Optical Supramolecular Sensing of Creatinine

Article abstract of DOI:10.1021/jacs.9b12071

Calix[4]pyrrole phosphonate-cavitands were used as receptors for the design of supramolecular sensors for creatinine and its lipophilic derivative hexylcreatinine. The sensing principle is based on indicator displacement assays of an inherently fluorescent guest dye or a black-hole quencher from the receptor's cavity by means of competition with the creatinine analytes. The systems were thermodynamically and kinetically characterized regarding their 1:1 binding properties by means of nuclear magnetic resonance spectroscopy (1H and 31P NMR), isothermal titration calorimetry, and optical spectroscopies (UV/vis absorption and fluorescence). For the use of the black-hole indicator dye, the calix[4]pyrrole was modified with a dansyl chromophore as a signaling unit that engages in F?rster resonance energy transfer with the indicator dye. The 1:1 binding constants of the indicator dyes are in the range of 107 M-1, while hexylcreatinine showed values around (2-4) × 105 M-1. The competitive displacement of the indicators by hexylcreatinine produced supramolecular fluorescence turn-on sensors that work at micromolar analyte concentrations that are compatible with those observed for healthy as well as sick patients. The limit of detection for one of the systems reached submicromolar ranges (110 nM).

Full text of DOI:10.1021/jacs.9b12071

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Article
Optical Supramolecular Sensing of Creatinine
Andrés F. Sierra, Daniel Hernández-Alonso, Miguel Angel Romero,
Jose Antonio Gonzalez Delgado, Uwe Pischel, and Pablo Ballester
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b12071 • Publication Date (Web): 11 Feb 2020
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Optical Supramolecular Sensing of Creatinine.
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Andrés F. Sierra1,4, Daniel Hernández-Alonso1, Miguel A. Romero2, José A. González-Delgado2, Uwe
Pischel2,* and Pablo Ballester1,3,*.
1Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology, Avgda. Països
Catalans 16, 43007 Tarragona (Spain).
2CIQSO - Center for Research in Sustainable Chemistry and Department of Chemistry, University of Huelva, Campus de El
Carmen s/n, E-21071 Huelva (Spain).
3Catalan Institution of Research and Advanced Studies (ICREA), Passeig Lluís Companys 23, 08018 Barcelona (Spain).
4Universitat Rovira i Virgili, Departament de Química Analítica i Química Orgànica, c/Marcelꞏlí Domingo, 1, 43007
Tarragona (Spain).
ABSTRACT: Calix[4]pyrrole phosphonate-cavitands were used as receptors for the design of supramolecular sensors for creati-
nine and its lipophilic derivative hexylcreatinine. The sensing principle is based on indicator displacement assays of an inherently
fluorescent guest dye or a black-hole quencher from the receptor’s cavity by means of competition with the creatinine analytes. The
systems were thermodynamically and kinetically characterized regarding their 1:1 binding properties by means of nuclear magnetic
resonance spectroscopy (1H and 31P NMR), isothermal titration calorimetry, and optical spectroscopies (UV/vis absorption and
fluorescence). For the use of the black-hole indicator dye, the calix[4]pyrrole was modified with a dansyl chromophore as signaling
unit that engages in Förster resonance energy transfer with the indicator dye. The 1:1 binding constants of the indicator dyes are in
the range of 107 M-1, while hexylcreatinine showed values around (2-4)  105 M-1. The competitive displacement of the indicators
by hexylcreatinine produced supramolecular fluorescence turn-on sensors that work at micromolar analyte concentrations that are
compatible with those observed for healthy as well as sick patients. The limit of detection for one of the systems reached submi-
cromolar ranges (110 nM).
well-known receptors for the effective binding of neutral polar
INTRODUCTION
species, as well as mono- and poly-atomic anions.15 The supe-
rior binding properties of these receptors are inherent to the
structural elements that relate to their cone conformation.
