Fluorescent acridine-based receptors for H2PO4 -

Article abstract of DOI:10.1021/jo202077v

Two new pseudopeptidic molecules (one macrocyclic and one open chain) containing an acridine unit have been prepared. The fluorescence response of these receptors to a series of acids was measured in CHCl₃. Receptors are selective to H₂

Full text of DOI:10.1021/jo202077v

Article
pubs.acs.org/joc
−
Fluorescent Acridine-Based Receptors for H2PO4
Vicente Martí-Centelles,†,‡ M. Isabel Burguete,† Francisco Galindo,† M. Angeles Izquierdo,†
D. Krishna Kumar,‡ Andrew J.P. White,‡ Santiago V. Luis,*,† and Ramon Vilar*,‡
́
†Department of Inorganic and Organic Chemistry, Universitat Jaume I, E-12070 Castellon
́
, Spain
‡Department of Chemistry, Imperial College London, London SW7 2AZ, U.K.
S
* Supporting Information
ABSTRACT: Two new pseudopeptidic molecules (one macro-
cyclic and one open chain) containing an acridine unit have been
prepared. The fluorescence response of these receptors to a
series of acids was measured in CHCl3. Receptors are selective
−
−
to H2PO4 versus HSO4 , and an even higher selectivity is
found over other anions such as Cl−, Br−, CH3COO−, and
CF3COO−. We show that the macrocyclic receptor is more
selective for H2PO4− than the related open chain receptor. The
supramolecular interactions of triprotonated receptors with
different anions have been modeled in silico and have been
studied by different experimental techniques. Optimized geometries obtained by computational calculations agree well with
experimental data, in particular fluorescence experiments, suggesting that the selective supramolecular interaction takes places
through coordination of the anions to the triprotonated form of the receptor.
acridine moiety and their fluorescence response to a series of
acids in CHCl3. These results show that both receptors are able
to selectively recognize H2PO4− over other anions, with the
macrocycle 2 being more selective for this anion than the open-
chain compound 1.
INTRODUCTION
■
Fluorescent receptors have important applications in a wide
range of areas such as supramolecular, synthetic, bioorganic,
medicinal, and biological chemistry.1−14 Processes such as
protein binding to specific ligands or molecular recognition of
DNA can be studied with the help of fluorescent systems.15−20
The recognition of acids is an interesting field that has been
widely investigated.21−32 However, in most cases, the recogni-
tion event is based on the interaction between the hydrogen
ions generated upon acid dissociation and the molecular
receptor. A direct and specific interaction of the receptor with
the corresponding conjugate base is often lacking in the
recognition process. In those cases, Coulombic interactions
are predominant and a poor selectivity is generally found,
usually with the recognition of the species having the highest
charges.33−35
Chemical receptors containing acridine fragments have
attracted interest due to the luminescent properties of this
species. For example, some macrocyclic and open chain
structures containing the acridine scaffold have been found to
bind to DNA.36,37 On the other hand, Kim et al. have prepared
an imidazolium acridine derivative as a fluorescent chemo-
sensor for pyrophosphate and dihydrogen phosphate.38
In recent years, some of us have focused on the preparation
and study of pseudopeptidic macrocyclic structures. A central
concept for the development of these compounds is the
generation of well-defined three-dimensional multifunctional
structures allowing them to selectively host small molecules and
ions.39−44
RESULTS AND DISCUSSION
■
Synthesis of the Receptors. Receptors 1 and 2 contain
amide and amine groups that can interact with anions via
hydrogen bonding and, if the amine is protonated, also via ion-
pairing.45,46 On the other hand, the acridine unit provides the
compounds with fluorescent properties, and additionally, its
protonation at the heterocyclic nitrogen atom can be used to
add additional interactions with anions and regulate the
fluorescent behavior of the system. Finally, the macrocyclic
structure in compound 2 can provide an appropriate scaffold
for a proper preorganization that can result in enhanced
recognition properties.47−51
Receptors 1 and 2 were prepared as shown in Scheme 1. The
4,5-bis(aminomethyl)acridine dihidrochloride 3 was prepared
according to previously reported procedures.52 Pseudopeptidic
compound 5 was obtained by reaction of 4,5-bis(aminomethyl)-
acridine with the corresponding amino acid activated as its
hydroxysuccinimide ester using dimethoxyethane (DME) as
solvent. Best results were obtained when the reaction was
stirred for 8 h at room temperature and then for 5 h at 50 °C.
The Cbz protecting group was then removed with the use of
HBr/AcOH to afford the amine hydrobromide, from which the
In this paper, we report on the synthesis of two pseudo-
peptidic compounds, 1 and 2 (Chart 1), containing a functionalized
Received: October 11, 2011
Published: November 11, 2011
© 2011 American Chemical Society
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Chart 1. Pseudopeptidic Receptors Containing Acridine Fragments Considered in This Work
a
Scheme 1. Synthesis of Pseudopeptidic Receptors 1 and 2
a
Key: (i) DME, rt; (ii) HBr/AcOH 33%, rt then aq NaOH; (iii) TBABr, DIPEA, CH3CN, temperature gradient from 50.0 to 81.6 °C.
free amine (open-chain receptor 1) was obtained upon
neutralization with NaOH. The key step for the preparation
of macrocycle 2 was the final macrocyclization reaction. In this
case, a careful control of the temperature with a temperature
gradient, using conditions similar to those previously reported
for related pseudopeptidic macrocycles,53 provided signifi-
cantly better yields than when the reaction was performed at
reflux. Thus, macrocycle 2 was prepared from the reaction
of bis(amidoamine) 1 with 1,3-bis(bromomethyl)benzene in
CH3CN, in the presence of tetrabutylammonium bromide
(TBABr) and N,N-diisopropylethylamine (DiPEA), using a
temperature gradient from 50.0 to 81.6 °C. The crude product
was purified by column chromatography to afford the expected
macrocycle in 85% yield. To the best of our knowledge, the
preparation of pseudopeptidic structures containing acridine
fragments has not been reported previously. It is clear that the
relatively high yields obtained for the macrocyclization reaction
involve a high degree of preorganization of the open-chain
intermediate and the corresponding transition state, as has been
demonstrated in other cases using this methodology54−57 and
open the way for the preparation of new families of macrocyclic
receptors containing this important moiety.
