Design, synthesis, and biological evaluation of abiotic, non-lactone modulators of LuxR-type quorum sensing

Article abstract of DOI:10.1016/j.bmc.2011.06.072

Quorum sensing (QS) is a cell-cell signaling mechanism that allows bacteria to monitor their population size and alter their behavior at high cell densities. Gram-negative bacteria use N-acylated L-homoserine lactones (AHLs) as their primary signals for QS. These signals are susceptible to lactone hydrolysis in biologically relevant media, and the ring-opened products are inactive QS signals. We have previously identified a range of non-native AHLs capable of strongly agonizing and antagonizing QS in Gram-negative bacteria. However, these abiotic AHLs are also prone to hydrolysis and inactivation and thereby have a relatively short time window for use (～12-48 h). Non-native QS modulators with reduced or no hydrolytic instability could have enhanced potencies and would be valuable as tools to study the mechanisms of QS in a range of environments (for example, on eukaryotic hosts). This study reports the design and synthesis of two libraries of new, non-hydrolyzable AHL mimics. The libraries were screened for QS modulatory activity using LasR, LuxR, and TraR bacterial reporter strains, and several new, abiotic agonists and antagonists of these receptors were identified.

Full text of DOI:10.1016/j.bmc.2011.06.072

Bioorganic & Medicinal Chemistry 19 (2011) 4812–4819
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of abiotic, non-lactone
modulators of LuxR-type quorum sensing
⇑
Christine E. McInnis, Helen E. Blackwell
Department of Chemistry, University of Wisconsin–Madison, 1101 University Ave., Madison, WI 53706, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Quorum sensing (QS) is a cell–cell signaling mechanism that allows bacteria to monitor their population
Received 20 May 2011
Revised 22 June 2011
Accepted 26 June 2011
Available online 1 July 2011
size and alter their behavior at high cell densities. Gram-negative bacteria use N-acylated L-homoserine
lactones (AHLs) as their primary signals for QS. These signals are susceptible to lactone hydrolysis in bio-
logically relevant media, and the ring-opened products are inactive QS signals. We have previously iden-
tified a range of non-native AHLs capable of strongly agonizing and antagonizing QS in Gram-negative
bacteria. However, these abiotic AHLs are also prone to hydrolysis and inactivation and thereby have a
relatively short time window for use (ꢀ12–48 h). Non-native QS modulators with reduced or no hydro-
lytic instability could have enhanced potencies and would be valuable as tools to study the mechanisms
of QS in a range of environments (for example, on eukaryotic hosts). This study reports the design and
synthesis of two libraries of new, non-hydrolyzable AHL mimics. The libraries were screened for QS mod-
ulatory activity using LasR, LuxR, and TraR bacterial reporter strains, and several new, abiotic agonists
and antagonists of these receptors were identified.
Keywords:
N-Acylated L-homoserine lactone
Gram-negative bacteria
Heterocycles
LuxR-type receptors
Pseudomonas aeruginosa
Quorum sensing
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
bacterial QS signal. Our laboratory has made recent contributions
in this area through the design and synthesis of non-native small
Bacteria can monitor their population densities using
a
molecules that agonize and antagonize QS in Gram-negative
language of low molecular weight chemical signals in a process
called quorum sensing (QS).1,2 Depending on the environment,
these signaling molecules accumulate in proportion to bacterial
cell density. Once a critical population density is achieved,3 the
bacteria can alter their gene expression levels in order to initiate
behaviors that benefit the multicellular community.4–6 These
behaviors are extremely diverse, and include biofilm formation,
virulence factor production, root nodulation, swarming, antibiotic
production, and bioluminescence.7–14 Many of these behaviors
play important roles in mediating both pathogenic and symbiotic
relationships with eukaryotic hosts.15–18 Notably, several of the
most notorious human pathogens (e.g., Staphylococcus aureus and
Pseudomonas aeruginosa) use QS to control virulence.19,20 As such,
there is significant interest in understanding the role and mecha-
nisms of QS in pathogenesis with the goal of developing novel
anti-infective strategies.21,22
bacteria.23,29
Gram-negative bacteria largely use N-acylated L-homoserine
lactones (AHLs) as their QS signals.6,11,12,25,30 In general, these
signals are cell permeable and are generated by a synthase protein
(a LuxI-type synthase). The AHLs are then sensed by a cognate,
cytoplasmic receptor (a LuxR-type receptor) at sufficiently high
intracellular AHL concentrations.31 The AHL–receptor complex
typically dimerizes, binds specific DNA promoter sequences, and
activates the transcription of genes that the bacteria utilize at high
cell density.32 Over ꢀ100 different LuxI/R-type QS circuits have
been indentified in bacteria,7 and many species appear to utilize
multiple LuxI/R-type circuits in tandem to control QS.33
With regard to the QS signals, there are ꢀ25 known naturally
occurring AHLs, and they only differ in the structure of their respec-
tive acylgroups.6 These groupsare typically aliphatic (4–18carbons)
and have differing levels of oxidation at the 3-position.34,35 LuxR-
type proteins are generally highly selective for their native AHL,
and the AHLs of other species can act as inhibitors.36 Therefore, acyl
chain composition dictates species specificity for AHL signals.
