Tuning the emission properties of fluorescent ligands by changing pH: The unusual case of an acridine-containing polyamine macrocycle

Article abstract of DOI:10.1021/jp4015756

Synthesis and characterization of a new macrocyclic compound, composed by a triethylentetraamine chain linking the 4 and 5 positions of an acridine moiety, are reported. The molecule, devised as a fluorescent chemosensor for anions, has revealed an intriguing pH-dependent spectroscopic behavior, whose features are the specific object of this article. Ligand protonation in aqueous solution has been analyzed by means of potentiometric, ¹H NMR, UV-vis, and fluorescence emission measurements. The molecule binds up to four protons in the pH range 2-11. Protonation takes place on the aliphatic tetraamine chain, while the acridine nitrogen does not participate to proton binding even at very low pH. Differently from acridine, the UV-vis spectra are almost unaffected by the pH. On the opposite, the emission spectra are strongly pH-dependent. In fact, at low pH values, the spectra show a blue-shifted emission, resembling that of unprotonated acridine, while at slightly acidic and alkaline pH the fluorescence features a red-shifted band similar to that of acridinium cation. This unusual behavior occurs in the mono-, bi-, and triprotonated forms of the compound and is interpreted as due to an excited state proton transfer from an aliphatic ammonium group adjacent to the acridine moiety to the acridine nitrogen. In the fully protonated state, this process is prevented owing to unfavorable molecular arrangements mainly determined by electrostatic repulsions. This interpretation is supported by quantum mechanical calculations as well as molecular dynamics simulations.

Full text of DOI:10.1021/jp4015756

Article
pubs.acs.org/JPCA
Tuning the Emission Properties of Fluorescent Ligands by Changing
pH: The Unusual Case of an Acridine-Containing Polyamine
Macrocycle
Stefano Puccioni,† Carla Bazzicalupi,† Andrea Bencini,† Claudia Giorgi,*,† Barbara Valtancoli,†
Greta De Filippo,‡ Vito Lippolis,‡ Pier Remigio Salvi,†,§ Giangaetano Pietraperzia,†,§ Riccardo Chelli,*,†,§
and Cristina Gellini*,†,§
†Dipartimento di Chimica “Ugo Schiff”, Universita
̀
di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy
‡Dipartimento di Scienze Chimiche e Geologiche, Universita
̀
di Cagliari, S.S. 554 bivio per Sestu, 09042 Monserrato, Cagliari, Italy
§European Laboratory for Nonlinear Spectroscopy (LENS), Via Nello Carrara 1, 50019 Sesto Fiorentino, Firenze, Italy
S
* Supporting Information
ABSTRACT: Synthesis and characterization of a new macrocyclic compound,
composed by a triethylentetraamine chain linking the 4 and 5 positions of an
acridine moiety, are reported. The molecule, devised as a fluorescent chemosensor
for anions, has revealed an intriguing pH-dependent spectroscopic behavior, whose
features are the specific object of this article. Ligand protonation in aqueous
1
solution has been analyzed by means of potentiometric, H NMR, UV−vis, and
fluorescence emission measurements. The molecule binds up to four protons in
the pH range 2−11. Protonation takes place on the aliphatic tetraamine chain,
while the acridine nitrogen does not participate to proton binding even at very low
pH. Differently from acridine, the UV−vis spectra are almost unaffected by the pH.
On the opposite, the emission spectra are strongly pH-dependent. In fact, at low
pH values, the spectra show a blue-shifted emission, resembling that of
unprotonated acridine, while at slightly acidic and alkaline pH the fluorescence
features a red-shifted band similar to that of acridinium cation. This unusual behavior occurs in the mono-, bi-, and triprotonated
forms of the compound and is interpreted as due to an excited state proton transfer from an aliphatic ammonium group adjacent
to the acridine moiety to the acridine nitrogen. In the fully protonated state, this process is prevented owing to unfavorable
molecular arrangements mainly determined by electrostatic repulsions. This interpretation is supported by quantum mechanical
calculations as well as molecular dynamics simulations.
strands is therefore often accompanied by changes of the
emission wavelengths and/or fluorescence intensity.22,23 As a
consequence, acridine-based compounds, in particular 3,6-
diamino derivatives, such as acridine-orange and proflavine, are
used as fluorescent probes for DNA and RNA in a number of
biological procedures.28,29 Luminescence is also strongly
affected by protonation of the acridine nitrogen.6−9,15 For
instance, protonation of the acridine nitrogen in aqueous
solution occurs at acidic pH and gives rise to a 40 nm red-shift
of the original emission band of acridine at 430 nm.