Fluorescence Response to Acids. The electronic
absorption spectra and the steady-state fluorescence emission
spectra of 1 and 2 were recorded in CHCl3. As expected,58
compounds 1 and 2 have similar properties and both show
absorption maxima at 357 nm and present a maximum of
emission at 420 nm when excited at 357 nm (Figure 1).
Different experiments were carried out to analyze the
potential interaction of both receptors with acids in CHCl3.
Thus, the interactions with H3PO4, H2SO4, HCl, HBr, acetic
acid, and trifluoroacetic acid in CHCl3 were studied. As can be
seen in Figure 2, upon addition of acids there is an increase of
emission in the case of H3PO4, which is more significant than in
the case of H2SO4. The response toward HCl, HBr, acetic acid,
and trifluoroacetic acid is practicably negligible. It must be
noted that the recorded fluorescence corresponds to a new
band centered at 510 nm, whereas the original fluorescence at
420 nm disappears when the new compound is formed. Titra-
tion fluorescence spectra did not show an isosbestic point, and
therefore curves did not allow us to calculate the corresponding
binding constants (see the Supporting Information). The much
larger emission enhancement observed for H3PO4 suggested
that compound 2 could be used as potential fluorescent
chemical sensor for the detection of this acid.59−67
Compounds 1 and 2 were characterized by spectroscopic and
1
This is more clearly highlighted when the normalized
emission values for the maxima of the corresponding emission
bands are represented for both compounds. Figure 3 compares
the fluorescence measured after the addition of 25 equivalents
of each acid. We can see that both the open chain and the
macrocyclic compound display preferred recognition toward
H3PO4, being the affinity toward this acid higher with the
macrocyclic receptor 2.
analytical techniques. Interestingly, in CDCl3 solution, the H
NMR signals of the CH2 groups at positions 4 and 5 of the
acridine moiety are considerably different between compounds
1 and 2. In the open-chain derivative 1, these protons appear as
doublets, whereas in compound 2 they appear as double
doublets. This suggests the presence of an appreciable coupling
constant with the amide hydrogen atom in 2 because of the
higher rigidity provided by the macrocyclic structure.
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Figure 3. Normalized fluorescence intensity at 510 nm of compounds
1 and 2 (10 μM) upon addition of 25 equiv of each acid in CHCl3.
λex = 357 nm.
receptor with H2PO4− can be considered in highly acidic media.
Thus, the origin of the recognition of H3PO4 by 1 and 2 could
rely on the protonation of the receptor and the stabilization of
−
the triprotonated species by the H2PO4 anion, which is not
−
accomplished with HSO4 or the other anions assayed. This
process would involve the protonation of the basic nitrogen
atom of the acridine moiety, leading to a strongly fluorescent
acridinium substructure.68
Figure 1. Normalized absorption (left axis) and emission (right axis)
spectra for compounds 1 and 2 (10 μM) in CHCl3. λex = 357 nm: (a)
compound 1, (b) compound 2.
The participation of a complex species involving the
−
protonated receptor interacting with H2PO4 was confirmed
by the following experiments. First, no change in fluorescence
was observed when compound 1 or 2 were titrated with
tetrabutylammonium dihydrogen phosphate (TBAH2PO4).
Similarly, the addition of an excess of a strong acid such as
TFA did not produce significant changes in the fluorescence,
therefore the enhanced fluorescence cannot be only due to a
protonation event but to a combined effect of proton and
anion. To corroborate this 50 equivalents of TFA were added
to receptors 1 and 2 followed by a titration with TBAH2-
PO4. As can be seen in Figure 4, this led to an increase in
fluorescence.
In order to fully characterize the photophysical and acid−
base properties of compounds 1 and 2, fluorescence measure-
ments in water were carried out. To better understand the
results obtained from these studies, the corresponding
photophysical parameters describing the fluorescence of
compounds 1 and 2 in this solvent were initially determined
with the use of both steady-state and time-resolved fluorescence
(time correlated single photon counting technique, TCSPC)
measurements. The fluorescence parameters for the parent
acridine were also determined for comparison. The results
obtained are shown in Table 1. Variations in the pH of the
solutions were achieved by adding small aliquots of H2SO4 or
NaOH. The samples were purged with nitrogen before any
measurement.
Values of quantum yield and fluorescence lifetime obtained
for acridine at acid and basic pH correspond with those
reported in the literature.69−73 The quantum yields obtained for
1 and 2 at acidic pH were very similar (0.43 and 0.38 respec-
tively) and slightly lower than those calculated for acridine itself
(0.65). The quantum yield seems to undergo an important
decrease upon basification of the medium, although the low
solubility of the pseudopeptidic receptors only allowed carrying
out the measurement in a small pH range. Values for the
different fluorescence (τF) lifetimes were obtained by
Figure 2. Emission spectra of compounds 1 and 2 (10 μM) after
addition of 25 equiv of each acid in CHCl3. λex = 357 nm: (a)
compound 1, (b) compound 2.
According to the expected acid−base properties of the
compounds involved, the interaction of the triprotonated
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Figure 5. Fluorescence decay curves of compounds 1, 2, and acridine
(20 μM in water, pH adjusted with H2SO4) in acidic aqueous solution
(λexc = 372 nm/ λem = 475 nm). The incident light pulse is also shown
(open triangles).