Over the past seven years, our laboratory has designed a range
of AHLs that contain non-native acyl groups for use as chemical
tools to study QS in Gram-negative bacteria.23 We have screened
these compounds for activity in a range of species, including
P. aeruginosa (for LasR and QscR),37,38 Vibrio fischeri (LuxR),39
Agrobacterium tumefaciens (TraR),36 Pectobacterium carotovora
The use of abiotic small molecules23–26 and macromolecular
probes27,28 to modulate QS pathways has emerged as a viable strat-
egy to study QS and control bacterial group behaviors with both
spatial and temporal control. Most of these non-native molecules
affect QS pathways by either intercepting or inactivating the native
⇑
Corresponding author. Tel.: +1 0 608 262 1503; fax: +1 0 608 265 4534.
E-mail address: blackwell@chem.wisc.edu (H.E. Blackwell).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.072

C. E. McInnis, H. E. Blackwell / Bioorg. Med. Chem. 19 (2011) 4812–4819
4813
(ExpR1/ExpR2),40 and Chromobacterium violaceum (CviR).41 Careful
2. Results and discussion
study of the active AHLs has revealed many structure–activity
relationships (SARs) that dictate receptor selectivity for non-native
AHL agonists and antagonists.42 Representative LuxR-type receptor
agonists and antagonists reported by our laboratory are shown in
Figure 1.
2.1. LasR structural considerations
We utilized OdDHL from P. aeruginosa as a scaffold for the de-
sign of our first set of non-lactone derivatives (Library A). Similar
to Suga’s earlier work (see above),56 we decided to retain the 3-
oxo-dodecanoyl group from OdDHL in each library member, and
simply replace the lactone with a non-native head group. In our
choice of head groups, we scrutinized prior work that showed that
a rigid aromatic or conjugated moiety adjacent to an aliphatic acyl
chain could yield compounds active in LasR.43,48–55 We thus fo-
cused largely on aromatic head groups in our design of Library A.
We also studied the X-ray crystal structure of the ligand-binding
domain of LasR bound to OdDHL (Fig. 2B) to ascertain molecular
interactions essential for binding (making the assumption that
non-lactone analogs could target the same ligand-binding site).60
For example, there is a water-mediated hydrogen bond between
the 3-oxo group of OdDHL and the Arg61 guanidinium in the
ligand-binding pocket. The acyl tail then packs into a hydrophobic
cleft extending away from the lactone. We reasoned that these
interactions would likely be maintained in Library A, with all mem-
bers have 3-oxo dodecanoyl tails. A key hydrogen bond is also
made between the OdDHL lactone carbonyl and Trp60 side chain
NH. We sought, where possible, to retain and/or probe this hydro-
gen bonding contact in Library A.
The heterocycles, carbocycles, and other head group mimics
that we selected for incorporation into Library A are shown in
Figure 3A. Fluorine substituted aromatic rings (16 and 17) were
chosen as lactone carbonyl mimics to probe Trp60 interactions,
due to fluorine’s ability to accept hydrogen bonds. Multiple fluo-
rine substitutions were investigated in 18 and 19 to determine if
Trp60 could hydrogen bond to multiple atoms given the correct
spatial orientation. Non-hydrogen bond donors (in 14, 20, 21,
and 24) were included to examine the effects of other electrostatic
interactions. Library A also contained non-functionalized carbocy-
cles (12, 13, 15, 22, and 25) to explore the necessity of the Trp60
binding interaction for LasR modulation.
In addition to these cyclic head group replacements, we also
incorporated glycine ethyl ester (26) and alanine methyl ester
(27) as acyclic head groups into Library A (Fig. 3A). These groups
were selected to explore the effects of opening the lactone at bonds
(typically) unavailable for cleavage in Nature; our design process is
shown in Figure 4. The glycine ethyl ester analog was designed by
conceptually breaking the bond between carbons 2 and 3. In turn,
the alanine methyl ester analog originated from conceptually
breaking the bond between carbons 3 and 4. Such non-native,
alternatively ‘hydrolyzed’ head groups have yet to be probed in
AHL analogs, to our knowledge. We do note, however, these com-
pounds will also be prone to hydrolysis, as they retain ester func-
tionalities.61 We included them in this study nonetheless, as they
represented novel head group replacements.
