β-Cyclodextrin Encapsulation of Synthetic AHLs: Drug Delivery Implications and Quorum-Quenching Exploits

Article abstract of DOI:10.1002/cbic.202000773

Many bacteria, such as Pseudomonas aeruginosa, regulate phenotypic switching in a population density-dependent manner through a phenomenon known as quorum sensing (QS). For Gram-negative bacteria, QS relies on the synthesis, transmission, and perception of low-molecular-weight signal molecules that are predominantly N-acyl-l-homoserine lactones (AHLs). Efforts to disrupt AHL-mediated QS have largely focused on the development of synthetic AHL analogues (SAHLAs) that are structurally similar to native AHLs. However, like AHLs, these molecules tend to be hydrophobic and are poorly soluble under aqueous conditions. Water-soluble macrocycles, such as cyclodextrins (CDs), that encapsulate hydrophobic guests have long been used by both the agricultural and pharmaceutical industries to overcome the solubility issues associated with hydrophobic compounds of interest. Conveniently, CDs have also demonstrated anti-AHL-mediated QS effects. Here, using fluorescence spectroscopy, NMR spectrometry, and mass spectrometry, we evaluate the affinity of SAHLAs, as well as their hydrolysis products, for β-CD inclusion. We also evaluated the ability of these complexes to inhibit wild-type P. aeruginosa virulence in a Caenorhabditis elegans host infection study, for the first time. Our efforts confirm the potential of β-CDs for the improved delivery of SAHLAs at the host/microbial interface, expanding the utility of this approach as a strategy for probing and controlling QS.

Full text of DOI:10.1002/cbic.202000773

Combining Chemistry and Biology
Accepted Article
Title: β-Cyclodextrin encapsulation of synthetic AHLs: drug delivery
implications and quorum-quenching exploits.
Authors: Eric W Ziegler, Alan B Brown, Nasri Nesnas, Chris Chouinard,
Anil K Mehta, and Andrew George Palmer
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000773
Link to VoR: https://doi.org/10.1002/cbic.202000773
01/2020

10.1002/cbic.202000773
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FULL PAPER
-Cyclodextrin encapsulation of synthetic AHLs: drug delivery
implications and quorum-quenching exploits.
Eric W. Ziegler,[a] Alan B. Brown,[a] Nasri Nesnas,[a] Christopher D. Chouinard,[a] Anil K. Mehta,[b]
and Andrew G. Palmer,[a][c]
*
We dedicate this manuscript to Dr. J. Clayton Baum, an advisor, mentor, colleague, coauthor, scientist (in the noblest sense of the
word), and also — perhaps most importantly — a friend to all at Florida Institute of Technology. We dedicate this work to the effect
that Dr. Baum had on our lives and those of his students while serving as a professor and researcher of physical chemistry
throughout his tenure at Florida Tech. We dedicate this work to his thoughtfulness and his careful curation of experiments that EWZ
performed as a student and TA under his direction that are foundational to the work presented here. Lastly, we dedicate this
manuscript to the memories of and lessons learned from Dr. Baum that we will cherish and hold with us for the many years to come.
Dr. Baum, ad astra per scientiam!
[a]
[b]
[c]
E.W. Ziegler, Dr. A.B. Brown, Dr. N. Nesnas, Dr. C.D. Chouinard, Dr. A.G. Palmer
Department of Biomedical and Chemical Engineering and Sciences
Florida Institute of Technology
150 W. University Boulevard, Melbourne, FL 32901
Dr. A.K. Mehta
National High Magnetic Field Laboratory, McKnight Brain Institute
University of Florida
1149 Newell Drive, Gainesville, FL 32610
Dr. A.G. Palmer
Department of Ocean Engineering and Marine Sciences
Florida Institute of Technology
150 W. University Boulevard, Melbourne, FL 32901
E-mail: apalmer@fit.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: Many bacteria, such as Pseudomonas aeruginosa,
regulate phenotypic switching in a population density-dependent
manner through a phenomenon known as quorum sensing (QS). For
Gram-negative bacteria, QS relies on the synthesis, transmission, and
perception of low molecular weight signal molecules that are
predominantly N-acyl-L-homoserine lactones (AHLs). Efforts to
disrupt AHL-mediated QS have largely focused on the development
of synthetic AHL analogues (SAHLAs) that are structurally similar to
native AHLs. However, like AHLs, these molecules tend to be
hydrophobic and are poorly soluble under aqueous conditions. Water-
soluble macrocycles, such as cyclodextrins (CDs), that encapsulate
hydrophobic guests, have long been used by both agricultural and
pharmaceutical industries to overcome solubility issues associated
with hydrophobic compounds of interest. Conveniently, CDs have also
demonstrated anti-AHL-mediated QS effects. Here, using
Introduction
Since its discovery in 1970, population density-dependent gene
expression—typically referred to as quorum sensing (QS)— has
been widely observed among bacteria and is frequently
associated with phenotypes that, if expressed by a lone bacterium,
would be ineffective.1,2 Common examples of these phenotypes
include biofilm and virulence factor production by Pseudomonas
aeruginosa, an opportunistic pathogen known for its adverse
effects on patients suffering with cystic fibrosis or nosocomial (i.e.,
healthcare-related) infections. When it was observed that
pathogenic bacteria in which the underlying QS molecular
circuitry was disrupted exhibited less virulence, QS inhibition
became an attractive target for anti-infective and anti-virulence
strategies, necessitated by the increasing frequency of multidrug
resistant bacterial infections and dwindling arsenal of viable
antibiotics with which to treat them.3,4
QS relies on the synthesis, transmission, and perception of low
molecular weight signal molecules generically classified as
autoinducers (AIs). Presently, at least a half-dozen different QS
strategies have been identified, each differentiable by the
molecular structure of the AI employed, their cognate receptor,
and the mechanism of signal transduction within the cell.5 In
Gram-negative bacteria, such as P. aeruginosa, the predominant
AIs are N-acyl-L-homoserine lactones (AHLs, Figure 1).6
fluorescence
spectroscopy,
nuclear
magnetic
resonance
spectrometry, and mass spectrometry, we evaluate the affinity of
SAHLAs, as well as their hydrolysis products, for -CD inclusion. We
also evaluated the ability of these complexes to inhibit wildtype P.
aeruginosa virulence in a Caenorhabditis elegans host infection study,
for the first time. Our efforts confirm the potential of -CDs for the
improved delivery of SAHLAs at the host:microbial interface,
expanding the utility of this approach as a strategy for probing and
controlling QS.
