SMC 010

Competition Experiments
Lei You and Eric V. Anslyn

The University of Texas at Austin, Austin, TX, USA
1 Introduction
1
2 Single Analyte Sensing Versus Differential Sensing
Using IDAs
2
3 Anionic IDAs Using Organic Hosts
3
4 Metal Complexing IDAs for Anions
7
5 Sensing of Cations Using IDAs
14
6 Sensing of Neutral Analytes Using IDAs
16
7 IDAs Using Biological Hosts
19
8 Enantioselective IDAs Introduction
21
9 Summary and Conclusion
25
References
25
be used to evaluate binding strength and selectivity. One

major disadvantage of this approach is the requirement of
a dedicated, sometimes difficult, synthesis.
An IDA is based on the competition between an indicator
and an analyte for the binding site. An indicator is
allowed to bind to a receptor, and then displaced upon
the introduction of an analyte. Normally, the free and
bound indicators have different colorimetric or fluorescent
properties, causing a signal change upon displacement. The
major requirement for an IDA with a desired sensitivity is
that the binding affinity of the analyte to the receptor is
comparable to that of the indicator to the receptor.
KI

H:I
KG
H+G

H:G
KG /KI
H:I + G

H:G +
H+I
INTRODUCTION
Competitive binding assays are well established in bioanalysis.1 Over the past decade, substantial efforts have
been made to use competition experiments in other research
areas, especially supramolecular chemistry.2 Instead of covering all types of competition experiments, this chapter
focuses on a unique competition assay, the so-called indicator displacement assay (IDA), which has become a standard strategy for molecular recognition and sensing.3, 4
In the traditional indicatorspacerreceptor approach, an
indicator (i.e., the signaling unit) is covalently attached
to a receptor (i.e., the binding and recognition motif)
through a spacer (Figure 1).5, 6 The binding of an analyte
will induce a spectroscopic change in the sensing motif.
On the other hand, these spectroscopic measurements can
The interactions between the indicator or analyte and the

receptor can be either covalent or noncovalent. Reversible
covalent bonds are widely used in IDAs. The interaction of vicinal diols and arylboronic acids to form cyclic
boronate esters is one of the most explored. Many indicators contain a catechol unit, which is an ideal binding target
for boronic acids. Another well-studied reversible covalent
interaction is the dynamic imine formation, an equilibrium
between primary amine and carbonyl host. This interaction
is also widely used in the creation of dynamic combinatorial
libraries. The common noncovalent interactions between
an indicator or analyte and the receptor are electrostatic
interaction, hydrogen bonding, salt bridge, and metal complexation. These interactions are dependent on many factors
such as the geometry of the guest, hydrophobicity, charge
state, and the solvent system.
IDAs display many advantages. First, because the indicator is not covalently linked to the host, the synthetic efforts
Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc010
Concepts
Analyte
(a) Signaling
unit
Recognition
unit
Analyte
(b) Receptor indicator

complex
Figure 1
Receptoranalyte
complex
Indicator
Schematic illustration of the indicatorspacerreceptor approach (a) and indicator displacement assay (b).
can be diminished significantly. Second, a broad range of

receptors and indicators can be screened to modulate the
sensitivity as well as the selectivity of the assay. For the
development of an IDA for a specific analyte, the host is
generally designed and synthesized first and then an appropriate indicator is chosen. Third, the IDA works well in
both aqueous and organic media. As a result, the binding
constant of the indicator or analyte can be fine-tuned by
simply changing the solvent system. Finally, the assay is
readily adapted to platforms for quick analysis, such as
plate readers and flow injection analysis (FIA). An IDA
is also simple to implement with pattern-recognition-based
multicomponent array sensing. The major drawback of an
IDA-based experiment is the intrinsic release of dye, which
in some cases may make them incompatible with living tissue or cells, because the free indicator is present and can
diffuse away the desired signal.
There are different types of IDAs. Depending on the
detection method, colorimetric IDAs use colorimetric indicators, while fluorescent IDAs utilize fluorescent indicators.
Actually, in some cases the IDA can utilize either a colorimetric or a fluorescent indicator. A fluorescent IDA can
have higher sensitivity than a colorimetric IDA because
changes in emission of an indicator are measured rather
than absorbance. Sometimes the color change after indicator displacement is so apparent that naked-eye detection is
possible, which is extremely useful for a quick qualitative
analysis. The receptors used in an IDA can be synthetic
or natural. IDAs are also useful in exploring the interactions between substrates and biological hosts, including
proteins and nucleic acids. Alternatively, biological hosts
can be employed as a sensing platform through IDAs for
small molecules, which are challenging to target using other
methodologies. Examples of different types of IDAs are
discussed in this tutorial.
SINGLE ANALYTE SENSING VERSUS

DIFFERENTIAL SENSING USING IDAs
In traditional IDAs, one receptor is specifically designed

for a particular analyte based on the well-known lock
and key principle. In this scenario, it is always crucial to
increase selectivity and decrease the interference from other
structurally similar analytes. The selectivity is generally
achieved by the design of elegant receptors. However,
other factors, such as indicators, solvents, and pH can
also be used complementarily to fine-tune the selectivity.
Significant progress has been made in the design of
selective receptors for sensing and detection of small
molecules using IDAs. Despite tremendous success with
small molecules, it remains very challenging to design
selective molecular receptors for macromolecules such as
proteins and nucleic acids. Moreover, it is difficult to use
selective receptors to analyze complex mixtures such as
wine and perfume, which generally contain components
with subtle structural differences.
A highly selective receptor for each analyte is not necessary in many cases. Natural protein receptors used in the
mammalian senses of smell and taste have inspired chemists
to use an array of receptors for various analytes.7 The
receptors are differential instead of selective for a single
analyte. Each receptor may bind a series of analytes, but
with different affinity. Contrary to single analyte sensing,
lack of specificity and selectivity is desired for differential
receptors.8 An array could be constructed from multiple
receptors, multiple indicators, and multiple analytes. The
signals generated from multiple differential binding interactions (i.e., multiple IDAs) are normally analyzed by pattern
recognition protocols, such as principal component analysis
(PCA) and artificial neural networks (ANN). The resulting
DOI: 10.1002/9780470661345.smc010
Competition experiments
fingerprint provides characteristic diagnostic patterns for the
individual analyte or complex mixtures comprising multiple analytes. Differential sensing with synthetic receptors is
a rapidly growing research area within the supramolecular
community. Creation of synthetic receptors can be facilitated by the use of combinatorial chemistry. Examples of
IDAs for both single analyte sensing and differential sensing are discussed in the following sections.
0.450
0.050
(a)
ANIONIC IDAs USING ORGANIC

HOSTS
The majority of IDAs have been developed for anions.

Anionic motifs are present in most biological molecules,
including amino acids, nucleic acids, and fatty acids.
Anions play important roles in numerous physiological
processes and phenomena such as protein biosynthesis,
transport of neurotransmitters, sugar metabolism, and regulation of enzyme activity. Many geochemical processes
also involve inorganic anions. As a result, the development
of receptors and methodologies for anion recognition and
sensing is one of the major current goals in supramolecular chemistry. In the design of anion receptors,9 ion-pairing
interactions,10, 11 Lewis acid (including metal ions) coordination,12 and hydrogen bonding13 are the main binding
forces. Anion inclusion complexes have also been explored.
Purely organic receptors for anionic IDAs are discussed
here. Receptors based on metal complexation are discussed
in a later section.
Citrate is an intermediate in the well-known citric acid
circle (Krebs cycle) and is also found in soft drinks and
sports drinks. Citrate has a charge state of 3 at physiological pH. Our design of citrate receptor 1 focused on
the 1,3,5-trisubstituted-2,4,6-triethylbenzene.14 The sterics
imparted by three ethyl groups ensures that six substituents
point up and down alternatively around the benzene ring.
The resulting preorganized guanidinium groups facilitate
the binding of citrate to 1 through H-bonds and salt-bridge
interactions. The sensing ensemble for citrate (3) contains
receptor 1 and the anionic indicator 5-carboxyfluorescein
(2).15 Indicator 2 was chosen because it has two carboxylates and is a pH indicator. Its absorbance or fluorescence
response is very sensitive to changes in pH. When receptor 1 was added to the indicator solution, the absorbance at
498 nm increased, suggesting that more indicator molecules
became bound. Upon addition of citrate to the solution of
1 and 2, the absorbance decreased as the indicator was displaced from the host (Figure 2). The binding constant for
3 with 1 was found to be 2.9 105 M1 , which was determined by UVvis spectroscopy. An order of magnitude
520
l (nm)
0.450
420
0.050
420
(b)
520
l (nm)
Figure 2 Absorbance spectrum of 2: addition of 1 to a solution

of 2 at 14 M (a) and addition of 3 to a solution containing 74 M
of 1 and 14 M of 2 (b). (Reproduced from Ref. 15. WileyVCH, 1998.)
increase in binding constant was observed when the solvent was changed from water to 3 : 1 (v/v) methanolwater.
This is consistent with previous results showing that a
decrease in polarity and H-bonding ability of the solvent
leads to increases in binding. Calibration curves were generated from a titration and the sensing ensemble exhibited
selectivity for citrate over succinate and acetate (Figure 3).
The concentration of citrate in various beverages was measured, and the results were consistent with those determined using gravimetry and nuclear magnetic resonance
(NMR). Schmuck and coworkers also reported a similar
pinwheel-scaffold-based tricationic guanidiniocarbonyl pyrrole receptor for naked-eye sensing of citrate using 2 as an
indicator.16, 17
O
HN
H
N
HN
NH
N
H
H
N
1
N
H
HO
O
H
N
N
H
O
O
3Na
O
O 2
O
3
In addition to carboxylateguanidinium ion-paring interactions, an IDA was successfully extended to other anionic
substrates, such as tartrate,18 glucose-6-phosphate,19
DOI: 10.1002/9780470661345.smc010
Concepts
OH
B OH
0.70
HN
HN
NH
H
N
0.60
0.50
NH
OH
O
N
H
N
HH
N
HO
OH
OO
O
OH
5
0.40
0.5
104c
(mol
1.0
As expected, it is very challenging to design selective synthetic receptors for structurally similar analytes
such as tartrate and malate. Our group has developed a
protocol based on multicomponent indicator displacement
assays (MIDAs) to sense tartrate and malate simultaneously.23 Two guanidinium and boronic acids containing
hexasubstituted hosts (4 and 7), both with affinities for
tartrate and malate, were used. Two indicators with different binding affinities for the two receptors were chosen: pyrocatechol violet (8) and bromopyrogallol red (9).
The receptors and indicators were placed in a single
cuvette, and the absorbance spectra of this four-component
7
6
4
3
2
0
0.4
0.8
1.2
104c (mol L1)
Figure 3 UV calibration curve for citrate (75 M 1, 14 M