Figure 4. Fluorescence emission in CHCl3 of 1 and 2 (10 μM)
without and with TFA in the presence of TBAH2PO4. λex = 357 nm:
(a) compound 1, (b) compound 2.
Figure 6. Fluorescence decay curves of compounds 1 and acridine
(20 μM in water, pH adjusted with NaOH and H2SO4) in less acidic
aqueous solution (λexc = 372 nm/ λem = 426 nm). The incident light
pulse is also shown (open triangles).
deconvolution of the experimental curves (Figures 5 and 6).
The lifetime values determined for the pseudopeptidic receptor
1 were smaller than those for the parent acridine. In the case of
compound 1 at pH = 4.16 the experimental curve indicated the
participation of a second lifetime, being the contribution of the
shorter lifetime much higher than the contribution of the
longer lifetime.
The variation in the fluorescence of the receptors upon
modification of the pH was used to determine the cor-
responding pKa values for the protonation of the acridine
moiety. Figure 7 shows the main differences in the fluorescence
spectra for 1 and 2 at two different pH values and the resulting
titration curves are shown in Figure 8. The pKa values obtained
for compounds 1 and 2, and shown in Table 1, revealed some
interesting trends. The pKa of the third protonation of the
macrocyclic compound 2 (pKa ∼ 1.7), which leads to the
formation of strongly fluorescent acridinium species, is lower
than that of the open chain compound 1 (pKa ∼ 2.7). This
trend can be explained by taking into account that in macro-
cycle 2 the two initial ammonium groups are closer to the third
protonated site due to the rigidity of the macrocyle. In con-
trast, in the case of the open-chain compound 1, its higher con-
formational flexibility can allow to adopt conformations in
which the two ammonium groups are located as far as possible
from the acridinium unit to minimize the electrostatic repul-
sion. Therefore, it is more difficult to protonate the acridine
unit in the macrocyclic compound 2 justifying the difference
of 1 order of magnitude in the acidities. Nevertheless, once the
Table 1. Fluorescence Properties for Compounds 1 and 2 in Water (Measurements Made with a Compound Concentration of
20 μM in Water Using H2SO4 and NaOH To Adjust the pH)
d
e
f
f
pH
λabs (nm)
λexc (nm) λem (nm) ES1 (kcal/mol)
ΦF
τ1 (ns)
α1 (%)
τ2 (ns) α2 (%)
χ2
pKa
a
g
i
acridine
0.92
12.65
0.55
354, 398
355
354
354
359
359
477
427
502
501
65.9
72.7
62.7
62.6
0.65
0.24
0.38
0.43
0.05
31.6
1.03
1.22
1.15
1.15
0.96
5.42
b
a
a
c
h
9.5
j
g
2
359, 397
358, 398
357
23.9
1.67 0.02
2.74 0.01
g
1
0.68
26.6
h
h
4.16
356
432
71.8
1.3
95.43
12.2
4.57
a
b
c
d
e
λem = 540 nm. λem = 470 nm. λem = 450 nm. λexc = 366 nm. Quinine sulfate standard (aqueous H2SO4 0.1 M, air). ΦF = 0.53 (taken from
f
g
h
i
j
ref 73). λexc = 372 nm. λem = 475 nm. λem = 426 nm. Taken from ref 69. It was not possible to measure this sample at pH 4 due to precipitation
of the product.
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conclude that the fluorescent moiety in the supramolecular
−
−
complex formed between 1 or 2, and H2PO4 or HSO4 in
CHCl3 is always the acridinium fluorophore.
The overall protonation scheme for the open-chain
compound 1 and macrocycle 2 must consider three steps as
shown in Figure 9 for receptor 2. First, protonation of the
Figure 9. Overall protonation scheme for the macrocycle 2.
Figure 7. Fluorescence spectra at different pH: (a) compound 1,
λex = 366 nm; (b) compound 2, λex = 368 nm. Probe concentration:
10 μM in aqueous solution (1% DMSO).
amines takes place, followed by that of the acridine unit,
accordingly to the bigger basicity of the secondary amines as
compared to the acridine. The isosbestic point observed in the
titrations confirms that the observed fluorescence change is due
to the equilibrium between the diprotonated (LH22+) and
triprotonated (LH33+) species; see the Supporting Information.
A similar protonation scheme can be drawn for the open-chain
compound 1. It must be pointed out that solvent has an impor-
tant effect on the recognition process. In water the solvation
energy is larger than the host−guest binding energy. Thus,
spectra from titrations in water show an isosbestic point due
to equilibrium observed between the triprotonated and the
diprotonated receptor and no supramolecular species are
detected. In contrast, in chloroform, solvation energy is not as
important and supramolecular interactions play an important
role. In this case, the spectra obtained from titrations did not
show an isosbestic point indicating the participation of different
species in the recognition process.
1H NMR Spectroscopic Experiments. NMR experiments
were also carried out in order to confirm the suggested
protonation sequence. NMR titration experiments of com-
pound 2 showed that the addition of 2 equiv of TFA protonates
the two secondary amines. The protonation of the acridine, as
indicated by a downfield shift of ca. 1 ppm for the acridine
signal at 8.75 ppm, is more difficult. As shown in Figure 10, a
large concentration of TFA (350 mM), much above 3 equiv,
has to be added to protonate the acridine in compound 2
(Figure 10c).
Figure 8. Fluorescence pH titration of compounds 1 and 2,
monitoring emission at 500 nm. Probe concentration: 10 μM in
aqueous solution (1% DMSO). λex = 368 nm for 2 and λex = 366 nm
for 1. For compound 1 pKa = 2.74 0.01; for compound 2 pKa =
1.67 0.02.
acridine nitrogen atom is protonated, the photophysical behavior
is quite similar.