These compounds are composed of a -butyrolactone `head
group’ that participates in highly conserved molecular recognition
interactions (i.e., hydrogen bonding) with cognate receptors
1
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(LuxR-type proteins), and an acyl tail that encodes species and/or
signal specificity through exclusive interactions with the cognate
receptor facilitated by the chain length and oxidation state at the
-position.7,8 AHL-mediated QS inhibition, to date, has largely
focused on using small molecule ligands capable of emulating the
molecular recognition interactions between native AHLs and their
receptor proteins (i.e., receptor antagonism). Example classes of
AHL receptor antagonists include halogenated furanones,9,10
anthropogenic AHLs,11–16 and homocysteine analogues (i.e.,
thiolactones).17
line the narrow (1) and wide (2) brims while the inner cavity is
lined by hydrogens, imbuing it with hydrophobic character. This
highly organized architecture makes the macrocycle both water-
soluble and capable of hosting poorly soluble, hydrophobic
species; the latter of which is driven by the hydrophobic effect.
Indeed, many of the molecules used for acyl chain substitutions
in SAHLAs are known to associate with -CD. Notably, -CD are
biocompatible, do not elicit the adverse side effects experienced
by patients exposed to DMSO, and have, therefore, been utilized
in both agricultural and pharmaceutical formulations to enhance
solubility, stability, and bioavailability of high-value hydrophobic
molecules, such as chemotherapeutic agents.26
Figure 1. Chemical structures of bioactive, native N-acyl-L-homoserine
lactones and synthetic antagonists that demonstrate bioactivity against the
QscR-mediated QS in P. aeruginosa.
Figure 2. (a) Cyclodextrin geometry and (b) a possible orientation of a
{CD:SAHLA} complex with hydrophobic tail docking within CD cavity.
Anthropogenic AHLs, sometimes known as synthetic AHL
analogues (SAHLAs), have been remarkably successful at
modulating QS.11,18–22 Blackwell and coworkers, since reporting a
rapid solid-phase supported method for the synthesis of AHLs and
SAHLAs, have made significant contributions to this class of QS
modulators by: (a) generating an extensive synthetic AHL library
of variable lactone stereochemistry and acyl tail composition, (b)
screening that library for bioactivity against well-characterized
AHL-mediated QS circuits, and (c) evaluating the effects of
SAHLAs on prospective host organisms. Such screens have
identified many effective receptor antagonists (for examples, see
Figure 2), many of which possess native lactone stereochemistry
and acyl tails containing aromatic groups that are highly
hydrophobic.13–16,23,24 Though effective antagonists, these
compounds, like native AHLs, are often poorly soluble under
aqueous conditions as a consequence of their hydrophobic tails
and, as such, frequently must be introduced into biological assays
using carrier solvents such as dimethyl sulfoxide (DMSO). DMSO
is commonly used in medical formulations; however, it is known
to alter membrane permeability, complicating assay results, and,
physiologically, can induce adverse effects in patients (e.g.,
nausea and vomiting.25
Apart from their potential utility in solubilizing poorly soluble
SAHLAs, which would presumably increase SAHLA bioactivity
(via improved delivery), - and -CDs (along with their
derivatives) — by themselves — have demonstrated efficacy as
QS inhibitors through the formation of inclusion complexes with
naturally occurring AHLs. In effect, inclusion complex formation
reduces the concentration of free AHLs in the bacterial
environment.27–32 These preliminary studies underscore both the
potential of this approach, as well as the need for further biological
and chemical studies to clearly determine the true benefits.
Biologically, these prior studies have focused on the use of QS
reporter lines, which are unable to synthesize AHLs on their
own,27–29 and often behave differently than the wildtype strains
from which they are derived.18 Furthermore, these assays are
typically performed in idealized culture conditions in which no host
is present, further simplifying the assay but also distancing it from
biological relevance. Evaluating the effectiveness of these
complexes under native conditions (i.e., during a host-infection
assay) is crucial to the successful development of the SAHLA:CD
approach as an anti-virulence strategy.
Improved delivery systems for SAHLAs could significantly
contribute to efforts to advance QS regulation as a viable anti-
virulence strategy. We propose that, in place of DMSO as a carrier
solvent to facilitate solubilization, the hydrophobicity of these
antagonists could be exploited to form inclusion complexes with
macrocyclic hosts, such as cyclodextrins (CDs, Figure 3).
CDs are commonly composed of six (-CD), seven (-CD), or
eight (-CD) glucopyranosyl subunits bound by  linkages. As
a result, CDs adopt a toroid geometry such that the alcohol groups
Chemically speaking, previous efforts to obtain binding
constants for {CD:AHL} complexes have also encountered certain
limitations. For example, binding constants have been determined
and/or estimated in either unbuffered or biologically irrelevant
aqueous environments (e.g., D2O,27 distilled water,28 and
carbonate buffer29). Many of these studies have been conducted
under conditions where AHLs hydrolyze rapidly (pH 9.529), giving
rise to binding constants that likely represent statistical mixtures
of AHLs and their hydrolysis products; with considerable error.28
2
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Finally, these studies are often performed at relatively high AHL
concentrations, which is problematic because AHLs are known to
form micelles33 and the formation of these supramolecular
architectures has been suggested as a source of deviation of
expected {AHL:receptor} complex formation (as monitored by
fluorescence quenching) in at least one binding study.34 The
formation of micelles is particularly problematic in binding assays,
two SAHLA hydrolysis products, noting differences in association
behavior.