The luminescence properties of acridine and its derivatives
and their sensitivity to substrate coordination make them
promising candidates to develop new fluorescent chemosensors
for analytes in aqueous solution.30−32 Chemosensors are
normally constituted by a binding moiety capable to recognize
a targeted substrate, linked via a spacer to a signaling units for
1. INTRODUCTION
In the last years, increasing interest has been devoted to
acridine and its derivatives, due to their peculiar chemical1−3
and photophysical properties2−15 as well as to the biological
activity16,17 imparted by the planar tricyclic structure. From this
point of view, the known capability of acridine to intercalate
between DNA base pairs has been largely exploited to develop
new agents with anticancer activity.16−25 The first and most
cited example is amsacrine,20,22 which contains a 4-amino-3-
methoxyphenyl-methanesulfonamide function linked to the 9
position of acridine. This molecule, used in clinics since 1976, is
the first totally synthetic drug of DNA intercalating type, the
beneficial effect in cancer therapy being due to its interaction
with Topo-II-DNA complex.20,22,26,27 Its success has led to the
synthesis of a number of similar compounds for pharmaco-
logical purposes and presently acridine derivatives are used not
only as antitumor drugs, but also as antibacterial and
antimicrobial agents.16−18 Acridine-based compounds normally
display an intense fluorescence, which is strongly sensitive to
the polarity of the medium. Interaction within DNA/RNA
Received: February 13, 2013
Revised: April 11, 2013
Published: April 15, 2013
© 2013 American Chemical Society
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example, a fluorogenic molecule whose fluorescence emission is
quantitatively altered by the binding event.33 From this point of
view, polyamine macrocycles have been revealed to be versatile
binding units for metal cations33−42 and, more recently, also for
anionic species.43,44 In fact, they can easily protonate affording
polyammonium cation at neutral pH values, capable to bind
anions via charge−charge or hydrogen bonding (H-bonding)
interactions. Furthermore, compared to open-chain polyamines,
their cyclic structure leads to an increased density of positive
charge on the receptor, strengthening the overall host−guest
interaction. In this context, the insertion of a signaling unit,
most often a fluorescent heteroaromatic fragment, as an integral
part of the polyamine macrocyclic structure, represents one of
the approaches to the development of anion chemo-
sensors.43−57
selective anion binding requires an optimal stereochemical
fitting between the binding sites of the two partners in the
host−guest interaction. At the same time, protonation of the
receptor can strongly affect its luminescence properties.
Therefore, the investigation of the structural and photophysical
properties of the polyammonium cations generated by
protonation is a necessary preliminary analysis in the study of
the anion chemosensing capability of new receptors. In the
present case, we noted that at strongly acidic pH the L receptor
displays an emission band peaked at 430 nm, while in slightly
alkaline medium the fluorescence emission is red-shifted at 460
nm, which is the opposite behavior observed for acridine.7,10
Stimulated by these unexpected observations, we decided to
focus our efforts on a multidisciplinary study of the structural
and photophysical properties of the receptor L at different pH
values, by coupling potentiometric, 1H NMR, UV−vis
absorption and fluorescence emission measurements in
aqueous solution with quantum mechanical (QM) calculations
and molecular dynamics (MD) simulations.
With this in mind, we have synthesized the new receptor 1-
(6,7)-acridina-3,6,9,12-tetraazatridecaphane (from now on
denoted as L), composed by a triethylentetraamine chain
linked to the 4,5 positions of an acridine moiety through two
methylene spacers (Figure 1). It is now accepted that the
2. EXPERIMENTAL SECTION
2.1. Synthesis of the Ligand. 4,5-Dibromomethylacridine
1 has been synthesized using a modification of the procedure
developed by Di Giorgio et al.58 1,4,7,10-Tetratosyl-1,4,7,10-
tetraazadecane 2 was obtained as previously reported.59 The
synthetic pathway to obtain L is depicted in Figure 2.
3,6,9,12-Tosyl-1-(6,7)-acridina-3,6,9,12-tetraaza-
tridecaphane (Compound 3 in Figure 2). The tosylated amine
2 (500 mg, 0.655 mmol) and K2CO3 (1.5 g, 11.0 mmol) were
suspended in refluxing CH3CN (20 mL). A solution of 1 (240
mg, 0.655 mmol) in CH3CN (40 mL) was then added
dropwise in 2 h to the resulting mixture. After completing the
addition, the suspension was refluxed for 22 h and then filtered.
The solution was evaporated in vacuum to yield the crude
product which was chromatographated on activated alumina
(II/III) CH2Cl2-ethyl acetate 10:1 mixture. The eluted fractions
were collected and evaporated to dryness to afford 3 as a
colorless solid (310 mg, 49%). Anal. Calcd for C49H51N5S4O8
C, 60.91; H, 5.32; N, 7.25. Found C, 60.83; H, 5.33; N, 7.22.
1H NMR (CDCl3) δ 2.38 (s, 6H), 2.42 (s, 6H), 2.54 (t, 4H),
2.70 (t, 4H), 3.39 (t, 4H), 5.18 (s, 4H), 7.21 (d, 4H), 7.32 (d,
4H), 7.42 (d, 4H), 7.54 (dd, 2H), 7.76 (d, 4H), 7.89 (d, 2H),
Figure 1. 1-(6,7)-Acridina-3,6,9,12-tetraazatridecaphane (L) structure
with atom labeling for the purpose of the present paper.
binding affinity for anions of a polyamine receptor does not
only depend on the facile protonation of the amino groups in
solution, but also on the structural organization of the
ammonium groups within the macrocyclic structure. In fact,
Figure 2. Synthesis scheme of L.
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7.95 (d, 2H), 8.72 (s, 1H). 13C NMR (CDCl3) δ 146.99,
143.53, 143.44, 137.17, 135.66, 135.19, 134.84, 132.69, 129.98,
129.95, 129.77, 129.74, 128.78, 127.71, 127.69, 127.47, 126.60,
126.06, 49.55, 49.44, 49.18, 48.22, 21.67, 21.64.
motive force data were treated by means of the computer
program HYPERQUAD.64
1
2.4. NMR Measurements. H (400 MHz) and 13C (100
MHz) in CDCl3 and D2O solutions at different pH values were
recorded at 25 °C on a 400 MHz Bruker Avance II
spectrometer. To adjust the pD, small amounts of 0.01 M
NaOD and DCl were added to the solution containing L. The
pH was calculated by the measured pD values by using the
relationship65 pH = pD − 0.40.
