Acid-Base Sensors Based on Novel Quinone-Type Dyes

Article abstract of DOI:10.1002/chem.200305206

We present a detailed study on the acid-base behaviour of a family of "potentially antiaromatic" p-benzoquinonediimine ligands. These 12π electron molecules can be considered as constituted of two chemically connected but electronically not conjugated 6π-

Full text of DOI:10.1002/chem.200305206

FULL PAPER
Acid Base Sensors Based on Novel Quinone-Type Dyes
Mourad Elhabiri,[a] Olivier Siri,[b] Alejandra Sornosa-Tent,[a] Anne-Marie Albrecht-
Gary,*[a] and Pierre Braunstein*[b]
Abstract: We present a detailed study
on the acid base behaviour of a family
of ™potentially antiaromatic∫ p-benzo-
quinonediimine ligands. These 12p
electron molecules can be considered
as constituted of two chemically con-
nected but electronically not conjugat-
ed 6p-electron subunits. Upon succes-
™double∫ cyanine-type chromophores
are generated in solution and allow a
precise and sensitive spectrophotomet-
ric detection. These molecules repre-
sent a new class of tunable quinones
whose electronic and structural proper-
ties can be triggered by proton input,
as established by a complete physico-
chemical study involving a combination
of potentiometric and spectrophoto-
metric methods (absorption and emis-
sion).
Keywords: absorption
¥
benzoquinonediimine ¥ fluorescence
spectroscopy ¥ protonation ¥ sensors
sive
protonation,
™mono∫
and
Introduction
aqueous media. Therefore, synthetic efforts are currently in
progress in order to overcome these limitations and to pro-
duce new and stable spectrophotometric sensors based on
cyanine-type dyes. Other interesting candidates as spectro-
photometric chemosensors are molecules exhibiting a quino-
noid structure, which have attracted the interest of a large
community for decades[7] owing to their chemical and physi-
cal properties. More specifically, research on the relation be-
tween colour and structure has, since a long time, involved
benzoquinonediimines such as L, which are used as colo-
rants for various applications. These 12p electron molecules
should actually be better considered as constituted of two
6p subsystems,[8] chemically connected by CꢀC single bonds
but electronically not conjugated (Scheme 1).[9,10]
Spectrophotometric chemosensors constitute a large class of
™reporter∫ molecules able to bind to other molecules or ions
and signal their presence.[1] In particular, acid base sensors
capable of indicating the presence or absence of protons are
of great importance in for example life sciences, detectors
responding to environmental changes, and memories or
logic gates in nanotechnology.[2,3] Fluorescent sensors are es-
pecially interesting due to the high sensitivity of this method
and numerous fluorescent systems displaying bimodal re-
sponse ™on∫ and ™off∫ at near-neutral pH 4.5 8 have been
reported and have led to practical applications for intracel-
lular pH measurements.[4] Cyanine dyes, which constitute a
subgroup of the polymethine dyes, have been used as sensor
molecules for the fluorescent detection of analytes in diag-
nostics.[5,6] Despite their success, only few examples of fluo-
rescent cyanine biosensors sensitive to proton concentration
have been reported,[6] mainly due to difficulties associated
with photodecomposition, self-aggregation or hydrolysis in
Scheme 1. Resonance forms in 2,5-diamino-1,4-benzoquinonediimines.
[a] Dr. M. Elhabiri, A. Sornosa-Tent, Dr. A.-M. Albrecht-Gary
Laboratoire de Physico-Chimie Bioinorganique
UMR 7509 CNRS, ECPM, Universitÿ Louis Pasteur
25 rue Becquerel, 67200 Strasbourg (France)
Fax : (+33)390-24-26-39
We have previously shown that N-neopentyl p-benzoqui-
nonediimine[9] L1 undergoes significant spectrophotometric
changes as a function of pH.[10] Upon successive protonation
of L1, ™mono∫ and ™double∫ cyanine-type chromophores are
generated in solution.
