Neutral urea-based receptors for phosphates: synthesis and spectrophotometric studies

Article abstract of DOI:10.1016/j.tet.2016.09.042

A series of five fluorescent ligands (L1–L5) for anions were designed and synthesized. Urea, thiourea, and cyanoguanidine moieties were chosen as the binding groups, while 3,5-bis(trifluoromethyl)phenyl, coumarin, and BODIPY were used as the chromophores. The cyanoguanidines L3–L5 were promptly synthesized from corresponding thioureas using EDCI followed by reaction with cyanamide in the presence of triethylamine. The absorption and emission properties of the ligands L1–L5 were studied in CH₃CN; their coordination properties towards phosphate, pyrophosphate, and hydroxide anions, together with binding constants were evaluated.

Full text of DOI:10.1016/j.tet.2016.09.042

Accepted Manuscript
Neutral urea-based receptors for phosphates: synthesis and spectrophotometric
studies
Mauro Formica, Vieri Fusi, Luca Giorgi, Giovanni Piersanti, Michele Retini, Giovanni
Zappia
PII:
S0040-4020(16)30958-9
10.1016/j.tet.2016.09.042
TET 28113
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 26 May 2016
Revised Date: 7 September 2016
Accepted Date: 20 September 2016
Please cite this article as: Formica M, Fusi V, Giorgi L, Piersanti G, Retini M, Zappia G, Neutral urea-
based receptors for phosphates: synthesis and spectrophotometric studies, Tetrahedron (2016), doi:
10.1016/j.tet.2016.09.042.
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ACCEPTED MANUSCRIPT
Graphical Abstract
3-
3-
HP2O7
HP2O7
CN
N
HN
N
H
N
N
B
F
F

ACCEPTED MANUSCRIPT
Receptors for Phosphates:
Neutral
Urea-based
Synthesis
and
Spectrophotometric Studies
Mauro Formica,a Vieri Fusi,a,* Luca Giorgi,a Giovanni Piersanti,b Michele Retinib and
Giovanni Zappia.b,*
a Department of Pure and Applied Sciences, Chemistry Section, University of Urbino
“Carlo Bo”, P.zza Rinascimento 6, I-61029 Urbino, Italy.
bDepartment of Biomolecular Sciences, University of Urbino “Carlo Bo”, P.zza
Rinascimento 6, I-61029 Urbino, Italy.
Corresponding author: Prof. Vieri Fusi, e-mail: vieri.fusi@uniurb.it
Abstract
A series of five fluorescent ligands (L1–L5) for anions were designed and
synthesized. Urea, thiourea, and cyanoguanidine moieties were chosen as the binding
groups, while 3,5-bis(trifluoromethyl)phenyl, coumarin, and BODIPY were used as
the chromophores. The cyanoguanidines L3–L5 were promptly synthesized from
corresponding thioureas using EDCI followed by reaction with cyanamide in the
presence of triethylamine. The absorption and emission properties of the ligands L1–
L5 were studied in CH3CN; their coordination properties towards phosphate,
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pyrophosphate, and hydroxide anions, together with binding constants were
evaluated.
Keywords: BODIPY / Coumarin / Fluorescence / Urea / Phosphates
1. Introduction
Anions are ubiquitous species in nature, where they play a number of key roles
in living organism; in biological systems, the majority of enzyme substrates and
cofactors are in fact anionic.1 Among Amongst all anions, phosphates are particularly
important, as they are involved in many biological processes so that their
measurements are also crucial for clinical diagnosis of various disorders. Moreover,
phosphates are present in detergents and fertilizers and their extensive worldwide use
is related with environmental problems. Therefore, selective and sensitive receptors
and sensors for anions are useful in a number of scientific disciplines, including
biology, physiology, and environmental chemistry.2 The importance of anion
receptors in the field of organocatalysis has also been recently recognized.3
Consequently, new synthetic anion receptors that can be used as novel signaling and
recognition moieties are highly desired.
Typically, an anion sensor is generally designed to contain both a recognition
moiety, which is responsible for the selective reaction/interaction with the anion and
,
a spectroscopic or signaling moiety, which has optical properties that change upon
reaction/interaction with the anion of interest.
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It is interesting the Anion recognition driven by noncovalent interactions, such
as electrostatic interactions,4 anion-π interactions,5 halogen bonding,6 and hydrogen
bonding,7 is interesting because it allows the use of metal-free receptors in contrast
with the classic metallo-receptors. Hydrogen bonding, due to its relative strength and,
most importantly, directionality, occupies an important place in receptor design.
Various functional groups, such as amide,8 urea,9 and guanidinium,10 are commonly
encountered as hydrogen-bond donors.
