Efficient synthesis and evaluation of quorum-sensing modulators using small molecule macroarrays

Article abstract of DOI:10.1021/ol901871y

A method for the synthesis of small molecule macroarrays of N-acylated L-homoserlne lactones (AHLs) Is reported. A focused library of AHLs was constructed, and the macroarray platform was found to be compatible with both solution and agar-overlay assays u

Full text of DOI:10.1021/ol901871y

ORGANIC
LETTERS
2009
Vol. 11, No. 20
4600-4603
Efficient Synthesis and Evaluation of
Quorum-Sensing Modulators Using
Small Molecule Macroarrays
Thanit Praneenararat, Grant D. Geske, and Helen E. Blackwell*
Department of Chemistry, UniVersity of WisconsinsMadison, 1101 UniVersity AVenue,
Madison, Wisconsin 53706-1322
blackwell@chem.wisc.edu
Received August 11, 2009
ABSTRACT
A method for the synthesis of small molecule macroarrays of N-acylated L-homoserine lactones (AHLs) is reported. A focused library of AHLs
was constructed, and the macroarray platform was found to be compatible with both solution and agar-overlay assays using quorum-sensing
(QS) reporter strains. Several QS antagonists were discovered and serve to showcase the macroarray as a straightforward technique for QS
research.
Bacteria can assess their local population densities and
environment using a cell-cell signaling process called
quorum sensing (QS).1 This phenomenon allows bacteria to
coordinate group behavior and is widespread in bacteria.
Interest in QS is intense and growing due to the critical role
of this signaling process in pathogenic and symbiotic host/
bacteria relationships. Since its discovery, QS has sparked
the attention of chemists due to its reliance on a chemical
“language” of small molecule and peptide signals, or
autoinducers, and their cognate signal receptors.2 Indeed, this
reliance provides unique opportunities for chemists to
manipulate and study QS at the molecular level.
productive autoinducer-receptor binding occurs, and this
binding event controls the expression of myriad genes
involved in bacterial group behaviors. Pertinent QS pheno-
types include virulence factor production, biofilm formation,
and swarming in pathogens, and bioluminescence and root
nodulation in symbionts. Methods to block or promote QS
would allow for the attenuation of these diverse phenotypes,
and considerable recent research has focused on the design
of non-native molecules that can intercept QS signaling
pathways.3 Much of these efforts have been directed at QS
in Gram-negative bacteria,4 and our laboratory has reported
several families of non-native autoinducer mimics capable
In general, bacterial cell density correlates with autoinducer
concentration. Once a threshold concentration is reached,
(3) (a) Bjarnsholt, T.; Givskov, M. Phil. Trans. R. Soc. B 2007, 362,
1213–1222. (b) George, E. A.; Muir, T. W. ChemBioChem 2007, 8, 847–
855.
(1) For reviews of QS, see: (a) Camilli, A.; Bassler, B. L. Science 2006,
311, 1113–1116. (b) Fuqua, C.; Parsek, M. R.; Greenberg, E. P. Annu. ReV.
Genet. 2001, 35, 439–468.
(4) For recent reviews, see: (a) Geske, G. D.; O’Neill, J. C.; Blackwell,
H. E. Chem. Soc. ReV. 2008, 37, 1432–1447. (b) Welch, M.; Mikkelsen,
H.; Swatton, J. E.; Smith, D.; Thomas, G. L.; Glansdorp, F. G.; Spring,
D. R. Mol. Biosyst. 2005, 1, 196–202.
(2) Eberhard, A.; Burlingame, A. L.; Eberhard, C.; Kenyon, G. L.;
Nealson, K. H.; Oppenheimer, N. J. Biochemistry 1981, 20, 2444–2449.
10.1021/ol901871y CCC: $40.75
Published on Web 09/10/2009
 2009 American Chemical Society