Moreover, aryl-extended calix[4]pyrroles can be synthetically
elaborated both at their upper and lower rims. These structural
modifications of the receptor scaffold are translated into mod-
ulations of solubility and binding properties. Fluorescent ca-
lix[4]pyrrole chemosensors for the recognition of ani-
The level of creatinine (Cr, see Figure 1) in human bodily
fluids is a widely applied clinical indicator for monitoring
,
kidney and renal functions.1 2 Creatinine is a metabolic waste
product, being related to muscle activity, and any anomalous
secretion due to organ malfunction leads inevitably to higher
concentrations of Cr in blood serum and/or urine. Methods for
molecular biosensing of creatinine using simple fluorescent
probes or in combination with bioconjugates have been report-
,
,
,
,
,
,
,
,
,
,
ons16 17 18 19 20 21 22 23 24 25 and cations26 27 are known in the litera-
ture. In contrast, the development of fluorescent ca-
lix[4]pyrrole chemosensors for the detection and quantifica-
tion of relevant polar biomolecules remains practically unex-
plored.28
, , , ,
ed.2 3 4 5 6 While these latter approaches rely on unspecific
interactions between creatinine and the probe molecule, su-
pramolecular sensing offers distinct benefits for the design of
selective and specific sensors by building on size, shape and
function complementarity between receptor and analyte. Un-
fortunately, most synthetic receptors designed for creatinine
binding are flat and edge-functionalized with divergent hydro-
In 2016, we introduced an ion-selective-electrode (ISE) suita-
ble for the detection of Cr in bodily fluids.29 This ISE hinged
on the use of a novel mono-phosphonate calix[4]pyrrole 1 as
ionophore (Figure 1). Receptor 1 displayed high affinity for
Cr and its protonated form Cr•H+. In contrast to the previous-
ly published plane creatinine receptors,7,10,11,12 macrocycle 1
features a deep aromatic cavity that is functionalized with a
single phosphonate group at the open end (upper rim) and four
pyrrole NHs at the closed end.
, , ,
,
gen bonding motifs.7 8 9 10 11 These characteristics make them a
non-ideal choice for supramolecular sensing design. Hence, it
is not surprising that supramolecular approaches to creatinine
sensing, using synthetic receptors, are very scarce in litera-
,
ture.12 13
The selection of the synthetic receptor unit is a key element in
the design of supramolecular sensors. In this respect, aryl-
extended calix[4]pyrroles14 and their cavitand derivatives are
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Scheme 1. Schematic approaches to optical supramolecu-
lar sensing of creatinine (analyte) using a calix[4]pyrrole
scaffold (receptor). Top: Displacement of a black-hole
quencher (BHQ) and deactivation of FRET quenching of a
receptor-appended fluorophore (signaling unit). Bottom:
Displacement of a fluorescent dye.
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Figure 1. Top: molecular structures of receptor 1, creatinine Cr, the 1:1
inclusion complex Cr⊂1 and its binding equilibrium. Bottom: structure of
the dansyl-modified calix[4]pyrrole receptor 2.
In both cases, we choose pyridyl-N-oxides as binding motifs
of the indicator guest. Pyridyl-N-oxides are known to form
host-guest complexes with calix[4]pyrroles having binding
constants (106-107 M1)32 that are comparable with or slightly
higher than those of Cr and its derivatives. On the one hand,
receptor 2 could be combined with a black-hole quencher
guest (3, Figure 2) which on binding deactivates the dansyl
fluorescence by Förster resonance energy transfer (FRET).
The competitive displacement of the indicator by HexCr (as
lipophilic Cr analogue) is expected to be signaled by the ob-
servation of fluorescence turn-on. On the other hand, receptor
1 could be combined with a fluorescent guest (4, Figure 2). On
binding by the receptor, the guest would then undergo fluores-
cence quenching which is inverted on competitive displace-
ment with HexCr. This again yields a fluorescent turn-on
sensor. Based on these approaches highly sensitive optical
supramolecular sensors for creatinine derivatives are present-
ed.
The cavity dimensions of 1 are ideal for the inclusion of Cr by
surrounding most of its surface. The polar groups of 1 offer com-
plementary hydrogen-bonding, π-π, CH-π interactions with the
included Cr. These attributes translate into an efficient binding
for Cr in organic solvents. Moreover, the 1:1 Cr⊂1 inclusion
complex is kinetically stable on the 1H NMR chemical shift time-
scale.
RESULTS AND DISCUSSION
Synthesis
Figure 2. Molecular structures of HexCr, the para-substituted pyridyl-N-
oxides 3 and 4 and the dansyl reference model 5.
The synthesis of receptor 2 involved four reaction steps start-
ing from the known α,α,α,α-tetra-hydroxy-calix[4]pyrrole 6
(Scheme 2).33 The nitro mono-aryl bridged intermediate 7 was
prepared using reaction conditions previously reported for a
structurally related compound.34 The nitro-diol-cavitand 7 was
isolated, as a racemic mixture, after column chromatography
purification in 32 % yield. The incorporation of the bridging
phosphonate group was achieved by reacting the nitro-diol 7
with phenylphosphonic dichloride using triethylamine as base.
