacuum and aqueous HCl (5 mL, 10% v/v
solution) was added dropwise with stirring. The aqueous and
organic layers were separated and the aqueous layer extracted
with CH2Cl2. The organic extracts were combined and washed
with aqueous HCl (10% v/v solution). The organic extracts were
washed with sat. aqueous NaHCO3, dried (MgSO4) and the
solvent removed under reduced pressure. The crude product
material was purified by column chromatography (SiO2, 5 : 5
petroleum ether : EtOAc) to yield the title compound as a light
brown solid (44%). Rf 0.5 (5 : 5 petroleum ether : EtOAc); νmax
(neat)/cm−1 3295 (C–H), 2968 (N–H), 1639 (CvO), 1587,
1545, 1455; δH (500 MHz; CDCl3) 7.36–7.07 (5H, m, aryl CH),
5.86 (1H, br s, NH), 3.47 (2H, dt, J = 7.0 Hz, 6.5 Hz, NHCH2),
2.79 (2H, t, J = 7.0 Hz, NHCH2CH2), 2.29 (1H, hept, J = 7.0
Hz, C(O)CH((CH3)2)), 1.09 (6H, d, J = 7.0 Hz, C(O)CH
((CH3)2)); δC (125 MHz; CDCl3) 177.1 (CvO), 139.0 (C),
128.8 (CH), 128.6 (CH), 126.4 (CH), 40.5 (CH2), 35.7 (CH2),
35.5 (CH), 19.6 (CH3); HRMS (ESI+) m/z found [M + H]+
192.1391, C12H18NO+ requires 192.1383; m.p. 88–91 °C (5 : 5
petroleum ether : EtOAc).
81–85 °C (7 : 3 petroleum ether : EtOAc).
(S)-4-(1H-Indol-2-yl)-N-(2-oxotetrahydrofuran-3-yl)butanamide
(19). EDC (282 mg, 1.5 mol equiv.) and DMAP (72 mg,
0.6 mol equiv.) were added to a stirred solution of (S)-(−)-(α)-
amino-γ-butyrolactone hydrobromide (23, 178 mg, 0.98 mmol)
and indole-3-butyric acid (200 mg, 0.98 mmol) in anhydrous
CH2Cl2 (10 mL) under a nitrogen atmosphere. The reaction
mixture was stirred at room temperature for 18 hours and then
diluted with CH2Cl2, washed with HCl (10% v/v solution), dried
(MgSO4) and the solvent removed under reduced pressure. The
resulting orange oil was purified by column chromatography
(SiO2, gradient 6 : 1 CH2Cl2 : EtOAc to 1 : 1 CH2Cl2 : EtOAc) to
give the title compound as a slightly opaque oil (180 mg, 64%).
Rf 0.21 (1 : 1 EtOAc : CH2Cl2); νmax (neat)/cm−1 3377 (NH
indole), 3301 (NH amide), 2916 (CH), 1765 (CvO ester), 1649
(CvO amide), 1522 (CvC aromatic); δH (500 MHz; CDCl3)
8.14 (1H, br s, NH indole), 7.58 (1H, d, J = 8.0 Hz, aryl CH),
7.34 (1H, dt, J = 8.0 Hz and 1.0 Hz, aryl CH), 7.18 (1H, ddd,
J = 8.0 Hz, 7.0 Hz, 1.0 Hz, aryl CH), 7.10 (1H, ddd, J = 8.0 Hz,
7.0 Hz and 1.0 Hz, aryl CH), 6.95 (1H, d, J = 2.0 Hz, aryl CH),