1-(6,7)-Acridina-3,6,9,12-tetraazatridecaphane Tetrahy-
drobromide Pentahydrate (L·4HBr·5H2O). Tetratosylated
macrocycle 3 (310 mg, 0.321 mmol) and phenol (3.5 g, 37
mmol) were dissolved in HBr/CH3COOH (33%, 15 mL). The
solution was stirred at 90 °C for 22 h. The resulting suspension
was filtered, and the solid was washed with CH2Cl2 for several
times. The yellowish solid was recrystallized from an ethanol/
water mixture (20 mL) in the presence of few drops of 48%
HBr to give L as the tetrahydrobromide salt L·4HBr·5H2O (92
mg, 38%). Anal. Calcd for C21H41N4O5Br4 C, 33.05; H, 5.42;
N, 9.18. C, 33.12; H, 5.43; N, 9.34. 1H NMR (D2O, pH 1.9) δ
9.21 (s, 1H), 8.30 (d, 2H), 8.05 (d, 2H), 7.71 (dd, 2H), 5.07 (s,
4H), 3.57 (t, 4H), 3.35 (t, 4H), 3.13 (s, 4H). 13C NMR (D2O,
pH 1.9) δ 145.84, 139.06, 133.91, 131.51, 128.08, 126.82,
126.17, 59.92, 47.94, 44.61, 43.92, 43.48. MS (Supporting
Information, SI) m/z 350 (M + H)+.
L·4HBr·2H2O Crystal. Crystals suitable for X-ray analysis of
the dihydrate salt L·4HBr·2H2O were prepared in 89% yield by
slow evaporation at room temperature of an aqueous solution
of L (0.01 M), acidified with 1 M HBr up to pH 2, in the
presence of a fifty-fold excess of NaBr. Anal. Calcd for
C21H35N5O2Br4 C, 33.75; H, 5.00; N, 9.93. Found C, 33.82; H,
5.14; N, 9.73.
2.5. Absorption and Emission Spectra. The absorption
spectra of L (3 × 10−5 M) and acridine (8 × 10−6 M) in water
solution at various pH have been measured with a Cary 5
spectrophotometer in the spectral range 200−700 nm. The
spectral bandwidth was set to 2 nm and the optical path length
was 1 cm. Fluorescence emission has been measured on the
same solutions by means of a Perkin-Elmer LS55 spectrophoto-
fluorimeter, exciting at 345 nm and collecting the emission in
the range 350−680 nm. Attenuating the sample emissions by
using a T = 1% neutral density filter allows to measure all of the
samples in the same experimental conditions, i.e., excitation and
emission slits fixed at 10 nm. Emission spectra have been
corrected for the response of the photomultiplier. The absence
of exciplexes or aggregation effects have been checked by
diluting the solutions down to 8 × 10−7 M without observing
any change both in the absorption and emission features. In
recording the spectra at different pH, the examined pH values
for L are 1.1, 2.1, 5.0, 8.2, and 12.0, while for acridine they are
2.5, 5.0, and 8.0. The pH values have been chosen to roughly
maximize the presence of only one species in solution, as
established from potentiometric experiments. Each pH value
has been adjusted adding concentrated NaOH or CH3SO3H
solutions.
2.6. Computational Methods. QM calculations have been
performed on neutral L and protonated forms of L, HnLn+ with
n = 1, 2, 3, 4, the protonation sites being established according
to potentiometric and NMR measurements. In the HL+ and
H2L2+ species the acidic protons are localized, respectively, on
one and both the amino groups adjacent to the acridine moiety
(N2 and N2′; see Figure 1 for labeling), while in H3L3+ and
H4L4+ the additional acidic protons are positioned on the
remaining N3 and N3′ aliphatic amine functions. The L and
HnLn+ configurations subject to structural QM optimization
have been obtained from a quality threshold clustering66
analysis applied to a collection of conformations taken from
serial-generalized ensemble MD simulations67−69 of L and
HnLn+ water solutions at room conditions. For each compound,
the configuration representative of the most populated cluster
has been taken. Details of clustering analysis and MD
simulations are reported in the Supporting Information. MD
simulations have been performed using the ORAC program.70
The ground state structures of L and HnLn+ have been achieved
through energy minimization using density functional theory
with the B3LYP functional and 6-31++G(d,p) basis set. The
minimum-energy structures of the species with acridine in its
protonated form have also been determined at the same level of
theory. Their initial structures correspond to the ground-state L
and HnLn+ species, by moving a proton from the N2 or N2′
amino group to the N1 nitrogen. Electronic energies for the S0
→ S1 vertical transition are calculated on chain-protonated and
acridine-protonated structures of HnLn+ using time-dependent
density functional theory71,72 with the same functional and
basis set. The excitation energies have been calculated
promoting electrons from the 27 highest-energy valence
2.2. X-ray Structure Determination of L·4HBr·2H2O.
Crystal data for L·4HBr·2H2O: (C21H35Br4N5O2)2, MW =
1418.36, orthorhombic, Pbca, a = 22.535(1) Å, b = 13.0247(9)
Å, c = 39.473(3) Å, V = 11586(1) Å3, Z = 8, T = 150 K, μ(Cu
Kα) = 6.986 mm−1, 60477 reflections measured, 6837 unique
(R(int) = 0.0884). The final agreement factors were R1 =
0.0785 (5146 data with I > 2σ (I)) and wR(F2) was 0.2460 (all
data). Due to the somewhat low quality of the crystal,
reflections were used only up to 0.9 Å resolution (2θmax
=
118°) in structure solution and refinement. The structures were
solved by direct methods (SIR2004)60 and refined using
SHELX-97.61 All of the non-hydrogen atoms were anisotropi-
cally refined, while the hydrogen atoms were introduced in
calculated positions and their coordinates and thermal
parameters refined according to the linked atom. The oxygen
atoms belonging to the solvent molecules were refined using
partial population parameters. Residual electron density near a
disordered water oxygen remains at the end of refinement.