The signaling moiety is usually a chromophore or fluorophore, which is able to
efficiently change his its photophysical properties in the presence of anions. In
particular, fluorescent anion sensors have been recently investigated in many fields
due to their powerful ability to improve analytical sensitivity; in addition, they are
used as a diagnostic tool for in vivo imaging studies.11 Moreover, processes such as
protein binding to specific ligands or molecular recognition of DNA are commonly
studied and followed with the help of fluorescent systems.12-16
Among different fluorophores, difluoroboradiaza-s-indacene (BODIPY) dyes
is are one of the most appealing thanks to their excellent photophysical and
photochemical properties such as high fluorescent quantum yields, large extinction
coefficients, narrow emission profiles, and good photostabilities.17 In addition to the
numerous applications in biological labeling and cell imaging, they have also been
investigated as artificial light harvesters, sensitizers for solar cells, molecular
photonic wires, solid state dye lasers,18-20 and photodynamic therapy agents.21 This
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particular class of fluorophores has attracted particular attention because their optical
properties are tunable through chemical modifications on the dye core.22
Recent works work within our group has focused on the synthesis and study of
a series of both macrocyclic and open-chain fluorescent anion receptors bearing the
urea moiety as the a hydrogen-bond donor and different units such as 4-nitrophenyl,23
3,5-bis(trifluoromethyl)phenyl,24 and coumarin25 as the signaling moiety. In this
paper, we report the syntheses and signaling results of the five novel urea-derived
ligands L1–L5 (Figure 1), which have different acidities of the ureidic functional
group together with different signaling units. Ligands L1, L2, and L3 incorporate the
3,5-bis(trifluoromethyl)phenyl group as the chromophore, while the urea, thiourea,
and cyanoguanidine group, respectively, are the binding unit. Ligands L4 and L5
present two different signaling units, the coumarin and BODIPY moieties,
respectively, preserving the cyanoguanidine binding unit constant. The
cyanoguanidine unit was chosen as a nontoxic isostere to thiourea,26 owing to its
comparable acidity and structural features: pKa = 15 (thiourea) and 14
(cyanoguanidine); C-N bond length = 1.34 Å for both thiourea and cyanoguanidine;
N-C-N angle = 119º (thiourea) and 124º (cyanoguanidine). Moreover, the
cyanoguanidine binding unit is present in the drug Cimetidine, which has been
approved by the FDA for inhibition of gastric acid secretion.27 The cyanoguanidines
L3–L5 were easily prepared from the corresponding thioureas, obtained from
available and/or readily prepared primary amines and isothiocyanates, followed by
treatment with EDCI, NEt3, and cyanamide in warm polar solvent such as
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acetonitrile or DMF. Reasonably, the cyanoguanidine formation supports a
mechanism whereby EDCI-mediated desulfurization is accompanied by double
deprotonation of the thiourea by NEt3 to yield a transient carbodiimide which is
subsequently attacked by the poor nucleophile cyanamide. The ability of ligands L1-
L5 in binding and sensing anions were investigated considering phosphates
-
3-
phosphate anions, such as H2PO4 and HP2O7 , and OH- in CH3CN.
CF3
CF3
S
O
F3C
N
N
F3C
N
N
H
H
H
H
L2
L1
CF3
CN
N
CN
N
HN
N
H
F3C
N
N
H
H
L3
CN
N
N
F
N
F
O
O
N
N
H
B
H
L4
L5
Figure 1: Structures of the five novel urea-derived ligands analyzed in this study.
2. Results and discussions
2.1. Synthesis
Urea L128 and thiourea L229 were prepared by direct reaction of benzylamine
with 1-isocyano-3,5-bis(trifluoromethyl)benzene (2) and 1-isothiocyano-3,5-
bis(trifluoromethyl)benzene (3), respectively. The three novel cyanoguanidines L3–5
were obtained from the corresponding thioureas via activation of the thiourea group
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with EDCI followed by reaction with cyanamide in the presence of triethylamine in
CH3CN or DMF.30
Thus, as shown in Scheme 1, thiourea 3 was treated with EDCI (1.2 equiv.),
triethylamine (1.3 equiv.), and cyanamide (1.5 equiv.) in CH3CN under refluxing
conditions overnight. Cyanoguanidine L3 was obtained in 41% yield as a white solid.
According to this synthetic approach, cyanoguanidine L4 was obtained by first
treating 7-amino-4-methyl-2H-chromen-2-one (4) with isothiocyanomethyl benzene
(5) and then with EDCI/triethylamine and cyanoamide to obtain L4 in 42% overall
yield. On the other hand, cyanoguanidine L5 was prepared starting from the readily
available aminophenyl-BODIPY derivative 7,31 which was first converted into the
corresponding isothiocyanate and then treated with benzylamine to give 8 in 73%
yield. Finally, the thiourea was converted into L5, as reported for L4, by reaction
with EDCI/triethylamine and cyanoamideguanidine in 54% yield. The ligand L5 was
thus obtained as a red solid in 40% overall yield from 7.
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X
N
CF3
CF3
2 X = O
3 X = S
CF3
X
F3C
NH2
N
N
H
H
CH2Cl2
L1 X = O; 57%
L2 X = S; 64%
1
CF3
NC
N
EDCI,TEA
CH3CN
N
H
N
CF3
L2
H
N
NH2
L3; 41%
Me
O
N
Me
O O
S
S
5
N
H
N
O
DMF
H
H2N
6; 72%
4
Me
O
NC
EDCI,TEA
CH3CN
N
N
N
O
N
NH2
H
H
L4; 57%
S
N
NH2
HN
N
H
1) CSCl2, TEA, CH2Cl2
NH2
N
N
F F
N
B
B
2)
, CH2Cl2/CH3CN
F F
8; 73%
7
CN
N
HN
N
H
EDCI,TEA
CH3CN
N
NH2
N
B
N
F F
L5; 54%
Scheme 1. Synthesis of the five ligands analyzed in this study.