An X-ray crystal structure analysis of the monoprotonat-
ed derivative (L1)H+ established that protonation occurred
at one of the imine functions and generated an iminium ion
stabilised by intramolecular delocalisation, so that the posi-
E-mail: amalbre@chimie.u-strasbg.fr
[b] Dr. O. Siri, Prof. P. Braunstein
Laboratoire de Chimie de Coordination
UMR 7513 CNRS, Universitÿ Louis Pasteur
4, rue Blaise Pascal, 67070 Strasbourg (France)
Fax : (+33)390-24-13-22
E-mail: braunst@chimie.u-strasbg.fr
134
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/chem.200305206
Chem. Eur. J. 2004, 10, 134 141

134 141
tive charge is delocalised over the NPCPCPCPN subunit.[10]
Therefore (L1)H+ contains in the solid state two different
conjugated p-systems, with and without delocalisation. The
X-ray structure analysis of the diprotonated species
Herein, we wish to report the synthesis and full character-
isation of the new N-substituted p-benzoquinonediimine L2
which contains benzyl groups as the light emitting compo-
nents and four nitrogen atoms as the protonation sites. By
analogy with aryl-containing polyamines receptors,[19,20] the
use of benzyl groups as N-substituents is expected to lead to
fluorescent sensors for which photophysical properties could
be triggered by proton input, thus allowing ™on/off∫ switch-
ing of the luminescence.[6,21] The stability of L2 and of its
neopentyl analogue L1 in aqueous media allowed their phys-
ico-chemical and electronic properties to be determined in
methanol/water (80/20 w/w). Their protonated forms have
been fully characterised by a combination of spectrophoto-
metric methods (absorption and/or emission) and potenti-
ometry.
2+
(L1)H2 similarly showed that each positive charge is delo-
calised, leading to a double cyanine-type chromophore for
Results and Discussion
which the 6p-subsystems are connected to each other by
two CꢀC single bonds.[10] Since L1 appeared to be the first
molecule of its kind with two separated, conjugated and lo-
calised 6p electron systems that can be tuned by protonation
to become delocalised, this prompted us to evaluate such
systems as spectrophotometric sensors for proton detection.
The electronic situation in our molecules is novel with re-
spect to previously reported cyanine or cyanine-type dyes.[7,
Synthesis and characterisation: The N,N’,N’’,N’’’-tetrabenzyl-
2,5-diamino-1,4-benzoquinonediimine base, noted L2, was
prepared in two steps similarly to the neopentyl analogue L1
(Scheme 2).[9,10] Tetraaminobenzene was first reacted with
benzoyl chloride in acetonitrile and NEt3, to afford the tet-
rabenzamidobenzene (4) which was fully characterised. This
intermediate was then reduced with LiAlH4 in THF yielding
L2 as an orange solid after aerobic workup.
11]
In related systems 1 3 involving a quinoxalino-phenazine
backbone, the two ™face-to-face∫ p-systems are not fully in-
dependent owing to a ™peripheral∫ C=C connection be-
tween the endocyclic protonations sites which allows conju-
18]
gation between the two 6p electron systems.[12
Scheme 2. i) PhC(O)Cl/NEt3/CH3CN/Ar; ii) LiAlH4/Ar, THF/aerobic
workup.
The successive protonations of L (L = L1 or L2) led to
the formation of ™mono∫ and ™double∫ cyanine-type sys-
tems LH+ and LH22+, respectively (Scheme 3). Compounds
2+
L2, (L2)H+ and (L2)H2
were fully characterised by
1H NMR spectroscopy and elemental analyses. Further pro-
3+
4+
tonation leads to LH3 and LH4 which will be character-
ised by spectrophotometric methods.
UV/Vis absorption: Compounds L1 and L2 are soluble in
dichloromethane, tetrahydrofuran or acetonitrile, sparingly
soluble in methanol and insoluble in water whereas their
protonated forms are more soluble in methanol and soluble
Scheme 3. Protonation of N,N’,N’’,N’’’-tetrasubstituted-2,5-diamino-1,4-benzoquinonediimine-type ligands (L1: R=benzyl and L2: R=neopentyl).
135
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A.-M. Albrecht-Gary, P. Braunstein et al.