Attempts to introduce the residual density as an additional
crystallization water molecule were unfruitful. The CIF file for
this structure has been deposited to the Cambridge Crystallo-
graphic Data Center, CCDC deposition no. 911103.
2.3. Potentiometric Measurements. Potentiometric
measurements were carried out in degassed 0.1 M N-
(CH3)4NO3 aqueous solutions, at 25 °C by using equipment
and procedure described in ref 62. The combined Ingold 405
S7/120 electrode was calibrated as a hydrogen concentration
probe by titrating known amounts of HNO3 with CO2-free
N(CH3)4OH solutions and determining the equivalent point
by the Gran’s method,63 which allows us to determine the
standard potential Eo, and the ionic product of water (pKw =
13.83(1) at 25 °C in 0.1 M solution of N(CH3)4NO3). The
concentration of L was generally 10−3 M. At least three
measurements (about 100 data points each one) were
performed in the pH range 2−11, and the relevant electro-
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orbitals to all 555 virtual orbitals. All calculations have been
performed using the Gaussian09 program,73 whether in vacuum
or using the polarizable continuum model with dielectric
constant of water.74
conformation. The two acridine units are partially overlapped
and parallel, accounting for a face-to-face π-stacking interaction.
The angle between the acridine planes is 1.6° and the
interplanar distance 3.3 Å, while the centroids of the central
rings of the two acridine units are 1.6 Å shifted to each other.
The two aliphatic chains point in opposite directions and are
involved in a dense H-bonding network with bromide ions and
water molecules. Although the Fourier difference map does not
allow to determine the position of hydrogen atoms, it is to be
noted that all the aliphatic nitrogens are involved in two or
more strong H-bond interactions with bromide ions and, in the
case of N3a, N3′a, N2′b, and N3b, with water molecules.
Acridine is not involved in H-bonding either with bromide ions
or water molecules. This suggests that all aliphatic nitrogen
atoms are likely to be in their protonated form, while the
acridine nitrogen remains not protonated. Only a weak H-bond
between the acridine nitrogen and a single benzylic ammonium
group of each macrocyclic unit is formed. In fact, both
N2a···N1a and N2b···N1b distances are 3.07 Å, while the N2a-
H···N1a and N2b-H···N1b angles are 122.9(6)° and 121.8(5)°,
respectively, far from the optimal value of 180°. Furthermore,
both the aliphatic N2a and N2b amino groups give rise to two
additional interactions with two bromine ions (N2a···Br3 =
3.30(1) Å, N2a···Br4 = 3.61(1) Å, N2b···Br2 = 3.233(8) Å,
N2b···Br1 = 3.328(8) Å), accounting for their protonation and
ruling out protonation of acridine.
3. RESULTS AND DISCUSSION
3.1. Synthesis. Macrocycle L was obtained by using a
modification of the Richman and Atkins procedure75 (Figure
2). Reaction of the acridine dibromomethyl derivated 1 with
the tosylated tetraamine 2 in CH3CN in the presence of K2CO3
as base, affords, after purification by chromatography, the
tosylated macrocycle 3. Finally, removal of the tosyl groups was
performed in HBr/CH3COOH in the presence of phenol as
antioxidant, as the commonly used deprotection in concen-
trated H2SO4 leads to the decomposition of the acridine
moiety.75 The macrocycle L was then precipitated as
tetrahydrobromide salt by addition of CH2Cl2 to its solution
in HBr/CH3COOH. Crystals of the L·4HBr·2H2O salt, suitable
for crystallographic analysis, were obtained by slow evaporation
of an aqueous solution of L at pH 2 in the presence of a large
excess of NaBr.
3.2. Crystal Structure of L·4HBr·2H2O. The crystal
structure consists of H4L4+ cations, bromide ions, and water
molecules. The asymmetric unit contains two H4L4+ cations (a
and b units in Figure 3), coupled by π-stacking interactions
1
3.3. Potentiometric and H NMR Study of the Ligand
Protonation. Although acridine suffers poor solubility in
water, the presence of a hydrophilic tetraamine chain in L
ensures high solubility and allows for the determination of its
protonation constants via potentiometric titrations. The
stepwise protonation constants of L (log K), potentiometrically
determined at 25 °C in 0.1 M solution of N(CH3)4NO3, are
reported in Table 1. As normally found in polyamine-based
Table 1. Protonation Constants (log K) Related to the
Various Equilibria Involving Neutral and Protonated Forms
a
of L
equilibrium
log K
L + H+ = HL+
10.24 (6)
9.44 (5)
5.91 (9)
3.36 (9)
HL+ + H+ = H2L2+
H2L2+ + H+ = H3L3+
H3L3+ + H+ = H4L4+
Figure 3. ORTEP drawing of the L·4HBr·2H2O X-ray structure,
displaying the dense H-bonding network formed by the two cations,
bromide ions and water molecules. Symmetry operators: ′−x + 1/2, +y
+ 1/2, +z; ″x, −y + 1/2, +z − 1/2. Relevant distances (Å) are:
N2a···Br3 = 3.30(1), N2a···Br4 = 3.61(1), N2a···N1a = 3.07(1),
N3a···O10 = 2.87(3), N3a···Br6 = 3.26(1), N3a···Br8″ = 3.39(1),
N3′a···O6 = 2.91(2), N3′a···O1 = 3.40(1), N3′a···Br6′ = 3.37(1),
N2′a···Br7 = 3.26(8), N2′a···Br3′ = 3.24(8), N2′b···O3 = 2.79(2),
N2′b···Br2′ = 3.34(9), N3′b···Br8 = 3.29(1), N3′b···Br5′ = 3.24(1),
N3b···Br5 = 3.24(1), N3b···O2 = 3.00(2), N2b···Br2 = 3.233(8),
N2b···Br1 = 3.328(8), N2b···N1b = 3.07(1).