2.2. Solution Studies
The binding properties of host ligands L1–L5 with guest anions G
-
3-
(G = H2PO4 , HP2O7 , and OH-) were studied by UV-vis and fluorescence techniques
in CH3CN solutions solution. Table 1 summarizes the addition constants of the anions
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studied to the ligands L1–L5, which were obtained by processing the
spectrophotometric data derived from the titration experiments.
Table 1. Logarithms of the addition constants of the G guests (G = AcO-, F-, Cl-,
3-
HP2O7 , and OH-)a to L1–L5 as determined by UV-Vis spectroscopy in CH3CN
solution at 298 K.
log K(c)
L1
L2
L3
L4
L5
Reactionb
L + PPi = [L(PPi)]
L + H2PO4 = [L(H2PO4)]
L + OH = [LOH]
LOH + OH = [L(OH)2]
4.9(1)
6.7(1)
5.0(1)
4.1(1)
5.0(1)
5.1(1)
4.8(1)
4.0(1)
6.2(1)
6.0(1)
6.3(2)
5.9(2)
5.4(1)
4.6(1)
5.2(1)
5.2(2)
5.9(1)
5.0(2)
5.4(1)
5.2(2)
a Anions were added as tetrabutylammonium salts; b Charges are omitted for clarity;
c Values in parentheses are the standard deviation to the last significant figure.
The UV-Vis absorption spectra of all ligands L1–L5 were affected by the
addition of guests. For example, Figure 2(a) shows the titration spectra of L1 with the
3-
anion HP2O7 (PPi). In the absence of anion, L1 showed a strong UV absorption in
the range of 225–325 nm, with two main bands with a λmax at 248 nm (ε = 24900 cm-
1mol-1dm3) and 291 nm (ε = 2100 cm-1mol-1dm3). The addition of increasing amounts
of PPi to L1 shifted the two λmax towards lower energies; after the addition of
approximately 2 equiv. of PPi (see the inset of Figure 2(a)), the λmax shifted to 250 nm
(ε = 24000 cm-1mol-1dm3) and 294 nm (ε = 2350 cm-1mol-1dm3). This spectral change
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was attributed to the formation of L1–PPi adducts, which affects the chromophore in
this solvent. According to the UV–Vis experiments, the deprotonation process was
not observed in the presence of a large amount of the guest. The presence of the 3,5-
bis(trifluoromethyl)phenyl group makes the ligand L1 fluorescent; in fact, free L1
shows an emission band in CH3CN with a λem centered at 333 nm (λex = 248 nm). The
addition of interacting anions, however, induced very small changes in the
fluorescence spectrum (Figure S1, ESI), thus preventing the possibility to exploit this
technique in signaling the interaction with phosphates phosphate guests.
(a)
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(b)
Figure 2: (a) UV-vis titration of L1 (2.52 × 10-5 mol dm-3) with PPi (0–2.52 × 10-4
mol dm-3) in CH3CN solution at 298 K; (b) UV-vis titration of L2 (2.54 × 10-5 mol
dm-3) with PPi (0–2.54 × 10-4 mol dm-3) in CH3CN solution at 298 K. Inset: variation
of the λabs at 248 nm (a) of L1 as a function of PPi added.
The ligand L2, a thio analog of L1, has a thiourea moiety as the binding group
and the 3,5-bis(trifluoromethyl)phenyl group as the chromophore. L2 exhibited an
intense absorption band in the UV spectrum centered at 250 nm. As observed for L1,
addition of the PPi anion to L2 induced a red shift of the λmax at 250 nm (ε = 20500
cm-1mol-1dm3) to 255 nm (ε = 18500 cm-1mol-1dm3); adding more than 20 equivalent
of PPi the formation of a new absorption band at 315 nm, attributable to the
formation of the deprotonated form of the ligand, is observable (Figure 2(b)). When
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present in a large excess (>20-fold), the PPi anion is able to deprotonate the ligand
L2 but not L1 and this can be ascribed to the higher acidity of the thiourea with
respect the urea moiety.32 The deprotonation process, however, did not hamper
determination of the association constant with this anion because it occurs only at
high PPi concentrations. Free L2 is not fluorescent probably due to photoinduced
electron transfer (PET) quenching because of the presence of the sulfur atom,33 and it
also remains quenched in the presence of anions.