FULL PAPER
in water (ca. 10ꢀ4 m). L1 is much more soluble in organic sol-
vents than L2, which facilitates purification by crystallisation
of its protonated forms. The acido basic properties were
thus examined in a mixture methanol/water (80/20 w/w). p-
Benzoquinonemonoimine and p-benzoquinonediimine de-
rivatives are known to undergo rapid hydrolysis in water
and fast mixing techniques (e.g. stopped-flow) are generally
required to determine their protonation constants.[22,23] Inter-
estingly, L1 and L2 were found to be stable under our experi-
mental conditions and not altered by reversible variations of
pH. The UV/Vis absorption spectra of L1 and L2 were re-
corded in a large span of pH values (Figure 1).
zoquinonediimine (logb1 =5.75; logb2 <7.25),[22] L1 and L2
clearly shows a drastic increase of the respective protonation
constants mainly due to inductive and mesomeric effects. In
a statistical model, a difference between two successive pro-
tonation constants of two identical and independent sites of
a molecule is expected to be DlogK=0.6.[28] However, a
much larger DlogK value (ꢂ2.2 for L1 and ꢂ2.5 for L2) was
observed between K1 and K2, that could be explained by
strong electrostatic repulsions between the two independent
2+
2+
conjugated subunits in (L1)H2 and (L2)H2 (Scheme 3
and Table 1).[28] Under very acidic conditions, low constants
could be determined for further protonation of L1 and L2
(Table 1). For L2, b3 was easily
attributed to the protonation of
one of the four equivalent ni-
trogen sites of (L2)H2
2+
(Scheme 3). Its low value
(logb3 =14.6ꢁ0.2) could be ex-
plained by the resulting loss of
the optimal delocalisation and
symmetry occurring in (L2)-
2+ [10,29]
H2
.
Owing to the de-
crease of conjugation, protona-
3+
tion of (L2)H3 could be ex-
pected to follow a mechanism
with less interactions and then
closer to a statistical process.[28]
The influence in the chemical
structure of the N-substituents,
neopentyl in L1 and benzyl in
L2, respectively, does not signif-
icantly affect the protonation
constants owing to the presence
of a common methylenic spacer
in L1 and L2, which is a poor
electronic relay.
Figure 1. Absorption spectra of L1 and L2 as a function of pH. Solvent: methanol/water (80/20 w/w); T=
25.0ꢁ0.28C; I=0.5m (N(nBu)4ClO4); l=1.0 cm; a) [L1]tot =5.76î10ꢀ5 m, 1) pH 2.66, 2) pH 10.19; b) [L1]tot
=
5.23î10ꢀ5 m, 1) pH 2.20, 2) pH 1.31; c) [L2]tot =4.23î10ꢀ5 m), 1) pH 2.86, 2) pH 8.03; d) [L2]tot =4.35î10ꢀ5 m,
1) pH 2.52, 2) pH 1.30.
As an example, the corre-
sponding
distribution
dia-
gram[30] of the species resulting
The processing[24,25] of the spectrophotometric and poten-
tiometric data led to the respective protonation constants of
L1 and L2 (Table 1).
from protonation of L1 versus pH is given in Figure 2. It
shows that under suitable conditions of acidity, L1, (L1)H+
2+
and (L1)H2 can be isolated almost quantitatively as pure
species.[10]
Table 1. Protonation constants of ligands L1 and L2.[a]
The electronic spectra of the protonated species of L1 and
L2 are given in Figure 3.
Equilibrium
L1
L2
logbn ꢁ3s
logbn ꢁ3s
b1
+
L + H+
LH
8.28ꢁ0.01[b]
8.09ꢁ0.09[c]
14.03ꢁ0.09[b]
13.7 ꢁ0.2[c]
7.96ꢁ0.05[b]
ƒ!
L + 2H+
ƒ
7.5 ꢁ0.2[c]
b2
2+
ƒ!
LH
13.76ꢁ0.09[b]
12.9 ꢁ0.2[c]
14.6 ꢁ0.2[c]
ƒ
2
3
b3
3+
4+
L + 3H+
L + 4H+
LH
ƒ!
ƒ
b4
ƒ!
ƒ
LH4
16.5 ꢁ0.2[c]
[a] methanol/water (80/20 w/w); T=25.0
ꢁ
0.28C. [b] I=0.1m
bn
(NEt4ClO4). [c] I=0.5m (N(nBu)4ClO4). [d] L + NH+
and L=L1 or L2.