We currently seek to improve the potency of our lead AHL ago-
nists and antagonists. It is well known that the AHL head group is
prone to hydrolysis at pH values of 7 and above, and the hydro-
lyzed compound is inactive.43–45 For example, the native AHL for
P. aeruginosa, N-(3-oxo)-dodecanoyl HL (OdDHL, Fig. 2A), has a
half-life of approximately two days in growth medium at 37 °C,
while shorter chain AHLs hydrolyze in as little as 6 h.46,47 Our
non-native AHLs share this same hydrolytic instability, and this
can limit their application in biological experiments, particularly
those over prolonged time periods.36 We reasoned that replacing
the lactone head group in our lead AHLs with a non-hydrolyzable
motif could engender heightened compound stability, and there-
fore, activity.
Others have previously examined lactone replacements in AHL
analogs and uncovered a small set of compounds with moderate
agonistic and antagonistic activities in LuxR-type proteins.43,48–55
For example, the Suga group designed a 96-member library of
OdDHL and N-butanoyl HL (BHL) analogs that contained a variety
of heterocyclic and aromatic head groups, yet retained the native
acyl groups, and evaluated the library for activity in both LasR
and RhlR in P. aeruginosa.56,57 Several active agonists and antago-
nists were uncovered, most notably analogs with phenolic and
cyclohexanol head group replacements.58 Spring and co-workers
have studied AHL analogs that contain cyclopentanone head
groups, and shown that these compounds can strongly activate
QS phenotypic responses in P. aeruginosa, Serratia ATCC39006,
and Erwinia carotovora.30,50 In a related effort, Greenberg and
co-workers have screened large, unbiased libraries of small mole-
cules and identified several structurally unrelated, non-lactone
compounds that can strongly modulate LasR.49,59 Inspired by this
recent work, we sought to examine the QS modulatory activity of
chimeric compounds that contained (1) non-hydrolyzable head
groups, and (2) our previously identified non-native acyl groups
from lead AHLs.
Here we report the design and synthesis of a first- and second-
generation library of AHL mimics containing non-lactone head
groups. The first library (A) was designed to target LasR in P. aeru-
ginosa. Active head groups from this library were then utilized for
the design of chimeric ligands in a second-generation library (B).
Both libraries were tested in bacterial reporter strains for activity
in LasR, LuxR, and TraR. A set of new agonists and antagonists were
found for these QS receptors. Several of these AHL mimics could be
useful for biological experiments that require long time periods or
elevated pH. Further, they also provide insights into which aspects
of the lactone head group are necessary for LuxR-type receptor
agonism and antagonism.
Figure 1. Selected non-native AHLs reported by our laboratory that can agonize and antagonize LuxR-type receptors.

4814
C. E. McInnis, H. E. Blackwell / Bioorg. Med. Chem. 19 (2011) 4812–4819
O
O
O
A
B
N
H
7
O
Figure 2. (A) The natural ligand for LasR in P. aeruginosa, OdDHL. (B) View of OdDHL bound in the ligand-binding pocket in LasR (from the reported X-ray crystal structure of
the OdDHL:LasR N-terminal ligand-binding domain complex).60 Hydrogen bonds are shown as dashed lines.
R =
A
OMe
F
O
12
13
16
20
24
25
F
Cl
N
N
17
18
21
F
OMe
OMe
O
O
F
14
15
22
23
26
27
O
F
O
NO2
O
F
19
B
O
O
7
NH2
R
O
O
+
DMAP
CH2Cl2
O
O
O
O
CH3CN
R
O
O
O
N
Cl
7
rt, 12 h
H
7
2 h, rt -->
5 h, 82 °C
Figure 3. (A) Non-lactone head groups (R) incorporated into Library A. (B) Synthesis and overall structure of Library A members. DMAP = dimethyl aminopyridine. R is a novel
head group shown in part A.

C. E. McInnis, H. E. Blackwell / Bioorg. Med. Chem. 19 (2011) 4812–4819
4815
We used our previously reported bacteriological assay protocols
for small molecule screening, which allowed for comparisons to be
made between the assay data reported here and our past work.42,65
All compounds were screened at 10 lM in both agonism and
antagonism assays. No effects on bacterial growth were observed
over the time course of the reporter gene assays (4–16 h).