Taking advantage of the above observations as well as desiring a sensor with a
binding unit more acidic than urea (as obtained with L2) but able to signal the
coordination of a guest by fluorescence, we designed and synthesized the new ligand
L3, which contains the cyanoguanidine moiety as the binding group instead of
thiourea. Therefore, PET quenching due to the sulfur atom of the thiourea moiety
should be prevented. N,N’,N’’-trisubstituted guanidine groups often adopt a trans
orientation of the two hydrogen atoms of the guanidine subunit; hence, they are not
preorganized to bind anionic guests.34 However, in the presence of strongly
interacting anions, such receptors usually are flexible in solutions, thus orienting both
NH groups towards the anion. In contrast, weaker binding of guests may result in an
interaction with only a single NH group.35 The ligand L3 is able to coordinate both
-
H2PO4 and PPi anions as revealed by UV-Vis and fluorescence titrations. The UV-
Vis spectrum of L3 shows the formation of a new intense band centered at 304 nm (ε
= 17200 cm-1mol-1dm3) when interacting with the PPi anion (Figure 3). This band
increased in intensity up to one equivalent of guest, thus indicating the formation of a
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stable L-PPi complex with a 1:1 stoichiometry (Table 1). As depicted in the proposed
model for the interaction between L3 and pyrophosphate (Scheme 2), the host-guest
interaction occurs via H-bonding among NH and O atoms giving rise to the
photochemical change. The ligand L3 is fluorescent and shows an intense emission
band centered at 286 nm (λex = 252 nm); however, as observed in the case of L1, the
addition of interacting guests did not influence significantly the emission spectrum
(Figure S2, ESI). A large excess of the PPi anion gives a quenching of the emission
because of a deprotonation of the ligand.
Figure 3: UV-vis titration of L3 (2.53 × 10-5 mol dm-3) with PPi (0–2.53 × 10-4 mol
dm-3) in CH3CN solution at 298 K. Inset: variation of the λabs at 304 nm as a function
of PPi addition.
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CF3
CN
N
F3C
N
H
N
H
O
O
O
OH
P
P
O
O
O
Scheme 2: Proposed binding mode of L3 with PPi.
Next, with the aim to preserve of preserving the effective cyanoguanidine
binding site for PPi and improve the fluorescent optical response, the ligands L4 and
L5 were designed and synthesized. These two ligands incorporate two well-known
chromophoric units, the coumarin and BODIPY dyes in L4 and L5, respectively;
these chromophores were chosen for their excellent absorption and emission
properties. In the case of L4, the absorption spectra recorded during the titration with
-
H2PO4 and PPi anions are reported in Figure S3(a) (ESI) and Figure 4(a),
respectively. As shown, the UV-Vis spectrum of the ligand alone shows strong
absorption bands in the range of 265–385 nm, with two main bands centered at 292
nm (ε = 9000 cm-1mol-1dm3) and 324 nm (ε = 12900 cm-1mol-1dm3). The addition of
guests caused a shift of the two λmax of the coumarin moiety towards lower energies;
however, the spectra obtained in the two cases are quite different. The spectra
recorded during PPi anion addition show the formation of a new band centered at 378
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nm with the disappearance of the band at λmax = 324 nm. This new absorption band is
attributable to the formation of the deprotonated form of the ligand; in fact, the UV-
Vis spectrum obtained by titrating L4 with a strong base, such as Bu4NOH, shows the
same profile, with a main absorption band centered at 380 nm (ε = 15500 cm-1mol-
1dm3) (Figure S4, ESI). The deprotonation process observed for both PPi and
hydroxide anions, however, did not hamper the determination of the association
-
constants with these guests. In contrast, the interaction with the H2PO4 anion did not
give rise to this new band, resulting only in a slight red-shift of the λmax at 324 nm
(Figure S3(a), ESI). Similar considerations were made by observing the fluorescence
emission spectra of L4. This ligand is highly fluorescent due to the presence of the
coumarin dye, and its emission properties significantly change when interacting with
anions. The fluorescence spectra obtained during the titration of L4 with the PPi
anion in CH3CN are reported in Figure 4(b). The free ligand shows an intense
fluorescence emission with a λmax centered at 390 nm (λex = 339 nm). Adding PPi
anion, the intensity of this band decreased together with the formation of a new band
at λmax = 466 nm. As observed in the absorption spectra, this new band can be
ascribed to the fluorescence emission of the deprotonated form of the ligand; in fact,
the fluorescence spectrum of L4 recorded in the presence of an excess of a strong
base, such Bu4NOH, shows an intense emission band centered at 469 nm (λex = 339
-
nm) (Figure 5). When L4 interacted with the H2PO4 anion, the emission spectrum of
the ligand only changed slightly (Figure S3(b), ESI). Furthermore, the typical
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emission band of the deprotonated ligand at 469 nm was observed when a large
-
excess (100-fold) of H2PO4 was added to the solution.
(a)
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(b)
Figure 4: (a) UV-vis titration of L4 (2.45 × 10-5 mol dm-3) and (b) fluorescence
titration of L4 (5.55 × 10-6 mol dm-3; λex = 520 nm) with PPi in CH3CN solution at
298 K. Insets: variation of the λabs at 324 nm (a) and variation of emission intensity at
565 nm (b) as a function of PPi addition.
