Biguanides, anion receptors and sensors

Article abstract of DOI:10.1039/c7cc05012g

Biguanides are strong bases (pK_a > 10), their protonated forms bind anions and may therefore act as receptors for anions. We report on easy-to-make anion receptors and fluorescence-based sensors utilizing the biguanide moieties that respond to the presence of anions with a change in fluorescence. The observed changes in fluorescence are anion-specific and even though the biguanide receptors are cross-reactive, these sensors may be used to identify various anions (halides, carboxylates, phosphates). Paper-based analytical arrays were used to assess the discriminatory ability of the sensors in the qualitative and quantitative analysis of multiple anions.

Full text of DOI:10.1039/c7cc05012g

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Biguanides, anion receptors and sensors.
Mariia Pushinaa and Pavel Anzenbacher, Jr.a,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Biguanides are strong bases (pKa>10), their protonated forms bind
anions and may therefore act as receptors for anions. We report
on easy-to-make anion receptors and fluorescence-based sensors
utilizing the biguanide moieties that respond to the presence of
anions with a change in fluorescence. The observed changes in
fluorescence are anion-specific and even though the biguanide
receptors are cross-reactive, these sensors may be used to identify
various anions (halides, carboxylates, phosphates). Paper-based
analytical arrays were used to assess the disciminatory ability of
the sensors in the qualitative and quantitative analysis of multiple
anions.
Guanidine
Biguanide
NH NH
NH
H2N
NH2
H2N
N
H
NH2
Compounds used in this study
-
BF4
H
N
H
H
H
N
N
-
H
N
H
N
Ar
N
H
N
H
N
H
S1, S2, S3
S4, S5, S6
Ar
N
N
H
H
H
H
BF4
H
N
H
N
H
N
Ar
-
N
N
Efforts aimed at the discovery of anion receptors and sensors
have risen tremendously over the last decades.1
BF4
H
H
H
H
,2,3
These
endeavors have been driven by the ascent in demand of new
(bio)analytical and environmental survey methods. Here, the
need for new anion-binding materials led to the utilization of
hydrogen bond donors and positively charged species as the
binding moieties. Examples of such functional groups are
S1, S4 Ar=
S2, S5 Ar=
S3, S6 Ar=
,
5
,6
,9
guanidinium,4
ammonium,7
amide,8
urea10
and
Figure 1. Structures of the compounds of this study.
thiourea,11 pyrrole,13 15 and imidazolium16 17 to name a few.
Given their basicity (pKa 12-13) and protonation at or close to
neutral pH, and a combined binding modes based on ion
pairing and hydrogen bonding guanidinium derivatives have
been shown to be particularly effective anion-binding
receptors.18
,
12
,14
,
,
Here, we report fluorescent compounds utilizing the biguanide
moiety and their binding and sensing of anions. The
compounds comprise two biguanide moieties and are similar
in structure to chlorhexidine (Compounds S1
set of compounds (Compounds S4 S6) comprises only one
biguanide unit. This design consideration was made on the
assumption that the bifunctional molecules S1 S3 would be
able to form clefts capable of binding dicarboxylates while the
monofunctional molecules S4 S6, would bind preferentially
-S3). The second
-
Recently, we investigated the potential utility of biguanide
(Figure 1) moiety as a building block for anion receptors.
Similarly to guanidines, biguanides are also bases (pKa 11-12)
and are capable of polyvalent binding due to multiple-site
hydrogen bonding sites.19 Furthermore, drugs such as
metformin and the important antiseptics chlorhexidine and
polyhexanide also comprise biguanide moieties (Figure 1).
-
-
monoanions. The aromatic units (2-anthryl, 1-naphthyl,
phenyl) serve as fluorophores and exhibit varying propensity
to aggregation.
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-
Compounds S1
literature procedures.20 As expected, in the 1H-NMR spectra of
S1-S3) we have seen the broadening of peaks associated with
-S6 BF4 salts were synthesized following
(
intermolecular aggregation. Upon heating to 70o C, the peaks
become sharper, presumably due to disruption of the
aggregates (Figure 2).21 Interestingly, some resonances
associated with the N–H moieties coalesced, suggesting
efficient proton exchange with the solvent.
Figure 3. Top (A): Stacked spectra of S3(BF4)2 (16 mM) upon titration with chloride (TBA
salt) at 70o C in DMSO-d6 – water (10%). Bottom (B): Titration isotherm corresponding
to the lowest field resonance (9.4 ppm in the initial spectrum).
Figure 2. 1H-NMR spectra of S3 (16 mM) at 25o C and 70o C show aggregation at room
temperature in DMSO-d6 – water (10%).