a
The L concentration is 10−3 M in 0.1 M aqueous solution of
N(CH3)4NO3 at 25 °C. Values in parentheses are errors on the last
significant digit.
compounds, facile protonation of the amino groups can occur
in aqueous solutions. Accordingly, L can bind up to four acidic
protons in the investigated pH range 2−11, affording different
protonated species from alkaline to acidic pH values (Figure 4).
L possesses four amino groups and the acridine nitrogen as
potential binding sites for protons. On the other hand, the first
two protonation constants are in the range normally observed
for aliphatic amines76 and far higher than that observed for
acridine7,76,77 (log K = 5.4), suggesting that the corresponding
protonation steps occur on the polyamine chain. Conversely,
the values of the remaining protonation constants are similar or
lower than that of acridine (Table 1) and, in principle, the
corresponding protonation steps may involve either the amino
groups of the aliphatic chain or the acridine nitrogen.
between the two acridine moieties. In Figure 3, an ORTEP
drawing of the crystal structure is reported including symmetry
related bromide ions (Br3′, Br2′, Br5′, Br6′, and Br8″). Each
H4L4+ unit is almost planar as revealed by the angle between
the mean planes of acridine and macrocycle chain. The latter is
defined by the C and N atoms of the aliphatic chain plus those
of acridine closing the cycle. The angle results 4.0° for the a
unit and 5.2° for the b unit, indicating a nearly flat
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protons, in particular for the H1 signal. It is known that in
macrocyclic polyamines, including those containing aromatic or
heteroaromatic rings, the acidic protons are often delocalized
over the whole set of nitrogens or shared via hydrogen bonding
between different amino groups.78 This probably also occurs in
the present case. However, the structural characteristics of the
macrocycle and the nature of the aromatic ring may influence
the distribution of the acidic protons within the cyclic
framework. For instance, in polyamine macrocycle containing
phenanthroline79 or dipyridine80 units, the first protonation
steps often occur on the amino groups far from the
heteroaromatic moieties, whereas preferential protonation of
the benzylic amines has been observed in benzene-81,82 or
thiophene-containing83 polyazamacrocycles. In the present
case, the larger shift observed for the signals of the methylene
groups H5 and H6 leads us to propose that in the H2L2+ species
the two acidic protons are located, to a greater extent, on the
N2 and N2′ nitrogens. This would also ensure a reduction of
the electrostatic repulsions between the two ammonium
groups, which result far apart from each other, separated by
the large acridine unit. However, a minor localization of
positive charge on the N3 and N3′ amino groups and/or on the
acridine nitrogen cannot be ruled out. Binding of the third and
fourth acidic protons below pH 6 gives rise to the formation of
the H3L3+ and H4L4+ forms. This process is accompanied by a
relevant downfield shift of the signals of the methylene groups
H7 and H8, adjacent to the N3 and N3′ nitrogens, while only
minor shifts are observed for the remaining signals, including
those of acridine. Therefore, the last two protonation steps are
likely to mainly involve the two central amino groups of the
aliphatic chain.
Figure 4. Distribution diagram of the protonation states of L (10−3 M)
in 0.1 M solution of N(CH3)4NO3 at 25 °C.
A qualitative description of the successive protonation steps
1
of L can be gained by H NMR spectra recorded at different
pH values. It is known, in fact, that proton binding is
1
accompanied by marked downfield shifts of the H resonances
of the methylene groups in α-position with respect to the
aliphatic nitrogen involved in the protonation process. These
spectral changes can be used as diagnostic tool to monitor the
1
protonation process. The H NMR spectrum of L at pH 12,
where the free amine predominates, features a set of eight
signals, four for the acridine moiety [a singlet at 7.95 ppm
(assigned to the H1 hydrogen, see Figure 1 for labeling), two
doublets at 7.73 and 7.48 ppm (the hydrogen atoms H2 and
H3) and a doublet of doublets at 7.28 (H4)], and four for the
aliphatic chain [two singlets at 3.80 and 2.45 ppm (H5 and H8)
and two multiplets at 2.60 and 2.45 ppm (H6 and H7)]. These
spectral features account for a time-averaged C2v symmetry of
the molecule, which is preserved throughout all the investigated
pH range. As shown in Figure 5, formation of the mono- and
These data indicate that, throughout all of the considered pH
range, acridine is never directly involved in proton binding,
even at strongly acidic pH, probably due to the presence of a
high density of positive charge on the side chain that prevents
acridine protonation. This finding is supported by the crystal
structure of the H4L4+ cation, which shows that acridine is
unprotonated in this form, at least in the solid state.