2, and 498 nm) in 3 : 1 methanolwater at pH 7.4 (a) and
fluorescence calibration curves for citrate (), succinate (), and
acetate () (excitation at 490 nm and emission at 525 nm) at
identical conditions (b). (Reproduced from Ref. 15. WileyVCH, 1998.)
inositol-1,4,5-trisphosphate (IP3),20 and gallate21 by our

group via the use of boronic acids, which are known
to rapidly and reversibly form boronate esters with
vicinal diols and 2-hydroxycarboxylates.22 Receptor 4 was
designed for the analysis of tartrate (6), which is a natural product present in grape juices and wines. The boronic
acid and guanidinium motifs were incorporated into the
receptor to bind diol and carboxylates respectively. The
indicator employed was alizarin complexone (5), which
is a pH-sensitive dye. Upon addition of 4 to a solution of indicator 5 in 3 : 1 methanol/water at pH 7.4, the
absorbance at 450 nm increased, while the absorbance at
525 nm decreased. The absorbance change was reversed
after the addition of tartrate (Figure 4). A binding constant of 5.5 104 M1 between 2 and tartrate was obtained.
This sensing ensemble is selective for tartrate over other
mono- and bis-carboxylates, with the exception of malate
(Figure 5).
(b)
1.5
L1)
(a) 350
700
0
(a)
350
(b)
700
l (nm)
Figure 4 Absorbance spectrum of 5: addition of 4 to a solution

of 5 at 180 M (a) and addition of 6 to a solution containing
180 M of 4 and 180 M of 5 (b). (Reproduced from Ref. 18.
Wiley-VCH, 1999.)
DOI: 10.1002/9780470661345.smc010
1.55
Tartrate
(mM)
1.45
1.35
1.25
Malate
(mM)
1.15
1.05
0.2 0.4.....1.2
0
0.2
.....
0.4
0.95
1.2
0.85
Figure 6 A schematic representation of one experiment performed where UVvis spectra were recorded at different concentrations of tartrate and malate. (Reproduced from Ref. 23.
Wiley-VCH, 2003.)
0.75
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
CAnal. (mM)
Figure 5 Calibration curves at 450 nm for tartrate (), malate

(), ascorbate (), lactate (), succinate (), and glucose ().
(Reproduced from Ref. 18. Wiley-VCH, 1999.)
chemosensing ensemble span a large wavelength range. The

concentrations of the receptors and indicators were kept
constant, and the concentrations of two analytes were varied (Figure 6). A series of UVvis spectra were recorded,
and data at 27 wavelengths from each spectrum was used as
a training set for multilayer perceptron artificial neural networks (MLP-ANN). The concentrations of unknowns that
were left out during the training process were predicted
successfully with an error of <6%. After further training, the error was reduced to <2% (Table 1). A similar
single-vessel, differential-sensing assay was used to measure citrate and calcium simultaneously.24
chemosensing ensemble is composed of receptor 10 and

the indicator pyrocatechol violet (8) in 1 : 1 water/methanol
solution at pH 7.4. Upon binding of 8 with the receptor
10, the absorbance at 430 nm (yellow) decreased, while
the absorbance at 526 nm (purple) increased. Addition of
HEP to the sensing ensemble reversed the color change,
indicating that 8 was displaced from the binding site by the
analyte (Figure 7). A binding constant of 3.8 104 M1
was obtained between receptor 10 and HEP. Compound
10 has a lower binding affinity for analytes with lower
anionic charge density, such as chondroitin 4-sulfate (ChS)
and hyaluronic acid (HA), revealing that ion-pairing electrostatic interactions play a major role in the binding. Moreover, the receptor lacking the boronic acids moiety has a
very low affinity for HEP.
HO
OH
OH
HN
B OH
HN
NH
H
N
4
HO
B OH
N
HH
N
HN
NH
H
N
H
N
N
H
N
H
O
Br
B(OH)2
HN
NH
NH3
O
OH
OH
H3N
NH
Br
O3S
HO
OH
9
HN
O
OSO3
O
OH
OH
OH
Using the similar pinwheel scaffold described above,

receptor 10 was designed for colorimetric sensing of heparin (HEP) (11).25 HEP is used in anticoagulation therapy
during bypass surgery to prevent clotting in the extracorporeal circuit and to mediate activation of the haemostatic
system. HEP is a highly negatively charged oligosaccharide.
A novel positively charged amino acid with a boronic
acid side chain was incorporated into the receptor. The
H3N
O
H HN
N
10 O
OH
OH
B(OH)2
HN
SO3
B(OH)2
CO2
OH
O O
NHSO3
OSO3
11
ATP is involved in most biochemical metabolism processes. The importance of ATP has inspired chemists
to design chemical sensors for ATP and other phosphates. Owing to its polyanionic nature, receptors with
DOI: 10.1002/9780470661345.smc010
Concepts
Table 1
The results and error from artificial neutral network analysis (concentration in mM).
Training
NN1
Validation
Validation
Validation
Validation
Real [malate]
case
case
case
case
no.
no.
no.
no.
1
2
3
4
Predetermined [malate]
Real [tartrate]
0.0731
0.568 (3%)
1.034 (4%)
1.136 (5%)
1.00
0.80
0.22
1.00
1.00
0.53
0.24
0.000
0.59
0.99
1.19
NN2
Validation case no. 1
0.2
0.2
0.2
Predetermined [tartrate]
0.971
0.791
0.212
1.013
(3%)
(1%)
(5%)
(1%)
0.995 (0.6%)
0.527 (0.0%)
0.238 (1.3%)
Emission intensity (a.u.)
Reproduced from Ref. 23. 2003.
Absorbance
0.8
0.6
0.4
0.2
400
450
500
550
600
ADP
AMP
ATP
NAD
NADP
Citrate
Succinate
Fumarate
PPi
350
300
250
200
150
100
0.1
HEP
ChS
HA
0.2
2
400
600
800
1000
Concentration (M)
Figure 8 Increase in fluorescence intensity of HPTS by titrating

various anions into a solution of 12 and HPTS. (Reproduced from
Ref. 26. Elsevier, 2004.)
0.0
200
650
Wavelength (nm)
(a)
Absorbance change at 526 nm
400
350
(b)
450
10
12
When HPTS was displaced from 12 by polyanionic ATP at

pH 7.5, the fluorescence emission increased. The binding
constant for ATP with 12 was found to be 2.87 104 M1 .
Figure 8 demonstrates that other anionic species with lower
negative charge density, such as ADP, AMP, NADP, NAD,
and pyrophosphate, have a lower binding affinity toward the
host.
[Analyte]/[receptor]
Figure 7 UVvis spectra of 8 (54 M) in the presence of

0.16 mM 10 and 01.75 mM 11 (a). Absorbance change at 526 nm
of 8 upon addition of HEP, chondroitin 4-sulfate (ChS), or
hyaluronic acid (HA). (Reproduced from Ref. 25. American
Chemical Society, 2002.)
O3S
N
N
N
12
positive charges would be desired for ion-pairing interactions. The tetracationic calixpyridinium receptor 12
designed by Akkaya and coworkers resembles calixarenes
to some extent.26 The tetraanionic indicator 8-hydroxy1,3,6-pyrenetrisulfonate (HPTS, 13) was chosen for fluorescent detection. HPTS fluorescence intensity decreased upon
addition of receptor 12. Fluorescence pH titration of HPTS
in the presence of the host revealed approximately one unit
decrease in pKa of HPTS. The binding constant between the
host and HPTS was found to be 3.9 105 M1 at pH 7.5.
SO3
O3S
OH
13
The sensing of halogen anions has also been extensively

studied. An example of great interest is the receptor mesooctamethylcalix[4]pyrrole (14) developed by Sessler and
coworkers.27 This receptor is particularly easy to prepare
and has a higher affinity for fluoride than other halides.
It coordinates the anions in acetonitrile through pyrrole
NH/anion hydrogen bonds (Figure 9).28 The complexation
of indicator 4-nitrophenolate (15) to 14 causes a color
change, and the displacement of the indicator with fluoride
restores the yellow color. Other dyes such as Brookers
DOI: 10.1002/9780470661345.smc010
NO2
NO2
14
O
CH3
CH3 H
CH3 H
H
N
CH3
N
N
CH3
CH3 H
N
CH3
O
15
Yellow
Colourless
F
F
NO2
CH3
Yellow
Figure 9
CH3 H
N
CH3
H
N
14 15
H
CH3 H
N
N
CH3
CH3
CH3
Complex formation between 14 and 15 or fluoride.
merocyanine (16) were also explored in the displacement

assay using the same receptor.29 Machado and coworkers
reported an IDA based on the interaction of phenylboronic
acid (17) and nucleophilic anions.30 The interaction of
receptor 17 with indicator 16 generates a colorless covalently linked complex. Fluoride reacts with this complex,
displaces 16 through a bimolecular substitution mechanism,
and colors the solution (Figure 10).
NH
N
19
N
HN N
O
NH
O2 C
O
HN
N
O
15
B
OH
OH
16
17
0.4
0.3
0.2
0.1
3.0
4.0
5.0
6.0
METAL COMPLEXING IDAs FOR