As can be deduced from Table 1 the behavior of compounds
1 and 2 in acidic medium is comparable to the one displayed by
acridine; i.e., the emitting species at long wavelength is clearly
the acridinium cation which displays fluorescence (ES1 ∼ 62−
66 kcal/mol and τ1 ∼ 24−32 ns) properties clearly different
to those of the parent acridine (ES1 ∼ 72 kcal/mol and τ1 <
10 ns). Hence, from these data in aqueous medium we can
Titration of compound 1 could not be carried out due to
precipitation of the product. Nevertheless, the triprotonated
1
compound 1 is soluble in CHCl3, and the H NMR spectrum
could be recorded (Figure 11). It can be seen that in
triprotonated compound 1 the acridine CH2 protons are non
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Figure 10. 1H NMR titration of 2 in CDCl3 in the presence of
different concentration of TFA. Probe concentration, 5 mM: (a) TFA
0 mM, (b) TFA 10 mM (2 equiv) (a precipitate was formed), (c) TFA
350 mM (all the precipitate was redissolved). Arrows show the
variation of the 9H of the acridine moiety in compound 2.
Figure 12. Optimized geometries at the B3LYP/6-31G* level of
−
−
3+
theory: (a) 6H33+, (b) 7H33+, (c) H2PO4 ·6H33+, (d) HSO4 ·6H3
,
−
−
3+
(e) H2PO4 ·7H33+, (f) HSO4 ·7H3
.
−
−
H2PO4 ·6H33+, −209.40 kcal/mol for HSO4 ·6H33+, −215.74
kcal/mol for H2PO4 ·7H33+, and −203.85 kcal/mol for
−
−
HSO4 ·7H33+. Therefore, those results suggest that for
compound 1 the binding energy of its triprotonated form
−
Figure 11. 1H NMR titration of 1 in CDCl3 with TFA. Probe
concentration, 5 mM: (a) TFA, 0 mM; (b) TFA, 350 mM. An
expansion of the signals corresponding to the acridine-CH2 protons of
each trace is given.
with H2PO4 can be 10.07 kcal/mol more favorable than that
−
for HSO4 , while for compound 2 the difference is slightly
higher (11.89 kcal/mol more favorable). These computational
results are in good agreement with the experimental fluores-
cence studies in CHCl3 and can be used to rationalize the ob-
served selectivity.
equivalent (see expansions in Figure 11) suggesting that for this
protonation state the conformation is more rigid.
A hydrogen bonding analysis of the structures of the different
supramolecular complexes formed between triprotonated 6 and
triprotonated 7 with the different anions is also useful for a
better understanding of the stabilities found.74 For this purpose,
a Monte Carlo conformational search with the MMFF force
field, as implemented in Spartan’08, was carried out for each
protonated species in order to have a wide range of conformers.
The most stable conformer for each species was fully optimized
at the B3LYP/6-31G* level of theory with the Gaussian
03 software, and then the H-bond distances were measured
(see Supporting Information). It can be observed that the open
chain compound 6 and its protonated species, and accordingly
the related receptor 1, are able to form a higher number of
intramolecular hydrogen bonds than the macrocyclic com-
pound 7 and its protonated derivatives (see the Supporting
Information). Considering the last protonation step, it can be
observed that the diprotonated open-chain compound 6 is
predicted to form two hydrogen bonds while the triprotonated
6 can form four hydrogen bonds. In contrast, for macrocycle
Computational Calculations. In order to investigate the
−
−
potential interaction of anions H2PO4 and HSO4 with
triprotonated 1 and 2 species, computational studies were
performed. To reduce computational cost, the benzene ring of
the amino acid side chain in structures 1 and 2 was replaced by
an H atom, yielding structures 6 and 7, respectively (see the
Supporting Information). First, the minimum conformers of
the L, LH+, LH22+, and LH3 species (with L being 6 and 7)
3+
were located performing a Monte Carlo conformational search
with Spartan ’08 at the MMFF level of theory. The same
calculation was then performed for the complexes
−
3+
−
H2PO4 ·LH3 and HSO4 ·LH33+. The resulting geometries
were minimized with Gaussian 03 at the B3LYP/6-31G* level
of theory. Computational models (Figure 12) show that the
−
−
anions H2PO4 and HSO4 interact with the triprotonated
receptor with the participation of an H-bond between the H of
the acridinium fragment and the anion. The calculated Gibbs
free energies for the interaction are −219.46 kcal/mol for
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7 both the monoprotonated and the triprotonated species
can form three hydrogen bonds (see Table 2). Therefore, the
a
Table 2. Hydrogen Bonds Found in the Optimized
Geometries for Reactants and Products at the Different
Protonation Steps for 6 and 7
6
7
reaction
reactants
products
reactants
products
H + L = HL
6
5
2
5
2
4
2
3
3
3
3
3
H + HL = H2L
H + H2L = H3L
a
Number of hydrogen bonds detected as defined by a H···X distance <
2.65 Å.
potential stabilization of the 6H33+ species through the presence
Figure 13. Molecular structure of a crystal of [2-H2](H2PO4)2
highlighting the presence of the phosphate groups.
of those two additional hydrogen bonds after protonation of
2+
6H2 supports the difference of the pKa observed exper-
imentally between 1 and 2.
one of the terminal phenyl rings (with centroid···centroid and
mean interplanar separations of ca. 3.91 and 3.60 Å,
respectively). The two amide groups have markedly different
orientations with respect to the nitrogen of the acridine moiety,
the N(16)- and N(31)-based amides adopting anti and syn
conformations respectively. The latter allows for an intra-
molecular N−H···N hydrogen bond [N(31)···N(1) 2.873(12)
Å], though the location of the hydrogen atom could not be
determined; modeled as being located on N(31) with an N−H
distance of 0.90 Å, the H···N separation and N−H···N angle are
2.24 Å and 127°, respectively). This intramolecular N···N
contact precludes the protonation of both nitrogens involved as
this would result in an H···H separation of ca. 1.52 Å. The
conformation of the macrocycle also places the N(16)-based
amide unit proximal to the 1,3-xylenyl ring with a C···C
separation between the amide carbon and the proximal
substituted carbon of the aryl ring of ca. 2.98 Å.