of antagonizing and agonizing QS in these organisms.5 Potent
QS modulators still remain scarce, however. The continued
growth of this area demands the development of efficient
design, synthesis, and screening strategies for the identifica-
tion of new QS modulators. Here, we report the application
of the small molecule macroarray platform to the synthesis
and screening of non-native N-acylated L-homoserine lac-
tones (AHLs) for QS modulators. A macroarray of AHLs
was constructed using an efficient cyclization-cleavage
strategy and demonstrated to be compatible with on- and
off-support QS reporter gene assays. This work provides an
expedient new method for QS probe discovery.
reaction to release the AHLs from the support.10 Careful
optimization revealed that a dichlorophenol-type linker
facilitated such a cleavage step, and our AHL synthesis route
is outlined in detail below (Schemes 1 and 2).
Scheme 1.
Planar Support Modification and Linker Installationa
Our laboratory has been developing the small-molecule
macroarray as a tool for chemical biology research for several
years.6 This technique involves the spatially addressed
synthesis of small molecules on planar cellulose supports
(0.3 cm2 spots). Libraries of ∼10-1000 can be made
routinely in a day, typically on a 10-100 µg/compound scale.
The benefits of the small molecule macroarray include ease
of manipulation, reduced reagent use, inexpensive support
systems, and compatibility with a diverse range of biological
assays either performed on or off of the support. We recently
demonstrated the use of macroarrays for the discovery of
antibacterial small molecules; this was facilitated by straight-
forward bacterial agar-overlay assays performed directly on
the array surface.7 We sought to broaden the scope of the
macroarray approach, and in the current study investigated
the feasibility of non-native AHL synthesis and screening
on the array platform.
AHLs are the primary class of QS signaling molecules
used by Gram-negative bacteria, and are sensed by LuxR-
type receptors.8 Non-native analogs of these ligands were
among the first synthetic molecules evaluated for QS
activity.3a,4 The majority of these analogs retain the native
L-HL headgroup yet contain non-native acyl groups. We
previously discovered that phenylacetyl derivatives of this
type (PHLs) were extremely potent QS modulators in a range
of species.5 Cinnamyl HLs (CHLs), while less studied, have
also been found to have interesting QS activity profiles.9 We
therefore decided to focus largely on PHLs and CHLs in
this proof-of-concept study.
a TsCl ) tosyl chloride; DIC ) N,N′-diisopropylcarbodiimide; HOBt
) 1-hydroxybenzotriazole; DMF ) N,N′-dimethylformamide.
Macroarray synthesis commenced with readily available
Whatman 1Chr filter paper (1; Scheme 1). Grids (1.5 × 1.5
cm) were drawn on paper sheets, and the sheets were
subjected to blanket tosylation using previously reported
methods.7,11,12 Blanket displacement of the primary tosyl
groups by subjection to neat 4,7,10-trioxa-1,13-tridecanedi-
amine 2 (with gentle heating in a laboratory oven) yielded
spacer-functionalized support 3. This support was synthesized
routinely on a multisheet scale and found to be stable at room
temperature (rt) for months. Immediately prior to use, the
linker unit, 3,5-dichloro-4-hydroxybenzoic acid 4, was
coupled to support 3 in a spatially addressed format (spotting
at each grid intersection)12 using standard carbodiimide
conditions (DIC) to give linker-derivatized support 5.
The first step in AHL synthesis was the spatially addressed
esterification of N-Fmoc-OTrt-L-homoserine (Hse; 6) with
support 5 using CDI and NMI at rt (spotting repeated 3×;
Scheme 2).13 “Capping” of any unreacted phenols (and
residual alcohols or amines) via blanket acetylation yielded
support 7. Thereafter, the loading of Hse (6) onto the support
was evaluated by UV Fmoc quantification. Test spots of the
support were subjected to 4% DBU in DMF (15 min at rt),
and UV analysis revealed Hse (6) loadings of ∼100 nmol
In designing our solid-phase synthesis strategy on planar
cellulose support, we desired a “traceless” linker that would
provide AHLs after cleavage that contained no structural
feature originating from the linker. In addition, we needed a
cleavage procedure that yielded products with only volatile
byproducts in order to permit on-support bacteriological
assays. These requirements led us to select a linker compat-
ible with an acid-mediated, tandem lactonization-cleavage
(5) (a) Geske, G. D.; O’Neill, J. C.; Miller, D. M.; Wezeman, R. J.;
Mattmann, M. E.; Lin, Q.; Blackwell, H. E. ChemBioChem 2008, 9, 389–
400. (b) Geske, G. D.; O’Neill, J. C.; Miller, D. M.; Mattmann, M. E.;
Blackwell, H. E. J. Am. Chem. Soc. 2007, 129, 13613–13625. (c) Geske,
G. D.; O’Neill, J. C.; Blackwell, H. E. ACS Chem. Biol. 2007, 2, 315–320.
(6) Blackwell, H. E. Curr. Opin. Chem. Biol. 2006, 10, 203–212.
(7) Bowman, M. D.; O’Neill, J. C.; Stringer, J. R.; Blackwell, H. E.
Chem. Biol. 2007, 14, 351–357.
(10) For other examples of cyclization-cleavage routes to γ-lactones,
see: (a) Reference 5. (b) LeHetet, C.; David, M.; Carreaux, F.; Carboni,
B.; Sauleau, A. Tetrahedron Lett. 1997, 38, 5153–5156.
(11) (a) Bowman, M. D.; Jeske, R. C.; Blackwell, H. E. Org. Lett. 2004,
6, 2019–2022. (b) Lin, Q.; O’Neill, J. C.; Blackwell, H. E. Org. Lett. 2005,
7, 4455–4458
(12) “Blanket” reactions involve full submersion of the support in
reagents. “Spotted” reactions involve delivery of reagents onto support
.
(8) For a review of AHL-LuxR-type QS, see ref 4 and: Lazdunski, A. M.;
Ventre, I.; Sturgis, J. N. Nat. ReV. Microbiol. 2004, 2, 581–592.
(9) Palmer, A. G.; Blackwell, H. E. Nat. Chem. Biol. 2008, 4, 452–
454.
.
(13) This method is modified from the following report. Ay, B.;
Volkmer, R.; Boisguerin, P. Tetrahedron Lett. 2007, 48, 361–364.
Org. Lett., Vol. 11, No. 20, 2009
4601