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Figure 5: UV-vis (solid line) and fluorescence (dotted line) spectra of ligand L4
recorded in the presence of a large excess (100-fold) of Bu4NOH
Ligand L5 replaces the coumarin group of L4 with the highly sensitive
BODIPY dye. The UV/Vis spectrum of the free ligand shows an intense absorption
band at λmax = 522 nm (ε = 71220 cm-1mol-1dm3), a shoulder at 493 nm that
corresponds to the S0 → S1 (π-π*) transitions of the BODIPY chromophore, and a
weaker broad band at around 370 nm that can be assigned to an S0 → S2 (π-π*)
-
transition.36 The addition of the anionic guests H2PO4 , PPi, and OH-, however,
induced only small changes in the UV-Vis spectra. As an example, the interaction
with PPi resulted in a hypochromic shift of the maximum absorption wavelength to λ
= 518 nm and a slight increase of molar absorptivity (ε = 80200 cm-1mol-1dm3) of the
17

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BODIPY chromophore (Figure 6(a)). Unfortunately, the modifications observed in
the absorption spectra, both in terms of the wavelength maxima and absorptivity, are
very small, possibly due to the distance between the signaling chromophoric unit and
the receptor binding site. Of note, in the ligand L5, a phenyl group is present as a
spacer between the BODIPY core and the cyanoguanidine moiety. However, the
presence of the BODIPY moiety confers to the ligand a high fluorescent emission
that can be monitored in the presence of anions. The free L5 exhibits a broad and
intense emission band centered at 535 nm (λex = 520 nm). The addition of anions
decreased the emission intensity, although without total fluorescence quenching; thus,
titration experiments were carried out to evaluate the stability constants by this
technique. As an example, Figure 6(b) reports the emission spectra recorded during
the titration of L5 with PPi; as shown, the fluorescence emission intensity was
significantly reduced upon interaction with this anion. Similar behaviors were
-
observed in the cases of H2PO4 and OH- anions, thus revealing the formation of
stable L5-G adducts with these guests as well. The addition of a large excess (100-
fold) of a strong base, such as Bu4NOH, resulted in a complete quenching of the
emission (Figure S5(b), ESI); moreover, the same behavior was observed after the
addition of a 100-fold excess of PPi. These data can be explained by considering that
L5 undergoes deprotonation in the presence of a large excess of strong base and that
the deprotonated form of the ligand is not emitting.
2.3. Addition constants
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Table 1 reports the stability constants obtained after processing the data
derived from the UV-Vis titration experiments in CH3CN. All ligands L1–L5 were
-
3-
able to bind guests G (H2PO4 , HP2O7 , and OH-) to form stable adducts having an L
to G stoichiometry of 1:1, with the exception of hydroxide anion that provided the
LG2 species.
The addition association constants of ligands L1–L5 with various guests are
-
rather similar. For example, for the interaction of the H2PO4 anion with L1 and L4,
respectively, the log K values ranged from 6.7 to 4.6. These results can be explained
by considering that the binding unit is always the ureidic functional group and that
the ligands are not preorganized molecules. However, ligand L3, which contains the
cyanoguanidine group as the binding unit, was a better receptor for the PPi anion in
CH3CN, with the highest addition constant (log K = 6.2) among all the ligands
studied. Unfortunately, insertion of a stronger signaling unit, as in L4 and L5, slightly
decreased the binding capabilities of these receptors towards this anion, probably due
to the higher steric hindrance of the coumarin (L4) and BODIPY (L5) chromophores
with respect to the 3,5-bis(trifluoromethyl)phenyl group (L3). The phosphate anion,
instead, interacted more strongly with the urea-based ligand L1, and the addition
constant value determined was >2 logarithmic units higher than that of the
cyanoguanidine-based ligand L4 (log K = 6.7 and 4.6 for L1 and L4, respectively).
For this anion, the increase in acidity of the ureidic functional group of the receptor
did not result in a stronger interaction with the phosphate anion; in fact, the addition
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constants for L1 and L2, containing urea and thiourea moieties, were log K = 6.7 and
5.1, respectively (Table 1).
The hydroxide anion deprotonated the ureidic functional group of the ligands
only when it was present in a large excess; therefore, it was possible to safely
evaluate the addition constants for G = OH- by titrating ligands L1–L5 with
Bu4NOH. All the ligands formed stable [LOH]- and [L(OH)2]2- complexes, and the
values of the formation constants for these species are reported in Table 1. The log K
values for the addition of the first and the second hydroxide anions are almost
identical for each ligand, suggesting an independent coordination process for each
NH group. In particular, these findings were observed for the ligands L3–L5, which
contain the cyanoguanidine group; moreover, the trans orientation of the two
hydrogen atoms of the guanidine subunit that N,N’,N’’-trisubstituted guanidine
groups often adopt could favor two distinct coordination processes.
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PPi
a)
PPi
b)
Figure 6: (a) UV-vis titration of L5 (9.9 × 10-6 mol dm-3) and (b) fluorescence
titration of L5 (1.39 × 10-6 mol dm-3) with PPi in CH3CN solution at 298 K.
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3. Conclusions
The syntheses and the coordination properties of five novel fluorescent anion
receptors are reported. The ligands have different binding units with different
signaling chromophores. The aim of this work was to evaluate the proper
combination of an effective binding group for anions and a suitable fluorescent
signaling unit. To this end, three different urea-based derivatives were synthesized
and their binding behavior toward selected anionic guests was evaluated. Afterwards,
by keeping the binding unit constant, different chromophores were explored to
identify the best sensor among those synthesized. The ligands L1–L3 have the same
3,5-bis(trifluoromethyl)phenyl signaling moiety and the binding unit is either urea,
thiourea, or cyanoguanidine, respectively. Since the cyanoguanidine-based ligand L3
was revealed to be the most promising one, L4 and L5 were synthesized, which
contained this binding unit as well as the coumarin or BODIPY moiety, respectively.