Lastly, and building on these inclusion complex studies, we
confirm, for the first time, the utility of {SAHLA:CD} inclusion
complexes for improved anti-virulence under native (i.e., host
infection) conditions. Specifically, we establish increased host
survival during infection assays of Caenorhabditis elegans by
wildtype P. aeruginosa PAO1. We discuss our findings with
regards to their potential benefits to QS regulation and anti-
virulence.
29
most notably those involving indicator displacement methods,
because their presence introduces another hydrophobic effect
driven association phenomenon (i.e., indicator incorporation into
the micelle), which may result in observed spectral changes
consistent with displacement from the CD cavity by analyte but
not necessarily indicative of it. Indicator displacement assays rely
on two competing equilibria; the introduction of additional
competing equilibria further complicates assay results and their
interpretation.
Figure 4. Molecular structures of the three fluorescent synthetic AHLs used in
this study to evaluate inclusion complex formation of Blackwell Library
constituents with -CD.
Figure 3. Cartoon of proposed SAHLA-CD synergistic action where (a)
SAHLA:CD are deployed, SAHLA is released, and SAHLA binds to AHL
receptors; (b) free native AHL is captured by the now-vacant CD cavity, which
leads to (c) fewer free AHLs in the chemical environment.
Results and Discussion
SAHLA ASSOCIATION WITH -CD
Our investigation into the association behavior of synthetic AHLs
with -CD began with 1, principally because the association of its
carboxylic acid precursor (4-biphenylacetic acid, BPAA) with -
CD is well-known through both solubility studies and X-ray
crystallography.39–41 The crystal structure of the {-CD:BPAA}
inclusion complex, solved by Wang and coworkers, indicated that
the biphenyl region of BPAA docks within the host’s hydrophobic
cavity.41 This SAHLA also has biological relevance as a broad
spectrum QS antagonist, capable of inhibiting the TraR15
(Agrobacterium tumefaciens, Ti plasmid replication and
conjugation),42–44 LuxR15 (Vibrio fischeri, bioluminescence),45,46
and QscR47 (P. aeruginosa, ``anti-virulence'')48. Conveniently,
biphenyl is also a fluorophore and as fluorescence behavior is
highly sensitive to the chemical environment experienced by the
fluorophore, we hypothesized that such an approach could
provide a relatively facile and rapid way to monitor the association
of 1 with -CD.
As expected, titration of 1 with -CD altered its fluorescence
behavior (Figure 5, top), specifically reducing fluorescence
intensity, inducing a bathochromic shift (2 nm), and sharpening
spectral features. We interpreted the change in fluorescence
exhibited by 1 at increasing concentrations of -CD to indicate
inclusion complex formation. Application of the Benesi-Hildebrand
equation to the fluorescence at max (315 nm) afforded a linear
Because of the inherent anti-virulence capabilities exhibited by
CDs and their ability to solubilize poorly soluble compounds, we
hypothesized that the co-application of these two species could
actually work in concert to elicit additive if not synergistic effects.
As illustrated in Figure 3, SAHLAs could be solubilized by, and
delivered to, the bacterial environment by a CD. Once CD-
encapsulated SAHLAs were released, they would compete with
native AHLs for receptor binding, while the now free CDs could
encapsulate native AHLs, further impeding QS-activation.
In the present study, we have evaluated the association of three
representative SAHLAs (1-3, Figure 4) with -CD in biologically
relevant phosphate buffered saline (pH 7.3), where AHLs
hydrolyze significantly slower than at pH 9.5.35–38 We selected β-
CDs for the present study as they are the most economical of the
three CDs and more importantly, posses the appropriate
dimensions to provide a ‘snug’ fit for the aromatic moieties of
SAHLAs 1-3.
These ligands were selected both for their physical properties
as well as biological utility as QS antagonists. As the acyl tail of
all three of these previously synthesized compounds possess
fluorophores, it was possible to directly obtain binding constants
for complex formation.
Inclusion complexes were further
characterized by nuclear magnetic resonance (NMR) and mass
spectrometries. To demonstrate why the conditions — especially
pH — for AHL binding constant determination and/or estimation
is highly relevant, we also determined the binding constants of
double reciprocal plot that suggested
a 1:1 host/guest
complexation stoichiometry and an association constant Kbind of
3750  160 M-1. Notably, the Benesi-Hildebrand method requires
that either host or guest is present in excess and that, as a result,
3
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10.1002/cbic.202000773
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isotherm and afforded a statistically similar association constant
of 3850  170 M-1. The agreement between the results was not
surprising given that the assumption made by the Benesi-
Hildebrand method — in this case: that the host concentration
was significantly greater than that of the guest — was satisfied.
It is reasonable to infer that the biphenyl moiety of 1 docks within
the macrocycle’s hydrophobic cavity, especially given that
fluorescence behavior is highly sensitive to changes in chemical
environment and the known {-CD:BPAA} complex crystal
structure.
65
60
55
50
45
1
70
60
50
40
30
20
10
0
8
0
2
4
6
[
-CD] / mM
However, the fluorescence titration experiment does not
explicitly provide structural information regarding the {-CD:1}
complex. To this end, we performed 2D-ROESY NMR, which
identifies nuclei that are located in close spatial proximity to one
another. For a CD host:guest complex, 2D-ROESY would indicate,
by the presence of cross peaks, that the internal protons of the
CD cavity (i.e., H3 and H5) are in close proximity to the protons
on the docking region of the guest. Shown in Figure 5 (middle),
the aromatic resonances of 1 exhibit cross-peaks with the internal
hydrogens of -CD, indicating that the biphenyl region is
responsible for complex formation. This not only confirmed that
the fluorophore docked within the CD cavity, but also suggested
that, given the similar structure of the {-CD:BPAA} complex,
molecules that possess similar complex-forming regions or
complexophores demonstrate similar modes of association.