Further quantitative measurements were performed using UV-
vis and fluorescence spectroscopy. Compared to the NMR
titrations, such measurements are less prone to suffer from
effects of aggregation. To further decrease the extent of
possible aggregation, a nonionic surfactant (Triton X-100) was
added into the solvent. The UV-vis spectra show several
maxima, whose intensity changed throughout the titration
with anions, while forming clearly defined isosbestic points
(Figure 4). The corresponding isotherms show a distinct non-
monotonous behaviour and a clear saturation. In general, the
UV-vis spectra, collected at a lower concentration (20 µM), did
not reveal significant signs of obvious aggregations, while the
fluorescence spectra displayed the excimer emission only in
the case of the anthracene compounds S1 and S4. This
suggests that, with exception of compounds S1 and S4, at low
concentrations (<20 µM) compounds S2,S3 and S5,S6 do not
aggregate in an appreciable way. To gain a qualitative insight
into the anion binding event of the anions with the biguanide
compounds, we performed 1H-NMR titrations of S1
-S6 (see
biguanide receptors S1
affinity constants for S1
S6. The values of corresponding binding constants for S1
-S6 show a similar behaviour albeit the
ESI, S55-S60). The simplest case, titration of S3(BF4)2 with
chloride is shown in Figure 3. As expected, the NH resonances
involved in hydrogen bonding with the anion show a concerted
downfield shift against the C-H resonances of aryl substituents.
No deprotonation was observed within the concentration
range studied. This observation confirms our expectation that
biguanide moiety and the corresponding compounds may act
as receptors for anions.
-S3 are slightly higher than those of S4
-
-S3
are shown in Table 1. These binding constants were
determined from fluorescence titrations.
Table 1. The affinity constants (Ka, M-2 × 104) [a] are obtained from
fluorescence titrations of S1-S3 with anions.
Guest
Fluoride
Chloride
Acetate
Oxalate
Malonate
Glutamate
Benzoate
Phthalate
H2Phosphate
S1
S2
S3
10.0
5.80
51.0
41.0
14.0
1.30
7.80
0.48
35.0
6.90
0.12
11.0
12.0
5.50
0.87
5.00
3.80
ND
1.40
NRb
3.30
4.10
2.60
0.57
0.66
0.30
0.48
However, NMR titrations using multi-valent anions such as
dicarboxylates or dihydrogen phosphate revealed that multiple
NH resonances disappeared from the spectrum. Two
possibilities were considered: rapid exchange of the NH
protons or a multiple deprotonation of the biguanide moiety.
The latter possibility was explored by treatment of the sensor
S3 with tetrabutylammonium hydroxide, which resulted in
different spectra than those obtained with anions. Thus, we
conclude that the disappearance of the NH resonances is due
to an acceleration of the proton exchange of the NH fragments
with the solvent, promoted by the binding of the anion.
a The titrations were recorded in DMSO:CHCl3 (3:7 v/v) with addition of Triton X-
100 and Kas are calculated based on the change in fluorescence intensity upon
addition of each guest. The errors of the curve fitting were < 20%. b Ka could not
be calculated due to small change in fluorescence in the presence of chloride.
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0.8
addition of aliquots of malonate (TBA salt) to a solution of S1
,
Ka = (2.9 ± 0.6)×104 M-2
1.0
0.8
0.6
0.4
0.2
0.0
further aggregation was observed, as confirmed by the
analysis of the emission spectra. With increasing concentration
of malonate anion, the band at 450 nm, originating from
excited states of anthracene monomer decreases and is
accompanied by an increase in intensity of the excimer band at
520 nm.23,24,25,27 With simple monoanions such as dihydrogen
phosphate, only the quenching of the intensity of the parent
monomer peak was observed (Figure 6).
A
0.6
0.4
0.2
0.0
A
A
A
317 nm
0
20
40
60
80 100 120
[Malonate], µM
250
300
350
400
450
500
Ka = (1.4 ± 0.2)×105 M-2
Wavelength, nm
5x105
1.0
I
0.8
I
0.6
4x105
I
Ka = (1.5 ± 0.2)×105 M-2
0.4
I
2.0
1.5
1.0
0.5
0.0
0.2
1.0
3x105
2x105
1x105
0.0
451 nm
0.8
A
0
10 20 30 40 50 60 70 80
A
0.6
0.4
0.2
0.0
[Malonate],
µM
A
A
420 nm
0
100
200
300
400
500
[Dihydrogen phosphate],
µM
450
500
550
600
Wavelength, nm
250
300
350
400
450
500
550
Ka = (3.5 ± 0.9)×105 M-2
6x105
5x105
4x105
3x105
2x105
1x105
1.0
Wavelength, nm
I
0.8
I
0.6
Figure 4. Top: Absorption titration spectra and isotherm of S2 (20 μM) upon addition of
malonate anion. Bottom: Absorption titration spectra and isotherm of S1 (20μM) upon
addition of dihydrogen phosphate anion. Both titrations were acquired in DMSO:CHCl3
(3:7, v/v).