Absorption and emission spectroscopy was used to study all of the ligands. Ligands
-
L1–L5 were able to bind the guests G examined (PPi, H2PO4 , and OH- anions), and
the evaluated stability constants accounted for the formation of [LG] species; when
the guest was the OH- anion, the dinuclear species [LG2] was also observed with all
five ligands. The cyanoguanidine-based ligand L3 provided higher addition constants
-
with both PPi and OH- anions, while H2PO4 afforded the most stable [LG] species
with the ligand L1. Insertion of the coumarin and BODIPY chromophores in L4 and
L5, respectively, resulted in sensors with better signaling. Of note, the complexes
formed with the guests examined had the maximum absorption in the visible region
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and both ligands were fluorescent, with the emission intensity strongly decreasing
upon interaction with the anions examined. However, the addition constants
determined were less than that of L3, probably due to the steric hindrance of the
signaling unit. Thus, taking all the results into consideration, the ligands L4 and L5
show the most promise as anion sensors.
Acknowledgments
We acknowledge financial support from Department of Base Sciences and
Foundations Pure and Applied Sciences, University of Urbino.
4. Experimental Section
4.1. General Methods
IR spectra were recorded on a Shimadzu FTIR-8300 spectrometer. Melting points
were determined on a Buchi melting point B 540 apparatus and are uncorrected. The
HPLC system (Waters Alliance 2795) was coupled with a photodiode array detector
(Waters 2996 PDA), followed by an electrospray mass spectrometer detector (ESI-
MS) (Waters-Micromass ZQ) worked by Mass Lynx 4.1 SP4 software. The ESI-
MS analyses were performed in a positive mode under the following conditions:
source and desolvation temperature 100 and 250 °C; capillary and cone voltage 2.5
kV and 25 V; cone and desolvation flow (nitrogen gas) 40 and 400 L/h. HPLC
analyses were performed on a 25 cm x 4.6 mm Discovery C-18, 5 cm column
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(Supelco, Bellefonte, PA) equipped with a Supelguard Discovery C-18 guard column
(2 cm x 4 mm, 5 cm). Solvents A (MeOH) and 5 (C, 5nM ammonium acetate) were
run at a flow rate of 1 mL/min. The running gradient was adjusted to 10% A (2 min),
increasing to 90% A (18 min), and then 100% C (2 min), followed by a
reequilibration at 10% A (15 min). All solvents were HPLC-grade (Aldrich-Sigma)
and water was purified via a Millex Q-plus system (Millipore). Elemental analyses
were performed on a Carlo Erba analyzer, and the results are within ±0.4 of the
theoretical values (C,H,N).
All chemicals were purchased from Aldrich, Fluka and Lancaster in the highest
quality commercially available. All the solvents were dried prior to use.
Chromatographic separations were performed on silica gel columns by flash
chromatography (Kieselgel 60, 0.040–0.063 mm, Merck). TLC analyses were
performed on precoated silica gel on aluminium sheets (Kieselgel 60 F254, Merck).
4.2. Synthesis
1-Benzyl-3-[3,5-bis(trifluoromethyl)phenyl]urea (L1)
To a well stirred solution of benzylamine 1 (0.55 ml, 5.0 mmol) in dry CH2Cl2 (10
ml) was added 3,5-bis(trifluoromethyl)phenyl isocyanate 2 (1.24 ml, 6 mmol) under
N2. The reaction mixture was stirred at r.t. overnight, concentrated to reduced
pressure and purification by column chromatography (SiO2; CH2Cl2 100% to
CH2Cl2:MeOH = 98:2) provided L1 (1.03 g, 57%) as a white solid.
TLC (CH2Cl2) = Rf : 0.35 (UV, KMnO4); m.p.: 189-190 °C;
1H-NMR (200 MHz, DMSO-d6, δ in ppm): 4.32 (d, J= 5.7 Hz, 2H), 7.03 (t, J=5.7 Hz,
13
1H), 7.30 (s, 5H), 7.53 (s, 1H), 8.10 (s, 2H), 9.39 (s, 1H). C-NMR (50 MHz,
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DMSO-d6, δ in ppm): 43.2, 126.5, 127.2, 127.5, 128.7, 130.7, 131.4, 140.4, 143.0,
155.3.
FTIR (film, cm-1): 3327, 1655, 1560, 1272, 1168, 877.
MS (ESI): 361.1 (100%) [M-H]-;
Anal. Calcd. For C16H12F6N2O (362.09): C, 53.05; H, 3.34; N, 7.73; Found: C 53.01;
H, 3.41; N, 7.75.
1-Benzyl-3-[3,5-bis(trifluoromethyl)phenyl]thiourea (L2)
To a well stirred solution of benzylamine 1 (0.55 ml, 5.0 mmol) in 10 ml dry CH2Cl2
(10 ml) was added 3,5-bis(trifluoromethyl)phenyl isothiocyanate 3 (1.1 ml, 6.0
mmol) under N2. The reaction mixture was stirred at r.t. overnight, concentrated to
reduced pressure and purified by column chromatography (SiO2; cyclohexane:Ac-
OEt = 80:20) to provide L2 (1.18 g, 64%) as a white solid.