6.14 (1H, d, J = 6.0 Hz, NH amide), 4.50 (1H, ddd, J = 11.5 Hz,
9.0 Hz and 6.5 Hz, NHCHC(vO)), 4.39 (1H, td, J = 9.0 Hz and
1.5 Hz, CHH′OC(vO)), 4.20 (1H, ddd, J = 11.0 Hz, 9.0 Hz and
6.0 Hz, CHH′OC(vO)), 2.85–2.74 (2H, m, C(vO)CH2), 2.69
(1H, dddd, J = 12.5 Hz, 9.0 Hz, 6.0 Hz and 1.5 Hz, CHH′
CHNH), 2.25 (2H, dd, J = 8.5 Hz and 7.0 Hz, CH2), 2.09–1.98
(3H, m, CHH′CHNH and CH2); δC (100 MHz; CDCl3) 175.7
(CvO ester), 173.7 (CvO amide), 136.3 (C), 127.4 (C), 121.9
(CH), 121.8 (CH), 119.2 (CH), 118.8 (CH), 115.4 (C), 115.2
(C), 111.2 (CH), 66.1 (CH2), 49.0 (CH), 35.4 (CH2), 25.6
(CH2), 24.3 (CH2); LCMS (ES−) (MeCN) 285 (M − H)−. This
data is in accordance with that previously reported.47
3-Methyl-N-phenethylbutanamide (22). A solution of isovale-
ric acid (0.22 mL, 2.0 mmol), EDC (0.462 g, 2.41 mmol),
DMAP (0.03 g, 0.27 mmol) and 2-phenylethylamine (24,
0.25 mL, 2.0 mmol) in anhydrous CH2Cl2 (6 mL) was stirred at
room temperature under nitrogen for 12 hours. The semi-solid
mixture was re-dissolved in CH2Cl2 and aqueous HCl (5 mL,
10% v/v solution) was added dropwise with stirring. The
aqueous and organic layers were separated and the aqueous layer
extracted with CH2Cl2. The organic extracts were combined and
washed with aqueous HCl (10% v/v solution). The organic
(S)-2-(4-Bromophenyl)-N-(2-oxotetrahydrofuran-3-yl)acetamide
(20). EDC (267 mg, 1.5 mol equiv.) and DMAP (68 mg,
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extracts were washed with sat. aqueous NaHCO3, H2O dried
(MgSO4) and the solvent removed under reduced pressure. The
crude product material was purified by column chromatography
(SiO2, 3 : 7 petroleum ether : EtOAc) to yield the title compound
as a yellow crystalline solid (44%). Rf 0.5 (3 : 7 petroleum ether :
EtOAc); νmax (neat)/cm−1 3301 (C–H), 2962 (N–H), 1637
(CvO), 1541, 1496, 1454; δH (500 MHz; CDCl3) 7.28–7.08
(5H, m, aryl CH), 6.20 (1H, br s, NH), 3.46 (2H, td, J = 7.0 Hz,
6.0 Hz, NHCH2), 2.78 (2H, t, J = 7.0 Hz, NCH2CH2),
2.10–1.95 (3H, m, C(O)CH2 and CH((CH3)2)), 0.88 (6H, d, J =
7.0 Hz, CH((CH3)2)); δC (125 MHz; CDCl3) 173.0 (CvO),
138.9 (C), 128.7 (CH), 128.6 (CH), 126.4 (CH), 45.9 (CH2),
40.6 (CH2), 35.7 (CH2), 26.1 (CH), 22.4 (CH3); HRMS (ESI+)
m/z found [M + H]+ 206.1543, C13H20NO+ requires 206.1539;
m.p. 61–65 °C (3 : 7 petroleum ether : EtOAc).
22.1 (CH2), 14.0 (CO2CH2CH3); LCMS (EI+) (MeCN) 325
(M + H)+ .