The reaction produced a mixture of two diastereoisomers, 8in
and 8out, which differ in the relative spatial orientation of the
P=O group with respect to the aromatic cavity. Diasteroiso-
mers 8in and 8out were isolated as pure compounds after
column chromatography purification of the reaction crude in
46% and 22% yield, respectively.
Drawing on calix[4]pyrrole 1 we present herein optical supra-
molecular sensors for creatinine and derivatives. In an expan-
sion, the scaffold of 1 was modified with a dansyl fluorophore,
yielding receptor 2 (Figure 1). Using fluorescence emission as
output signal of a molecular sensor provides a convenient way
of analyte detection, even with the naked eye. In addition,
fluorescence can be monitored for relatively low concentra-
tions (sub-micromolar) of the sensor, improving the sensitivity
further.
A priori two photophysical design strategies (see Scheme 1)
based on the popular and functionally flexible competitive
,
indicator-displacement-assays (IDA),30 31 could be followed
with receptors 1 and 2.
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Scheme 2. Synthetic scheme for the preparation of the
tion of the synthesized pyridine was performed using m-
chloroperoxybenzoic acid (m-CPBA).37
fluorescent mono-phosphonate cavitand 2. The structures
of the cavitands 7 and 8in are depicted as single enantio-
mers for simplicity.
The fluorescent N-oxide 4 was isolated after column chroma-
tography purification, using neutral alumina, in 58% yield.
This strategy was not implemented in the synthesis of 3 owing
to the presence of additional nitrogen atoms susceptible to N-
oxidation with the peroxyacid treatment.38 Instead, we synthe-
sized 3 following a reported procedure that involves the prepa-
ration of 4-amino-pyridine-N-oxide, its diazotization and
subsequent coupling with dimethyl-aniline.39 Starting from 4-
Boc-amino-pyridine, we isolated 3 in high purity and 11%
overall yield.
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Finally, due to the reduced solubility of Cr in organic sol-
vents, we prepared the lipophilic derivative HexCr. The com-
pound was obtained, following a known protocol, 40 by treating
methyl N-cyano-N-hexyl-aminoacetate with aqueous ammonia
at room temperature. HexCr proved to be soluble up to 1.5
mM in dichloromethane and chloroform, thereby enabling
binding studies by NMR spectroscopy (see below).
Binding studies of 2 with HexCr
1
Gratifyingly, single crystals of 8in and 8out (Figures S63 and
S64), that were suitable for X-ray diffraction analysis, were
obtained from acetone and dichloromethane solutions, respec-
tively (Figure 3). In the solid state, 8in showed the aryl-
extended calix[4]pyrrole core in cone conformation with one
molecule of acetone included in the aromatic cavity. The four
pyrrole NHs interact with the acetone guest via hydrogen
bonding. The nitro-aryl bridging group adopted a pseudo-
equatorial conformation.35
We initially probed the interaction of 2 with HexCr using H
1
and 31P NMR spectroscopy. The H NMR spectrum of 2 at
mM concentration in CDCl3 solution showed sharp and well-
resolved proton signals (Figure 4a, left panel).
The addition of 0.5 equiv. of HexCr to a mM solution of 2 in
CDCl3 produced the appearance of a new set of signals that
were assigned to the protons of the bound host (Figure 4b, left
panel). Remarkably, the bound pyrrole NHs resonated as four
sharp singlets around 9.8 ppm. The significant downfield shift
(∆δ = +1.5-1.7 ppm) experienced by the NH protons testifies
their involvement in hydrogen bonding interactions with the
oxygen atom of the bound HexCr. Moreover, we detected two
new signals in the aliphatic region of the spectrum. These
signals were assigned to the protons H1 and H7 of the bound
HexCr. The H1 proton experienced a large upfield shift (∆δ =
2.71 ppm) with respect to its chemical shift value in the free
HexCr (Figure S47). This observation supports the deep in-
clusion of HexCr in the aromatic cavity of 2, where the H1
proton experiences the shielding effect exerted by the four
meso-phenyl groups of the receptor.
The addition of incremental amounts of HexCr produced a
gradual increase in intensity of the proton signals assigned to
bound 2 at the expenses of those of the free counterpart. In the
presence of 1 equiv of HexCr, only the proton signals corre-
sponding to the bound host 2 were observable (Figure S47).