2.4. Biological screening of Library A
We first evaluated Library A for LasR agonistic and antagonistic
activities using the E. coli and P. aeruginosa reporter strains. The as-
say data are shown in Table 1. Several compounds were active in
both strains, yet their relative activities were muted overall in
the P. aeruginosa reporter relative to the E. coli reporter. We reason
that this could be due to the P. aeruginosa strain containing com-
peting LuxR-type receptors and/or the lowered cell permeability
of P. aeruginosa relative to E. coli. P. aeruginosa is well known to
have numerous efflux pumps that hamper cellular uptake of com-
pounds.66 In general, sterically small and sparsely functionalized
head groups appended to the 3-oxo-dodecanoyl group were the
most active. We discuss trends in activity for the six most active
compounds (12, 13, 17, 23, 24, and 26) below.
The unsubstituted phenyl (i.e., aniline) analog 12 was the stron-
gest LasR antagonist identified in the E. coli LasR strain, with the
meta-nitro benzyl (23) and ortho-fluoro benzyl (17) analogs being
the second and third most active LasR antagonists, respectively.
Additional fluoro substituents (18 and 19) or a para-fluoro substi-
tuent (16) yielded less active compounds. The antagonistic activi-
ties of 12, 17, and 23 corroborated earlier findings by Greenberg,
Suga, and Kim demonstrating that related aromatic analogs are
LasR antagonists (albeit in alternate strains for Suga and
Kim).49,56,67 As aniline derivative 12 was the most active overall,
this finding does question the proposed critical nature of the AHL
lactone carbonyl for LasR binding with Trp60 (assuming these ana-
logs target the same site via their antagonism mechanism). In turn,
the relatively high activity of aromatic analogs overall suggests
Figure 4. Conceptual design of the unnatural, ring-opened forms of the lactone ring
utilized in Library A. (A) The glycine ethyl ester analog (26). (B) The alanine methyl
ester analog (27).
2.2. Synthesis of Library A
Library A was synthesized in parallel using standard solution-
phase methods (Fig. 3B). The head groups were incorporated as pri-
mary amines. A Meldrum’s acid derivative was used as a common
dodecanoyl intermediate. Reaction of Meldrum’s acid with deca-
noyl chloride afforded the Meldrum’s acid derivative, which was
subsequently refluxed with the desired head group amines to yield
Library A. The 16 library members (12–27) were isolated in 35–70%
yields with purities of 92–99% (see Supplementary data).
2.3. Bacteriological assays
Small molecules are typically screened for LuxR-type agonism
or antagonism using bacterial reporter strains, and we used such
cell-based assays in the current study.23 These bacterial reporter
strains lack a functional LuxI-type synthase, yet retain a functional
LuxR-type receptor. They typically contain a QS promoter fused to
a reporter gene, and exogenous native AHL must be added to acti-
vate the system. Agonism or antagonism assays therefore can be
performed by adding the compound alone or in competition with
the native AHL ligand (at its EC50 value), respectively.
We utilized four bacterial reporter strains in this study to exam-
ine the activity of Library A (and eventually Library B) in LasR,
LuxR, and TraR. Two strains were selected for the LasR screens:
that
in LasR binding.
p–p stacking or hydrophobic interactions could play a role
Cyclopentyl analog 13 was found to be a strong LasR agonist in
both reporter strains. Again, this high level of activity suggests that
either the hydrogen bond to the native AHL lactone carbonyl is not
as crucial as originally thought, or that 13 binds LasR in an alterna-
tive manner. We note that Ishida et al. have reported a cyclopentyl
compound similar to 13, yet containing a simple (non-3-oxo) deca-
noyl tail;51 interestingly, they found that this derivative was a LasR
antagonist in a P. aeruginosa strain, as opposed to an agonist like 13.
These opposite activities highlight the importance of the 3-oxo
group and chain length in LasR modulatory activity.
The para-methoxy benzyl analog (24) displayed conflicting
activities between the two reporter strains: it is a strong LasR
agonist in the E. coli reporter, but a weak LasR antagonist in the
P. aeruginosa reporter. These data suggest that 24 may not solely
target LasR in the P. aeruginosa strain. The differing activities of
24 relative to its meta-nitro analog 23 (see above) suggest that sub-
tle steric and electronic changes to the benzyl head group can have
significant effects on ligand activity in LasR, at least in this E. coli
reporter strain. Benzyl analog 24 represents an excellent candidate
for further testing in additional heterologous reporter strains con-
taining QscR and RhlR, which also play a role in regulating the over-
all P. aeruginosa QS response.68,69 We note, again, that we cannot
discount compound permeability differences between the strains
also contributing to these disparate activity profiles (see above).
