Synthesis and application of an N-acylated l-homoserine lactone derivatized affinity matrix for the isolation of quorum sensing signal receptors

Article abstract of DOI:10.1016/j.bmcl.2011.04.062

The design and synthesis of an agarose resin functionalized with a Gram-negative quorum sensing (QS) signaling molecule analogue is described. The modified resin was utilized in affinity pull-down assays to successfully isolate QscR, a LuxR-type QS receptor from Pseudomonas aeruginosa. This resin may facilitate the identification of novel QS signal receptors using affinity chromatography techniques.

Full text of DOI:10.1016/j.bmcl.2011.04.062

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5054–5057
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and application of an N-acylated L-homoserine lactone
derivatized affinity matrix for the isolation of quorum sensing signal receptors
⇑
Thanit Praneenararat, Teresa M. J. Beary, Anthony S. Breitbach, Helen E. Blackwell
Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue, Madison, WI 53706-1322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of an agarose resin functionalized with a Gram-negative quorum sensing (QS)
signaling molecule analogue is described. The modified resin was utilized in affinity pull-down assays to
successfully isolate QscR, a LuxR-type QS receptor from Pseudomonas aeruginosa. This resin may facilitate
the identification of novel QS signal receptors using affinity chromatography techniques.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 17 March 2011
Revised 8 April 2011
Accepted 12 April 2011
Available online 22 April 2011
Keywords:
Affinity chromatography
AHL
N-Acylated L-homoserine lactone
LuxR-type receptors
QscR
Quorum sensing
Bacteria can regulate specific phenotypes as a function of their
cell density by sensing the concentration of small molecule chem-
ical signals. This small molecule signaling process is known as
quorum sensing (QS), and has attracted considerable interest
from the microbiology, chemical biology, and drug discovery
Chemical tools for evaluating the biochemical interactions be-
tween AHLs and LuxR-type proteins, as well as for the identifica-
tion of novel AHL receptors, would be useful for a broad range of
experiments.16,22,23 LuxR-type proteins have proven challenging
to characterize using standard biochemical and structural tech-
niques (largely due to their instability in the absence of AHL li-
gands), and this has limited our understanding of how they
perceive both native and nonnative AHL signals.9,24 In addition,
increasing evidence suggests that AHLs from one bacterial species
can induce phenotypic changes in neighboring species, including
other Gram-negative bacteria25 and potentially even their eukary-
otic hosts.26 This implies that bacteria may have more extensive
networks of communication pathways, both at the inter-species
and the inter-kingdom levels. Therefore, identifying new receptors
for AHLs may lead to a deeper understanding of these phenomena.
Affinity chromatography could provide solutions for several of
the challenges described above.27 In this methodology, small mol-
ecules of interest are chemically modified and covalently bound to
an insoluble solid support, typically agarose or sepharose resin.
These modified resins are then incubated with samples containing
communities.1–5 Gram-negative bacteria use N-acylated
L-homo-
serine lactones (AHLs, Fig. 1A) as their primary QS signals,6–8
which are produced by synthase proteins (LuxI-type proteins)
and are perceived by cytoplasmic transcription factors (LuxR-type
proteins).9 In general, AHLs are cell permeable and their concen-
tration increases with increasing cell density. Once a threshold
AHL concentration is achieved, productive AHL:LuxR-type recep-
tor binding occurs. These complexes typically dimerize, associate
with DNA, and initiate the transcription of the genes critical for
density-dependent phenotypes. Among the diverse phenotypes
regulated by this process are biofilm formation in pathogens like
Pseudomonas aeruginosa,10,11 bioluminescence in the symbiont
Vibrio fischeri,12,13 and root nodulation in mutualists like the
Rhizobia spp.14 Due to the impact of these QS-related phenotypes
in fields from healthcare to agriculture, there is considerable
interest in developing new approaches for modulating and
evaluating the chemical dialogue among bacteria.15,16 Chemical
approaches to intercept bacterial QS, whether by blocking
AHL:LuxR-type receptor binding or sequestering/degrading AHL
signals, have become prominent techniques in the field.17–21
Figure 1. (A) Generic structure of an N-acylated
L
-homoserine lactone (AHL); (B)
⇑
Corresponding author. Tel.: +1 608 262 1503; fax: +1 608 262 4534.
E-mail address: blackwell@chem.wisc.edu (H.E. Blackwell).
N-(3-oxo-dodecanoyl)-
P. aeruginosa.
L-homoserine lactone (OdDHL), primary QS signal in
a
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.062