The same analysis can be carried out to rationalize the
stability of the complexes formed. Considering the formation of
LH33+X− species, we must bear in mind that for compound
6H33+ four hydrogen bonds are formed, whereas in macrocyclic
compound 7H33+ (due to conformational restrains conferred by
the macrocyclic geometry) only three hydrogen bonds can be
formed. In the calculated structure for the complex
−
3+
H2PO4 ·6H3 there are five intermolecular hydrogen bonds
−
between the H2PO4 anion and 6H33+, as well as one
intramolecular hydrogen bond. In the structure for the complex
−
3+
HSO4−·6H3 there are four hydrogen bonds between the
3+
HSO4 anion and 6H3 and two intramolecular hydrogen
bonds. These data clearly support the selective recognition of
H2PO4− in the presence of HSO4 . Besides, energy calculations
−
for both complexes indicate an energy difference of about 10
kcal/mol in favor of the complex with H2PO4 . In the
−
−
3+
optimized structure for the complexes H2PO4 ·7H3 and
−
HSO4 ·7H33+ there are five hydrogen bonds between the anion
The PO4 units (whose protonation could not be
unambiguously determined) sit outside the macrocycle, but
on account of the disorder in the structure75 only the
heteroatom separations are worth considering. The N(16)
amide nitrogen is approached by the oxygen of the 50%
occupancy O(81)-based water molecule at ca. 2.74 Å, and
alternatively by one of the oxygen atoms of the 50% occupancy
P(70)-based H3PO4 unit at ca. 3.20 Å. The N(19) amine
and the triprotonated ligand, and no intramolecular hydrogen
bonds are observed. On the other hand, it must be noted that
for all five hydrogen bonds the anion is acting as the hydrogen
acceptor, the hydrogen atoms of the anions being located at the
outer surface of the complex. Thus, the atoms bearing the
negative charge are located much closer to the cationic
ammonium groups. The situation is different to that found
for the complexes formed by 6H3 for which the anions act
both as hydrogen donors and acceptors. Thus, the calculated
structures for the complexes from 7H3 suggest that the
3+
−
nitrogen is linked to an oxygen of the P(50)-based H2PO4
anion at ca. 2.76 Å and to an oxygen of the major occupancy
3+
−
orientation of the P(60)-based H2PO4 anion at ca. 2.82 Å.
preorganization of the macrocyclic structure provides an
excellent complementarity between this triprotonated receptor
Similarly, the N(28) amine nitrogen is linked to an oxygen of
−
the P(50)-based H2PO4 anion at ca. 2.71 Å and to an oxygen
−
−
and H2PO4 or HSO4 anions, leading to an optimization of
electrostatic interactions and hydrogen bonding. Again, energy
calculations reveal a higher stability (ca. 10 kcal/mol) for the
of the major occupancy orientation of the P(60)-based H2PO4
anion at ca. 2.74 Å.
−
As indicated above, from the X-ray crystal structure it was
not possible to unambiguously locate the protons on the
amines or acridine group. Therefore, fluorescence experiments
in the solid state were also carried out by putting a few crystals
in a glass surface. The results obtained are displayed in Figure 14
and Table 3. Both the qualitative and quantitative analysis
of the spectra reveal an excellent agreement with those cor-
responding to a 20 μM solution of 2 at pH = 4. According
to previous experiments, this pH value corresponds to a re-
gion for which the nitrogen atom of the acridine moiety is not
protonated.
complex with the H2PO4 anion.
X-ray Studies. Single crystals of [2-H2](H2PO4)2 suitable
for X-ray diffraction were grown from the slow evaporation of a
solution of 2 with 10 equiv of H3PO4 in a water, methanol and
acetonitrile mixture (Figure 13). Under these conditions, the
acridine unit of compound 2 should not be protonated. This is
consistent with the protonation patterns for the acridine unit
detected with the other techniques used.
The solid-state structure75 shows the macrocycle to have
adopted an essentially self-filling conformation stabilized by an
intramolecular π−π interaction between the acridine unit and
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Article
Figure 15. S1−S0 energy difference for the fundamental and first exited
state of model compound 4,5-dimethylacridine interacting with a
proton at different N···H distances.
−
of H2PO4 in chloroform in acidic media. Macrocyclic com-
pound 2 was found to be more selective toward H2PO4− than
the open-chain compound 1 versus other anions such as HSO4−,
acetate, trifluoroacete, chloride, and bromide. Under these con-
−
ditions, a selective detection of H2PO4 can be accomplished
Figure 14. Normalized excitation and emission spectra for macrocycle
2 in a concentration 20 μM (water, 2% DMSO, and pH adjusted with
HClO4, and NaOH) at (a) pH 4.00, (b) pH 1.00, and (c) for the
crystals obtained from compound 2 and 10 equiv of phosphoric acid.
through fluorescence signaling. The process has been studied in
detail both experimentally and with the help of high level
calculations at the B3LYP/6-31G* level. The whole set of data
reveal that both ligands (1 and 2) show, when triprotonated,
−
a high level of complementarity to the H2PO4 anion, being
−
Table 3. Fluorescence Measurements 20 μM in Water (2% of
DMSO) and pH Adjusted with NaOH/HClO4
able to provide a stronger interaction than that for HSO4 or
other anions. The differences observed between 1 and 2 must
be associated to the higher level of preorganization of the
macrocyclic structure of 2. It has been demonstrated that the
observed fluorescence in the presence of the guest is associated
to the conversion of the acridine moiety of the receptor into a
strongly emitting acridinium fluorophore.