Additionally, we observed cross peaks between the aromatic
resonances of 1 with CD’s H6 resonances, which line the primary
rim, suggesting that the biphenyl may dock well within the CD
cavity to position these nuclei in close spatial proximity. Notably,
these through-space interactions could arise through either the
lactone projecting from the primary or secondary faces. To further
support the existence of the {-CD:1} complex, we performed ESI
mass spectrometry with a methanolic mixture of 1 and -CD in
hopes of observing free 1, free -CD, and the {-CD:1} complex
as their sodium adducts. All three of these species were readily
detected as shown in Figure 5 (bottom).
Encouraged by the comparable inclusion complexes of 1 and
BPAA with -CD, we synthesized 2, a naphthalene substituted
SAHLA known to inhibit RhlR16 (P. aeruginosa, host evasion
andbiofilm development).49,50 We hypothesized that 2 would
emulate 2-naphthyloxyacetic acid (NOA) in terms of its
association with -CD.51 The fluorescence of 2, like NOA, was
enhanced in the presence of -CD (Figure 6, top).
The mixture of 2 with -CD also afforded a slight bathochromic
shift (2 nm) of the fluorescence peak at 325 nm and sharpening
of spectral features. As with 1, the linearity of the resulting double
reciprocal plot suggested a 1:1 complex stoichiometry. NLR
treatment of the fluorescence intensities at max (334 nm) also
yielded a 1:1 binding isotherm to which the experimental data was
well-fitted with an association constant of 395  15 M-1. Similar to
1, the existence of the {-CD:2} complex and 1:1 stoichiometry
was further supported by mass spectrometry (Figure 6, bottom)
300
320
340
360
380
400
Wavelength / nm
a
b
O
rem
b
b
ppm
3.70
3.75
3.80
3.85
3.90
3.95
4.00
a
H5
H6
H3
7.65
7.60
7.55
7.50
7.45
ppm
318.1111
[M+Na]+
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
350.1343
[M+MeOH+Na]+
613.2302
[2M+Na]+
1452.4778
[CD+Na]+
1157.3555
[CD+Na]+
1
2
3
4
5
6
7
8
9
1
1
1
1
1
4
1
0
0
0
0
0
0
0
0
0
0
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
m/z
Figure 5. (top) Fluorescence titration of 1 (1.8 M) with increasing concentration
of -CD (0 mM to 5.3 mM; solutions 1 through 8) in phosphate buffered saline
(pH 7.3) with (inset) fitting to 1:1 binding isotherm determined by non-linear
regression analysis at max = 315 nm. (middle) 2D-ROESY NMR cross-peaks
between biphenyl moiety aromatic protons of 1 (0.2 mM) and hydrogens (H3
and H5) internal to the macrocycle’s inner cavity ([-CD] = 2 mM) in D2O,
calibrated against methanol. Cross-peaks between resonances at 7.525 and
3.75 ppm arise from 1 aromatic resonances and acyl -protons. (bottom) Mass
spectrum obtained for -CD:1 mixture by ESI-MS indicating free 1, free -CD,
and {-CD:1} as their sodium adducts.
the concentration of the excess reagent is constant throughout
the complexation process. Application of non-linear regression
(NLR) methods (see Experimental), which do not suffer this
assumption, also indicated the data was well-fitted to a 1:1 binding
4
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recognition interactions (i.e., hydrogen bonding). Such
interactions include the lactone carbonyl oxygen, amide carbonyl
oxygen, and amide hydrogen that are only possible if the lactone
remains intact.7,8 Under aqueous conditions, however, lactones
(and thus AHLs) are prone to abiotic hydrolysis, resulting in loss
of biological activity.53,54 Our group and others have investigated
the stability of native AHLs under different acidities/alkalinities.35–
37
The hydrolysis of synthetic AHLs has been reported on by
Bertucci et al., specifically how the strength of an intramolecular
interaction (i.e., n*) influences hydrolysis rate.38
Hydrolysis, though, does not chemically alter the
complexophore of either native or synthetic AHLs. As such, we
reasoned that the AHL hydrolysis products should also form
inclusion complexes with -CD. This would unfortunately allow for
the potential formation of unproductive complexes, i.e., the
binding and delivery of inactive SAHLAs.
Having already synthesized AHLs 1 and 2 — which, like native
AHLs, have acyl chains that are carbon-based — and prepared
aqueous stock solutions for our binding studies, we allowed these
AHLs to undergo hydrolysis under neutral conditions, monitoring
progress by TLC (4:1 ethyl acetate/hexanes, vis. on UV active
TLC plates). Notably, AHLs migrate off the baseline (Rf ~ 0.4)
while their homoserine hydrolysis products do not (Rf ~ 0). AHLs
could not be detected after two weeks and we proceeded with
binding studies, noting similar changes in fluorescence spectra
morphology.
Figure 6. (top) Fluorescence titration of 2 (2.9 M) with increasing concentration
of -CD (0 mM to 7.4 mM; solutions 1 through 8) in phosphate buffered saline
(pH 7.3) with (inset) fitting to 1:1 binding isotherm determined by NLR analysis
at max = 333 nm. (bottom) ESI-MS spectrum of free 2, free -CD, and {-CD:2}
complex as sodium adducts.
While the acyl tails of many bioactive synthetic AHLs are
primarily all carbon; a number of heterocycles (e.g., furan, indole,
oxazole, thiophene) with potential biological activity in bacterial
systems have been developed. We, therefore, selected SAHLA 3
as a representative substrate for this class because its acyl tail
bears an indole and it is an antagonist for the LuxR receptor that
controls bioluminescence in V. fischeri.15 Exposure of 3 to
increasing concentrations of -CD afforded both fluorescence
enhancement and a hypsochromic shift (7 nm). This is consistent
with the behavior reported for its carboxylic acid precursor, 3-
indolebutyric acid, which is known to form an inclusion complex
with -CD.52 Similar to compounds 1 and 2, Benesi-Hildebrand
treatment of fluorescence intensities at max (360 nm) indicated
1:1 host/guest complex stoichiometry and non-linear regression
afforded a 1:1 binding isotherm to which our experimental data
was well fit (Kbind = 374  10 M-1). ESI-MS supported the existence
of the {-CD:3} complex with 1:1 stoichiometry.