products (11 and 12; Scheme 2). We sought to perform the
latter step using acid vapor, as this would yield the PHLs
and CHLs as spatially addressed (yet non-covalently bound)
arrays on the support after cleavage and amenable to
bacteriological agar overlay assays. Further, our previous
studies had shown that such vapor-phase cleavage steps on
intact macroarrays are operationally straightforward.6,7 Sig-
nificant optimization work revealed that both the deprotection
and the cleavage could be performed sequentially using TFA
vapor in a vacuum desiccator. A 10 min treatment was
sufficient to cleave the Trt group of support 9 with minimal
premature lactonization. The support (10) was then washed
and dried prior to a longer TFA vapor treatment (18 h, rt) to
generate AHLs 11 and 12 (Figure 1). Any residual TFA was
neutralized by subjecting the array to NH3 (generated from
NH4OH) and subsequent drying at rt.
Scheme 2.
AHL Synthesis on Planar Support 5a
The purities of the AHL library members were assessed
by punching out spots, elution, and either GC-MS or HPLC
analysis. Compound purity was excellent (g93%), reflecting
the advantage of the cyclization-cleavage strategy. To
analyze the isolated yields of AHLs, 4-bromo PHL (11b)
was selected as a representative compound. Comparison of
the quantity of 11b cleaved from the macroarray to a
calibration curve generated from an authentic sample of 11b
indicated 40 nmol (∼10 µg) of product was formed per spot
(∼40% yield based on Hse Fmoc analysis; see the Supporting
Information). Qualitative analysis of the GC and HPLC data
suggested that this conversion level could be approximated
for all 26 library members. The cleavage step was determined
to be the primary reaction reducing the overall product yield.
However, longer reaction times, or resubjection of the array
to cleavage conditions, failed to give higher yields of
product.14 In comparison to our previous solid-phase syn-
thetic route to AHLs (incorporating a CNBr-mediated
lactonization-cleavage),5 the macroarray route gave compa-
rable if not higher product purities yet reduced yields (by
∼10-20%). The absence of toxic CNBr in the current route,
however, coupled with the ease of planar support manipula-
tion versus beads, is advantageous. Importantly, the quantities
of AHLs isolated from the macroarray were more than
sufficient for biological assays (see below).
a CDI ) 1,1′-carbonyldiimidazole; NMI ) N-methylimidazole; DBU
) 1,8-diazabicyclo[5.4.0]undec-7-ene.
per spot. This loading level was comparable to other small-
molecule macroarray syntheses.6,7,11
Support 7 was next subjected to blanket Fmoc deprotection
with DBU/DMF, preparing it for acylation with various
carboxylic acids (8; Scheme 2). We chose 26 phenylacetic
and cinnamic acids (with one exception) as our acid building
blocks, which were readily coupled onto the support in a
spatially addressed manner using DIC at rt to generate
support 9. Notably, we selected a subset of acids in this step
that would yield several PHLs and one CHL previously
studied by our laboratory (Figure 1); this choice was
For quality control, we first examined the biological
activities of a subset of the cleaved PHLs (11e, 11i, 11l and
11m) in solution-phase bacterial reporter gene assays. Stock
solutions (∼0.5 mM) were prepared in DMSO, and examined
in a reporter strain of the marine symbiont Vibrio fischeri
(ES114 (∆-luxI)) that only luminesces in the presence of QS
activators.5c Antagonistic QS activities (versus the native
AHL signal for V. fischeri, N-3-oxo-hexanoyl HL (OHHL))
were assessed at ∼5 µM and compared to authentic samples5
of each PHL at 5 µM. The activities of the macroarray-
derived PHLs were comparable ((5%) to that of authentic
PHL samples (see Figure S-4, Supporting Information),
indicating both that our estimation of macroarray compound
yields was reasonable and that the samples were chemically
identical. Further, these results demonstrate the compatibility
Figure 1. Structures of the PHL (11) and CHL (12) macroarray
products. **For 11, n ) 1 except for 11m (n ) 2).
advantageous for two reasons: it would (1) allow us to
directly compare the macroarray synthesis route to our earlier
AHL synthetic route that utilized traditional, polystyrene solid
support,5 and (2) provide built-in controls for our later
development of QS bioassays performed on and off of the
array surface.
The final steps in AHL macroarray synthesis were the acid-
mediated stepwise deprotection of the Trt group of 9,
followed by cyclization-cleavage to give the PHL and CHL
(14) For comparison, solution-phase cleavage of spots (50% TFA in
CH2Cl2 for 18 h) gave a 31% isolated yield of 11h.
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Org. Lett., Vol. 11, No. 20, 2009