3.4. Absorption and Fluorescence Spectroscopy. The
acid−base equilibria of L have been characterized by absorption
and fluorescence spectra. It is convenient for their interpreta-
tion the comparison with equivalent data on acridine in
aqueous solution, whose dependence on pH is well-known.6,7
Our own results on acridine, obtained for the purpose of the
present paper and shown in Figures 6a and 7a, agree
satisfactorily with past reports.8,9 The lowest energy absorption
spectrum of acridine has been discussed in terms of two
transitions, the first of nπ* and the second of ππ* character,
whose relative energies are sensitive to the environment
polarity. In polar solvents the lowest transition has been
conclusively reported to be of ππ* nature.10 The absorption
onset shifts from ∼380 nm at alkaline and neutral pH, where
the unprotonated form predominates, to ∼430 nm at pH 2.5
where the acridinium ion is in the largest concentration. The
mirror symmetry between fluorescence and absorption spectra
and the comparable red shift observed in the fluorescence
spectra are good indications for the assignment to acridine and
acridinium ion of the two emissions7,11,13 (see Figure 7a). The
occurrence of the isosbestic point at 440 nm in the fluorescence
spectra points to the acid−base equilibrium of acridine in
solution.12,13
1
Figure 5. H-NMR signals of L as a function of pH (see Figure 1 for
labeling).
diprotonated species, HL+ and H2L2+, in the 7−11 pH range is
accompanied by marked downfield shift of the signals of the
methylene protons H5 and H6, adjacent to the amino groups
N2 and N2′. A minor shift is observed for the resonances of H7
and H8, close to central amino groups of the aliphatic chain, N3
and N3′. Finally, the formation of the HL+ and H2L2+ species
also induces downfield shifts for the resonances of the aromatic
The absorption spectra of L, measured in the 250−500 nm
wavelength interval at different pH values, are shown in Figure
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with origin at 404 nm, a broad maximum at ∼430 nm and
extension up to 540 nm. In slightly acidic and alkaline solutions,
pH 5.0 and 8.2, where the tri- and biprotonated species,
respectively, are in the largest concentration, as evidenced in
the inset of Figure 7b, the fluorescence emission is enhanced
and shifted to the red. Finally, in a strongly alkaline solution the
same spectrum is observed, though largely depressed.
Summarizing, varying the pH conditions (1) the L absorption
is unaffected, while (2) the L fluorescence is reversed with
respect to acridine in the sense that the spectrum at mildly
acidic and alkaline pH is similar to that of acridine at acidic pH
and vice versa. A second result is the absence of isosbestic
points in the L fluorescence spectra, in further contrast with the
acridine behavior. In agreement with potentiometric results,
this indicates that the acridine chromophore is not involved in
the acid−base equilibria in the ground state, and as a
consequence, the acridinium form of the chromophore is not
formed at any pH. Being the aromatic chromophore
responsible of the absorption spectrum and the acridine form
of the ligand occurring all over the investigated pH range, the
independence of the absorption spectrum on pH easily follows.
As to point (2), we start with the consideration that acid−
base properties of acridine in the lowest ππ* state differ from
those of the ground state. Acridine is significantly more basic
upon excitation, pKa = 9.2, than in the ground state, pKa =
5.4.6,7,9 Second, the pKa value of the excited state is comparable
to that of aliphatic amino groups in the ground state.76 This
suggests that a similar pKa increase may occur also in the
acridine unit of L favoring the intramolecular excited state
proton transfer from the ammonium group of the aliphatic
chain to the nitrogen of the acridine unit, as it has been
reported for other acridine derivatives.6,7,14,15 With the
indication from now on of the “neutral-like” and “protonated-
like” structures of the ligand at the acridine nitrogen as A and B,
respectively, the process, likely to involve the protonated amino
groups N2 and N2′ (see Figure 1 for atom labeling), can be
described schematically as excitation of the ligand in the
“neutral-like” form, A → A*, followed by proton transfer to the
“protonated-like” form in the excited state, A* → B*, and
fluorescence emission, B* → B (see Figure 8). A is recovered
Figure 6. UV−vis absorption spectra of aqueous solution of acridine
(panel a, C = 3 × 10−5 M) and L (panel b, C = 8 × 10−6 M) in
aqueous solution at different pH values.
Figure 7. Fluorescence emission spectra of aqueous solution of
acridine (panel a, C = 3 × 10−5 M) and L (panel b, C = 8 × 10−6 M) in
aqueous solution at different pH values. The excitation wavelength is
345 nm. In the inset, the fluorescence emission intensity measured at
456 nm as a function of pH has been overlapped to the distribution
diagram of the protonation states of L.
Figure 8. Schematic representation of the excited state proton transfer
from the A* to B*. The relevant parts of the A and B geometries are
also sketched, highlighting the role of the N2 amino group in the
proton transfer process.
6b. The absorption is due to the aromatic chromophore.