Finally, an excess of HexCr produced the appearance of the
proton signals corresponding to free guest (Figure 4c, left
panel).
An analogous behavior was observed in the acquired 31P NMR
spectra of the solutions. The 31P NMR spectrum of 9in showed
a somewhat broadened singlet resonating at 15.5 ppm corre-
sponding to the phosphorous atom of the inwardly directed
P=O group (Figure 4a right panel). In the presence of incre-
mental amounts of HexCr, the intensity of this phosphorous
signal decreased and a new sharp singlet, resonating at 13.7
ppm, was observed. In the presence of 1.5 equiv of HexCr,
only the phosphorus signal assigned to the P=O group in
bound 2 was detected (Figure 4c right panel).
Figure 3. X-ray structure of the CH3COCH3⊂8in inclusion com-
plex, thermal ellipsoids for C, N, O and P atoms set at 50% prob-
ability; H atoms are shown as spheres of 0.20 Å diameter.
The catalytic hydrogenation of the nitro-compound 8in, using
10% Pd/C, yielded quantitatively the corresponding amino-
derivative, which was used without further purification in the
coupling reaction with dansyl chloride. The fluorescent dansyl
amide mono-phosphonate cavitand receptor 2 was obtained in
98% yield as a racemic mixture. All attempts to separate the
enantiomers of 2, using chiral HPLC, were unsuccessful.
However, because creatinine is non-chiral, no difference for
the sensing is expected for the two stereoisomers. Receptor 2
was characterized by a full set of high-resolution spectra (Fig-
ures S9 to S18).
In turn, the precursor of the pyridyl-N-oxide dye 4 was pre-
pared following a reported procedure.36 The N-oxidation reac-
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Figure 5. Energy-minimized structure of the HexCr⊂2 complex
(PB86-D3/def2-SVP, CPM/chloroform). The non-polar hydrogen
atoms of receptor 2 were removed for the sake of clarity. The
encapsulated HexCr molecule is shown as CPK model.
In order to assess the accurate value of the stability constant of
the HexCr⊂2 complex, we performed isothermal titration
calorimetry (ITC) experiments. The successive addition of
aliquots of a chloroform solution of receptor 2 (2.2 mM) to a
solution of HexCr (0.3 mM) in the same solvent produced a
gradual release of heat corresponding to the binding event
(Figure 6, top). The normalized integrated heats were correct-
ed for dilution effects. This provided a sigmoidal binding
isotherm with an inflection point close to a host: guest ratio of
1 (Figure 6, bottom), as expected for 1:1 binding.
Figure 4. Left panel, selected regions of the 1H NMR spectra (500
MHz, 298 K) of CDCl3 solutions containing fluorescent receptor
2 and HexCr in different molar ratios: a) Free 2; b) 2 + 0.5 equiv.;
1
c) 2 + 1.5 equiv.; d). Trace shows one region of the H NMR
spectrum of HexCr. Right panel, corresponding 31P NMR spectra
(202 MHz, CDCl3, 298 K). Primed letters and numbers corre-
spond to the proton signals in the HexCr⊂2 complex. Proton
assignment of HexCr is shown in the inset structure. For the
proton assignment of 2 see Figure 1. * Residual methanol peak.
See supporting information for a more detailed preparation of the
analyzed solution mixtures.
In summary, these results indicate that receptor 2 and HexCr
exclusively formed a 1:1 inclusion complex, HexCr⊂2. In this
complex, the HexCr is deeply immersed in the aromatic cavi-
ty of the calix[4]pyrrole 2 in its cone conformation. The bind-
ing process displayed slow chemical exchange dynamics in the
1H and 31P NMR chemical shift timescales between free and
bound counterparts.
The kinetic stability of the complex was accompanied by a
high thermodynamic stability. Thus, the exclusive observation
1
of the signals of the bound host 2 in the H and 31P NMR
spectra acquired in the presence of 1 equiv of HexCr, allowed
us to estimate that the stability constant of the HexCr⊂2 com-
plex should be larger than 104 M-1.
The HexCr⊂2 complex was fully characterized using 2D
NMR experiments (COSY, ROESY Figure S48 to S51). To
gain additional insight into the three-dimensional structure of
the HexCr⊂2 complex, the putative 1:1 inclusion was energy
minimized (according to frequency calculation) at the
,
,
BP8641 42-D343 44/def2-SVP45 level of theory using a polariza-
ble continuum solvent model (PCM) for chloroform as imple-
mented in Gaussian 09.46 In analogy to the previously de-
scribed mono-phosphonate methylene-bridged receptor 1, the
energy-minimized structure of the HexCr⸦2 complex showed
that the aromatic cavity of 2 nicely complements in terms of
size, shape and functionalization the included HexCr (Figure
5). The average distance between the centroids of HexCr and
the dansyl group is in the range of 12 Å.