E(S1) − E(S0) (kcal/mol)
acridine pH 1.00
acridine pH 9.84
2 pH 1.00
65.9
72.5
62.7
71.4
62.8
71.7
67.6
2 pH 4.00
1 pH 1.00
EXPERIMENTAL SECTION
1 pH 4.00
crystal 2H22+·2H2PO4
■
−
Materials and Methods. All commercially available reagents and
solvents were used as received, without further purification. Acridine
was recrystallized from an ethanol−water mixture as described
previously.70
In order to rationalize and confirm these results, computa-
tional studies for the model compound 4,5-dimethylacridine
were carried out in the gas phase at the B3LYP/6-31G* level of
theory using the Gaussian 09 software. The geometry of the
4,5-dimethylacridine molecule interacting with one proton was
optimized for several N···H distances at the fundamental and
first excited state. The S1−S0 energy gap was then obtained as
the difference in energy of the optimized structures for the
fundamental and first exited state. A final reference structure
was optimized for 4,5-dimethylacridine considering the proton
at an infinite distance (see Figure 15).
From these calculations, we can conclude that the interaction
of the N of the acridine unit with one proton has a big effect on
the S1−S0 energy gap. As can be seen in Figure 15 the
experimental energy gap (67.6 kcal/mol) corresponds to the
presence of a large N(acridine)···H distance, which is more
appropriate for an acridine nitrogen acting as a hydrogen bond
acceptor than for an acridinium group acting as a hydrogen
bond donor.
Steady-State Fluorescence Spectroscopy. For fluorescence titra-
tions in water, steady-state fluorescence spectra were recorded in a
fluorimeter equipped with a 450 W xenon lamp. Fluorescence spectra
were recorded in the front face mode. For fluorescence titrations in
chloroform the emission spectra were recorded between 367 and 700
nm with an excitation wavelength of 357 nm.
Time-Resolved Fluorescence Spectroscopy. Time-resolved fluo-
rescence measurements were done with the technique of time cor-
related single photon counting (TCSPC). Samples were excited with
an IBH 372 nm NanoLED with a fwhm of 1.3 ns and a repetition rate
of 100 kHz. Data were fitted to the appropriate exponential model
after deconvolution of the instrument response function by an iterative
deconvolution technique, using the IBH DAS6 fluorescence decay
analysis software, where reduced χ2 and weighted residuals serve as
parameters for goodness of fit.
Nuclear Magnetic Resonance. 1H and 13C NMR spectra were
recorded on a 500 MHz spectrometer (500 MHz for 1H and 125 MHz
1
for 13C) or a 300 MHz spectrometer (300 MHz for H and 75 MHz
for 13C).
Fluorescence Measurements. pH Titrations in Chloroform.
Compounds were dissolved in CHCl3 to obtain 10 mM stock
solutions, then were diluted to 10 μM. These solutions were titrated
by adding increasing volumes of stock solutions of the acids of 0.005−
0.05 M in CHCl3 with a 2% of methanol.
CONCLUSIONS
■
We have designed, synthesized, and characterized two pseudo-
peptidic receptors for the efficient and selective complexation
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pH Titrations in Water. Compounds were dissolved in dimethyl
sulfoxide (DMSO) to obtain 1 mM stock solutions and then were
diluted to 10 μM with water containing a mixture of several buffers to
facilitate titrations between pH 9 and 1 (40 mM of each sodium salt:
acetate, phosphate, borate, and carbonate). The small percentage of
DMSO (1−2%) was required to avoid the precipitation of the
compounds due to low solubility in water. Slight variations in the pH
of the solutions were achieved by adding the minimum volumes of
0.1−1.0 M NaOH or 0.1−1.0 M HCl (typically 10 μL added to
10 mL), in such a way that dilution effects were negligible.76 pKa values
where obtained from the nonlinear curve-fitting of the data with
Origin 6.1.
Fluorescence Lifetime Determination. Compounds were dis-
solved in water and H2SO4 to obtain stock solutions (1 mM), and
then they were diluted to 20 μM with water. Variations in the pH of
the solutions were achieved by adding H2SO4 or NaOH. Samples were
purged with nitrogen.
800.3463. Anal. Calcd for C49H45N5O6: C, 73.57; H, 5.67; N, 8.76.
Found: C, 73.43; H, 5.82; N, 8.75.
Synthesis of Compound 1. Compound 5 (2.167 g, 2.71 mmol)
was placed into a 100 mL round-bottom flask, and then a 33% HBr/
AcOH solution (50 mL) was added. After complete dissolution of the
solid, a yellow solution was formed. The reaction was stirred under a
nitrogen atmosphere for 1 h. The resulting colorless solution was
poured into a 250 mL beaker containing diethyl ether (150 mL).