Taken together, these findings support the potential for CDs to
form inclusion complexes with SAHLAs, improving their solubility
and, as a result, their potential utility for QS modulation.
ASSOCIATION OF HYDROLYSIS PRODUCTS
Figure 7. (top) Fluorescence titration of 3 (8.7 M) with increasing concentration
of -CD (0 mM to 7.4 mM; solutions 1 through 8) in phosphate buffered saline
(pH 7.3) with (inset) fitting to 1:1 binding isotherm determined by NLR analysisat
AHLs, both native and synthetic, are only active in bacteria if they
interact with an AHL receptor through highly conserved molecular

max = 360 nm. (bottom) ESI-MS spectrum of free 3, free -CD, and {-CD:3}
complex as sodium adducts.
5
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Summarized in Table 1, the hydrolysis products of 1 and 2,
respectively 1h and 2h, demonstrated weaker affinity towards
association with -CD than their lactone precursors, meaning that
-CD preferentially binds active AHLs (and synthetic analogues)
over their corresponding inactive, hydrolyzed forms. In terms of
SAHLA solubility and delivery, this translates to CDs preferentially
forming productive complexes with bioactive molecules rather
of the AHL and its hydrolysis product. In that report, we predicted
that the hydrolysis product would likely have a lower affinity for
associating with the macrocycle due to its additional hydrogen
bonding sites and formal negative charge. This assertion has
been demonstrated here by comparing the binding constants of 1
with 1h and 2 with 2h. The effect of hydrolysis on binding affinity
is profound and underscores the importance of performing AHL-
than unproductive complexes with hydrolysis-inactivated SAHLAs. centric assays under near neutral or acidic conditions, where
We assert that this reduction in binding affinity is directly
attributable to molecular composition. Explicitly, AHL hydrolysis
products bear a formal negative charge (i.e., carboxylate) under
the conditions tested and three additional hydrogen bonding sites
relative to their respective AHLs. This difference not only affects
the polarity of the molecule (as seen by TLC), but also likely
increases the hydrolysis products’ solvation — particularly about
the carboxylate — making it more hydrophilic. Indeed, Dong et al.
noted that lactone hydrolysis products are in fact more hydrophilic
than their respective lactones using HPLC.55 Additionally, binding
constant decrease upon charge adoption is consistent with the
observed behavior of many species, including BPAA, as a
function of pH. Below its pKa, BPAA exhibits a significantly higher
binding constant with -CD than above its pKa; upon conversion
to its carboxylate, BPAA affinity towards association decreases.56
AHLs are stable.
Additionally, the decreased binding affinity of acyl homoserines
as compared to their lactones is a characteristic shared by the
AHL lactonase, AiiA, which is deployed by the Gram-positive
bacteria Bacillus thurengiensis to attenuate AHL-mediated QS.
Given CDs can be readily modified, we hypothesize that, in the
future, a -CD based artificial enzyme could be synthesized to
accomplish AHL hydrolysis — similar in action to AiiA. Such a
catalyst would naturally exploit the desired substrate’s propensity
for binding over its hydrolysis product to establish a natural
turnover cycle.
CD:SAHLA COMBINATION IMPROVES ANTI-VIRULENCE
From these experiments, it is apparent that -CD can form
inclusion complexes with synthetic AHLs and, as a result, can be
exploited to better solubilize these molecules. The high propensity
of 1 for inclusion is on the same order of magnitude as that of
Imatinib (logKbind = 3.18), a potent and selective inhibitor of protein
tyrosine kinase used in the treatment of chronic myelogenous
leukemia.58 The Captisol® formulation of Imatinib uses a -CD
derivative to enhance the compound’s solubility 50-fold. On the
other hand, as evidenced by the relatively low binding constants
that describe the inclusion complexes of 2 and 3 with -CD,
derivatization of the macrocycle (e.g., permethylation) may be
necessary to enhance solubility and make -CD more viable for
drug delivery; however, the association constants of 2 and 3 are
of the same magnitude as the protonated forms of Imatinib.
Perhaps more interestingly, though, the use of -CD in the context
of QS may have even greater effects than simply acting as a
delivery vehicle.
As noted previously, CDs and their derivatives demonstrate
efficacy as quorum sensing inhibitors by presumably forming
inclusion complexes with active AHL signal molecules, thereby
reducing the concentration of free AHLs in the bacterial
environment.27–32 We hypothesized that using a combination
treatment of CD and SAHLA, there would by some synergistic
effects in that CD would help solubilize the SAHLA and increase
its bioavailability and aid in its delivery. Because SAHLAs
outcompete native AHLs for receptor binding – they are receptor
antagonists – the vacated CDs could uptake the native free AHLs
and further impede the QS-circuit (Figure 3). To test this
Table 1. Binding Constants (Kbind
) of SAHLAs and hydrolysis products
determined in this study.
Substrate
1
Binding Constant, Kbind (M-1) [a]
3850  170
1h
2
2920  180
395  15
337  16
374  10
2h
3
[a] aReported binding constants represent the error-weighted mean obtained
from five independent fluorescence titration experiments. Errors reported
represent 3 SE (99.7% confidence) about the error-weighted mean.
As an alternative explanation, the hydrolyzed homoserine lactone
has more conformational freedom and is therefore more likely to
interact with the alcohol groups that line the primary or secondary
CD faces. Because there is greater electron density on the
homoserine than on its corresponding lactone, electrostatic
repulsion between the homoserine and CD alcohols could also
decrease the propensity of the hydrolysis products to form
inclusion complexes.
As we noted in our report on AHL hydrolysis kinetics,36 the
conditions under which AHL-centric assays are performed –
especially pH – are crucial to evaluate whether assays are
appropriate and how the results should be interpreted. In that
report, we noted the binding constant estimation performed by
Morohoshi et al.,29 who used Buvari57 and coworkers’
phenolphthalein displacement assay to determine the binding
constant of N-hexanoyl-L-homoserine lactone with various CD
derivatives, was seemingly inappropriate because, under those
conditions (pH 9.5), the AHL readily hydrolyzes (t1/2 < 20 min) and
the resulting binding constant likely describes a statistical mixture
hypothesis, we synthesized 4,
a moderate antagonist of
Pseudomonas’ LasR-circuit,15 and evaluated whether it could
form an inclusion complex with -CD by ESI-MS. Notably,
SAHLAs 1-3 are not significant inhibitors of LasR.15 To our delight,
4 formed a complex with 1:1 stoichiometry (Figure 8, top).