T. Praneenararat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5054–5057
5055
putative biological targets. Theoretically, small molecule moieties
on the resin should only bind to their specific protein partners, per-
mitting the latter to be retained throughout resin washing steps.
Isolated proteins can then be eluted, analyzed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and identi-
fied using mass spectrometry (MS) or other techniques. This strat-
egy has successfully identified several small-molecule target
proteins, including histone deacetylase28 and the FK-506 binding
protein (FKBP).29 Follow-up studies with these molecular targets
have contributed to the overall understanding of their associated
biological processes.
To date, the design of affinity matrices to assist in the identifi-
cation of QS-related protein targets has been limited. Certain
matrices and related chemical tools are relevant, however, and
are introduced briefly here. Spring and co-workers have utilized
an azithromycin-modified sepharose resin to identify its biological
targets in P. aeruginosa.30 Sub-bacteriocidal doses of azithromycin
have been shown to inhibit biofilm formation in P. aeruginosa,
and because biofilm formation and QS are linked in this pathogen,
QS pathways would appear to be a plausible target for azithromy-
cin. Interestingly, analysis of the azithromycin–sepharose resin
showed that many of the captured targets were ribosomal pro-
teins, which suggests P. aeruginosa biofilm inhibition via azithro-
mycin occurs through the ribosome as opposed to QS pathways.
Concurrently, Spring and co-workers have developed an elegant
3D small-molecule microarray system31 that allows for the print-
ing of AHL derivatives in a spatially addressable format.32 These ar-
rays consist of reactive polymer gel matrices, and can be readily
derivatized with AHL analogues containing nucleophilic groups at
their acyl chain termini. The 3D arrays have proven useful for the
screening of new AHL analogues for LuxR-type protein binding.
In related work, the Meijler group has synthesized an AHL probe
equipped with both a photoactive diazirine and ‘click’ chemistry
reactive alkyne.22 In this proof-of-concept work, they demon-
strated that their AHL probe binds to LasR, a prominent LuxR-type
protein in P. aeruginosa, with only a slight decrease in activity rel-
ative to LasR’s native AHL, N-(3-oxo-dodecanoyl)-
L
-homoserine
lactone (OdDHL; Fig. 1B). The authors also demonstrated that UV
exposure leads to covalent attachment of the AHL probe to LasR
through the diazirine moiety. These results suggest that the alkyne
moiety could be used to further react the AHL-LasR complex with
an affinity resin via click chemistry, and the authors highlight this
possibility as a goal for future work. Lastly, motivated by reports of
the immunomodulatory effects of OdDHL in humans,33 Seabra
et al. prepared two OdDHL-derivatized resins and applied them
for the identification of human receptors for AHLs.34 The OdDHL li-
gand was appended to the matrix through either the acyl chain ter-
mini or a mid-chain branch point using a piperazine linker. Two
proteins were isolated using these matrices, but neither appeared
to have immunological relevance.
Inspired by these recent studies, we sought to apply the affinity
chromatography technique in our own QS research with the ulti-
mate goal of developing a new tool to aid in the discovery of novel
QS targets. To the best of our knowledge, the application of
AHL-derivatized affinity resins for the identification of LuxR-type
receptors has not been reported to date. Herein, we describe our
initial studies toward the design, synthesis, and application of an
AHL-derivatized agarose matrix. Affinity pull-down assays using
this new AHL-functionalized matrix confirmed the binding of QscR,
an ‘orphan’ LuxR-type protein from P. aeruginosa,35 to the matrix.
These results serve to demonstrate the feasibility of utilizing
AHL-derivatized affinity matrices for LuxR-type receptor isolation
and characterization.
To guide our design of an appropriate ligand for matrix derivati-
zation, we first scrutinized the AHL:receptor binding interactions in
the three reported structures of AHL:LuxR-type proteins (TraR, LasR,
and SdiA).36–40 Each of these structures shows a series of conserved
hydrogen-bonding interactions between the AHL lactone ring and
residues in the binding pocket of the receptor. To prevent disruption
of these key interactions, we decided that attachment of an AHL to
the resin matrix should occur through the acyl tail as opposed to
the lactone head group. Such an attachment strategy was success-
Scheme 1. Synthesis of OdDHL-derivatized resin 7. Abbreviations: Boc = tert-butoxycarbanoyl, DCC = N,N0-dicyclohexylcarbodiimide, DMAP = 4-(dimethylamino)pyridine;
TFA = trifluoroacetic acid; DIEA = diisopropylethylamine.