ANIONS
Transition metalligand coordination has been well

explored in supramolecular systems, and, as expected, this
interaction is also widely incorporated into IDAs. The
diversity of metalreceptor (ligand) combinations helps
expand the scope of IDAs significantly. Coordination of
a transition metal ion (M) with an indicator often changes
the charge state of the indicator and also gives rise to new
absorption bands that may lead to large color changes. In
an M-IDA, a metal is coordinated to a receptor and then an
indicator is allowed to bind to the metal or both the metal
and the receptor. Upon addition of an analyte to the mixture, the indicator is displaced from the system, causing an
optical change (absorbance or fluorescence).
0.5
Absorbance
O
N
18
2.0
NH
HN
HN
1.0
20
14
0.0
O
N
H
H
N
0.0
Our group also created a chemosensing ensemble for

nitrate in organic solvents.31 An amide-linked C3 symmetric
bicyclic cyclophane (18) was used as the receptor.32 Two
molecules of 1,3,5-tris-aminomethyl-2,4,6-triethylbenzene
are linked by three 2,6-pyridine diamides with six amide
hydrogens facing the center of the cavity. The receptor
is capable of binding planar anions, such as acetate and
nitrate, using neutral hydrogen bonds to the anions
system. Methyl red (19) and resorufin (20) were chosen
as indicators. Addition of host 18 into a solution of
indicators resulted in change of the absorbance spectrum
with isosbestic point at 492 nm and 513 nm for 19 and
20, respectively. Nitrate can compete with these two
indicators for the host. The binding constant between
host and nitrate is 380 M1 in 1 : 1 CH3 OHCH2 Cl2 and
500 M1 in 3 : 1 CH3 CNCH2 Cl2 . Figure 11 shows that
the chemosensing ensemble exhibits modest selectivity for
nitrate over bromide and perchlorate.
NO2
N
H
7.0
8.0
9.0
[F ] (105 mol dm3)
Figure 10 Absorbance change at 571 nm upon addition of

tetrabutylammonium fluoride into complex 1617 in acetonitrile.
(Reproduced from Ref. 30. Elsevier, 2007.)
4.1
Amino acids
Amino acids are the basic building blocks of proteins and

are also important in organic synthesis. Moreover, many
DOI: 10.1002/9780470661345.smc010
Concepts
(8) in a 1 : 1 methanol/water mixture at pH 7.4, the color
changed from yellow (445 nm) to deep blue (647 nm). The
addition of various amino acids to the host/indicator mixture
reversed the color from deep blue to yellow (Figure 12).
The highest affinity was observed for aspartate with a binding constant of 1.5 105 M1 . Indeed, another host, which
lacks the appended guanidinium, has almost identical binding constants with aspartate, glutamate, and phenylalanine.
This further confirms the contribution of the attached guanidinium to the observed selectivity for aspartate.
Absorbance at 576 nm
0.12
(a)
0.11
0.10
0.09
0.08
10
20
30
40
50
OH
N
0.0035
H2N
NH
NH2
0.001
10
15
20
25
30
35
[Anion] (mM)
Figure 11 Absorbance change upon addition of NO3 (), Br

(), and ClO4 () into 1820 complex in 1 : 1 CH3 OHCH2 Cl2
(a) and 1819 complex in 3 : 1 CH3 CNCH2 Cl2 (b). (Reproduced from Ref. 31. Royal Society of Chemistry, 1999.)
individual amino acids play crucial roles in physiological

processes. One notable example is glutamate, which is
known as a neurotransmitter. The design of synthetic receptors to bind and recognize amino acids under physiological
conditions is still challenging. However, amino acids are
well known to bind metals through bidentate chelation by
the carboxylate and the amine. An M-IDA for amino acids
takes full advantage of this property. Over the last decade,
a number of M-IDA-based sensors have been reported for
amino acids.
Most amino acids sensors were designed for a specific
amino acid. Selectivity was usually achieved by targeting
a unique functional group in the side chain (e.g., carboxylate of Asp or Glu, guanidinium of Arg, imidazole of His).
Our group developed a zinc-complex-based receptor 21 for
aspartate.33 The terpyridine motif is a known ligand for
many transition metals, and the two attached guanidinium
groups were used to impart selectivity for the carboxylate
side chain of aspartate and glutamate. The H-bond between
carboxylate and guanidinium has been extensively incorporated into various supramolecular ensembles. Upon addition
of the host to a solution of the indicator pyrocatechol violet
SO3
NH2
Na 8
OH
OH
Fabbrizzi and coworkers have successfully demonstrated

that a dicopper polyamine cage 22 can be used to selectively detect glutamate in aqueous solution.34 Carboxylates
show good affinity for Cu2+ ions. Each trigonal tetraamine
subunit can coordinate a copper ion in a trigonal bipyramidal geometry, leaving one coordination site open in each
copper ion. As a result, the two carboxylates in glutamate
could bridge the two metal centers, allowing the inclusion of glutamate into the polyamine cage. The fluorescent
1.6
Absorbance
0.0015
H2N
21
0.002
0.0005
(b)
Zn2+
HN
0.0025
Yellow
Blue
0.8
0
370
470
570
670
770
Wavelength (nm)
(a)
1.6
Yellow
Absorbance
N
0.003
Blue
0.8
0
370
(b)
470
570
670
Wavelength (nm)
770
Figure 12 Absorbance spectrum of 8: addition of 21 to a

solution of 8 at 60 M (a) and addition of aspartic acid to a
solution containing 80 M of 21 and 60 M of 8 (b). (Reproduced
from Ref. 33. American Chemical Society, 2001.)
DOI: 10.1002/9780470661345.smc010
I f (490 nm)
300
250
200
150
100
0
(a)
1e 4
2e4
3e4
4e4
800
I f (513 nm)
indicator 6-carboxy-tetramethyl rhodamine (23) was used

in this competition assay, and its fluorescence is quenched
by the copper ion when bound into the cage complex.
When L-glutamate was added to the chemosensing ensemble solution, the indicator was released from the cage and
fluorescence (571 nm) was turned on again (Figure 13). A
binding constant of 7.9 106 M1 was obtained for the
cage complex with glutamate. The selectivity for glutamate
is achieved because the length between two carboxylates
matches well with the CuCu distance within the cage.
Using a similar strategy, Fabbrizzi and coworkers reported
an off/on fluorescent chemosensor (24) for histidine.35 The
imidazolate motif bridges the two copper centers. Figure 14
shows that the selectivity for histidine was also significantly
improved by the choice of indicator eosine Y (25). This
same receptor was also successfully employed to detect
pyrophosphate in water using a fluorescent competition
assay, and the selection of indicator also played a crucial
role in improving the selectivity.36
600
400
200
0
0
(b)
1e5
2e 5
240
NH
NH
Cu2+
NH
HN
NH
CO2
O2C
Br
NH
Cu2+
23
40
Br
(c)
CO2
2Na
25
24
Besides IDAs for a particular amino acid, nonselective

receptors have also been explored to discriminate amino
acids in the context of differential sensing.37 The use of
600
I F (a.u.)
120
80
Br
NH
400
200
0
106
160
Cu2+ NH
NH
200
Br
O
NH
N
Cu2+
22
NH
I f (540 nm)
NH
105
104
Concentration (mol
103
102
dm3)
Figure 13 Fluorescence change at 571 nm for 23 when Lglutamate () or related amino acids was added to a solution
of 22 (2.5 M) and 23 (0.25 M) at pH 7.0. (Reproduced from
Ref. 34. Elsevier, 2004.)
1e 6
2e 6
3e 6
4e6
Amino acid concentration (M)
Figure 14 Calibration curves for various amino acids using

chemosensing ensemble composed of 24 and coumarin (a),
fluorescein (b), and eosine Y (c). Histidine (), glycine (),
alanine (), and phenylalanine (). (Reproduced from Ref. 35.
American Chemical Society, 2003.)
various combinations of receptors, indicators, and analytes

provides many distinct chemosensing ensembles that can be
readily constructed into an array format. This approach is
capable of distinguishing structurally similar, nonfunctionalized amino acids such as leucine and isoleucine. Severin
and coworkers took advantage of commercially available,
air-stable, and water-soluble organometallic rhodium complex (26) as the nonselective receptor for the colorimetric
identification of 20 natural amino acids using an IDAbased array.38 This rhodium complex shows relatively fast
exchange kinetics and has a high affinity for amino acids.
Three indicators, gallocyanine (27), xylenol orange (28),
and calcein blue (29) were utilized in the study. Various pHs
were also employed in the competition assay to further finetune the discrimination ability of the sensing array. A linear
discriminant analysis (LDA) of the absorbance data was
performed, and the predictive ability of this sensor array
DOI: 10.1002/9780470661345.smc010
Concepts
Ala
Arg
Gln
Glu
Gly
IIe
Leu
Lys
Phe
Pro
Ser
Thr
Trp
Tyr
Val
PC2
19.5% of variance
2
2
10 000
5000
10
0
1
2
PC1 78.5% of variance
0
0.01
Figure 15 PCA plot for the discrimination and identification of