of the macroarray products with solution-phase reporter gene
assays.15 Encouraged by these results, we examined the
activity of the entire structurally novel CHL library (12) in
analogous QS antagonism assays, and identified several
potent QS antagonists (most notably, F5-CHL 12f and 4-Br,
2-F-CHL 12l; Figure 2). These two CHLs were capable of
Figure 3. C. Violaceum QS antagonism overlay assays (against HHL
at 1 µM) performed on cleaved PHL (11a-m, left) and CHL (12a-
m, right) macroarrays. DHL ) decanoyl HL control; blank areas
were punched out and used for loading calculations.
and appeared to be a comparable inhibitor to the control DHL
in this overlay assay. Interestingly, in contrast to the V.
fischeri assay above, none of the CHLs 12 were active in
the overlay assay. To better quantitate the activities of 11k,
11l, and DHL in C. Violaceum, we determined their IC50
values using a solution-phase violacein production assay (see
the Supporting Information).17 These values indicated that
DHL and 11l had similar inhibitory activities, and 11k was
∼10-fold less active (IC50 ) 4.12, 4.98, and 40.5 nM,
respectively (vs 400 nM HHL)), a trend approximated by
visual inspection of the overlay assay data (Figure 3).
In summary, we have developed an efficient synthesis of
AHLs on the small molecule macroarray platform and
generated a focused library of AHLs in high purities. The
macroarray was found to be compatible with both on- and
off-support QS assays, and several new QS antagonists were
identified in V. fischeri (12f and 12l) and C. Violaceum (11k
and 11l). We anticipate that these methods are compatible
with the synthesis and screening of AHL macroarrays of
expanded structural diversity and size, and such studies are
ongoing in our laboratory.
Figure 2. V. fischeri QS antagonism solution-phase assay data for
CHLs 12. Assay performed at 1:1 against OHHL; concentration of
all compounds ∼5 µM. Pos ) positive control, OHHL at 5 µM.
Neg ) negative control, 1% DMSO.
inhibiting QS responses in V. fischeri by 50% at 1:1 against
OHHL and represent new leads for QS probe development.
We next examined the feasibility of performing QS
reporter gene assays on intact, cleaved macroarrays of 11
and 12 in an agar overlay format and focused again on
antagonism-type assays. The soil bacterium Chromobacte-
rium Violaceum (CV026) was selected for these studies, as
this organism produces a brilliant violet pigment (violacein)
upon QS activation, providing an excellent visual reporter.16
In this reporter strain, QS inhibitors cause a loss of
pigmentation in the presence of its native ligand, N-hexanoyl
HL (HHL). A molten agar suspension of C. Violaceum and
HHL (1 µM) was added to Petri dishes, the cleaved arrays
were submerged in the agar, and the dishes were incubated
until white zones were apparent (16-20 h). N-Decanoyl HL
(DHL), a known inhibitor of C. Violaceum QS,16 was also
synthesized on the arrays for use as an internal control.
The overlay assay procedure proved successful on the
macroarrays and revealed two new QS antagonists in C.
Violaceum (4-CF3 PHL 11k and 4-Ph PHL 11l; Figure 3).
PHL 11k has previously been shown to be a cross-species
QS inhibitor,5 and its activity in C. Violaceum further
supports this activity trend. However, 4-Ph PHL 11l has
exhibited only limited and muted activity in other strains5
Acknowledgment. Financial support was provided by the
NIH (AI063326-01), Greater Milwaukee Foundation, Bur-
roughs Welcome Fund, and Johnson & Johnson. H.E.B. is
an Alfred P. Sloan Foundation Fellow. T.P. thanks the
Anandamahidol Foundation (Thailand) for a predoctoral
scholarship. Professors Jo Handelsman and Ned Ruby
(UWsMadison) are thanked for bacterial strains.
Supporting Information Available: Full experimental
procedures and characterization data, structures of carboxylic
acids 8, and additional biological assay data. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL901871Y
(15) Sufficient compound was generated for ∼30 assays per spot.
(16) McClean, K. H.; Winson, M. K.; Fish, L.; Taylor, A.; Chhabra,
S. R.; Camara, M.; Daykin, M.; Lamb, J. H.; Swift, S.; Bycroft, B. W.;
Stewart, G. S.; Williams, P. Microbiology 1997, 143, 3703–3711.
(17) Blosser, R. S.; Gray, K. M. J. Microbiol. Methods 2000, 40, 47–
55.
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