A white precipitate was formed and was filtered and the solid washed
with ether. The solid was redissolved in distilled water (30 mL), and
the solution was washed twice with CHCl3 (30 mL) and, finally, was
basified with solid NaOH until pH 12−13. NaCl was added until
saturation, and the aqueous phase was extracted three times with
CHCl3 (30 mL). The organic phase was dried with anhydrous MgSO4,
and the solvent was vacuum eliminated to afford compound 1 as a
white solid (1.297 g, 2.439 mmol, 90% yield): mp 83−85 °C; [α]25
=
D
−47.98 (c 0.01, DMSO); IR (ATR) 3288, 1648, 1430 cm−1; 1H NMR
(500 MHz, CDCl3) δ 8.70 (s, 1H), 8.45 (t, 2H, J = 5.4 Hz), 7.87 (d,
2H, J = 8.5 Hz), 7.69 (d, 2H, J = 6.7 Hz), 7.43 (t, 2H, J = 7.6 Hz),
7.03−7.10 (m, 7H, m), 6.92−7.03 (m, 2H), 5.10 (d, 4H, J = 6.1 Hz),
3.58 (dd, 2H, J = 3.9, 8.8 Hz), 3.19 (dd, 2H, J = 4.0, 13.7 Hz), 2.62
(dd, 2H, J = 9.1, 13.6 Hz), 1.40 (s, 4H); 13C NMR (126 MHz, CDCl3)
δ 174.0, 146.7, 137.9, 136.7, 136.1, 129.5, 129.1, 128.3, 127.7, 126.6, 126.4,
125.7, 56.7, 41.1, 40.7; HRMS (ESI-TOF)+calcd for C33H33N5O2
(M + H)+ 532.2713, found 532.2717. Anal. Calcd for C33H33N5O2: C,
74.55; H, 6.26; N, 13.17. Found: C, 74.38; H, 6.37; N, 13.00.
Quantum Yield Determination. Solutions of the compounds were
prepared as described for the fluorescence lifetime determination.
Fluorescence quantum yields of compounds 1, 2, and acridine are
reported relative to quinine sulfate (aqueous solution H2SO4 0.1 M,
air); ΦF = 0.53. The experiments were done using optically matching
solutions. Emission spectra was recorded upon excitation at λexc
=
366 nm. The quantum yield was calculated using eq 1. In this
expression, it is assumed that the sample and the reference are excited
at the same wavelength so that it is not necessary to correct for the
different excitation intensities of different wavelengths.
2
2
Φ = Φ × (A F /A F ) × (n /n )
r s s r
Synthesis of Compound 2. Compound 1 (0.500 g, 0.941 mmol),
TBABr (0.1516 g, 0.470 mmol), and DiPEA (1.611 mL, 9.41 mmol)
were placed in a 250 mL round-bottom flask, and then dry acetonitrile
(134 mL) was added. The reaction mixture was stirred at room
temperature until complete dissolution of the reagents, and then α,
α′-dibromo-m-xylene (0.248 g, 0.941 mmol) dissolved into a small
amount of dry acetonitrile was added. The reaction was maintained
heated by a temperature gradient from 50.0 to 81.6 °C (reflux) for
24 h, under a nitrogen atmosphere. After cooling, the solvent was
vacuum eliminated. Purification of the crude was carried out by
column chromatography over flash silica gel (dichloromethane/
methanol from 100:0 to 100:5 with a few drops of aqueous ammonia
added). The macrocycle was obtained as a white solid (0.507 g, 0.800
(1)
f
r
s
r
Here, Φf is the quantum yield, F is the integrated intensity, A is the
optical density, and n is the refractive index. The subscript r refers to
the reference fluorophore of known quantum yield and subscript s to
the sample.
Computational Studies. Monte Carlo conformational searches
at MMFF force field were carried out with the Spartan ’08 software.77
Calculations of model compounds 5 and 6, and their supramolecular
complexes were performed at the B3LYP/6-31G* level of theory using
the Gaussian 03 software.78 All geometries were fully optimized and it
was checked to be true minima by the analysis of the vibration normal
modes. Calculations for the interaction of 4,5-dimethylacridine with a
proton were carried out in the gas phase at the B3LYP/6-31G* level of
theory using the Gaussian 09 software.79 No symmetry constraints
were used. First excited state optimizations were performed using the
TD = (NStates = 6, Root = 1) parameters.
mmol, 85% yield): mp 89−90 °C; [α]25 = +41.44 (c 0.01, CHCl3);
D
IR (ATR) 3317, 2919, 1653, 1430; 1H NMR(500 MHz, CDCl3)
δ 8.73 (s, 1H), 7.85−8.00 (m, 4H), 7.66 (d, 2H, J = 6.7 Hz), 7.47 (dd,
2H, J = 6.9, 8.3 Hz), 7.25 (s, 1H), 7.13 (d, 1H, J = 7.6 Hz), 7.04 (d,
4H, J = 7.3 Hz), 7.01 (dd, 2H, J = 1.3, 7.6 Hz), 6.93 (t, 4H, J = 7.6
Hz), 6.86 (d, 2H, J = 7.4 Hz), 4.95 (dd, 2H, J = 7.5, 14.9 Hz), 4.79 (d,
2H, J = 5.7 Hz), 3.82 (d, 2H, J = 12.8 Hz), 3.49 (d, 2H, J = 12.8 Hz),
3.44 (dd, 2H, J = 4.7, 8.6 Hz), 3.10 (dd, 2H, J = 4.7, 13.9 Hz), 2.70
(dd, 2H, J = 8.6, 13.9 Hz), 1.81−1.90 (m, 2H); 13C NMR (126 MHz,
CDCl3) δ 173.6, 146.6, 140.1, 137.2, 136.5, 136.1, 129.2, 129.0, 128.9,
128.8, 128.2, 127.5, 127.5, 126.6, 126.4, 125.6, 65.1, 53.7, 40.5, 39.5;
HRMS (ESI-TOF)+ calcd for C41H39N5O2 (M + H)+ 634.3182, found
634.3181. Anal. Calcd. for C41H39N5O2: C, 77.70; H, 6.20; N, 11.05.
Found: C, 77.51; H, 6.35; N, 10.82.
Experimental Procedures. Synthesis of Compound 5. 4,5-
Bis(aminomethyl)acridine·2HCl (0.200 g, 0.645 mmol) was placed in
a 50 mL round-bottom flask, and 1 M aqueous NaOH (10 mL) and
MeOH (1 mL) were added. After complete dissolution of the solid,
the solution was extracted three times with dichloromethane (15 mL).