15 amino acids. (Reproduced from Ref. 38. American Chemical
Society, 2005.)
was excellent. The clustering of the data was visualized

by performing a PCA. The data were well separated with
the exception of valine and isoleucine (Figure 15). Using
a similar strategy, cross-reactive sensing arrays were also
constructed for identification and discrimination of peptides39 and sugars.40
0.1
10
[P ] (mM)
Figure 16 Change in fluorescence intensity of 30 at 580 nm

(excited at 485 nm) in the presence of metal ions upon the
following transformation (substrates S to products P): N-acetyl-Lmethionine to L-methionine with Cu2+ (); L-leucinamide to Lleucine with Cu2+ (); L-leucinamide to L-leucine with Ni2+ ().
(Reproduced from Ref. 41. Wiley-VCH, 2001.)
enzyme assay.42 The assay is amenable to high-throughput

screening of enzymatic reactions generating amino acids.
NH2
O
Cl
Cl
Rh
Cl
HN
OH
COOH
HOOC N
HOOC HO
Rh
N
Cl
SO3Na
R
HO
O
HO
R = CH2N(CH2CO2Na)2
28
27 OH
26
4.2
N
NH2 H
N
O
COOH
COOH
30
31
Phosphate-related anion species
N
OH
OH
O
29
Reymond and coworkers designed a new enzyme assay

utilizing an M-IDA.41 The fluorescence of a quinacridone
indicator 30 is quenched by formation of a macrocyclic
chelate M30 (M = Cu2+ or Ni2+ ). Because of a large 17membered ring in the chelate, this complex is relatively
unstable. As a result, other weak metal chelators, such as
amino acids, can efficiently compete with 30 for metal complexation. After the metal is released from the indicator,
the fluorescence is restored. Two enzymatic reactions that
produce amino acids from noncoordinating substrates were
successfully monitored. Figure 16 shows the fluorescence
response of 30 in the presence of metal ions during enzymatic reactions. Another indicator, calcein (31), which is
commercially available, was also employed in a similar
Phosphate-related anion species, including phosphate (Pi),

pyrophosphate (PPi), and adenosine triphosphate (ATP),
play crucial roles in energy transduction and metabolic
processes. ATP hydrolysis is central to many biological
reactions. In addition, phosphate anion is one of the basic
building components of nucleic acids (DNA and RNA). As
a result, the sensing and discrimination of these phosphaterelated anion species have captured the attention of many
research groups.
Kim and coworkers explored a known receptor 2,6bis(bis(2-pyridylmethyl)aminomethyl)-4-methyl-phenol (Hbpmp) for naked-eye detection of phosphate anion in aqueous solution.43 Zn2+ coordination with bis(2-picolyl)amine
(Dpa) together with the formation of a phenoxo-bridged
dinuclear metal complex (32) affords the anion binding
pocket. The pH-sensitive dye pyrocatechol violet (8) was
chosen as the indicator for the competition assay. When
[Zn2 (H-bpmp)]3+ was titrated into a solution of 8 at pH 7.0,
the solution color changed from yellow (444 nm) to blue
DOI: 10.1002/9780470661345.smc010
1.0
1.0
0.8
0.8
[Receptor]
0.4
0.4
0.2
0.2
0.0
0.0
300
400
500
(a)
600
700
800
300
(b)
l (nm)
[Phosphate]
0.6
0.6
11
400
500
600
700
800
l (nm)
Figure 17 Absorbance spectrum of 8: titration of 32 into a solution of 8 at 50 M (a) and titration of HPO4 2 into a solution containing
50 M of 32 and 50 M of 8 (b). (Reproduced from Ref. 43. Wiley-VCH, 2002.)
1.0
SO42
0.8
Acetate, HCO3,
NO3, N3,
CIO4, Cl, Br
F
0.6
Only mixture
0.4
HPO42
0.2
0.0
300
400
500
600
l (nm)
700
800
Figure 18 Absorbance spectra of 8 (50 M) in the presence

of 32 (50 M) and various anions (250 M). (Reproduced from
Ref. 43. Wiley-VCH, 2002.)
functionalized with attached guanidinium groups designed

to complement tetrahedral oxyanions of phosphate. The
binding constant for phosphate with 33 was reported
to be 1.5 104 M1 in 1 : 1 water/methanol at pH 7.4.
The sensing ensemble of the receptor with the indicator 5-carboxyfluorescein (2) was employed to detect
phosphate in aqueous solution.45 Upon binding to 33,
the color of 2 changed from yellow to orange. After
2 was released from the binding pocket by the addition of phosphate, the color changed from orange back
to yellow (Figure 19). The phosphate concentration in
serum and human saliva was measured using this displacement assay, and the results were comparable with the
data determined using a commercially available inorganic
phosphorus kit.
HN
(624 nm), indicating binding of 8 to the metal center in the

host. The addition of phosphate then reversed the color from
blue to yellow (Figure 17). The binding constant for phosphate and the indicator with the host complex is 1.12 105
and 5.3 104 M1 , respectively. Figure 18 indicates that
this sensing ensemble displays selectivity for phosphate
over other anions such as sulfate and perchlorate. However, it is not clear from the report whether pyrophosphate
can interfere with the assay or not.
N
N
Zn2+ O
N
Zn2+
N
32
Our group developed a C3v symmetric receptor 33 with

high selectivity and affinity for phosphate anions in aqueous solution.44 The receptor is derived from a tris(2pyridylmethyl)amine motif designed for copper binding
NH
HN
HN
HN
HN
NH
NH
N
2+
N Cu
N
NH
33
The bis(2-picolyl)amine (Dpa) unit designed for zinc

complexation has also been incorporated into various receptors for pyrophosphate sensing and detection. Smith and
coworkers studied a series of dinuclear zinc receptors (34,
35, and 36) and found that receptor 35 shows high selectivity and affinity for pyrophosphate under physiological
conditions.46 Other receptors with only one appended Dpa
arm (34) or para-substituted Dpa arms (36) show no binding toward pyrophosphate. The binding constant for this
receptor with pyrophosphate and phosphate was determined to be 1.52 107 and 7.3 105 M1 , respectively.
A fluorescent IDA was developed for pyrophosphate using
a coumarin-derived indicator 37. A more recent study
DOI: 10.1002/9780470661345.smc010
Concepts
Absorbance
12
Churchill and coworkers recently reported a novel fluorescent dinuclear zinc complex (40).50 The receptor
was synthesized by an effective one pot reaction, in
which the Schiff base ligand was generated in situ from
salicylaldehyde and L-lysine hydrochloride. In the solid
phase, a dimer is formed, exhibiting a four-membered
phenoxo-bridged metallacycle, which was confirmed by
X-ray diffraction (Figure 21). The receptor exhibits strong
blue fluorescence in neutral aqueous solution, and the
fluorescence intensity turns off gradually upon the addition
of pyrophosphate (Ka = 4.06 105 M1 ). The colorimetric
IDA was also performed for pyrophosphate with pyrocatechol violet (8) as the indicator. Both fluorometric and
colorimetric indicator displacement assay (C-IDA) titration
results suggest that 40 is selective for phosphate anions with
the order PPi ATP > ADP.
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
400
420
(a)
440
460
480
500
520
540
Wavelength (nm)
1.4
Absorbance
1.2
1
0.8
0.6
Cl
0.4
Zn2+
0.2
400
(b)
0
420
440
460
480
500
520
Wavelength (nm)
N
N
34
Zn2+
N
N
Zn2+
N N
Zn2+
Zn2+
Zn2+
35
SO3
36
O
OH
HO
HO
N
Zn2+
N
N
N
N
Zn2+
N
O
37
O
39
HO
HO
38
Gunnlaugsson and coworkers developed a supramolecular sensing ensemble for flavin monophosphate (41) based
on functional hybrid gold nanoparticles (AuNP) in aqueous
solution (Figure 22).51 The sensing ensemble also took
advantage of unique photophysical properties of lanthanide
ion complexes. A heptadendate Eu3+ cyclen complex 42
has a terminal thiol unit, which facilitates the binding of 42
onto the surface of AuNP to form AuNP-42. The excited
states of Eu3+ in this binary complex are quenched by
OH vibrational deactivation from water ligands, and, as a
result, no significant Eu3+ emission is observed. The displacement of such water ligands by the -diketone antenna
43 affords a red luminescent ternary complex AuNP-4243 upon sensitization of the Eu3+ excited states by the
employed the same zinc-binding motif with an m-terphenyl

linker (38).47 The reported binding constant for Pi and PPi
is 3.22 104 and 4.17 105 M1 , respectively. Indicators
pyrocatechol violet (8) and esculetin (39) were used in
the colorimetric IDA, and calibration curves for pyrophosphate were generated (Figure 20). Hong and coworkers also targeted pyrophosphate using similar receptors
with covalently attached chromophore or fluorophore as
reporters.48, 49
NH3
40
Figure 19 UVvis spectra of 2 (24 M) binding to 33 (a) and