TLC (cyclohexane:AcOEt = 7:3) = Rf : 0.49 (UV, KMnO4); m.p.: 152-153 °C.
1H-NMR (200 MHz, DMSO-d6, δ in ppm): 4.78 (d, J= 5.3 Hz, 2H), 7.35 (s, 5H), 7.73
(s, 1H), 8.27 (s, 2H), 8.70 (s, 1H), 10.18 (s, 1H). 13C-NMR (50 MHz, DMSO-d6, δ in
ppm): 47.5, 126.4, 127.5, 127.9, 128.8, 130.2, 130.9, 138.8, 142.3, 181.3.
FTIR (film, cm-1): 3222, 3046, 1550, 1278, 1178, 891.
MS (ESI): 377.0 (100%) [M-H]-.
Anal. Calcd. For C16H12F6N2S (378.06): C, 50.79; H, 3.20; N, 7.40; Found: C 50.81;
H, 3.21; N, 7.45.
1-Benzyl-3-[3,5-bis(trifluoromethyl)phenyl]-2-cyanoguanidine (L3)
To a well stirred solution of 1-benzyl-3-[3,5-bis(trifluoromethyl)phenyl]thiourea L2
(0.3 g, 0.793 mmol) in 8 ml of dry CH3CN (8.0 ml) was added EDCI (1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide) (182.4 mg, 0.951 mmol), triethylamine (143.7
mg, 1.03 mmol) and finally cyanamide (50.0 mg, 1.19 mmol) under N2. The reaction
mixture was stirred at r.t. for 1 h, refluxed for 16 h, and then concentrated under
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reduced pressure. The residue was purified by column chromatography (SiO2;
cyclohexane:AcOEt = 70:30) to provide L3 (0.128 g, 41%) as a white solid.
TLC (cyclohexane:AcOEt = 1:1) = Rf: 0.53 (UV, KMnO4); m.p.: 92-95 °C.
1H NMR (200 MHz, CDCl3, δ in ppm): 4.54 (d, J=5.7 Hz, 2H), 6.27 (bs, 1H), 7.32
13
(m, 5H), 7.63 (s, 1H), 7.71 (s, 2H), 8.59 (bs, 1H). C-NMR (50 MHz, CDCl3, δ in
ppm): 29.70, 46.24, 127.50, 128.35, 129.12, 132.32, 132.99, 136.03, 138.30, 157.97.
FTIR (film, cm-1): 3276, 2990, 2176, 1571, 1376, 1278, 1133, 888.
MS (ESI): 385.2 (100%) [M-H]-.
Anal. Calcd. For C17H12F6N4 (386.10): C, 52.86; H, 3.13; N, 14.50; Found: C 52.81;
H, 3.11; N, 14.55.
1-Benzyl-3-(4-methyl-2-oxo-2H-chromen-7-yl)thiourea (6)
To a well stirred solution of 7-amino-4-methyl-2H-chromen-2-one 4 (0.176 g, 1.0
mmol) in dry DMF (5.0 ml) was added triethylamine (0.144 g, 1.03 mmol) followed
by phenyl isothiocyanate 5 (0.160 ml, 1.20 mmol) under N2. The reaction mixture
was stirred overnight at 60 °C and then concentrated under reduced pressure. The
residue was purified by column chromatography (SiO2; CH2Cl2:MeOH = 99:1) to
provide 6 (233.0 mg, 0.72 mmol, 71.8% yield) as slight yellow solid.
TLC (CH2Cl2:MeOH = 98:2) = Rf: 0.42 (UV, KMnO4); m.p.: 199-201 °C.
1H-NMR (200 MHz, DMSO-d6, δ in ppm): 2.42 (s, 3H), 4.84 (d, J= 6.0 Hz, 2H), 6.21
(s, 1H), 7.39 (m, 7H), 7.57 (d, J=2 Hz, 1H), 8.01 (d, J=8.6 Hz, 1H), 8.43 (s, 1H).
13C-NMR (50 MHz, DMSO-d6, δ in ppm): 18.44, 47.65, 108.5, 112.7, 115.6, 118.3,
125.9, 127.5, 128.1, 128.8, 138.9, 143.6, 153.5, 153.8, 160.4.
FTIR (film, cm-1): 3250, 3052, 1747, 1550, 1281, 1171, 891.
MS (ESI): 325.0 (100%) [M+H]+.
Anal. Calcd. For C18H16N4O2S (324.09): C, 66.64; H, 4.97; N, 8.64; Found: C 66.81;
H, 4.91; N, 8.55.
1-Benzyl-3-(4-methyl-2-oxo-2H-chromen-7-yl) -2-cyanoguanidine (L4)
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To a well stirred solution of the 1-benzyl-3-[4-methyl-2-oxo-2H-chromen-7-
yl]thiourea 6 (0.43 g, 1.33 mmol) in dry DMF (5.0 ml) was added EDCI (254.1 mg,
1.33 mmol), TEA (0.184 ml, 1.33 mmol), cyanamide (55.7 mg, 1.33 mmol) under N2.
The reaction mixture was stirred overnight at 60 °C, concentrated and purified by
column chromatography (SiO2; CH2Cl2:acetone = 90:10) to provide L4 (250.0 mg,
0.75 mmol, 56.8%) as a yellow solid.