PD-12. Ethyl 2-(2-dodecyl-2H-tetrazol-5-yl)acetate (27,
0.140 g, 0.43 mmol) was dissolved in 1.5 mL of EtOH and
stirred at room temperature for 5 min. To this was added pow-
dered KOH (0.029 g, 0.52 mmol in 1.5 mL H2O) was added and
the solution stirred for 4.5 hours. EtOAc was then added and the
aqueous layer extracted, this was acidified with 1 N HCl until
between pH 1–2 and EtOAc added. The organic phase was
extracted and dried (MgSO4) and the solvent removed under
reduced pressure to give the title compounds as a white solid
(0.069 g, 59%). νmax (neat)/cm−1 2917 (NH), 1740 (CvO),
1520, 1467; δH (500 MHz; CDCl3) 4.59 (2H, t, J = 7.0 Hz,
NCH2CH2), 4.04 (2H, s, CH2CO2H), 2.00 (2H, dd, J = 10.0 Hz,
5.0 Hz, NCH2CH2), 1.38–1.18 (18H, m, 9 × CH2), 0.88
(3H, t, J = 7.0 Hz, CH3); δC (125 MHz; CDCl3) 172.5
(CvO), 159.3 (N–CvN), 53.3 (NCH2CH2), 31.9 (CH2),
31.4 (CH2), 29.57 (CH2), 29.56 (CH2), 29.5 (CH2),
29.31 (CH2), 29.30 (CH2), 29.2 (CH2), 28.8 (CH2), 26.2 (CH2),
22.7 (CH2), 14.1 (CH3); LCMS (ES+) (MeCN) 297 (M + H)+;
m.p. 67–69 °C.
Ethyl 2-(2H-tetrazol-5-yl)acetate (26). Sodium azide (0.500 g,
7.69 mmol), ammonium chloride (0.411 g, 7.69 mmol) and
ethyl cyanoacetate (25, 0.790 g, 6.66 mmol) were dissolved in
7 mL of DMF and stirred for 5 min. The temperature was then
raised to 100 °C and the solution was stirred at this temperature
for 16 hours. The reaction mixture was allowed to cool to room
temperature and a small amount of methanol was added. The
solvent was removed under reduced pressure and the residue re-
dissolved in a minimum amount of H2O. Aqueous HCl (1 N)
was then added until pH 2, and the resulting white solid precipi-
tate collected by vacuum filtration. This was washed with small
portions of cold H2O and the solvent removed under reduced
pressure to yield the title compound as an off white solid
(0.501 g, 42%). νmax (neat)/cm−1 2910 (N–H), 1741 (CvO),
1563, 1454; δH (500 MHz, d6-DMSO) 4.13 (2H, s, C(O)CH2),
4.13 (2H, q, J = 7.0 Hz, OCH2CH3, 1.20 (3H, t, J = 7.0 Hz,
OCH2CH3); δC (125 MHz, d6-DMSO) 167.7 (CvO), 150.5 (N–
CvN), 61.2 (CH2CH3), 29.5 (COCH2C), 14.0 (CH2CH3);
LCMS (EI+) (MeCN) 157 (M + H)+.
Analysis of ligand binding to LasR in vivo. The ligand
binding domain encoded by lasR was PCR amplified from
PAO1 genomic DNA using primers NlasRFxSph (5′
GTTGGTACCATCGAGGGAAGGGCCTTGGTTGACGGTTTT-
CTTG 3′) and ClasRHDIIIN3 (5′ GTTAAGCTTATTTGCT-
GACCGGATGTTCG 3′). The NlasRFxSph primer anneals to
the 5′ end of the lasR open reading frame (ORF) directly after
the ATG start codon and incorporates a SphI cleavage site into
the DNA amplicon. This primer also generates a Factor Xa pro-
teolytic cleavage site near to the N-terminus of the encoded
protein. The ClasRHDIIIN3 primer anneals to the sequence
region just 5′ of nucleotide 519 in the lasR ORF and incorporates
a HindIII restriction enzyme site. The PCR amplicon (encoding
residues 2–173 of the lasR ORF) was end-polished with SphI
and HindIII and sub-cloned into SphI/HindII-digested depho-
sphorylated pQE80. The pQE80 vector incorporates an in-frame
hexa-histidine tag onto the N-terminal end of the encoded
protein. The N-terminal His-tag and Factor Xa sites were incor-
porated in the eventuality that the LasR-LBD might need to be
purified by column chromatography, although this did not prove
necessary. The pQE80(lasR-LBD) vector was introduced into E.