The ‘ring-opened’ lactone analogs 26 and 27 provided some
interesting activity trends. First, only the glycine ethyl ester analog
(26) showed appreciable agonistic activity in LasR, indicating that
the ring opening in 27 strongly demoted compound activity
Escherichia coli DH5
a
(pJN105L + pSC11)62 and P. aeruginosa PA01
MW1 (pUM15).49 E. coli DH5
a
(pJN105L + pSC11) is a heterologous
reporter strain containing one plasmid for the LasR gene and a
second plasmid containing the promoter region for LasI fused to
b-galactosidase (b-gal). LasR activity is readout using a standard
colorimetric assay with ortho-nitrophenyl-b-galactoside (ONPG)
as the substrate for b-gal. The PA01 MW1 (pUM15) strain is a LasR
reporter in P. aeruginosa that lacks a functional LasI and contains a
plasmid with a LasR responsive promoter for Yellow Fluorescent
Protein (YFP), which facilitates straightforward evaluation of LasR
activity using fluorescence. Examining the library in both strains
allowed us to study the effects of the AHL analogs on LasR in an iso-
lated system (E. coli) and then in the presence of P. aeruginosa’s
complex QS network (including RhlR and QscR) in the native
PAO1 background. (We note that E. coli and P. aeruginosa have
different compound uptake/efflux profiles, and this feature should
be taken into account when comparing small molecule screening
data between the two strains (see below)).
V. fischeri ESI 114 (D
-LuxI)63 and A. tumefaciens WCF (pCF372)64
were used to examine the activity of library compounds in LuxR
and TraR, respectively. The V. fischeri mutant strain lacks a func-
tioning LuxI synthase, but retains its native lux operon, allowing
a quantitative luminescent readout based on LuxR activity. Simi-
larly, A. tumefaciens WCF (pCF372)64 lacks a functioning TraI, yet
contains a plasmid with a TraR responsive promoter for the b-gal
gene, thereby allowing for direct quantitation of TraR activity using
absorbance.

4816
C. E. McInnis, H. E. Blackwell / Bioorg. Med. Chem. 19 (2011) 4812–4819
Table 1
ate activity profile would also be maintained with alternate head
groups. The unfunctionalized phenylacetanoyl tail (from 1), which
is inactive in most LuxR-type receptors when appended to a native
lactone head group, was included to test if this inactivity would be
maintained in Library B. Lastly, the para-chloro phenylpropanoyl
group from AHL (8), which is a strong LuxR, LasR, and TraR antag-
onist, was selected to probe the ability of non-lactone AHL mimics
to maintain such multi-receptor, or more ‘broad-spectrum’,
activities.
Antagonism and agonism assay data for Library A in LasRa
Compound
E. coli LasR
P. aeruginosa LasR
Antagonismb (%) Agonismc (%) Antagonismb (%) Agonismc (%)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
54
À75
13
3
84
1
29
À1
9
1
54
0
0
0
0
0
0
0
0
4
0
1
0
12
0
17
0
8
13
38
22
13
0
0
0
1
13
13
16
5
2.6. Synthesis of Library B
0
0
0
0
26
0
Library B was synthesized in solution via standard carbodiim-
ide-mediated amide bond coupling reactions (EDC) between the
three head group amines and the respective carboxylic acids
( Fig. 5B). The 16 library members (28–43) were isolated in
60–95% yields with purities of 89–99% (see Supplementary data).
10
41
À68
À4
À136
À4
0
0
73
0
51
2
À7
17
38
5
11
23
a
2.7. Biological screening of Library B
All synthetic compounds were screened at 10
l
M. All assays were preformed in
triplicate; error did not exceed 10%. Positive controls were OdDHL at its EC50 value
(in each strain) for antagonism assays, and at 100 times its EC50 value for agonism
assays. Negative controls contained neither thiolactone nor natural AHL, and were
subtracted from each sample to account for background. Negative inhibition values
indicate that the compound activates at the tested concentration. Shaded rows
highlight compounds of interest. See text for details of strains.
Library B was screened for LasR antagonism and agonism using
the same reporter strains and protocols as for Library A. The results
of these screens are shown in Table 2. In general, similar to the
LasR screening data for Library A, the E. coli LasR strain yielded
compounds with heightened activities relative to the P. aeruginosa
LasR strain. The most active compound indentified was the meta-
nitro phenylacetanoyl glycine methyl ester (28), which displayed
a novel activity profile (see below).
b
Antagonism assays were performed against OdDHL at its EC50 value in each
strain: E. coli DH5
M.
a (pJN105L + pSC11) = 10 nM; P. aeruginosa PA01 MW1 (pUM15) =
1
l
c
Agonism assays were normalized to the positive control (OdDHL) in each strain.