However, at variance with results on pure acridine, all of the L
spectra show nearly the same absorption profile in the range
300−450 nm, irrespective of pH. Two band systems are
recognized, the first with onset around 390 nm and the second
with a peak at 355 nm and the associated vibronic structure,
assigned as the second ππ* transition of the chromo-
phore.8,11,84
The fluorescence spectra are collected in Figure 7b. Exciting
at 345 nm, pH dependent emissions are observed. In strongly
acidic solutions (pH = 1.1 and 2.1), where the most abundant
species is the tetraprotonated ligand H4L4+, the fluorescence
spectrum closely resembles that of acridine in alkaline solution,
in the final B → A process if the reasonable assumption is made
that the ground state pKa values of L are not substantially
changed with respect to those of acridine and the aliphatic
amino groups. Looking at Figure 7b, it is seen that our model is
consistent with the fluorescence behavior at pH 5.0 and 8.2,
that is, exciting the tri- and biprotonated species, but less
convincing for the fluorescence spectra at high and low pH
values. In strongly alkaline solution, A*→ A fluorescence is
expected because unprotonated ligand is predominant and
hence no proton transfer can occur. On the contrary, this
fluorescence is not observed. The inhibition is related, as often
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reported for unprotonated polyamine-based fluorescent chemo-
sensors,30,41 to a photoinduced electron transfer from the
nonbonding lone pair of the amino nitrogen to the excited
chromophore. The emission at pH 12.0 is thus ascribed to the
presence of a minor amount of the HL+ cation fluorescing
according to the above-mentioned scheme, A → A* → B* →
B. On the other pH side, in strongly acidic solution the
tetraprotonated form largely outweighs all of the others so that
in principle the proton transfer may occur in the excited state.
However, in this case only the A*→ A fluorescence is observed,
and the absence of the B* → B emission has been tentatively
related to the activation energy of the A* → B* process,
sufficiently high to prevent the excited state proton transfer.
3.5. Ab Initio and MD Simulation Results. Our model
implies that the A form lies at energy lower than B for all
protonated species. In order to validate this hypothesis QM
calculations have been performed on L and HnLn+ species,
according to the procedure outlined in Section 2.6. The
optimized structures correspond to energy minima, given the
absence of imaginary frequencies in the calculated vibrational
modes. Due to the multitude of conformational isomers relative
to L and HnLn+, only the structures which, according to the MD
simulations, correspond to the most populated isomers at room
temperature will be presently considered as starting structures
for energy optimizations. The optimized configurations of L,
H2L2+, and H4L4+ calculated in the polarizable continuum
model approximation are reported in Figure 9 as representative
results. Starting with the calculations in vacuum shown in Table
2, the highly unsymmetrical A and B structures of HL+ and
H2L2+ are almost at the same energy, while the B forms of
H3L3+ and H4L4+ are markedly more stable than A. The result is
Table 2. Calculated Energy Differences ΔEBA = EB − EA (eV)
between A- and B-Type Ground State Structures in Vacuum
and in Water Polarizable Continuum Medium (See Text for
the Definition of the A and B Structures). Last Row: ΔEBA
Relative to the Water Complexes of A and B in the
Polarizable Medium
ΔEBA
vacuum
HL+
H2L2+
H3L3+
H4L4+
−0.02
0.23
0.04
0.20
0.36
−0.74
0.01
−1.09
−0.15
0.07
polarizable medium
water complex
0.42
0.21
reasonably related to the nonbonded interactions N···H
between nitrogen and hydrogen atoms of adjacent amino
groups in the A and B forms. The energy pattern drastically
changes when HnLn+ is immersed in a polarizable continuum
(see Table 2). A close inspection of HnLn+ structures (see
Figure 9) indicates that increasing the positive charge on the
polyamine chain the macrocycle expands,85−88 and as a
consequence, nonbonded interactions are weakened, reducing
the molecular folding between the acridine ring and the average
polyamine plane. The overall effect is to stabilize the A form,
being the solvent interaction stronger with the outer
ammonium group along the chain than with the inner NH+
acridine fragment. Referring to Table 2, all of the stabilization
energies, defined as ΔEBA(vacuum) − ΔEBA(polarizable
medium), shift in favor of A, as much as 0.94 eV in the case
of H4L4+. Thus, from the data of Table 2 it may be seen that, at
room temperature, B is in negligible abundance with respect to
A for both the mono- and biprotonated species. Solvation
effects are better appreciated when the solvent−solute
interaction is taken into explicit account, for instance in the
complex of HnLn+ with one molecule of water. Now both A and
B forms are in the polarizable continuum and the water
molecule interacts alternatively with the N2 ammonium group
(form A) or with the N2 amino group (form B). The energy
differences ΔEBA are found to be positive; that is, the water
complexes with A are more stable than those with B for all the
HnLn+ species. It may be concluded that the model, in spite of
its crudity, emphasizes the essential role of the interaction with
the solvent in determining the invariance with pH of the
absorption profiles. The QM calculated structures are available
upon request.
With the acridine unit of the cromophore being responsible
of the HnLn+ absorption, the vertical excitation energies A →
A* and B → B* from the A and B minima are only slightly
perturbed by the polyamine chain. The calculated A → A*
transition energies of Table 3, which are around 3.20 eV
(corresponding to 388 nm excitation wavelength), match nicely
the lowest energy absorption bands. The B → B* transition
energies shift to the red, ∼2.92 eV (corresponding to ∼424 nm
excitation wavelength), thus justifying the even larger shift of
the fluorescence emission of the bi- and triprotonated species.
On the contrary, the fluorescence spectrum in strongly acidic
solutions overlaps quite closely that of acridine at pH 8.0,
where the chromophore is unprotonated. As anticipated, we
argue that the fluorescence is likely to be due to the A* → A
transition ruling out the proton transfer from the aliphatic
ammonium group to the acridine nitrogen in the excited state.
The high energy barrier for the A* → B* proton transfer in the
case of H4L4+ can be roughly rationalized considering the
structural arrangement of the aliphatic polyamine chain. The
macrocycle cavity expands going from HL+ to H4L4+, as
Figure 9. Calculated structures of L and the A-type H2L2+ and H4L4+
cations (a: left, top view; middle, side view) and of the B-type H2L2+
and H4L4+ cations (b: right, top view). Significant distances (Å)
between non adjacent nitrogen atoms follow. A-type H2L2+ cation:
N2···N2′ = 4.41, N3···N2′ = 4.62, N2···N3′ = 4.56; A-type H4L4+
cation: N2···N2′ = 6.14, N3···N2′ = 5.77, N2···N3′ = 5.34.