UVvis spectra of addition of phosphate to 33 (24 M) and 2
(24 M) (b). (Reproduced from Ref. 45. American Chemical
Society, 2003.)
SO3
OH
OH
DOI: 10.1002/9780470661345.smc010
13
Relative intensity at 468 nm
250
200
150
y = 7.09x
R 2 = 0.983
100
50
0
0
10
[PPi]
15
(106
20
25
M)
Figure 20 Calibration curve for PPi measured by fluorescence change for the displacement of 39 from 38. (Reproduced from Ref. 47.
Wiley-VCH, 2009.)
N2
O1 C11
C11*
N1
C11
O1
Zn1
Zn1*
Zn1
O2
O3
N1
O1*
O2*
(a)
Figure 21
N
O
P
O
O2
Crystal structure of 40 (a) and its core structure (b). (Reproduced from Ref. 50. American Chemical Society, 2009.)
antenna. The AuNP-42-43 self-assembly was employed to

sense a series of coordinating ligands through displacement assay. It was found that flavin monophosphate (41)
could displace 43 from AuNP-42-43, and this resulted in
almost complete quenching of Eu3+ emission. Other biologically relevant phosphate containing species, including
AMP, ADP, ATP, cAMP, and NADP, only led to partial
quenching.
N1*
(b)
OH
O
O
OH
N
O
OH
N
HN
N
Eu
N
O
N
O
41
CF3
O
43
3Tf
OH2
OH2
42
SH
10
AuNP-42
Water-coordinated Eu
Switched off
AuNP-4243
Luminescent
AuNP
Switched on
AuNP-4241
Antenna 43 is
displaced
Switched off
Figure 22 Schematic demonstration of AuNP-42-43 selfassembly and displacement. (Adapted from Ref. 51. American
Pattern-recognition-based differential sensing has also

been explored to discriminate phosphate-related anions.52
Severin and coworkers used a MIDA for the identification
and quantification of different nucleotides in aqueous solution.53 By combining a rhodium complex 26 with three
indicators (Mordant yellow 10 (44), gallocyanine (27), and
Evans blue (45)), the absorbance changes were recorded
upon the addition of nucleotides. A two-dimensional LDA
plot, shown in Figure 23, was generated using absorbance
DOI: 10.1002/9780470661345.smc010
14
Concepts
O
Cl
Rh
Cl
Cl
OH
N
SO3Na
NH2
Rh
N
Cl
26
27
O
OH
OH
NaO3S
OH
NaO3S
45
OH
N
44
80
PPi
The metal ions and indicators in conjunction with the cross

reactivity created by the tripeptides in the receptors were
used to generate a suite of 45 IDAs. LDA analysis generated patterns with 100% classification for the six peptides
studied.
GTP
40
Score 2
ADP
0
AMP
40
H3N
ATP
R3
cAMP
80
HN
400
200
200
OH
NH2
N
R1
NH
HO
Cl
O
OH
N
47
HN
HN
data at five wavelengths, and all nucleotides investigated, including ATP, GTP, ADP, AMP, and cAMP,
were well separated. Moreover, it was demonstrated that
the MIDA allows the simultaneous measurements of the
concentrations of ATP and cAMP/PPi with a single colorimetric reading (Figure 24). The sensor ensemble also
displays selectivity toward nucleotides and pyrophosphate
over phosphate.
A more recent study by our group employed an arraysensing scheme to pattern peptide phosphorylation.54 The
receptors (46) were designed on the basis of a metal binding tris(2-pyridylmethyl)amine motif with appended guanidinium units for phosphate anion binding. Tripeptide arms
were incorporated into the hosts to increase the diversity
and cross reactivity. A library of hosts were prepared using
split-and-pool solid-phase synthesis starting from a tripodal
precursor prepared by solution phase synthesis. Eight metal
cations and nine indicators were screened using a previously
reported, similar, but simplified, receptor (33) and phosphoserine. Three metal ions (Co2+ , Ni2+ , and Cu2+ ) and three
indicators (pyrocatechol violet (8), celestine blue (47), and
gallocyanine (27)) were selected. After prescreening with
resin-bound receptors, five receptors were resynthesized.
HN
NH
H2N
NH
O
H3N
NH
Score 1
Figure 23 LDA plot for the analysis of five nucleotides and

pyrophosphate. (Reproduced from Ref. 53. Royal Society of
Chemistry, 2007.)
H3N
2
R1 R
400
OH
NH
R2
120
O3S
NH2
HN
H2N
NH
N
M2+
NH
N
HO
Cl
N
O
OH
27
46
SENSING OF CATIONS USING IDAs
A broad range of amine-containing molecules are involved

during biological processes, such as neurotransmitters in
signal transduction and products of biodegradation and
metabolism. As a result, the development of sensing and
detection systems for those amines has significant importance. Amines are normally protonated at physiological pH,
and the resulting ammonium cations are the sensing target.
Organic ammonium cations can form inclusion complex
with macrocyles such as calixarene55 and cucurbituril.56
Guest competition between cationic analyte and indicator
provides the basis for sensing.
Acetylcholine (50) is one of the most abundant neurotransmitters. Inouye and coworkers took advantage of
DOI: 10.1002/9780470661345.smc010
[PPi/cAMP] (mM)
0.8
0.6
0.4
0.2
0.0
0.0
0.2
0.4
0.6
0.8
1.0
ATP (mM)
Figure 24 Actual (circles) and predicted (crosses) concentrations for ATP and cAMP/PPi. (Reproduced from Ref. 53. Royal
Society of Chemistry, 2007.)
a resorcinol/acetaldehyde tetramer 48 (calix[4]arene) as a

receptor.57 The tetraphenolate formed in alkaline protic solvent can form an inclusion complex with alkylammonium
cations by electrostatic and/or cation interactions. The
N-alkylpyridinium-derived pyrene 49 was chosen as an
indicator. When bound to the receptor, its orange fluorescence was quenched through PET (photoinduced electron transfer) from the anionic oxygen in the receptor.
The addition of acetylcholine led to competition for the
binding cavity and, as a result, the dye was released
and its fluorescence was turned on. The dye displacement was also confirmed by NMR studies. The strong
interaction between 48 and the quaternary ammonium
cation in acetylcholine contributes to the high selectivity. One disadvantage of this approach is that the
strongly basic condition used causes substantial problems such as nucleophilic attack on the pyridinium and
solvolysis of the acetyl ester. Shinkai and coworkers
HO
OH
O
OH
HO
HO
X
49
OH
48
X
OH OH OH HO
51
OH
X
OH
X = SO3Na
2.5
1.5
Cl
50
0.5
[Acetylcholine or amino acid] / [52]
X
X
OH
HO
utilized calix[n]arene-p-sulfonates 51 (n = 4) and 52

(n = 6) as receptors for the sensing of acetylcholine in
neutral aqueous solution via a similar competition assay.58
Figure 25 shows the fluorescence regeneration upon addition of acetylcholine.
Schrader and coworkers developed a colorimetric IDA
for catecholamines.59 Catecholamines feature a phenylethylamine moiety with two ortho-hydroxy groups on the
phenyl ring. Common catecholamines include dopamine
(53), noradrenaline (54), and adrenaline (55). There are
three structural motifs in the designed receptors (56, 57, and
58): a xylylene bisphosphonate moiety for aminoalcohol
recognition, an ortho-aminomethylphenylboronic acid unit
for catechol binding, and an unnatural amino acidbased
linker. Alizarin complexone (5) was chosen as an indicator. When the host 58 was titrated into the dye in aqueous solution, the color changed from deep red to orange
with a clear isosbestic point. Upon addition of the competing catecholamine, the dye was released and the color
was reversed (Figure 26). All catecholamines studied gave
similar quantitative data under identical conditions. The
binding constants for noradrenaline and the indicator with
host 58 are 350 and 3440 M1 , respectively. The lower
affinity for catecholamines indicates that a large excess
of analytes is required to restore the original color of
the dye, and, as a result, the sensitivity of the assay is
decreased.
Relative fluorescence intensity
1.0
15
OH
OH
52
OH OH
Figure 25 Fluorescence response upon addition of various analytes into a solution of 49 (0.1 mM) and 52 (1.0 mM). Acetylcholine (), glycine (), L-aspartic acid (), L-proline (), Lphenylalanine ethyl ester hydrochloride (), and glycine methyl
ester hydrochloride (). (Adapted from Ref. 58. American
DOI: 10.1002/9780470661345.smc010
16
Concepts
OH
NH3
HO
HO
OH
CH3O
OLi
56: X =
57: X =
58: X =
55
(HO)2B
O
R
N XN
H
OH
54
NH2
HO
OH
53
OLi
OCH3
OH
NH3
NH
OH
O
C6H4 CH2 ; R = H
(CH2)3 ; R = H
(CH2)3 ; R = CH3
OH
5
O
O
Nau and coworkers designed a competition assay to monitor cationic species generated in amino acid decarboxylase catalyzed reactions.60 Two macrocyles, cucurbit[7]uril
(CB7, 59) and p-sulfonatocalix[4]arene (CX4, 51), were
Bound
2.5
Free
used as hosts. Two fluorescent dyes, Dapoxyl (60) and

aminomethyl-substituted 2,3-diazabicyclo[2.2.2]oct-2-ene
(DBO,61), were chosen as indicators. CB7 forms an inclusion complex with Dapoxyl, while CX4 forms an inclusion
complex with DBO (Figure 27). Their fluorescence modulation is different upon forming the complex. Both CB7 and
CX4 bind the enzyme substrate (amino acids) weakly, but
have higher affinity for the cationic alkylammonium products. As a result, only the enzymatic product can compete
with and displace the dye from the host. Once bound to
the cavity of CB7, the fluorescence intensity of Dapoxyl
increased 200-fold. The addition of amino acids did not
interfere with the formation of the inclusion complex CB7Dapoxyl. However, a significant decrease was observed in
fluorescence when the product amines were introduced to
the solution of CB7-Dapoxyl, indicating release of Dapoxyl
from the binding cavity (Figure 28). Similarly, DBO was
displaced from the complex CX4-DBO by the amine, but
turn on fluorescence was observed upon amine binding.
The sensing mechanism is similar to that of the chemosensing ensemble for acetylcholine described above. Using
these dye-displacement assays, label-free, nondestructive,
and real-time monitoring of amino acids decarboxylase
activity was achieved.
1.5
1
0.5
0
300
400
0.005
(a)
500
l (nm)
600
1.17
1.12
1.07
1.02
0.97
0.92
(b)
0.01
0.015
0.02
[Guest] (M)
Figure 26 Absorbance spectra of 5 upon addition of host 58