LH 1ꢃnꢃ4
nþ
ƒ!
ƒ
n
b1 and b2 were attributed to the protonation of the imine
functions, in agreement with literature data available for
closely related compounds.[22,26,27] A comparison between
1,4-benzoquinonemonoimine (logb1 =3.72ꢁ0.06),[22] 1,4-ben-
Figure 2. Distribution diagram of the species resulting from protonation
of L1. Solvent: methanol/water (80/20 w/w); T=25.0ꢁ0.28C; I=0.5m
(N(nBu)4ClO4). [L1]tot =5.00î10ꢀ5 m.
136
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Acid Base Sensors
134 141
Upon successive protonation of
the imine functions, the ligand-
centred fluorescence signifi-
cantly decreased to almost zero,
with
longer
a
concomitant shift to
wavelengths (lem
=
529 nm), when both imine re-
ceptors
are
protonated
(Figure 4).
The processing of the spec-
trofluorimetric data led to pro-
Figure 3. Electronic spectra of the different species resulting from protonation of a) L1 and b) L2; solvent:
methanol/water (80/20 w/w); T=25.0ꢁ0.28C; I=0.5m (N(nBu)4ClO4).
A comparison between the spectrophotometric data for
1,4-benzoquinonemonoimine (7) (lmax =262 nm)[22], 1,4-ben-
zoquinonediimine (12) (lmax =265.5 nm)[22]
,
L1 (lmax
=
338 nm) and L2 (lmax =345 nm) shows that substitution of
positions 2and 5 by amine functions leads to a bathochro-
mic shift of the intraquinone transitions of about 80 nm
(Table 2). N-Substitution of the imine functions of 7 and 12
by aryl groups leads to large bathochromic shifts (>
200 nm) of the maximum of the intraquinone p!p* absorp-
tion band[22,26,31,32] as shown in indoaniline (9, 10 and 11), N-
phenyl benzoquinonediimine (13, 14) or phenazine (15) type
compounds. This indicates the presence of more extended
p-systems most likely due to significant delocalisation
through the ™peripheral∫ N-substituents (Table 2). Further-
more, N-substitution of the amine and imine nitrogen atoms
by aryl groups (5, 6) similarly results in red shifts of the in-
traquinone p!p* absorption band compared to that of neo-
pentyl or benzyl derivatives L1 and L2. The presence of
methylenic spacers in the N-substituents of the latter two
molecules interrupts electronic communication and results
in confined systems with shorter wavelength absorptions.
The larger red shift for 6[12] compared with azophenine (5)[33]
is due to a ™peripheral∫ C=C connection between the endo-
cyclic protonations sites which allows conjugation between
the two 6p electron systems. The intraquinone p!p* transi-
Figure 4. Fluorescence spectra of L2 (7.58î10ꢀ6 m) as a function of pH.
Solvent: methanol/water (80/20 w/w); T=25.0ꢁ0.28C; I=0.1m
(N(C2H5)4ClO4); l=1 cm, lexc =358 nm. Spectra: 1) pH 3.34; 2) pH 10.53.
Excitation and emission slits: 3 nm.
tonation constants close to those obtained in the ground-
state by absorption spectrophotometry (Table 3).