5056
T. Praneenararat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5054–5057
fully utilized by Spring32 and Seabra34 (see above). For our AHL scaf-
fold, we chose to synthesize an analogue of OdDHL (Fig. 1B). We note
that this molecule is the native ligand for both LasR and QscR in
P. aeruginosa. QscR is a unique QS receptor, as it appears to bind
OdDHL, the native ligand for LasR, and repress the activity of LasR.35
Notably, QscR can be readily produced in full-length form (in P. aeru-
ginosa),41 and we have considerable experience in its manipula-
tion.42 We reasoned these capabilities would enable proof-of-
concept resin-QscR binding studies. We selected Affigel-10 agarose
resin (Bio-Rad; derivatized with N-succinimidyl (N-OSu) esters) as
our matrixdue to itswide and successful usage in other affinitychro-
matographyapplications.27 To facilitatecouplingto theactivated es-
ters of Affigel-10, we designed an OdDHL analogue (6) with a
primary amino group at the end of the acyl tail (Scheme 1).
The synthesis of AHL 6 commenced with nucleophilic substitu-
tion of NH3 onto 10-bromodecanoic acid 1 at ambient temperature,
followed by N-Boc protection to generate acid 2.23 Thereafter, 2
was allowed to react with Meldrum’s acid 3 under standard condi-
tions and coupled to homoserine lactone (4) to afford N-Boc AHL 5
in 78% yield over two steps. Cleavage of the N-Boc group with TFA
yielded amino-AHL 6 as the TFA salt in 56% yield after preparative
HPLC purification.
Figure 2. SDS–PAGE analysis of purified QscR with resin 7. L: protein molecular
weight ladders; P: purified QscR; R: protein denatured from resin after
incubation; S: flow-through solution from resin 7-QscR incubation; W1: 1st
washing solution; W2: 2nd washing solution. Data for both resin 7 (lanes 3–6)
and ethanolamine-capped control resin (lanes 7–10) are provided. The solid arrow
indicates the presence of QscR; the dashed arrow was a GroEL contaminant.45
7
Next, AHL 6 was mixed with Affigel-10 resin (N-OSu load-
ing = 7.5 lmol/mL of slurry) and DIEA at ambient temperature for
2 h to affect coupling. Using AHL 6 as the limiting reagent
(0.5 equiv relative to N-OSu available on resin) gave quantitative
resin coupling (i.e., 50% coverage), as indicated by LC–MS analysis
(see Supplementary data). Finally, ethanolamine was used to block
all remaining activated esters on the resin (to ensure that they
would not interfere with the subsequent experiments), and affor-
ded OdDHL-derivatized resin 7. To provide a control resin for pro-
tein binding studies, we also reacted Affigel-10 with ethanolamine
alone to generated a ‘capped’, AHL-free resin.
Figure 3. SDS–PAGE analysis of crude mixtures of QscR and resin 7. L: Protein
molecular weight ladders; C: crude lysate; R: protein denatured from resin 7 after
incubation; S: flow-through solution from resin 7-crude lysate incubation; W1: 1st
washing solutions; W2: 2nd washing solutions. Data for both resin 7 (lanes 3–6)
and ethanolamine-capped control resin (lanes 7–10) are provided. The solid arrow
indicates the presence of QscR.
We next tested the ability of resin 7 to bind to QscR. QscR was
produced in P. aeruginosa PAO-T7(pJLQhis), a QscR over-expression
strain, and purified by nickel affinity chromatography using re-
ported procedures.41 As this P. aeruginosa strain retains the syn-
thase for OdDHL (LasI), QscR is likely isolated as a mixture of
OdDHL-bound and OdDHL-free protein (and an equilibrium there-
of).43 We reasoned that this mixture would still be viable for bind-
ing experiments with resin 7. Samples of resin 7 and the control
the application of resin 7 to biologically relevant samples and the
potential identification of new LuxR-type proteins. Co-incubation