(a) and absorbance change at 460 nm in the presence of 5 and 58
upon addition of various analytes (b). Methoxytyrosine hydrochloride (black rhombus), fructose (gray triangle), catechol (gray
square), and noradrenaline (light gray rhombus). (Reproduced
from Ref. 59. Wiley-VCH, 2005.)
SENSING OF NEUTRAL ANALYTES

USING IDAs
Because electrostatic and reversible covalent interactions

are the main binding forces in the typical IDA, most analytes explored to date are charged. However, there are a
few examples of sensing neutral analytes using competition assay. One example is the supramolecular sensing
of caffeine reported by Waldvogel and coworkers.61 A
C3v symmetric, triphenylene ketal-based receptor 62 was
designed and synthesized. The aromatic motif allows
stacking with caffeine (63), while the appended urea provides a hydrogen bond interaction. A bathochromic shift
and 30% increase in quantum yield were observed when
caffeine was bound to 62. An NBD (nitrobenzoxadiazole)labeled caffeine analog T6BNF (64) was employed as
a competitive guest. The fluorescence intensity of 62 in
CH2 Cl2 decreased upon addition of T6BNF probably by
a Forster resonance energy transfer (FRET) mechanism.
The static quenching was confirmed by the relatively big
SternVolmer constant. When T6BNF was displaced by
caffeine, the fluorescence increased by a factor up to 4.
The advantage of the competition assay is evident because
a broad range of caffeine concentrations lead to signal
responses, while titration of 62 with caffeine reaches saturation rapidly (Figure 29).
DOI: 10.1002/9780470661345.smc010
SO3
SO3
NO
N
N
N
NO
N
17
O
N N
O
N N O
N N
O
O N N ON
N
N
N
NO N O N NN
O N
NO
N
N
N
O
O
N
O
H 3C
59
N+
CH3
H 3C
60
Weakly fluorescent
O 3S
O 3S
Strongly fluorescent
+
SO3
SO3
OHHO
CH3
NH3
NH3
N
N
N
N
HO
61
51
Strongly fluorescent
Weakly fluorescent
Figure 27 Inclusion complex formation between hosts (51, 59) and indicators (60, 61) and fluorescence response upon
association. (Reproduced from Ref. 60. Nature Publishing Group, 2007.)
R = C6H13
R
HN
HN
R
NH
O
H3C
O
O
R
HN
CH3
N
N
N
CH3
O
NH
O
HN
O
O
O
H3C
63
CH3
NO2
N
H
62
The second example focuses on an IDA for colorimetric

sensing of sulfur dioxide (SO2 ).62 SO2 is produced upon
burning sulfur-containing fuels and contributes to a series
of environmental problems such as acid rain. Because SO2
is colorless, the colorimetric sensing of SO2 is important.
Rudkevich and coworkers took advantage of noncovalent
interaction between amines and SO2 . It is well known that
secondary or tertiary amines and SO2 form 1 : 1 charge
transfer adducts. A zinctetraphenylporphyrin complex 65
was chosen as the indicator. When an amine (for example,
pyrrolidine) was added to a solution of 65 in CHCl3 , the
red solution changed to dark green and a bathochromic shift
was observed for the Soret band, indicating the formation
of 65amine adduct. Upon the introduction of SO2 , the color
was reversed and the Soret band was restored, indicating
that 65 was displaced by SO2 (Figure 30). The binding
constants for the complex between pyrrolidine and 65 and
for pyrrolidine and SO2 are 1.8 104 and 2.0 104 M1 ,
respectively. The sensing ensemble discriminates between
(CH2)6
N
O
64
SO2 and CO, CO2 , N2 O, NO2 , and H2 O. Lippard and

coworkers also utilized the Lewis acidbase interaction for
fluorescent detection of nitric oxide (NO).63 Fluorophores
(dansylimidazole or dansylpiperazine) bind to the axial
site of the metal center in a dirhodium tetracarboxylate
complex. Displacement of fluorophores by NO causes a
fluorescence change.
Ph
N
Zn2+
Ph
Ph
R3N :
+ SO2
Ph
65
DOI: 10.1002/9780470661345.smc010
R3N
SO2
18
Concepts
+ Arginine
NH2
Lysine
+
H3N
NH3
H2N
NH3 H N
2
H3N
(a)
NH3
N
H
Cadaverine
H3N
COO
Histidine
decarboxylase
+
NH3
NH3
COO
Ornithine
decarboxylase
+
NH3
NH3
H3N
HN
Agmatine
Lysine
(870 M1)
Ornithine
COO HN
Arginine
decarboxylase
NH2
NH3
N
H
COO
Lysine
decarboxylase
Histidine
Histamine
Putrescine
Histidine
(400 M1)
Arginine
(310 M1)
Ornithine
(380 M1)
Fluorescence (a.u.)
100
Putrescine
(3.7 105 M1)
Agmatine
(1.1 106 M1)
Cadaverine
(1.4 107 M1)
50
Histamine
(3.2 104 M1)
0
(b)
Cadaverine
(2.7 105 M1)
300
Fluorescence (a.u.)
5
10
0
25
Analyte concentration (m)
50
Agmatine
(7.1 106 M1)
10 15
Putrescine
(2.6 105 M1)
Histamine
(9.0 104 M1)
200
Arginine
(2.8 103 M1)
Lysine
(1.0 103 M1)
Histidine
(560 M1)
Ornithine
(210 M1)
100
(c)
100
200
100
200
100
200
100
200
Analyte concentration (M)
Figure 28 (a) Substrateproduct pair for four decarboxylases. (b) Fluorescence response upon titrations of amino acids and their
decarboxylation products into solution of Dapoxyl (2.5 M) and CB7 (10 M) at pH 6.0. (c) Similar titrations in the presence of DBO
(100 M) and CX4 (100 M). Binding constants are shown in parentheses. (Reproduced from Ref. 60. Nature Publishing Group,
2007.)
Neutral analytes containing vicinal diols, including sugars,64, 65 have also been the target of displacement assays.
The reversible covalent interaction between 1,2-diols and
phenylboronic acids to form cyclic boronate esters has
been extensively studied. The strength of this interaction
depends on the solvent, sterics, and pKa of boronic acid.
Our group has successfully developed an IDA for the
identification and analysis of threo diols.66 The receptors
(66, 67, and 68) were designed on the basis of orthoaminomethylphenylboronic acids, which were prepared
using a one-pot reductive amination reaction. Similar
structural motifs have also been incorporated into sensing
systems by Shinkai67 and others for sensing and detection
of saccharides.68 In our IDAs, the competition between the
diol and indicator for the boronic acid affords an equilibrium. The total concentration of threo diols was measured
DOI: 10.1002/9780470661345.smc010
19
4.5
4.0
F / F0 (392 nm)
3.5
3.0
2.5
1.5
Absorbance
1.5
1.0
0
50
100
150
200
250
300
[63] (M)
Ph
OH
HO
66
380
Ph
HO OH
67
O
OH
Ph
OH
68
O
Br
OH
HO
OH
Na 8
Br
Na
O
SO3
OH
OH
400
420
440
460
480
Wavelength (nm)
1.5
+ SO2
1.0
0.5
0.0
380
400
420
440
460
Wavelength (nm)
(c)
Figure 30 (a) Color change of 65 upon addition of pyrrolidine

and then SO2 in CHCl3 . (b) UVvis spectra of 65quinuclidine
upon addition of SO2 . (c) UVvis spectra of 65pyrrolidine
upon addition of SO2 . (Reproduced from Ref. 62. American
HO
O
69
0.5
Ph
1.0
(b)
Absorbance
by the use of an achiral receptor 66. The enantiomeric

excess was also determined after chirality was introduced
into the receptors (Figure 31, see more details in a later
section). By taking advantage of this competition assay, our
group was able to analyze the yield and enantiomeric excess
of an asymmetric sharpless dihydroxylation reaction.69 The
PCA demonstrated good chemoselective and enantioselective separation of the subtly different diols studied.
+ SO2
0.0
Figure 29 Fluorescence response of 62 (10 M) at 392 nm upon

caffeine addition in the presence of 50 M of 64 (), 110 M of
64 (), and in the absence of 64 (). (Reproduced from Ref. 61.
HO
+ Amine + SO2
(a)
2.0
O3S
OH
HO
9
OH
Boger and coworkers.70 The assay is rapid, nondestructive,

technically nondemanding, and compatible with highthroughput screening. The assay employs ethidium bromide
(70) or thiazole orange (71) as the indicator and the DNA
intercalator. Once bound to hairpin DNAs, the fluorescence
quantum yield of these molecules increases. Addition of a
DNA-binding reagent displaces the indicator and leads to a
decrease in fluorescence. The percentage of indicator displacement is directly related to the extent of binding. For
an individual DNA sequence, titration of a compound into
a DNA-indicator ensemble provides binding stoichiometry
and affinity. In an array format, the profile of displacement
in a library of DNA sequences establishes a compounds
IDAs USING BIOLOGICAL HOSTS