I
0.4
I
0.2
0.0
451 nm
0
2
4
6
8
10 12 14 16
[Dihydrogen phosphate],
µM
First, fluorescence responses were ascertained by naked eye
whereas the vials of sensors (DMSO:CHCl3, 3:7) were treated
with anions. Two types of response were observed (Figure 5):
First, weakly-basic anions such as chloride, nitrate and
benzoate did not show any change in colour, but a weak
amplification of fluorescence (~15%). Secondly, the more basic
anions such as fluoride and acetate (and other carboxylates),
showed change in colour emission from blue to yellow. The
latter change suggests an advanced proton transfer.22
450
500
550
600
Wavelength, nm
Figure 6. Top: Fluorescence titration spectra and isotherm of S1 (0.2 μM) upon addition
of malonate anion. Bottom: Fluorescence titration spectra and isotherm of S1 (0.2 μM)
upon addition of dihydrogen phosphate anion. Both titrations were acquired in
DMSO:CHCl3 (3:7, v/v).
The apparent affinity constants obtained from the
fluorescence titrations are shown in Table 1 for S1
-S3. For a
complete table showing also the results of S4-S6, see ESI.
A closer inspection of the photograph in Figure 5 and the data
in Table 1 suggests that the biguanide sensors are cross-
reactive. However, both the small changes, e.g. small increase
in fluorescence intensity in the presence of chloride or shift of
Figure 5. Naked eye observation of the emission of S1 upon addition of various anions emission wavelength in the presence of dihydrogen phosphate
(DMSO:CHCl3, 3:7, v/v) under black light illumination (365 nm).
may be successfully harnessed in differential arrays where
multiple sensors create a response patterns for each analyte,
The proton transfer issue in organic solvents led us to
transferring the test and assay into aqueous environment, see
below. Due to the issues with aggregation at room
temperature, we have included a surfactant Triton X-100. The
fluorescence spectra revealed an almost identical behaviour to
the one observed by naked eye. Here, the shape of the
spectrum of the 2-anthracenyl moieties in S1 displayed both
the monomer emission (λmax = 450 nm) and broad excimer
emission (λmax = 520 nm) (Figure 6). Depending on the nature
of the anion, two types of behaviour were observed. Upon
which then can be recognized by pattern analysis protocols.
To increase the applicability of the sensors presented here, we
performed the array-based test using paper microzone plates
by printing hydrophobic barriers on paper.28,29 Figure 7 shows
the paper microzone plate. Sensors S1-S6 were applied to the
paper in DMSO and dried. Analytes in the form of aqueous
solution were then applied to the sensor and the fluorescence
changes were recorded using an UV-scanner (See ESI for
details). Changes in fluorescence intensities were further
examined and classified using Linear Discriminant Analysis
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(LDA).30 We have observed similar trends in fluorescence as in
the organic solvents, with exception for weakly binding anions
such as chloride, which displayed an appreciable strong
response, see for example, the increased fluorescence
intensity for chloride (part of the photograph of the plate is
shown in Figure 7A) suggesting that the paper-based sensor
may be successful platform for biguanide-mediated anion
sensing. Using the paper plate method in conjunction with
pattern recognition analysis (LDA), we could correctly identify
11 anionic analytes (100% correct classification, for details see
ESI, S67). Importantly, we were also able to perform a
quantitative analysis of multiple anions (chloride, dihydrogen
phosphate, oxalate and pyrophosphate). In Figure 7B we show
the analysis of four different anions (for additional quantitative
analyses of multiple anions, see ESI, S74-S77).
P. A. gratefully acknowledges Bowling Green State University
(Building Strength Programme) for generous financial support.
Notes and references
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The successful recognition of multiple anions applied to the
sensor plate in the form of aqueous solution suggests that the
anion binding occurs via electrostatic attraction and multiple
hydrogen bonding as the main contributors to the sensor
response rather than sensor deprotonation.
In conclusion, we report that biguanides are easy-to-synthesize
hosts capable of binding various anions. The biguanide
moieties are prone to protonation at a neutral pH and are
capable of polyvalent binding due to multiple-site hydrogen
bonding sites, and delocalized positive charge. The presence of
fluorescent aromatic moieties enables the anion sensing, but
may also cause aggregation. The aggregation equilibria appear
to contribute to the differential response in fluorescence
caused by the anions. The present compounds show cross-
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capabilities in paper-based sensors where the analytes are
applied as aqueous solution. Paper based sensors enabled
recognizing anions in both qualitative and quantitative
manner. The biguanide moiety is a useful functionality for
recognition of anions in aqueous media, which is desirable in
the anion recognition.
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