Interestingly, many Library B compounds exhibited slight ago-
nistic activities in the antagonism assay in the P. aeruginosa LasR
strain, yet failed to agonize LasR in the agonism assay in this same
strain (i.e., in the absence of OdDHL). The meta-nitro and para-thi-
oether glycine ethyl ester chimeras (28 and 30), and the biphenyl
cyclopentyl chimera (40) showed greater than 20% agonistic activ-
ities only in the LasR antagonism assay. We have recently seen this
activity trend in the P. aeruginosa LasR reporter for another class of
non-lactone AHL mimics (i.e., thiolactones).70 Our current hypoth-
esis is that such non-native ligands are potentially capable of form-
ing heterodimeric complexes of LuxR-type proteins bound to the
native AHL and the non-native ligand (both present in the compet-
itive antagonism assay) that are more active than homodimeric
complexes formed from native AHL alone. In turn, these non-native
ligands cannot form active homodimers of the same receptors, and
thus are inactive in the agonism assay. While ongoing work in our
laboratory is directed at more fully understanding this potential
mode of action, analogs 28, 30, and 40 exhibit activity profiles in
LasR that are suggestive that they could operate via such a cooper-
ative agonism mechanism.
The majority of the glycine ethyl ester derivatives in Library B
(28–33) were moderate LasR antagonists (30–40%) in the E. coli re-
porter strain (Table 2). However, the meta-nitro chimera 28 was
also an excellent LasR agonist in this strain (when tested at the
same concentration in the absence of OdDHL), activating to 93%
of the level of OdDHL. One possible explanation for such a diver-
gent activity profile is the following: homodimers of LasR com-
plexed with either 28 or OdDHL are can agonize the system, yet
the heterodimeric complex of LasR–28/LasR–ODdHL is inactive.
An antagonistic effect would therefore be observed only when
the natural ligand is present. We have named compounds that
can behave in this manner putative ‘bimodal binders’ because of
their dual antagonistic and agonistic properties, and expound on
this pathway further in a related recent study.70 Additional studies
are certainly required to fully understand the mechanisms by
which 28 elicits its conflicting activities in LasR, and are ongoing.
Cyclopentyl derivatives 35–40 and aniline derivatives 41–43
were only weak antagonists of the E. coli LasR reporter strain and
relative to ‘ring closed’ OdDHL, while the ring opening in 26 had
less of an effect relative to OdDHL. Second, while 26 was a
moderate agonist of LasR in E. coli, it was only minimally active
in the P. aeruginosa strain. This trend is somewhat similar to that
observed for para-methoxy benzyl analog 24 above, so analogous
logic could apply for its possible mode of action.
Library A was also tested for activity in LuxR and TraR using the
reporter strains introduced above. However, compound activities
were low to modest in these species (see Supplementary data for
full data). These low activities were perhaps to be expected, how-
ever, as Library A was designed to target LasR in P. aeruginosa, and
reinforces prior work demonstrating that the length of the AHL
acyl tail, even in non-lactone derivatives, can give ligands receptor
selectivity.36
2.5. Design of Library B
Library B was designed around the most active head groups
uncovered in Library A (aniline, cyclopropyl amine, and glycine
ethyl ester in 12, 13 and 26, respectively; Fig. 5A). To these head
groups, we appended several acyl tails that have shown a range
of activities when incorporated in our previously reported AHL
analogs (see Fig. 1).36,39,42 We note that many of these earlier
AHL analogs were found to be active in LuxR, TraR, and/or LasR.
For example, the para-bromo and para-phenyl AHLs (3 and 6) are
strong antagonists of both LuxR and TraR, the meta-iodo phenyl-
propanoyl HL (7) is a strong antagonist of both LasR and LuxR,
and indole 9 targets LasR selectively. The receptor selectivity pro-
files of AHLs 3, 6, 7, and 9 prompted us to use their acyl groups
in Library B to examine if such selectivity trends would be retained
in chimeric AHL mimics. Likewise, the divergent activity of 2 (a
strong LuxR agonist and a strong LasR antagonist) made the
meta-NO2 phenylacetanoyl group an attractive choice for studying
contrasting receptor modulatory activity. We also included the
acyl tail from para-methoxy phenylacetanoyl HL (4), a more mod-
erate LasR antagonist, in order to investigate whether this moder-

C. E. McInnis, H. E. Blackwell / Bioorg. Med. Chem. 19 (2011) 4812–4819
4817
O
O
A
O
O2N
Br
N
O2N
N
H
O
H
28
36
HN
O
O
O
O
N
N
H
H
O
O
29
O
37
S
F3C
MeO
O
N
H
N
H
30
38
O
O
I
O
N
H
N
H
O
31
39
O
O
O
O
N
H
N
H
O
O
32
40
O
O
N
H
N
H
7
O
33
41
O
O
O
N
H
O2N
Br
N
H
O
Cl
34
42
Br
O
O
N
H
N
H
35
43
O
H
EDC, TEA
N
Z
B
Y
+
NH2
Y
HO
Z
CH2Cl2, rt, 12 h
O
Figure 5. (A) Structures of compounds in Library B. (B) Synthesis of Library B. Y represents the lead heterocycles identified from Library B. Z represents the acyl tails from
several of our previously reported non-native AHLs. EDC = 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). TEA = triethyl amine.
largely inactive in the P. aeruginosa LasR reporter strain (Table 2).