NH2
Various biological molecules, including peptides, proteins, and nucleic acids, generate vast chemical diversity that can be used as hosts in competition assays.
One well-known protocol is the fluorescent intercalator
displacement assay for targeting nucleic acids binding
properties of small molecules and proteins, pioneered by
S
H2N
N
71
70
DOI: 10.1002/9780470661345.smc010
(a)
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
(R,R )- Hydrobenzoin
(S,S )- Hydrobenzoin
Ph
(R )
N
(R )
B Ph
HO OH
KHG(R,R ) = 27 1.8
KHG(S,S ) = 4.9 1.3
0.001 0.002 0.003 0.004 0.005 0.006

[G ]I (M)
Concepts
20
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
(R,R )- Hydrobenzoin
(S,S )- Hydrobenzoin
Ph
(S )
N
(S )
B Ph
HO OH
KHG(R,R ) = 4.3 0.6

KHG(S,S ) = 28 2.2
0.001 0.002 0.003 0.004 0.005 0.006

[G ]I (M)
(b)
Figure 31 Binding isotherms for hydrobenzoin with 68 (a) and 67 (b). All binding constants are given in units of 102 M1 . The two
hosts display opposite chiral preference, indicating that they are cross reactive with each other. (Reproduced from Ref. 66. American
sequence selectivity. Alternatively, a library of compounds

can be screened in a high-throughput fashion for the
discovery of high-affinity binders for a defined sequence.
The DNA-binding affinity and selectivity of proteins can
also be studied using this strategy.
One class of oligonucleotide-based biological hosts
widely used in supramolecular sensing are RNA and
DNA aptamers. Aptamers can be selected for affinity to
most chemical entities including both small molecules and
macromolecules. The interactions between aptamers and
dyes have been well explored. Ellington and coworkers
explored various aptamerdye conjugates for targeting
small molecules and proteins. Stojanovic and coworkers developed an aptamer (72)-based colorimetric sensor
for cocaine (73).71, 72 The so-called anti-cocaine aptamer
MNS-4 binds cocaine via a hydrophobic pocket formed
by a noncanonical three-way junction. This aptamer has
a low micromolar dissociation constant and selectivity for cocaine. Thirty-five dyes were screened and a
cyanine dye, diethylthiotricarbocyanine iodide (74), displayed a large absorbance change in the presence of
the aptamer and micromolar concentrations of cocaine.
The dye is only sparingly soluble in the binding buffer.
However, complex formation with the added aptamer
resulted in significantly increased absorption. The two
peaks at 760 and 670 nm in the absorption spectrum
correspond to bound monomer and dimer indocyanine
respectively. Upon addition of cocaine, the monomeric
dye was released first, and as a result, the absorbance
at 760 nm decreased (Figure 32). A calibration curve
was generated from the change in absorbance at 760 nm
over various cocaine concentrations. Cocaine metabolites benzoyl ecgonine (75) and ecgonine methyl ester
(76) showed no interference in this competition assay.
The authors also described aptamer-based thiazole orange
displacement assay for tobramycin, theophylline, ATP,
GTP, and cAMP.73
NH O
OCH3
74
73 O
G
G G G
A
T
A A
7274
A
+
A T
T
C
7273
A
A
NH O
OCH3
O
75
T G A
G G
NH O
A
G
T
A
G
G
T G
OH
O
76
Our group recently explored the diverse binding capacity

of serum albumin (SA) and used it as a platform for differential sensing of terpenes.74 SA is a natural protein that
can bind a broad range of species, including metal ions and
hydrophobic molecules such as fatty acids, steroids, and
hormones. The proposed sensing ensemble is composed of
SA, the indicator 6-propionyl-2-dimethylaminonaphthalene
(PRODAN, 77), and/or hydrophobic additive (Figure 33).
Bovine serum albumin (BSA), human serum albumin
(HSA), and rabbit serum albumin (RSA) were employed
as hosts. A hypsochromic shift in fluorescence emission
was observed when SAs were added to aqueous solution
of PRODAN. The binding affinity of PRODAN to SAs
DOI: 10.1002/9780470661345.smc010
15
F2 (3.87%)
Absorbance
0.199
5
15
25
0.004
500.0
900.0
(nm)
(a)
15
15
25
15
25
F1 (95.44%)
(a)
15
F2 (17.45%)
100
90
A/A%
21
80
70
Linalool
a-Terpineol
Nerol
Geraniol
Citronellol
60
15
25
50
(b)
200
400
600
Concentration of cocaine (M)
800
Figure 32 UVvis spectra of 74 in the presence of 72 upon

addition of cocaine (a) and calibration curve for cocaine using
absorbance data at 760 nm (b). (Reproduced from Ref. 72.
Terpene
12
10
8
6
4
2
0
2
4
6
8
10
12
20
15
10
5
0
5
10
15
20
0 10
20
0
1
0
2
%)
2540 30
2.73
(c)
F1 (6
Hydrophobic
additive
Serum albumin
32%
(26,
PRODAN
F2
Figure 33 Schematic illustration of the proposed chemosensing

ensemble. (Reproduced from Ref. 74. American Chemical
Society, 2009.)
follows the order BSA > HSA > RSA. The binding profiles of five terpenes (linalool, nerol, geraniol, citronellol,
and -terpineol) were then defined. Upon addition of terpenes into the sensing ensemble, the fluorescence intensity
at the max for the free indicator increased, while the intensity at max for the bound indicator changed to different
directions depending on the SA used. These fluorescence
modulations result from allosteric changes in the binding
site of PRODAN together with some degree of indicator displacement. LDA showed discrimination of the five
terpenes studied; however, almost all discrimination took
place along one axis (Figure 34). The addition of deoxycholate to the sensing ensemble increased the discrimination
along the second axis. Terpenes in complex mixtures, such
as perfumes, could also be discriminated and identified
using a similar approach.
15
F1 (80.18%)
(b)
F3 (8.75%)
Linalool
a-Terpineol
Nerol
Geraniol
Citronellol
Perfume
Pertume-linalool
Pertume-a-terpineol
Perfume-nerol
Perfume-geraniol
Perfume-citronellol
30 40
Figure 34 (a) LDA plot for five terpenes studied. (b) LDA plot
with deoxycholate addition. (c) LDA plot for the terpenes in the
presence of deoxycholate and Masak perfume. (Reproduced from
Ref. 74. American Chemical Society, 2009.)
77
ENANTIOSELECTIVE IDAs
INTRODUCTION
The field of asymmetric synthesis based on either organic

or organometallic catalysts has been growing dramatically in the past decade. As a result, high-throughput
DOI: 10.1002/9780470661345.smc010
22
Concepts
OCH3
N
B
HO
OH
66
HO
OCH3
OH
78
HO
N
B
OH OCH3
HO
79
OH OCH3
80
O
OH
HO
HO
O
69
SO3
+
Na 8
determination of enantiomeric excess (ee) is in growing

demand. Traditional approaches for ee measurement include
chiral gas chromatography GC and high-performance liquid chromatography (HPLC). Our group has pioneered the
development of optical enantioselective IDAs with chiral receptors. Upon binding of an enantiomeric mixture
of a chiral analyte to an optically pure chiral receptor,
two diastereomeric hostguest complexes are formed. The
stability difference between two diastereomers leads to differing extents of indicator displacement, and, as a result, a
difference in optical signals (absorbance or fluorescence)
can be detected. A calibration curve of change in optical signal over ee can be generated and then the ee of
an unknown sample can be measured. The enantioselective indicator displacement assay (eIDAs) can be readily
performed in a plate reader, and, therefore, is particularly
suitable for high-throughput ee screening. Moreover, in
some cases, the concentration and enantiomeric excess can
R
N
B R
O O S
3
O
OH
OH
be determined in a single plate.
H : I +
Our first eIDA was developed for chiral -hydroxycarboxylates (Figure 35) and vicinal diols using chiral arylboronic acids as receptors.75, 76 The reversible interaction
between arylboronic acids and -hydroxycarboxylates or
diols has been described in the previous section. Our original design is based on the hypothesis that the incorporated
stereocenters near the postulated BN dative bond in the
receptors would induce enantioselectivity for the binding
between the receptor and two enantiomers of the analyte. However, extensive studies reveal that the proposed
BN dative bond does not exist to an appropriate extent
in protic solvents.7779 A small library of achiral and chiral phenylboronic acids (66, 7880) was synthesized, and
HO
OH
HO
O3S
HO
R
N
B
R
O O
R
O
+
OH
O
OH
(a)
O
R
N
B R
O
O
O
(b)
KR