coli strain DH5α by electroporation. The recombinant plasmid
insert was confirmed by sequencing. The resulting E. coli strain
was grown up overnight in LB medium (containing 50 μg mL−1
carbenicillin). The overnight culture was used to inoculate
50 mL cultures of LB/carbenicillin. After the cultures reached an
optical density (600 nm) of 0.5, IPTG (1 mM) was added (to
induce expression of lasR from the lac promoter on the pQE80
plasmid) to all of the flasks. At the same time, some flasks also
received OdDHL (10 μM), compound 3 (50 μM) or just DMSO
(solvent) as a control. After a further 2 hours of growth, the cul-
tures were harvested by centrifugation (5000g, 10 min, 4 °C)
and the cell pellets were resuspended in 5 mL of lysis buffer (0.1
M Tris-HCl, 0.2 M NaCl, 10 mM DTT (pH 7.5)) and sonicated
on ice. The sonicated samples were clarified by centrifugation
(12 000g, 5 min, 4 °C) and the soluble/insoluble (pelleted)
material was analysed by SDS-PAGE.
Ethyl 2-(2-dodecyl-2H-tetrazol-5-yl)acetate (27). Ethyl 2-(2H-
tetrazol-5-yl)acetate (26, 0.230 g, 1.49 mmol) and triethylamine
(0.180 g, 1.78 mmol) were dissolved in 3 mL of MeCN and
stirred to 90 °C over 15 min. 1-bromododecane (0.406 g,
1.63 mmol) was then added dropwise and the solution stirred at
reflux for 19 hours. The solution was cooled to room temperature
and filtered, and the solvent removed under reduced pressure to
give an oil. This was partitioned between H2O and EtOAc, with
the organic phase collected, and the aqueous layer washed a
further 2 times with EtOAC. The combined EtOAc washings
were dried (MgSO4) and the solvent removed under reduced
pressure. The crude product material was purified by column
chromatography (SiO2, gradient 9.5 : 0.5 hexane : EtOAc to 8 :
2 hexane : EtOAc) to yield the title compound as a colourless oil
(0.105 g, 22%). νmax (neat)/cm−1 2918, 1736 (CvO), 1469,
1342; δH (500 MHz, d6-DMSO) 4.64 (2H, t, J = 7.0 Hz,
NCH2(CH2)10), 4.11 (2H, q, J = 7.0 Hz, OCH2CH3), 4.03 (2H,
s, C(O)CH2C), 1.92–1.83 (2H, m, NCH2CH2) 1.33–1.12 (21H,
m, 9 × CH2 and OCH2CH3) 0.88–0.83 (3H, m, (CH2)11CH3); δC
(125 MHz, d6-DMSO): 168.4 (CvO), 159.9 (NCvN), 60.9
(CO2CH2CH3), 52.5 (CH2CO2CH2), 31.3 (CH2), 31.2 (CH2),
31.0 (CH2), 30.8 ((CH2)11CH3) 29.0 (CH2), 28.9 (CH2),
28.8 (CH2), 28.7 (CH2), 28.6 (CH2), 28.2 (CH2), 25.6 (CH2),
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26 A. M. Stevens, Y. Queneau, L. Soulere, S. von Bodman and
A. Doutheau, Chem. Rev., 2011, 111, 4–27.
Acknowledgements
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This work was supported by grants from the Engineering and
Physical Sciences Research Council, Biotechnology and Biologi-
cal Sciences Research Council, Medical Research Council,
Royal Society, Frances and Augustus Newman Foundation, and
Wellcome Trust. JTH is supported by a Medical Research
Council strategic priority studentship awarded to MW and DRS.
We also thank the reviewers for useful comments and
suggestions.
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