The only exception was the biphenyl cyclopentyl chimera 40,
which activated LasR only in the antagonism assay and thus exhib-
ited a potential cooperative agonistic profile (see above). Together
with the screening data for Library A in LasR, these results suggest
that the native OdDHL acyl chain generally engenders higher li-
gand activities in LasR relative to non-native acyl groups when ap-
pended to the non-native head groups in Library B.
We next screened Library B in LuxR and TraR reporter strains
(see above) for both antagonistic and agonistic activities. The
screening data are shown in Table 3. Four moderate to strong LuxR
modulators were indentified: 30, 31, 33, and 36. However, TraR
was largely unresponsive to Library B (data not shown). This is
perhaps expected, as TraR is believed to have a much more rigid
ligand-binding pocket relative to other LuxR-type proteins71,72
and is less tolerant in general to non-native AHL analogs.65
The most active LuxR modulator indentified in Library B was the
meta-iodo phenylpropanoyl glycine ethyl ester derivative 31,
which was a very strong (89%) LuxR antagonist. Notably, the AHL
analog of 31, AHL 7 (Fig. 1), displays analogous inhibitory activity
in LuxR,65 suggesting that antagonism is largely dependent on acyl
group structure, as opposed to head group structure, for these two
compounds. Aryl glycine ethyl ester derivates 30 and 33 were also
LuxR antagonists, yet were twofold less active (ꢀ40%).
The meta-nitro cyclopentyl derivative 36 was the only LuxR
modulator in Library B not based on glycine ethyl ester (Table 3).
Cyclopentyl analog 36 was a 62% LuxR antagonist, yet was also
capable of agonizing LuxR by 33%. We note that the AHL analog

4818
C. E. McInnis, H. E. Blackwell / Bioorg. Med. Chem. 19 (2011) 4812–4819
Table 2
acyl chain structure is critical for receptor selectivity, even if the
native lactone head group is removed.42
Antagonism and agonism assay data for Library B in LasRa
We next designed a 16-member second-generation library (B)
of chimeric molecules composed of the three lead head groups
from Library A appended to the acyl groups of a subset of our pre-
viously reported non-native AHLs. Several potent modulators were
uncovered, including the glycine ethyl ester analogs 28 and 31,
which were a strong LasR agonist and LuxR antagonist, respec-
tively. It was surprising that these novel ring-opened, achiral li-
gands were the most active in Library B, and motivates further
study of acyclic lactone head group replacements in autoinducer
analogs.
Additional work will be required to further improve the potency
of these lead compounds and to fully understand their mechanisms
of action, particularly with regard to potential cooperative agonism
and bimodal binding pathways.70 Nevertheless, the results of this
study suggest that the lactone head group is not required in small
molecule modulators of LuxR-type proteins, and corroborate sev-
eral previous reports of active non-lactone ligands. With regard
to LasR, our data for certain AHL mimics suggest that the lactone
hydrogen bond to Trp60 in LasR may not be essential for agonistic
or antagonistic activity, assuming that these compounds target the
OdDHL binding site. We of course cannot discount the possibility
that alternate hydrogen bond acceptors in selected AHL mimics
could replace the native AHL lactone carbonyl (e.g., esters in the
glycine ethyl ester derivatives) in this interaction.
Compound
E. coli LasR
P. aeruginosa LasR
Antagonism Agonism (%) Antagonism
Agonism (%)
(%)
(%)
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
41
33
33
38
38
36
38
11
29
20
24
11
4
93
0
0
0
0
0
0
0
0
0
6
0
0
3
0
3
À21
À15
À27
À17
À10
À6
À5
À11
À5
À10
À1
1
0
0
0
0
0
0
0
0
0
2
0
0
1
0
1
0
À26
20
12
20
5
0
12
a
See footnotes a–c in Table 1.
(3) of 36 is a highly potent agonist in LuxR.39 The dual activity of 36
in LuxR is reminiscent of the meta-nitro chimera 28 in LasR (see
above), and suggests that 36 may behave as a bimodal binder with
LuxR, forming inactive heterodimers of LuxR when the native li-
gand (OHHL) is present, yet active homodimers alone.
The new QS agonists and antagonists reported here could have
value as mechanistic probes to study QS. For example, certain non-
hydrolyzable analogs may be useful for controlled-release studies
or for experiments examining QS responses in relation to ligand
diffusibility and population density over time spans ranging from
several days to weeks.73 Further studies are warranted beforehand
to fully characterize their interactions with LuxR-type receptors,
and such experiments are ongoing in our laboratory.