The absolute quantum yield scarcely varies between
pH 9.8 and 6.6 (~0.18), whereas it significantly decreases
below pH 6.6 and amounts to 0.03 at pH 2.8 (Figure 5a), in
agreement with the relative quantum yield calculated from
the emission fluorescence titration (Figure 5b).[34]
1H NMR spectroscopy of L2 clearly showed that a fast in-
tramolecular double proton transfer, which involves two tau-
tomers, occurs in solution and leads to a structure of higher
symmetry (Scheme 4a). Upon the first protonation, a de-
crease of one quarter of the total fluorescence intensity
could be expected and this is consistent with our experimen-
tal observations (Figure 5b, Scheme 4a and Table 3). The
tions for (L1)H+ and (L1)H2 are similar and experience
2+
bathochromic shifts of about 25 30 nm compared to L1
(Figure 3 and Table 2). This is in agreement with recent
DFT calculations on L1 to be published elsewhere[10] as well
as with data obtained for 5 (Table 2). Compared to L2, the
2+
intraquinone p!p* transitions for (L2)H+ and (L2)H2 are
shifted to longer wavelengths by 7 and 24 nm, respectively
(Figure 3 and Table 2).[33]
Fluorescence: When the p-benzoquinonediimine system was
N-substituted by an alkyl (R = neopentyl, L1) or an aryl (R
= phenyl, 5) group, no significant emission signal was ob-
served. In contrast, compound L2 with benzylic moieties as
N-substituents revealed interesting emission properties and
the effect of pH on the fluorescence spectra of L2 was inves-
tigated. The absorbance at the chosen excitation wavelength
(lexc =358 nm) was kept lower than 0.1 to minimise resorp-
tion processes and remained constant throughout the fluo-
rescence titration (Figure 4). Upon excitation into the transi-
tions at 358 nm, a maximum of emission arising from the
1pp* state was observed at 480 nm at pH 10.53 (Figure 4).
Scheme 4. Tautomeric equilibria of a) L2 and b) (L2)H+ in solution.
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A.-M. Albrecht-Gary, P. Braunstein et al.
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Table 2. Spectrophotometric data (p!p* transition) for benzoquinone mono- and diimine derivatives.[a]
1H NMR spectrum of (L2)H+
2+
Ligand
L
LH+
LH2
Ref.
revealed a C2 symmetry in sol-
ution which could be explained
similarly by a fast proton trans-
fer involving two tautomers
(Scheme 4b). Further protona-
tion leads to a more symmetri-
max
max
max
lmax (logel ) [nm]
lmax (logel ) [nm]
lmax(log el ) [nm]
338 (4.40)
368 (4.30)
352 (4.23)
368 (4.32)
369 (4.29)
[a]
cal (L2)H2 species, for which
2+
the four equivalent light-emit-
ting benzylic groups are
switched off (Scheme 3, Fig-
ures 5 and 6).
345 (4.30)
344 (4.26)
[a]
[b]
According to the thermody-
namic Fˆrster cycle method,[35]
the frequency difference be-
tween absorption and emission
could nevertheless be used to
approximate the K* values for
L2.[36] logK1* and logK2* values
amount to 9.0ꢁ0.2and 8.8 ꢁ
0.3, respectively. These values
are consistent with earlier re-
sults which showed that the ni-
trogens of the imine functions
become more basic in the ex-
cited state S1.[37] Moreover,
these results suggest a decrease
of the repulsive interactions
between the two positively
389 (4.33)
[b], [33]
2+
charged halves of the (L2)H2
species in the excited state
(Scheme 4). For aryl-contain-
ing polyamine receptors, it has
been demonstrated that unpro-
tonated amine and/or imine
groups are in most cases effi-
cient intramolecular electron
transfer quenchers of the aro-
matic excited state (photoin-
505 (4.10)
530 (4.18)
[12]
[23]
262 (7)
275 (8)
duced
electron
transfer,
PET).[19,20,38,39] Our system
stands in interesting contrast,
which may be explained by an
intramolecular charge transfer
state (ICT)[40] from the amino
to the imino nitrogen functions
in L2 (Scheme 4). Theoretical
calculations were performed
on a model of L for which the
N-substituent is a hydrogen. Its
p molecular orbitals can be de-
duced from an interaction dia-
gram between p orbitals of
[HN-(CH)3-NH2] and/or [H2N-
554 (4.05)
[31]
[26]
511 (3.98) (10)
492(3.96) ( 11)
428 (3.55) (11)
257 (4.43)
265.5 (4.42)
[22]
[32]
(CH)3-NH2]+
fragments
(Figure 6).[10] A comparison be-
tween the p orbitals of the pro-
tonated fragment [H2N-(CH)3-
NH2]+ and the neutral [HN-
484 (3.91)
672(4.6)
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Acid Base Sensors
134 141
Table 2. (Continued)
Ligand
of the emission signal observed
2+
L
LH+
LH2
lmax(log el ) [nm]
Ref.