Figure 6. Raw data (heat vs. time) for the calorimetry titration of
HexCr (0.3 mM) with 2 (2.2 mM) performed at 288 K (top). Fit
of the integrated and corrected calorimetric data to the one-site
binding model (bottom). Note that the first two points were ex-
cluded from the fitting.
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The fit of the calorimetric titration data to a 1:1 binding model
This results in a significant spectral overlap with the dansyl
emission (J = 1.2 × 1010 cm6ꞏmol1), which translates into a
critical FRET radius of R0 = 43.7 Å. Taking into account the
modeled distance (~ 13 Å, Figure S66) between the dansyl
energy donor and 3 as the acceptor dye in the 3⸦2 complex, a
quantitative FRET process should result (FRET ca. 1). Hence,
we expected that on complexation of 3 by 2 ([2] = 5 µM) the
fluorescence of the dansyl chromophore was efficiently
quenched. Indeed, the fluorescence quenching was practically
quantitative (ca. 95%) when 2 equiv of 3 is added. The fluo-
rescence titration curve was fitted according to a 1:1 binding
model yielding a binding constant of Ka(3⊂2) = (1.2 ± 0.5) 
107 M-1 (corrected for minor inner-filter effects due to the
addition of a chromophoric titrator); see Figure 8.
returned an accurate value of the binding constant as
Ka(HexCr⊂2) = (4.5 ± 0.4)  105 M-1. In addition, the fit also
provided the binding enthalpy H = 10.9 ± 0.4 kJꞏmol-1,
which allowed us to calculate T∆S as 20.2 ± 0.6 kJꞏmol-1 (298
K). These results indicate that the binding of HexCr by recep-
tor 2 is mainly entropically driven. In accordance with previ-
ous interpretations, the de-solvation of the binding partners
and the solvation of the formed inclusion complex are thought
to play an important role in the complexation process of aryl-
extended calix[4]pyrrole cavitand receptors.47
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Interaction of receptor 2 with dye 3 probed by fluores-
cence spectroscopy and ITC experiments
The absorption spectrum of 2 in chloroform (blue line in Fig-
ure 7) shows an intense band with a maximum at 347 nm ( =
4300 M-1ꞏcm-1), typical for the dansyl chromophore (compare
with model 5; abs,max = 341 nm,  = 4400 M-1ꞏcm-1). Further,
an intense absorption band with a maximum at 249 nm, which
is characteristic for the ,*-transitions of the aryl-extended
calix[4]pyrrole, is present (see Figure S37). Excitation at 347
nm produces the characteristic and intense fluorescence emis-
sion of an intramolecular charge-transfer (ICT) excited state
with a maximum at fluo,max = 509 nm (green line in Figure 7).
An independent fluorescence lifetime quenching experiment
indicated that the quenching was not of dynamic nature (an
invariable lifetime of 14.8-14.9 ns was measured in the ab-
sence and presence of 3 as quencher). Hence, intermolecular
quenching can be ruled out in the investigated concentration
range.
600
600
500
500
400
The fluorescence quantum yield of 2 was determined as fluo
=
300
0.45 and the lifetime measured as fluo = 14.8 ns. These data
compare absolutely to the photophysical characteristics of the
dansyl model compound 5 (fluo,max = 495 nm, fluo = 0.46, fluo
= 13.3 ns) in the same solvent. Thus, it was concluded that the
calix[4]pyrrole has no significant quenching effect on the
appended fluorophore.
200
400
100
0
0
5
10
[3]/ M
15
20
300
200
100
0
For the realization of the IDA with the reporter pair 3⊂2 we
characterized the complexation of the pyridyl-N-oxide 3, an
analogue
of
the
well-known
acid
4-((4-
(DABCYL).
(dimethylamino)phenyl)azo)benzoic
400
500
600
/ nm
700
800
DABCYL is a very popular black-hole quencher in FRET-
,
based quenching pairs for nucleic acids and proteins.48 49 Dye
3 is an efficient light absorber between 400 nm and 550 nm
(abs,max = 480 nm,  = 39500 M-1ꞏcm-1); see Figure 7.