3. Conclusion
We have designed and synthesized two focused libraries of non-
lactone AHL mimics and evaluated their activities as agonists and
antagonists of LasR, LuxR, and TraR. This study was motivated by
our interest in enhancing the hydrolytic stability of such autoin-
ducer mimics in order to potentially increase their potency and
utility as chemical tools to study QS. Sixteen non-hydrolyzable
head groups were explored in the first-generation library (A), and
three head groups (aniline, cyclopentyl amine, and glycine ethyl
ester) yielded the most potent LasR modulators when derivatized
with the OdDHL acyl tail (i.e., in analogs 12, 13, and 26, respec-
tively). These compounds were largely selective for LasR over LuxR
and TraR, which reinforces previous work that demonstrated that
4. Experimental section
4.1. Compound synthesis
All chemical reagents were purchased from commercial sources
(Alfa-Aesar, Aldrich, and Acros) and used without further
purification. Solvents were purchased from commercial sources
(Aldrich and J.T. Baker) and used as obtained, with the exception
of dichloromethane (CH2Cl2), which was distilled over calcium
hydride immediately prior to use.
Table 3
Antagonism and agonism assay data for Library B in LuxRa
The Meldrum’s acid intermediate used in the synthesis of
Library A was generated by dissolving Meldrum’s acid (0.5 g,
3.5 mmol) in CH2Cl2 (50 mL) under nitrogen in an ice bath
(Fig. 3B). DMAP (0.86 g, 7 mmol) was added to the mixture and
allowed to completely dissolve. Dodecanoyl chloride (0.72 mL,
3.5 mmol) was then added slowly over an hour. The reaction mix-
ture was allowed to stir on ice for an additional hour, and then was
allowed to come to room temperature (rt) and stir overnight. The
reaction mixture was washed 2Â with 2 M HCl and 1Â with brine.
The organic layers were isolated, dried over MgSO4, filtered, and
concentrated to yield the crude Meldrum’s acid intermediate that
was used immediately in the next amide-coupling step.
Library A were synthesized by stirring the desired amine head
group (0.16 mmol) with the Meldrum’s acid intermediate from
above (48 mg, 0.16 mmol) in acetonitrile (15 mL) at rt under nitro-
gen for 2 h (Fig. 3B). The reaction mixture was then refluxed for an
additional 5 h to ensure complete reaction. The solvent was re-
moved in vacuo, and the reaction residue was redissolved in ethyl
acetate and washed 1Â each with saturated NaHCO3, 1 M NaHSO4,
and brine. The organic layers were combined, dried over MgSO4,
filtered, and concentrated to yield the Library A members. As
Compound
V. fischeri LuxR
Antagonismb (%)
Agonismc (%)
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
19
14
43
89
15
40
14
35
62
25
18
19
17
2
0
0
0
0
0
0
0
0
33
4
3
0
5
7
0
1
0
0
a
See footnote a in Table 1.
b
Antagonism assays were performed against OHHL at its the
-LuxI) strain = 2 M.
Agonism assays were normalized to the positive control
(100 M OHHL).
EC50 value in the V. fischeri ESI 114 (
D
l
c
l

C. E. McInnis, H. E. Blackwell / Bioorg. Med. Chem. 19 (2011) 4812–4819
4819
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The glycine ethyl ester head group in 26 was synthesized as
previously reported,74 and commercially available alanine methyl
ester was utilized to synthesize 27. Library B was synthesized
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4.2. Bacterial assays
Bacterial reporter gene assays for E. coli DH5a (pJN105L + pSC11),
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Financial support for this work was provided by the NIH
(AI063326), ONR (N00014-07-1-0255), Greater Milwaukee Foun-
dation Shaw Scientist Program, Burroughs Welcome Fund, Camille
& Henry Dreyfus Foundation, Research Corporation, and Johnson &
Johnson. C.E.M. was supported in part through a DOD (Air Force Of-
fice of Scientific Research) National Defense Science and Engineer-
ing Graduate (NDSEG) Fellowship (32 CFR 168a). We gratefully
acknowledge Professors Peter Greenberg (University of Washing-
ton), Stephen Winans (Cornell University), and Edward Ruby
(UW–Madison) for donations of bacterial strains and advice on
their manipulation. We also thank Karla Camancho for her assis-
tance in purifying members of Library A.
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Supplementary data
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Supplementary data (compound characterization data, biologi-
cal assay protocols, and additional screening data) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmc.2011.06.072.
53. Estephane, J.; Dauvergne, J.; Soulere, L.; Reverchon, S.; Queneau, Y.; Doutheau,
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