6
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000773
ChemBioChem
FULL PAPER
comes from the inhibition of QS-mediated virulence factor
production rather than any antimicrobial or host effect of these
compounds. To our knowledge, this is the first time that a
{SAHLA:-CD} complex has been employed to inhibit virulence in
a wildtype pathogen during a host-infection assay.
Conclusion
We have shown that synthetic AHLs, along with their hydrolysis
products form inclusion complexes with -CD using fluorescence
spectrophotometry along with NMR and MS spectrometries. We
propose that, as a result of that complexation, CDs can be used
to better solubilize and deliver SAHLAs to intended target sites.
However, as CDs alone demonstrate anti-QS effects, the
application of CDs as SAHLA delivery vehicles is a per se
combination therapy that elicits additive, if not synergistic, effects
that we have shown here using a C. elegans viability model. Lastly,
because there exists some differential in binding affinity of
SAHLAs and their hydrolysis products — and, by analogy, native
AHLs and their respective hydrolysis products — with -CD, we
assert that that differential could be exploited in the design of an
artificial AHL lactonase based on a -CD scaffold.
Experimental Section
Materials and Instrumentation.
L-homoserine (98%) was obtained from AABlocks and was converted,
as needed, to homoserine lactone hydrochloride by reflux with 4M
hydrochloric acid and evaporation to dryness. 4-Biphenylacetic acid (98%)
was purchased from Acros Organics. 2-Naphthylacetic acid (99%) and
indole-3-butyric acid (98%) were purchased from AlfaAesar. 3-
Phenylpropanoic acid (95%) was purchased from Enamine, LLC.
Triethylamine (99%) was purchased from Acros Organics. N-(3-
Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC-HCl) was
purchased from CreoSalus. Dichloromethane was purchased from Fisher
Scientific. All reagents were used without further purification.
Figure 8. (top) Molecular structure of synthetic AHL antagonist 4 that inhibits
LasR in P. aeruginosa and ESI-MS spectrum indicating 4 forms a 1:1 complex
with -CD. (bottom) Results of C. elegans viability assay indicating that when
10 M 4 and 50 M -CD are coapplied, viability at 12 (white bars) and 24 (grey
bars) hrs is dramatically increased, implying synergistic effects. Results are the
average of 3 trials (N=30 worms/treatment/trial). Error bars represent SE. ‘*’
indicates p-value < 0.05, ‘**’ indicates p-value < 0.01.
The identities of products were confirmed using 1H- and 13C-NMR, as
well as mass spectroscopy. All NMR spectra were collected using Bruker
Avance 400 MHz spectrometers at 22.0  0.5 C. Mass spectra were
collected using an Agilent 6560 IM-QTOF mass spectrometer (MS).
MS samples were prepared by dissolving samples in LC-MS grade
methanol (10 g/mL) and sonicating for 10 minutes. MS samples were
then direct infused at 15 L/min with positive mode electrospray ionization
(ESI). The samples were also analyzed after addition of 20 g/mL -CD.
Noncovalent complexes were identified by accurate mass as primarily the
[M+CD+Na]+ and [2M+CD+2Na]+2 adducts. Collision cross section (CCS)
was measured for all complexes.
Having confirmed the formation of the {-CD:4} complex, we
proceeded to test our hypothesis that they could function
synergistically to inhibit quorum sensing. Specifically, we
evaluated the virulence of wildype P. aeruginosa PAO1 with the
model eukaryotic host C. elegans. Briefly, mature C. elegans
(N=30) were transferred to plates containing P. aeruginosa in the
presence of -CD (50 µM) and/or SAHLA 4 (10 µM) and evaluated
for viability at 12 and 24 hours under a light microscope.
Nematodes were scored for viability by either watching them
move across the plate or by applying gentle pressue to the side
of the worms using a blunt glass tip to look for reduced motility
phenotypes. Shown in Figure 8 (bottom), treatment with either -
CD or 4 alone slightly increased C. elegans viability at 12 hours;
however, in both cases, this improvement was short-lived and did
not extend to the 24-hour benchmark. On the other hand, co-
application of 4 and -CD significantly increased C. elegans
viability beyond 24 hours, suggesting this combination treatment
indeed evokes additive, if not synergistic, effects. We note that
the addition of either -CD (50 µM) and/or SAHLA 4 (10 µM) had
no effect on P. aeruginosa growth in culture (Data not shown) or
C. elegans viability on pathogen-free plates arguing that this effect
Absorbance spectra were collected using
a
Jasco V-650
Spectrophotometer calibrated against a blank of phosphate buffered saline
(PBS, pH 7.3). Fluorescence measurements were performed using a Jobin
Yvon Horiba Fluoromax-3 fluorimeter and were corrected for the spectral
response of the instrument.
Melting points were observed visually using a MelTemp-II apparatus.
AHL Synthesis.
Following the procedure of Ruysbergh59 et al. for synthesizing native
AHLs with slight modification, compounds 1, 2, and 4 were prepared by
first dissolving freshly synthesized homoserine lactone hydrochloride (275
mg, 2 mmol) in deionized water (15 mL) and treating the resulting solution
with the corresponding carboxylic acid (2 mmol), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (380 mg, 2 mmol, 1 equiv.), and
7
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000773
ChemBioChem
FULL PAPER
triethylamine (300 mL, 2.15 mmol, 1 equiv.). The reaction mixture was
stirred vigorously overnight at room temperature, diluted with deionized
water (15 mL), and extracted with ethyl acetate (3 x 50 mL). The combined
organic phases were washed successively with saturated sodium
bicarbonate and brine solutions (30 mL each), dried over anhydrous
sodium sulfate, and concentrated in vacuo by rotary evaporation. Flash
silica chromatography (4:1 ethyl acetate/hexanes, visualized by UV and
ninhydrin staining) afforded the desired synthetic AHL in moderate yield as
a white solid.