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Table 3. Vertical Transition Energies A → A* and B → B* (eV, nm in parentheses) to the Lowest ππ* Excited State of L and
HnLn+ in Water Polarizable Continuum Medium (See Text for the Definition of A and B Structures)
L
HL+
H2L2+
H3L3+
H4L4+
ΔEAA*
ΔEBB*
3.24 (382)
3.19 (389)
2.96 (419)
3.20 (388)
2.95 (421)
3.22 (385)
2.95 (421)
3.20 (388)
2.84 (436)
reported in Table 4. The distances between N1 and the
aliphatic nitrogens are steadily growing from the mono- to the
two planes are almost parallel, in excellent agreement with the
indications from QM calculations.
Table 4. Distances (Å) between the Acridine Nitrogen N1
and the Amino Nitrogens N2, N3, N2′, and N3′ and between
N1 and the Closest H Atoms Bound to N2 and N2′ (See
Figure 1 for Atom Labeling). Structural Data from QM
Calculations on L and on A-type Forms of HnLn+ in Water
Polarizable Continuum Medium
4. CONCLUSIONS
Chemosensors are normally constituted by a signaling and a
binding unit, separated by appropriate spacers. The fluores-
cence properties of the signaling unit and their change upon
substrate binding are obvious factors which control the sensing
ability of the chemosensor. The present results indicate that the
fluorescence properties can be modified also by the structural
arrangement of the signaling unit with respect to the binding
unit. In fact, in the group of polyammonium-based fluorescent
ligands, one of the most common class of anion chemosensors,
macrocycle L represents an interesting case of pH controlled
molecular switch. By means of potentiometric and NMR
measurements the four acid−base equilibria of L have been
fully characterized. Thus, anion chemosensing may be tuned
along all the pH range by inducing the occurrence of one form,
out of several protonated species. Further, these data together
with the crystal results have been instrumental to highlight
fluorescence properties of L depending on the proton transfer
in the excited state from the ammonium group of the binding
unit to the signaling unit. Due to this process, the fluorescence
spectrum of L in slightly acidic and alkaline solutions, the most
relevant pH region for anion recognition and sensing, has been
correlated to that of the protonated acridine, normally not
present in solution in this pH range.
N2
H2
N3
N3′
N2′
H2′
L
3.22
3.38
2.87
3.18
3.80
2.62
2.98
2.03
2.55
3.51
5.54
5.12
5.25
5.34
5.38
5.09
5.02
5.47
5.72
5.82
3.41
3.38
3.71
3.75
3.98
2.87
4.00
3.53
3.44
3.81
HL+
H2L2+
H3L3+
H4L4+
tetraprotonated species, and on the other hand, the N1···H2
distances are remarkably much shorter for H2L2+ and H3L3+,
2.03 Å and 2.55 Å, respectively, than for H4L4+, 3.51 Å. The
peculiar properties of H4L4+ with respect to the other
protonated species come to strong evidence also recurring to
structural results obtained by MD simulations on the A forms
of HnLn+. These are reported in Figure 10. In the upper part of
Taking into account that anion binding often modifies the
charge localization on the receptors, L and its protonated forms
may represent a novel interesting tool to study anion
chemosensing. We are currently working in this direction.
ASSOCIATED CONTENT
■
S
* Supporting Information
Technical details of MD simulations, cluster analysis and crystal
structure CIF file. This material is available free of charge via
the Internet at http://pubs.acs.org.
Figure 10. Upper (a): distribution functions related to N2−H···N1
and N2′−H···N1 hydrogen−nitrogen distances (Å) in HnLn+ species.
Lower (b): distribution functions of the angle α formed by the mean
plane of the acridine ring and that of the macrocycle chain. The areas
of the distribution functions have been normalized to unity.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: claudia.giorgi@unifi.it; riccardo.chelli@unifi.it; cristina.
gellini@unifi.it.
the figure, the dependence of the distribution function relative
to the N2−H···N1 and N2′−H···N1 nonbonded distances is
plotted. Weak H···N1 bands peaked in the range 2.6−2.9 Å are
formed in the case of HL+, H2L2+, and H3L3+, whereas no such
band is found for H4L4+, the first peak of the distribution being
at much longer distance, ∼4 Å. A second distribution function
is shown in the lower part of Figure 10, that is, that relative to
the angle between the mean planes of the acridine ring and of
the macrocycle chain (see Section 3.2). This function may be
considered to give an estimate of the folding of the two planes
in HnLn+. The single peak around 20° is the distinctive feature
of the H4L4+ function, which confirms that in this species the
Author Contributions
Designed the research: Bencini, Gellini, Lippolis, Pietraperzia.
Wrote the paper: Bencini, Chelli, Gellini, Salvi. Synthesis and
characterization: De Filippo, Lippolis, Puccioni. Crystallog-
raphy: Bazzicalupi. Potentiometry and NMR titration: Giorgi,
Valtancoli. UV−vis and fluorescence: Gellini, Pietraperzia.
Calculations: Chelli, Salvi.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank the Italian Ministero dell’Istruzione, dell’Universita
della Ricerca for financial support (Project PRIN 2009,
2009Z9ASCA).
̀
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