Figure 8. Emission spectra of 2 (5 μM) registered on addition of
increasing amounts of 3 in chloroform; λexc = 317 nm. Inset: Plot
of the emission change at 505 nm (black circles) vs concentration
of 3. The red line corresponds to the fit of the titration data to a
binding model that considers the formation of the 1:1 complex,
3⊂2.
1.0
0.8
0.6
0.4
0.2
0.0
Next, we probed the interaction of 3 with receptor 2 by means
of ITC experiments (see Figure S59). The titrations produced
rectangular binding isotherms with the height corresponding
exactly to H and the sharp-increase occurring at the stoichi-
ometric equivalence point: host:guest ratio of 1. This is the
expected result for very tight binding and resulted only useful
to estimate that Ka(3⊂2) was larger than 107 M-1. The estimat-
ed magnitude of the binding constant value is in complete
agreement with the value calculated from the fluorescence
titrations. While the enthalpic contribution to binding, H,
could be accurately determined as 34.7 kJꞏmol-1 from the
height of the isotherm, the corresponding entropic term, which
should be positive, could not be derived owing to the estima-
tive nature of Ka(3⊂2). An accurate determination of Ka(3⊂2)
using ITC experiments required working in more dilute condi-
tions, that is reducing the “c“ value. This approach was not
300
400
500
/ nm
600
700
800
Figure 7. UV/vis-absorption spectra of 2 (blue) and 3 (red) and
the fluorescence spectrum of 2 (green). In black the spectral of
overlap is illustrated.
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experimentally feasible owing to reduced heat release of the
significantly (factor of 2.7 for 50 μM Cr). The limited solubil-
binding process, which dropped below the instrument’s sensi-
ity of Cr in chloroform (≤ 50 M) precluded the registration
of a complete titration curve. However, based on the obtained
data, it can be safely stated that a lower limit for the binding
constant of the Cr⸦2 complex is ca. 5  104 M-1. Hence, in
terms of its sensitivity range the herein developed fluorescent
IDA is fully compatible with Cr. The Cr⸦2 complex was
fully characterized by means of 1D and 2D high-resolution
NMR experiments (Figures S52-S54).
tivity (3-5 µcal per injection) at lower concentrations.50
Competitive IDA of the 3⸦2 ensemble with HexCr
We prepared an equimolar mixture of 3 and 2 (1 μM) in chlo-
roform solution. At this concentration complex formation is
reached to an extent of 83%. The solution was titrated with
incremental amounts of HexCr.51 According to the expecta-
tions, on displacement of 3, as quencher of the dansyl fluores-
cence, a turn-on effect was observed. For the addition of ca. 70
equiv HexCr a 4-fold fluorescence enhancement at 505 nm
was registered; see Figure 9. Noteworthy, HexCr does not
exercise any significant quenching effect on the dansyl fluoro-
phore when included in the cavity of receptor 2 (Figure S37).
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Finally, we tested the possible interference of other biological-
ly relevant analytes. For this purpose, urea and L-proline, as an
abundant amino acid, were chosen. The separate addition of
these compounds to the ensemble 3⸦2 (both components at 1
μM) yielded fluorescence increase factors of 1.4 (L-proline)
and 2.6 (urea); see Figures S38 and S39. These responses are
significant, however, smaller than those observed for HexCr
(see above).
350
300
Development of an IDA based on the non-fluorescent
receptor 1
300
250
200
250
150
The previously described receptor 1, which is known to be an
excellent host for Cr, is non-fluorescent.29 However, by intro-
ducing a fluorescent guest, receptor 1 can be integrated in an
optical IDA suitable for HexCr sensing. For this purpose, we
designed the guest dye 4, containing the pyridyl-N-oxide bind-
ing motif connected to a pyrene chromophore by an ethenyl
spacer. Dye 4 features an absorption maximum at 394 nm ( =
32600 M-1ꞏcm-1) and fluoresces with maximum at 474 nm
(fluo = 0.21, fluo = 1.11 ns). The rather broad emission band
and relatively large Stokes shift underpin the ICT character of
the fluorescent state.
100
200
0
10
20
30
40
50
60
70
150
100
50
[HexCr]/ M
0
400
500
600
700
/ nm
Figure 9. Emission spectra registered during the competitive IDA
of the 3⊂2 ensemble with HexCr; exc = 317 nm. The initial
fluorescence is due to the minor fraction (17%) of free 2 at the
used equimolar concentration (1 µM). Inset: Plot of the changes in
emission at 505 nm vs concentration of HexCr. The red line
corresponds to the fit of the titration data to a competitive binding
model that considers the formation of the 3⊂2 and HexCr⊂2
complexes.