Compound 3 was prepared by charging a round-bottomed flask in an
ice-water bath with freshly synthesized homoserine lactone hydrochloride
(275 mg, 2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (380
mg, 2 mmol, 1 equiv.), and 3-indolebutyric acid (410 mg, 2 mmol, 1 equiv.).
To these solids were added dichloromethane (15 mL) and triethylamine
(580 mL, 4 mmol, 2 equiv.). The reaction mixture was warmed to room
temperature and vigorously stirred overnight, then diluted with ethyl
acetate (30 mL) and extracted with deionized water (30 mL). The aqueous
phase was further extracted with ethyl acetate (2 x 30 mL). The combined
organic layers were washed successively with saturated sodium
bicarbonate and brine solutions (30 mL each), dried over anhydrous
sodium sulfate, and concentrated in vacuo by rotary evaporation. Flash
chromatography (4:1 ethyl acetate/hexanes, visualized by UV and
ninhydrin staining) afforded 3 as a white solid.
Binding Constant Determination.
Methanolic stock solutions of AHLs 1, 2, and 3 were prepared and
subsequently diluted with PBS (pH 7.3) to 90 M, 145 M, and 435 M,
respectively ([MeOH] = 2% v/v). An aqueous -CD stock (10.6 mM) was
prepared by dissolving the macrocycle in PBS. For binding constant
determination studies, 100 L aliquots of aqueous AHL stock solution were
transferred into 5 mL volumetric flasks charged with varying amounts of -
CD stock solution and filled to the mark with PBS ([MeOH] < 0.04% v/v).
Solutions were inverted and left to equilibrate for 15 minutes prior to
performing fluorescence measurements.
For 1, the excitation and emission wavelengths were selected to be 254
nm and 315 nm. For 2, the excitation and emission wavelengths were
selected to be 277 nm and 334 nm. For 3, the excitation and emission
wavelengths were selected to be 280 nm and 360 nm. All spectra were
collected with increments of 1 nm, integration times of 0.05 sec, and
bandwidths (both emission and excitation) of 3 nm.
Association constants were initially determined by application of the
Benesi-Hildebrand equation (Equation 1), which suggested
a 1:1
host/guest complex stoichiometry by a linear double-reciprocal plot. Here,
I indicates the intensity of a given solution, I0 is the intensity of the solution
without CD present, [CD] denotes the total concentration of CD present in
solution, complex denotes the change between a fluorescence quantum
efficiency factor of the free and bound fluorophore, and Kbind denotes the
binding constant.
N-(4-biphenyl)acetyl-L-homoserine lactone (1):
1H-NMR (400 MHz; CDCl3, ): 7.62-7.56 (4H), 7.48-7.42 (2H), 7.39-7.32
(3H), 6.05 (1H, d, J = 3.6 Hz), 4.54 (1H, ddd, J = 11.6, 8.7, 6.0 Hz), 4.44
(1H, ddd, J = 9.0, 9.0, 0.9 Hz), 4.26 (1H, ddd, J = 11.2, 9.3, 6.0 Hz), 3.67
(2H, s), 2.81 (1H, dddd, J = 12.7, 8.4, 5.9, 1.1 Hz), 2.12 (1H, dddd, J =
12.4, 11.5, 11.5, 8.9 Hz); 13C-NMR (100 MHz; CDCl3, ): 175.22, 171.62,
140.66, 140.61, 133.11, 129.98, 128.96, 127.96, 127.59, 127.19, 66.15,
49.54, 43.02, 30.43; Melting Point: 173-175 C; Mass (m/z, [M+Na]+):
(theor.) 318.11061, (found) 318.1115; Yield: 65%.
1
1
1
=
+
(1)
[
]
퐶퐷 ∆휙푐표푚푝푙푒푥 퐾푏푖푛푑 ∆휙푐표푚푝푙푒푥
퐼 − 퐼0
However, to overcome the limitations posed and circumvent assumptions
made by the Benesi-Hildebrand method, we fitted the data using non-linear
regression (NLR).
Briefly, at low enough concentrations (i.e., A()<0.05), the fluorescence
(F) of a solution can be thought of as the sum of the individual components
in that solution multiplied by that component’s “fluorescence efficiency
factor,” , which is proportional to its fluorescence quantum yield.
Assuming the macrocycle’s fluorescence is trivial, the fluorescence can be
written as a function of total guest concentration (G0, known) and the
concentration of the host:guest complex ([HG], unknowable given an
unknown association constant).
N-(2-naphthalene)acetyl-L-homoserine lactone (2):
1H-NMR (400 MHz; CDCl3, ): 7.87 (3H), 7.74 (1H, s), 7.53-7.46 (2H, ‘dd’),
7.39 (1H, dd, J = 8.4, 1.7 Hz), 6.00 (1H, d, NH, J = 4.2 Hz), 4.51 (1H, ddd,
J = 11.6, 8.6, 6.1 Hz), 4.41 (1H, ddd, J = 9.2, 8.9, 0.6 Hz), 4.23 (1H, ddd,
J = 11.1, 9.3, 6.0 Hz), 3.79 (2H, s), 2.76 (1H, dddd, J = 12.7, 8.5, 5.9, 1.2
Hz), 2.08 (1H, dddd, J = 12.3, 11.4, 11.4, 9.0 Hz); 13C-NMR (100 MHz;
CDCl3, ): 175.13, 171.62, 133.71, 132.78, 131.64, 129.13, 128.52, 127.91,
127.86, 127.27, 126.67, 126.35, 66.11, 49.49, 43.62, 30.34; Melting Point:
193-195 C; Mass (m/z, [M+Na]+): (theor.) 292.0956, (found) 292.09496;
Yield: 39%.