of resin 7 with crude PAO-T7(pJLQhis) lysates and analogous SDS–
PAGE analysis as described above again yielded one prominent
band (Fig. 3; lane 3, denoted ‘R’). This band was extracted from
the gel and subjected to trypsin digest followed by ESI-MS/MS
analysis, which confirmed the presence of QscR (see Supplemen-
tary data). Similar to the experiment with purified QscR, no signif-
icant binding of QscR to the ‘capped’ control resin was observed
(lane 7, denoted ‘R’). We note that PAO-T7(pJLQhis) is an overex-
pression strain and therefore the quantity of QscR in the cell lysate
is considerably higher than that in a wild-type strain.46 Even so,
these initial results demonstrate the exciting potential for resin 7
to be utilized in the isolation of LuxR-type proteins from more
complex biological mixtures.
resin were incubated with purified QscR (3.75 lmol resin-bound
AHL to 15 nmol QscR, or 250:1 AHL/protein) in buffer for 24 h at
4 °C,44 after which the resins were filtered and subjected to wash-
ing in order to remove any unbound/excess protein. These wash-
ings were collected, and the resin samples and washings were
each diluted with gel loading buffer and heat-denatured before
being loaded onto a 12% polyacrylamide gel for SDS–PAGE analysis
(see Supplementary data for full details).
Figure 2 shows the SDS–PAGE data for the resin 7/purified QscR
binding experiment. Resin 7 was found to cleanly bind QscR (lane
3, denoted ‘R’). Significant QscR was found in the collected super-
natant (lane 4, denoted ‘S’), with reduced quantities being ob-
In conclusion, we have designed and synthesized a new AHL-
modified affinity matrix, and demonstrated that this matrix can
bind to the QscR protein from P. aeruginosa. This OdDHL-deriva-
tized resin (7) was shown to bind QscR from both purified protein
samples and crude cell lysates using SDS–PAGE. These proof-of-
concept experiments illustrate how resin 7 could be used as a tool
to isolate and potentially identify new AHL receptor proteins in
bacteria. As mentioned above, a number of eukaryotes display sen-
sitivity to AHLs.26 Therefore, resin 7 and related derivatives could
also prove useful for the elucidation of such cross-kingdom sensing
pathways. Our progress towards these goals, along with further
optimization of resin 7, will be reported in due course.
served in the washing samples (lanes
5 and 6). The large
quantity of residual QscR in the supernatant could be due to a high
percentage of native OdDHL-bound QscR in our protein sample
(see above)43 and/or the reduced ability of QscR to bind the re-
sin-bound OdDHL analogue. Nevertheless, resin 7 bound sufficient
QscR for it to be clearly observable on the gel. Further, the control
resin showed no detectable binding (lane 7, denoted ‘R’), indicating
that the appended OdDHL analogue is essential for binding of QscR
to the resin. Collectively, these data show that resin 7 is capable of
binding to a purified LuxR-type protein.
Acknowledgments
Encouraged by these results, we then evaluated the ability of re-
sin 7 to bind and effectively remove QscR from a more complex
sample, that is, cell lysates. Such a capability would be useful for
We gratefully acknowledge the NIH (AI063326), Greater Mil-
waukee Foundation, Burroughs Welcome Fund, and Johnson &

T. Praneenararat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5054–5057
5057
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Johnson for financial support. T.P. thanks the Anandamahidol
Foundation (Thailand) for a pre-doctoral scholarship. A.S.B. was
supported in part by NIH Chemistry Biology Interface Training
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Supplementary data
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