1.0
0.5
0.0
Hence, the observed fluorescence enhancement corresponds to
the undisturbed response of the IDA. The fit of the competi-
tive titration data to a competitive binding model of two 1:1
complexes returned a stability constant for the HexCr⊂2
complex of Ka(HexCr⊂2) = (3.2 ± 0.4)  105 M-1. This value
is in excellent agreement with the one obtained in the ITC
experiment; (4.5 ± 0.4)  105 M-1 (see above).
The need for a large excess HexCr is explained by the differ-
ence in the binding constant values of the competitors; see
above. Gratifyingly, the concentration range that is required to
obtain a visible fluorescence response of the 3⸦2 IDA coin-
cides with or is well below the expected clinical levels of
creatinine in blood serum (60-420 M) or urine (3-25 mM) for
healthy individuals.2 In the case of kidney or renal dysfunction
these values are dramatically increased. We determined the
limit of detection (LOD) of the 3⸦2 IDA to be ca. 110 nM.
300
400
500
600
700
/ nm
Figure 10. UV/vis absorption (left) and fluorescence emission
(right) spectra of 4 on addition of receptor 1. The fluorescence
spectra were obtained for excitation at 350 nm.
The UV/vis absorption titration of 4 with incremental amounts
of receptor 1 (inverse titration) provided a set of spectra dis-
playing one quasi-isosbestic point at 350 nm (see Figure 10).
This is the expected result for the exclusive formation of a 1:1
complex in binding systems involving only two absorbing
species: the unbound dye 4 and its inclusion complex 4⊂1.52
The titration was also monitored by fluorescence spectrosco-
py; see Figure 10. In the course of the titration the fluores-
Noteworthily, very similar results were obtained when creati-
nine (Cr) was used instead of the lipophilic version HexCr
(Figure S42). On addition of Cr to the ensemble 3⸦2 (both
components at 1 μM) the fluorescence of the system increased
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cence was significantly quenched (ca. 80% on addition of 2.5
CONCLUSIONS
equiv 1) and at the endpoint a slightly blue-shifted emission
spectrum (Δλ = -11 nm) was observed. This the likely result of
the disturbance of the ICT state on interaction pyridine-N-
oxide moiety with the host, leading to the observed quenching.
The analysis of the emission changes of the dye vs the concen-
tration of 1, using a 1:1 binding model, provided the binding
constant value for the corresponding inclusion complex;
Ka(4⊂1) = (1.2 ± 0.2)  107 M-1 (Figure S40). ITC experi-
ments (Figure S61) confirmed the tight and strong 1:1 binding
with an exothermic enthalpic contribution of H = 38.0 ± 0.4
kJꞏmol-1. However, for the same reasons as discussed above
for the binding of 3 by host 2, only a lower limit of 107 M-1
can be stated for the binding constant value. Noteworthily, this
agrees nicely with the more accurate data obtained by fluores-
cence titration.
Calix[4]pyrrole-derived IDAs were designed to enable the
efficient sensing of Cr and its derivative HexCr. The ca-
lix[4]pyrrole receptors, 1 and 2, operates according to the
principles of size, shape and functional complementarity. Cr
and derivatives are included in the receptor’s cavities (Ka ca.
4.5  105 M-1), establishing five complementary hydrogen
bonds: four with pyrrole NHs and one with the upper rim
phosphonate group. In one version of the developed sensors,
compound 2, the receptor unit features a covalently attached
dansyl fluorophore. This provides a means for FRET-based
IDA through the displacement of a black-hole quencher 3 by
Cr or its lipophilic analogue HexCr. In another version, the
inherently fluorescent pyrene-based dye 4 formed a reporter
pair with receptor 1. In both developed IDAs, the indicator
dyes interact with the calix[4]pyrrole cavitand scaffolds
through complexation of the pyridyl-N-oxide motif (Ka ca. 107
M-1). The two developed IDAs work in organic chlorinated
solvents at a range of concentrations that coincide with those
in which Cr is present in bodily fluids (blood serum and
urine). Remarkably, the 3⊂2 IDA features a limit of detection
as low as 110 nM, drawing on the high sensitivity of the fluo-
rescence measurements and the significant affinity of receptor
2 for HexCr and Cr. The developed IDAs rely on a combina-
tion of hydrogen bonding, π-π and CH-π interactions, as driv-
en forces of the molecular recognition events. To the best of
our knowledge, the reported work describes unprecedented
examples of fluorescent supramolecular sensors for creatinine.