The organic layers were collected and dried with anhydrous MgSO4.
The solvent was then vacuum evaporated to afford 4,5-bis-
(aminomethyl)acridine (0.142 g, 0.600 mmol, yield 93%) as a
yellowish solid. This was dissolved in DME (15 mL) in a 50 mL
round-bottom flask. Cbz-L-Phe-OSuc (4) (0.476 g, 1.20 mmol) was
dissolved in DME (10 mL) and slowly added to the reaction flask.
A white precipitate was rapidly formed, and the mixture was stirred at
room temperature for 8 h and then at 50 °C for 5 h. After cooling,
the white precipitate was filtered and washed with cold water (50 mL)
and a small amount of methanol to afford the expected compound 5 as
a white solid (0.443 g, 5.55 mmol, 85% yield): mp 260−263 °C;
Crystal data for [2-H2](H2PO4)2: [C41H41N5O2](H2PO4)2·
0.5(H3PO4)·2.33(H2O), M = 920.79, rhombohedral, R3 (no. 146),
a = 30.4865(6) Å, c = 12.0383(4) Å, V = 9689.7(4) Å3, Z = 9,
Dc = 1.420 g cm−3, μ(Mo Kα) = 0.194 mm−1, T = 173 K, pale yellow
blocks, Oxford Diffraction Xcalibur 3 diffractometer; 10055 indepen-
dent measured reflections (Rint = 0.0270), F2 refinement, R1(obs) =
0.0868, wR2(all) = 0.2622, 6751 independent observed absorption-
corrected reflections [|Fo| > 4σ(|Fo|), 2θmax = 58°], 676 parameters.
The absolute structure of [2-H2](H2PO4)2 was determined by a
combination of R-factor tests [R1+ = 0.0868, R1− = 0.0871] and by use
of the Flack parameter [x+ = +0.00(15), x− = +1.11(15)]. CCDC
845873.
[α]25 = +34.12 (c 0.01, DMSO); IR (ATR) 3284, 1649, 1454 cm−1;
D
1H NMR (500 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.57 (t, 2H, J = 5.3
Hz), 8.00−8.10 (m, 2H, m), 7.60 (d, 2H, J = 8.4 Hz), 7.52 (d, 4H, J =
4.9 Hz), 7.09−7.36 (m, 20H), 5.09 (m, 4H), 4.93 (q, 4H, J = 12.7 Hz),
4.41 (dd, 2H, J = 9.0, 13.7 Hz), 3.07 (dd, 2H, J = 4.6, 13.5 Hz), 2.85
(dd, 2H, J = 10.4, 13.1 Hz); 13C NMR (75 MHz, DMSO-d6) δ 172.3,
156.6, 146.2, 138.7, 137.7, 137.3, 137.2, 129.9, 128.9, 128.7, 128.3,
128.1, 128.0, 127.8, 126.9, 126.6, 126.3, 66.0, 57.2, 39.4, 38.3; HRMS
(ESI-TOF)+calcd for C49H45N5O6 (M + H)+ 800.3448, found
498
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Article
(22) Caballero, A.; White, N. G.; Beer, P. D. Angew. Chem., Int. Ed.
2011, 50, 1845−1848.
ASSOCIATED CONTENT
■
S
* Supporting Information
(23) Gale, P. A. Chem. Commun. 2011, 47, 82−86.
(24) Gale, P. A. Chem. Soc. Rev. 2010, 39, 3746−3771.
(25) Joyce, L. A.; Shabbir, S. H.; Anslyn, E. V. Chem. Soc. Rev. 2010,
39, 3621−3632.
1H and 13C NMR spectra, 2D NMR spectra, and mass spectra
for compounds 1 and 2; fluorescent titration spectra for
compounds 1 and 2 with different acids; details of the DFT
calculations; X-ray crystallographic information. This material is
available free of charge via the Internet at http://pubs.acs.org
(26) Gale, P. A.; Gunnlaugsson, T. Chem. Soc. Rev. 2010, 39, 3595−
3596.
(27) Jordan, L. M.; Boyle, P. D.; Sargent, A. L.; Allen, W. E. J. Org.
Chem. 2010, 75, 8450−8456.
AUTHOR INFORMATION
■
(28) Duke, R. M.; Veale, E. B.; Pfeffer, F. M.; Kruger, P. E.;
Gunnlaugsson, T. Chem. Soc. Rev. 2010, 39, 3936−3953.
(29) Mullen, K. M.; Beer, P. D. Chem. Soc. Rev. 2009, 38, 1701−1713.
(30) Kubik, S. Chem. Soc. Rev. 2009, 38, 585−605.
(31) Coleman, A. W.; Perret, F.; Mousa, A.; Dupin, M.; Guo, Y.;
Perron, H. Top. Curr. Chem. 2007, 277, 31−88.
(32) Bazzicalupi, C.; Bencini, A.; Bianchi, A.; Cecchi, M.; Escuder, B.;
Corresponding Author
*E-mail: (S.V.L.) luiss@qio.uji.es; (R.V.) r.vilar@imperial.ac.uk.
ACKNOWLEDGMENTS
■
The UK’s Engineering and Physical Sciences Research
Council (EPSRC) is thanked for a Leadership Fellowship
to R.V. The Spanish Ministry of Science and Innovation
(CTQ2009-14366-C02-01) and UJI-Bancaixa (P1-1B-2009-
59) are thanked for support to S.V.L. The Spanish Ministry
of Science (FPU AP2007-02562) and EU seventh Framework
Program (PIIF-GA-2009-235411) are thanked for financial
support to V.M.C. and D.K.K., respectively. The support of
the SCIC of the UJI for the different instrumental techniques is
acknowledged.
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