TLC (CH2Cl2:MeOH = 8:2) = Rf: 0.45 (UV, KMnO4); m.p.: 214-217 °C.
1H-NMR (200 MHz, DMSO-d6, δ in ppm): 2.33 (s, 3H), 4.42 (s, 2H), 6.21 (s, 1H),
13
7.25 (m, 7H), 7.64 (d, J=9.3 Hz, 1H), 8.01 (bs, 1H), 9.40 (s, 1H). C-NMR (50
MHz, DMSO-d6, δ in ppm): 18.5, 45.4, 109.3, 113.1, 116.1, 116.9, 118.5, 126.4,
127.6, 127.7, 128.9, 138.8, 142.1, 153.5, 154.0, 158.1, 160.3, 163.3.
FTIR (film, cm-1): 3282, 2992, 2176, 1745, 1563, 1372, 1281, 1153.
MS (ESI): 333.0 (100%) [M+H]+.
Anal. Calcd. For C19H16N4O2 (332.13): C, 68.66; H, 4.85; N, 16.86; Found: C 68.71;
H, 4.81; N, 16.85.
1-Benzyl-3-[4-(4,4-difluoro-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-
diazaindacen-8-yl)phenyl]thiourea (8)
To a well stirred solution of 8-(4-aminophenyl)-4,4-difluoro-1,3,5,7-tetramethyl-2,6-
diethyl-4-bora-3a,4a-diazaindacene 731 (166.0 mg, 0.42 mmol) in dry CH2Cl2 (50.0
ml) at 0° C, was added triethylamine (0.6 ml) followed by a solution of thiophosgene
(0.056 ml, 0.74 mmol) (Caution! This reaction should be carried out in a good hood
because of the toxicity of thiophosgene) in 10.0 ml of dry CH2Cl2 under N2. The
reaction mixture was stirred for 15 min at r.t. followed by addition of H2O (30 ml).
The water phase was extracted with CH2Cl2 (2x20 ml) and the organic phases were
dried over Na2SO4 and evaporated under reduced pressure to give the crude 4-(4,4-
difluoro-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diazaindacen-8-yl)phenyl
isothiocyanate.
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The above isothiocyanate was dissolved in a 1:1 mixture of dry CH2Cl2 and CH3CN
(30 ml) followed by the addition of benzylamine (0.09 ml, 0.82 mmol). The reaction
mixture was stirred overnight at r.t., concentrated and purified by column
chromatography (SiO2; first with CH2Cl2:cyclohexane
=
8:2 then
CH2Cl2:cyclohexane = 9:1 and finally only CH2Cl2) to obtain 8 (164.0 mg, 0.30
mmol, 73%) as a red solid.
TLC (CH2Cl2:cyclohexane = 7:3) = Rf: 0.23 (UV); m.p.: 187-190 °C.
1H-NMR (200 MHz, DMSO-d6, δ in ppm): 0.95 (t, J=7.5 Hz, 6H), 1.34 (s, 6H), 2.30
(q, J=7.5 Hz, 4H), 2.43 (s, 6H), 4.79 (d, J=6.0 Hz, 2H), 7.30 (m, 7H), 7.78 (d, J=8.0
Hz, 2H), 8.27 (t, J=6.0 Hz, 1H), 9.80 (bs, 1H).
13C-NMR (50 MHz, DMSO-d6, δ in ppm): 12.0, 12.7, 14.9, 16.9, 47.5, 122.8, 127.5,
127.7, 128.0, 128.6, 128.8, 130.0, 133.0, 138.6, 139.1, 141.0, 153.5, 181.2.
FTIR (film, cm-1): 3213, 3012, 1546, 1261, 1183, 891.
MS (ESI): 543.0 (100%) [M-H]-.
Anal. Calcd. For C31H35BF2N4S (544.26): C, 68.38; H, 6.48; N, 10.29; Found: C
68.31; H, 6.41; N, 10.35.
1-Benzyl-3-[4-(4,4-difluoro-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-
diazaindacen-8-yl)phenyl]-2-cyanoguanidine (L5)
To a well stirred solution of the above thiourea 8 (0.1g, 0.18 mmol) in dry CH3CN
(5.0 ml) was added EDCI (42.3 mg, 0.18 mmol), TEA (0.033 ml, 0.24 mmol),
cyanamide (11.7 mg, 0.28 mmol) under N2. The reaction mixture was stirred
overnight at 60° C, concentrated and purified by column chromatography (SiO2; first
with CH2Cl2:cyclohexane = 9:1 then with CH2Cl2 and finally with CH2Cl2:MeOH =
(99.5:0.5) to provide L5 (55.0 mg, 54%) as a red solid.
TLC (CH2Cl2:MeOH 99:1) = Rf: 0.21 (UV); m.p.: 251-253 °C.
1H-NMR (200 MHz, DMSO-d6, δ in ppm): 0.94 (t, J=7.4 Hz, 6H), 1.32 (s, 6H), 2.29
(q, J=7.4 Hz, 4H), 2.43 (s, 6H), 4.55 (d, J=5.9 Hz, 2H), 7.41 (m, 9H), 7.87 (t, J=5.9
Hz, 1H), 9.26 (s, 1H).
28