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Finally, we tested the ensemble 4⸦1 in a competitive IDA
with HexCr.53 The sensing ensemble consisted of equimolar
amounts (each 1 M) of receptor 1 and indicator 4, assuring a
complexation degree of 80%. The incremental addition of
HexCr restored the emission of the dye (fluorescence en-
hancement factor of 2.3 at 50 M HexCr). The corresponding
titration curve was analyzed using a binding model of two
competitive complexes. The fit provided a binding constant of
(4.3 ± 0.5)  105 M-1 for the HexCr⸦1 complex. This value is
again in good agreement with the one determined in ITC ex-
periments; Ka(HexCr⸦1) = (1.5 ± 0.3)  105 M-1(Figure S60).
The ITC experiment provided further information on the en-
thalpic and entropic contributions to binding: ∆H = 7.8 ± 0.1
kJꞏmol-1 and T∆S as -19.8 ± 0.1 kJꞏmol-1 (298 K). This led to
the conclusion that the binding of HexCr by 1 is mainly en-
tropically driven.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures and characterization data of the synthe-
sized compounds, NMR, UV/vis and emission spectra of the
binding studies. X-ray crystals structures (CIF files) of HexCr, 7,
8in and 8out. Fits of the fluorescent titration data. Figures of the
raw data and normalized heats obtained in the isothermal titration
calorimetry (ITC) experiments. This material is available free of
charge via the Internet at http://pubs.acs.org.
Not surprisingly, the IDA with Cr yielded practically the same
switch-on response as for HexCr (Figure S43). On addition of
50 M Cr a fluorescence enhancement factor of 1.8 was ob-
tained. The fitting of the competitive binding titration returned
a binding constant of Ka(Cr⸦1) = (3.0 ± 1.0)  105 M-1.
Future challenges and developments
The future challenges of the described supramolecular design
include the improvement of their sensing selectivity and the
compatibility with aqueous media. On the one hand, as herein
demonstrated the current design shows interferences with
other biologically relevant analytes, such as proline and urea.
In practical terms, this limitation could be avoided by previous
extractive separation of creatinine. On the other hand, the
issue of water solubility of the calix[4]pyrrole receptor and/or
the dyes can be addressed by synthetic modifications involv-
ing the incorporation of ionizable and charged groups in their
scaffolds periphery. However, it is expected that the binding
constant of creatinine with the water-soluble versions of the
receptor would drop considerably owing to the polar nature of
the analyte and the competition of water for hydrogen bond-
ing. This drawback might be partially overcome by perform-
ing the sensing experiments in microheterogeneous media,
such as micelles and liposomes. We are currently expanding
our efforts in these directions and we expect to report our
findings in due course.
The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
* E-mail: pballester@iciq.es; uwe.pischel@diq.uhu.es
ACKNOWLEDGMENT
This work was supported by Gobierno de España MINECO and
FEDER funds (CTQ2017-84319-P for P.B. and CTQ2017-89832-
P for U.P.), CERCA programme/Generalitat de Catalunya, and
AGAUR (2017-SGR-1123). We also thank Dr Eduardo C. Es-
cudero-Adán, X-ray Diffraction Unit of ICIQ, for help with the
analysis of the X-ray crystallographic data and Dr Luis Martínez-
Crespo for experimental assistance and helpful discussions in the
initial phases of the project.
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50 The interaction of 2 with 3 was also probed using 1H NMR spec-
troscopy (Figure S56).
51
The release of 0.5 equiv of HexCr from the HexCr⸦2 complex
to the bulk solution induced by adding 0.5 equiv of 3 was evidenced
by 1H NMR spectroscopy (Figure S56). The reverse experiment is not
feasible to be performed at mM concentration owing to the reduced
solubility of HexCr in chloroform solution (~ 1.5 mM).
52
1
The interaction of 1 with 4 was also probed using H NMR spec-
troscopy (Figure S57).
53
The release of 0.5 equiv of HexCr from the HexCr⸦1 complex to
the bulk solution induced by adding 0.5 equiv of 4 was evidenced by
1H NMR spectroscopy (Figure S58). The reverse experiment is not
feasible to be performed at mM concentration owing to the reduced
solubility of HexCr in chloroform solution (~ 1.5 mM).
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52
53
54
55
56
57
58
59
60
10
ACS Paragon Plus Environment