[
]
[
]
퐹 = 퐺 휙퐺 + 퐻퐺 휙퐻퐺
(2)
(3)
(
[
])
[
]
퐹 = 퐺0 − 퐻퐺 휙퐺 + 퐻퐺 휙퐻퐺
N-(3-indole)butyryl-L-homoserine lactone (3):
1H-NMR (400 MHz; MeOD-d4, ): 7.53 (1H, d, J = 7.8 Hz), 7.31 (1H, d, J
= 8.1 Hz), 7.07 (1H, “t”), 7.03 (1H, s), 6.98 (1H, “t”) 4.58 (1H, dd, J = 10.8,
9.3 Hz), 4.41 (1H, td, J = 9.0, 1.8 Hz), 4.27 (1H, ddd, J = 10.4, 9.1, 6.6 Hz),
2.8 (2H, t, J = 7.4 Hz), 2.51 (1H, dddd, J = 12.3, 9.0, 6.7, 2.1 Hz), 2.30
(2H,t, J = 7.5 Hz), 2.23 (1H, dddd, J = 12.1, 10.7, 10.7, 9.3 Hz), 2.02 (2H,
p, J = 7.4 Hz). 13C-NMR (100 MHz; MeOD-d4, ):177.63, 176.45, 138.39,
128.94, 123.22, 119.56, 119.56, 115.75, 112.28, 67.30, 50.02, 36.52,
29.76, 27.65, 25.66. Melting Point: 63-65 C; Mass (m/z, [M+Na]+: (theor.)
309.12151, (found) 309.1221; Yield: 50%.
The concentration of the host:guest complex can rarely be known
experimentally; however, it can be written as a solution to the quadratic
equation resulting from the definition of the 1:1 host:guest association
constant Kbind, where [H] and [G] are the free host and guest
concentrations, respectively.
[
]
퐻퐺
퐾푏푖푛푑
=
(4)
[
][
]
퐻
퐺
[
]
퐻퐺
N-(3-phenylpropanoyl)-L-homoserine lactone (4):
1H-NMR (400 MHz; CDCl3, ): 7.32-7.25 (2H), 7.24-7.16 (3H), 6.07 (1H, d,
NH, J = 4.2 Hz), 4.53 (1H, ddd, J = 11.5, 8.5, 6.1 Hz), 4.43 (1H, t, J = 8.9
Hz), 4.26 (1H, ddd, J = 11.1, 9.4, 5.9 Hz), 2.97 (2H, t, ABX2, JAX = JBX
7.7 Hz), 2.78 (1H, dddd, J = 12.6, 8.3, 5.9, 1.0 Hz), 2.58 (1H, ABX2, JAB
퐾푏푖푛푑
=
(
[
])(
[
])
퐻0 − 퐻퐺 퐺0 − 퐻퐺
1
퐻퐺 2 − (퐻0 + 퐺0 +
) 퐻퐺 + 퐻0퐺0 = 0
[
]
[
]
퐾푏푖푛푑
=
=
14.9 Hz, JAX2 = 7.6), 2.53 (1H, ABX2, JAB = 14.9 Hz, JBX2 = 8.0 Hz), 2.03
(1H, dddd, J = 11.8, 11.8, 11.8, 9.1 Hz); 13C-NMR (100 MHz; CDCl3, ):
175.60, 172.87, 140.64, 128.78, 128.55, 126.58, 66.27, 49.42, 38.06,
31.62, 30.64; Melting Point:146-148 C; Mass (m/z, [M+Na]+): (theor.)
256.09496, (found) 256.0959; Yield: 44 %.
(5)
1
1
[
]
퐻퐺
=
((퐻0 + 퐺0 +
)
2
1
2
퐾푏푖푛푑
√
−
(퐻0 + 퐺0 +
) − 4퐻0퐺0)
퐾푏푖푛푑
Fitting the fluorescence data using Equations 3 and 5, optimized using the
Simplex algorithm on PSI-PLOT software, afforded the association
8
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000773
ChemBioChem
FULL PAPER
constants and fluorescence efficiency factor of the host:guest complex.60
The fluorescence efficiency factor of the guest was determined by dividing
the fluorescence of the null/guest only solution by analyte concentration.
Initial guesses for the association constants were the association
constants determined by the Benesi-Hildebrand method (using regression
in Microsoft Excel) while guesses for the host:guest complex fluorescence
quantum efficiency factor were input as the fluorescence of the highest -
CD concentration solution divided by the analyte concentration.
Binding constant determination studies were per-formed with five
replicates. The values reported reflect the weighted mean of those results
and the error indicates three SE (99.7% confidence interval) about the
reported average.
Keywords: Synthetic AHL Analogues • Host-Guest Chemistry •
Cyclodextrin • Fluorescence Titration • Quorum Sensing
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EWZ sincerely thanks Alexander Yonchak (University of Rhode
Island) for his insightful conversation at the ACS National Meeting
in Orlando, FL (Spring 2019). EWZ thanks ABB for providing
necessary chemicals for AHL synthesis. AGP thanks the Guisbert
Lab (Florida Institute of Technology) for the donation of C.
elegans and the Blackwell Lab (University of Wisconsin-Madison)
for the gift of P. aeruginosa PAO1. We thank the Florida Institute
of Technology for its financial support for a portion of this work. A
portion of this work was performed in the McKnight Brain Institute
at the National High Magnetic Field Laboratory’s Advanced
Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility,
which is supported by National Science Foundation Cooperative
Agreement No. DMR-1644779 and the State of Florida. This work
was also supported in part by an NIH award, S10 RR031637, for
magnetic resonance instrumentation. Purchase of NMR
spectrometers for AHL characterization was assisted by the
National Science Foundation (CHE 03 42251).
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Entry for the Table of Contents
Synthetic AHL analogues (SAHLAs) have demonstrated efficacy as quorum sensing inhibitors by receptor antagonism. However, like
native AHLs, these compounds are poorly soluble. To improve their solubility, we investigate the association of three representative
SAHLAs, along with their hydrolysis products, with -cyclodextrin by spectrofluorimetry and evaluate co-application of CDs with
SAHLAs using a C. elegans infection model.
11
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