H :GR + I
KS
GS

H : GS + I
H : I + GR
HO
OH
N
O
B
R
O O
R
O
HO
HO
Figure 35 Enantioselective indicator displacement assay for -hydroxycarboxylates with 8 (a) and 69 (b). (Adapted from Ref. 76.
DOI: 10.1002/9780470661345.smc010
fluorescent indicator 4-methylesculetin (69), provided a relatively broad dynamic range where eIDAs were employed
to determine the ee of -hydroxycarboxylates and vicinal
diols. For example, the association constants of receptor
79 with 69, D-phenyllactic acid (PL) and L-PL, were found
to be 3.02 104 , 4.35 103 , and 1.09 104 M1 , respectively, measured by fitting to a 1 : 1 binding isotherm or
competitive binding methods. Figure 36 shows the binding
isotherms of D-PL or L-PL in the presence of 8 and host
66 or 79. As expected, the achiral receptor showed no
chiral discrimination between two enantiomers, but can
be used to determine the total concentration of PL. The
average absolute error for ee determination is 10%.
-Amino acids ee determination using eIDAs has also
been achieved in our group (Figure 37).80 Copper complexes of C2 symmetric chiral 1,2-diamines were utilized
as receptors (8184), and chrome azurol S (CAS, 85) was
chosen as a colorimetric indicator. Coordination of CAS to
0
D-PL/79
L-PL/79
D-PL/66
L-PL/66
0.2
0.4
0.6
0.8
1
1.2
10
15
20
25
30
35
[Analyte (D or L-PL)]/[Receptor (66 or 79)]
Figure 36 Absorbance change at 520 nm for 8 and host 66 or

79 upon addition of D-PL or L-PL. (Adapted from Ref. 75.
several indicators were screened to improve the enantioselectivity and sensitivity of the assay. Specifically, the colorimetric indicator pyrocatechol violet (8), in conjunction with
L-
O
O
R
Cl
Cl
SO3
81-L-AA
CAS
More stable diastereomer

Cl
HO
+
N
H2
O
R
id
o ac
amin
CuII
H
CuII
H3 N +
O
O
O
D- a
SO3
Cl
O
min
o ac
81-CAS
id
O
O
N
Cu
O
H3 N +
HO
II
N
H2
O
R
Cl
Cl
R
SO3
CAS
81-D-AA
(a)
Less stable diastereomer
+
O
O
H3 N +
O
H
ino
acid
Cu
H
HO
N
N
H2
II
+
Cl
Cl
O
N
N
SO3
O
Cu
m
L-a
R
2
83-L-AA
II
Cl
CAS
More stable diastereomer
Cl
SO3
D-a
min
o ac
83-CAS
id
O
H3 N +
Cu
O
II
N
H2
HO
+
Cl
Cl
R
SO3
(b)
Figure 37
23
83-D-AA
Less stable diastereomer
CAS
Enantioselective indicator displacement assay for -amino acids using host 81 (a) or 83 (b) and indicator CAS.
DOI: 10.1002/9780470661345.smc010
24
Concepts
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
100%
50%
0%
ee
50%
100%
0%
20%
40%
60%
80%
100%
0%
20%
40%
60%
80%
100%
20%
40%
60%
80%
ee
100%
Figure 38 Enantiomeric excess calibration curve for isoleucine using 81 as the host and CAS as the indicator. (Reproduced from
Ref. 80. American Chemical Society, 2008.)
Valine
Aspartate
Histidine
Isoleucine
Valine test samples
Aspartate test samples
Histidine test samples
Isoleucine test samples
20%
40%
60%
80%
ee
100%
(a)
Valine
Tryptophan
Alanine
Serine
Valine test samples
Tryptophan test samples
Alanine test samples
Serine test samples
(b)
Figure 39 The 96-well plate used for -amino acids ee analysis. For valine, aspartate, histidine, and isoleucine, using 81 as the host
(a) and for valine, tryptophan, alanine, and serine, using 83 as the host (b). (Reproduced from Ref. 81. American Chemical Society,
2008.)
the Cu2+ center led to a color change from yellow (429 nm)
to intense blue (602 nm) in 1 : 1 water/methanol solution at
pH 7.5. Upon addition of -amino acids, the color change
was reversed due to the release of CAS from the receptor.
The steric hindrance between the side chain of -amino
acids and the chiral receptor results in a different stability
of the diastereomers formed, and, therefore, a different
extent of dye displacement. Those steric interactions were

confirmed by X-ray crystal structure analysis. Host 81 has
stronger affinity for L-amino acids, while its enantiomer
82 shows preference toward D-amino acids. The opposite
enantioselectivity for aspartate, asparagine, and histidine
was probably due to a different binding mode involving
side-chain coordination. Chiral discrimination of 13 of the
DOI: 10.1002/9780470661345.smc010
17 studied -amino acids was observed (Ala, Arg, Asn,
Asp, His, Ile, Leu, Met, Pro, Ser, Thr, Trp, and Val). Calibration curves were obtained, and the one for isoleucine is
shown in Figure 38. Enantiomeric excess of the unknown
samples was determined with an average error <14%. All
eIDAs for -hydroxycarboxylates, diols, and -amino acids
have been successfully transitioned into high-throughput
analysis using an array-sensing protocol.81 Figure 39 shows
a 96-well plate constructed for analysis of ee of -amino
acids.
O
O
N
Cu
H
2+
Cl
3Na
SO3
82
H
Cu
83
85
N
Cu2+
2. E. V. Anslyn, J. Org. Chem., 2007, 72, 687.

3. S. L. Wiskur, H. Ait-Haddou, J. J. Lavigne, and E. V.
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1609.
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14.
Cl
81
Cu2+
O
HO
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Competitive binding assays, IDAs, are a powerful and facile

technique for the creation of molecular sensors. Guest competition between an analyte and an indicator for a host
provides the rationale of IDA. As illustrated above, IDAs
have been used to target a broad range of analytes, including
anions, cations, and neutral species. Moreover, both synthetic and biological receptors have been employed as hosts
for IDAs. This method provides chemists a sensing strategy and paradigm complementary to the traditional indicatorspacerreceptor approach. IDAs can be employed
to create a detection and sensing scheme for individual
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DOI: 10.1002/9780470661345.smc010

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Competition Experiments

Lei You and Eric V. Anslyn

be used to evaluate binding strength and selectivity. One

The interactions between the indicator or analyte and the

(b) Receptor indicator

can be diminished significantly. Second, a broad range of

SINGLE ANALYTE SENSING VERSUS

In traditional IDAs, one receptor is specifically designed

ANIONIC IDAs USING ORGANIC

The majority of IDAs have been developed for anions.

Figure 2 Absorbance spectrum of 2: addition of 1 to a solution

104c (mol L1)

Figure 3 UV calibration curve for citrate (75 M 1, 14 M

inositol-1,4,5-trisphosphate (IP3),20 and gallate21 by our

Figure 4 Absorbance spectrum of 5: addition of 4 to a solution

Figure 5 Calibration curves at 450 nm for tartrate (), malate

chemosensing ensemble span a large wavelength range. The

chemosensing ensemble is composed of receptor 10 and

Using the similar pinwheel scaffold described above,

Emission intensity (a.u.)

Reproduced from Ref. 23. 2003.

Figure 8 Increase in fluorescence intensity of HPTS by titrating

When HPTS was displaced from 12 by polyanionic ATP at

Figure 7 UVvis spectra of 8 (54 M) in the presence of

The sensing of halogen anions has also been extensively

Complex formation between 14 and 15 or fluoride.

merocyanine (16) were also explored in the displacement

METAL COMPLEXING IDAs FOR

Transition metalligand coordination has been well

Our group also created a chemosensing ensemble for

[F ] (105 mol dm3)

Figure 10 Absorbance change at 571 nm upon addition of

Amino acids are the basic building blocks of proteins and

Figure 11 Absorbance change upon addition of NO3 (), Br

individual amino acids play crucial roles in physiological

Fabbrizzi and coworkers have successfully demonstrated

Figure 12 Absorbance spectrum of 8: addition of 21 to a

indicator 6-carboxy-tetramethyl rhodamine (23) was used

Besides IDAs for a particular amino acid, nonselective

Amino acid concentration (M)

Figure 14 Calibration curves for various amino acids using

various combinations of receptors, indicators, and analytes

Figure 15 PCA plot for the discrimination and identification of

was excellent. The clustering of the data was visualized

Figure 16 Change in fluorescence intensity of 30 at 580 nm

enzyme assay.42 The assay is amenable to high-throughput

Phosphate-related anion species

Reymond and coworkers designed a new enzyme assay

Phosphate-related anion species, including phosphate (Pi),

Figure 18 Absorbance spectra of 8 (50 M) in the presence

functionalized with attached guanidinium groups designed

(624 nm), indicating binding of 8 to the metal center in the

Our group developed a C3v symmetric receptor 33 with

The bis(2-picolyl)amine (Dpa) unit designed for zinc

employed the same zinc-binding motif with an m-terphenyl

Figure 19 UVvis spectra of 2 (24 M) binding to 33 (a) and

Relative intensity at 468 nm

antenna. The AuNP-42-43 self-assembly was employed to

Pattern-recognition-based differential sensing has also

The metal ions and indicators in conjunction with the cross

Figure 23 LDA plot for the analysis of five nucleotides and

SENSING OF CATIONS USING IDAs

A broad range of amine-containing molecules are involved