[27]
until pH ꢂ6.5. To explain the
max
max
max
lmax (logel ) [nm]
lmax (logel ) [nm]
unexpected
fluorescence
quenching with decreasing pH,
we suggest that the PET
quenching mechanism occurs
by protonation of the imine
units in the excited state with
the loss of the ICT character as
shown in the interaction dia-
gram between the p levels of
two [H2N-(CH)3-NH2]+ frag-
ment.[10] The small difference
between the K1* and K2* values
suggests a decrease of the p-
electron delocalisation. The
fluorescence quenching ob-
served under acidic conditions
is therefore mainly due to a
480 (3.51)
520 (3.93)
448 (3.80)
517 (3.99)
[27]
[a] This work; solvent: CH3OH/H2O (80/20 w/w); T=25.0ꢁ0.28C; I=0.5m. [b] This work; solvent: CH2Cl2;
T=25.0ꢁ0.28C. Ref. [12]: solvent: CH3CN/water (70/30 v/v); T=258C. Ref. [22]: solvent: water; T=258C; ki-
netic determinations. Ref. [23]: solvent: water; T=208C; kinetic determinations. Ref. [26]: solvent: water; T=
258C; kinetic determinations. Ref. [27]: solvent: water; T=258C; kinetic determinations. Ref. [31]: solvent:
water; T=258C; kinetic determinations. Ref. [32]: solvent: water; T=258C; kinetic determinations. Solvent:
CH2Cl2; T=25.0 ꢁ 0.28C. Ref. [33]: solvent: CH2Cl2; T=25.08C.
Table 3. Protonation constants and emission data of ligand L2.[a]
rel
Equilibrium
logbn ꢁ3s
lemmax [nm][b]
Q
/QLrel [c]
LHnnþ
L2
480
477
530
1.00
0.91
0.22
possible
PET
deactivation
b1
2
+
L2 + H+
L2 + H+
(L )H
7.9ꢁ0.3
ƒ!
ƒ
mechanism.
b2
2
2+
ƒ!
ƒ
(L )H
13.9ꢁ0.4
2
[a] Solvent: methanol/water (80/20 w/w); T=25.0ꢁ0.28C; I=0.1m (NEt4ClO4). [b] lexc =358 nm; excitation
and emission slits: 3 nm. [c] Determined from recalculated, normalised fluorescence spectra. The errors are es-
timated to 1 nm for the wavelengths and to 10% for the quantum yields.
Figure 5. Ligand-centred emission quantum yields (a) and recalculated,
2+
normalised fluorescence spectra of L2, (L2)H+ and (L2)H2 (b) Solvent:
methanol/water (80/20 w/w); T=25.0ꢁ0.28C; I=0.1m (NEt4ClO4).
a) Protonation constants of Table 1; [L2]tot =7.58î10ꢀ6 m; standard: qui-
nine sulfate in 0.05m H2SO4, Qabs =0.546.
Figure 6. Orbital interaction diagram obtained for a model of L with R=
H (Scheme 1) by means of EHT calculations. Imino nitrogen (left) and
amino nitrogen (right).
(CH)3-NH2] moiety shows a lowering of the symmetry from
C2v to Ci, respectively with a concomitant increase of the p-
orbital coefficient of the imine group in L.
Conclusion
Therefore, electronic excitation leads to an intramolecular
charge transfer from the donor (amino group) to the accep-
tor end (imino group), in agreement with the enhancement
Absorption and/or fluorescence spectrophotometric titra-
tions of p-benzoquinonediimine derivatives (ligands L1 and
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A.-M. Albrecht-Gary, P. Braunstein et al.
FULL PAPER
L2), over a wide pH range, have enabled the determination
of their acid base properties in the ground and excited
states. The structural changes observed in the solid state
upon protonation are retained in solution as demonstrated
by the complete physico-chemical studies presented here.
The resonance effects in the protonated forms of L1 and L2
are triggered by protonation and especially lead to a fluores-
cent ™on/off∫ switch L2 modulated by proton input. Fluores-
cent cyanine-type dyes can be used as markers for different
bio-applications,[6,21] and the detection of protons is of ongo-
ing interest for monitoring the acidity regulation in biologi-
cal media.[41] Valuable pH sensitive fluorescent dyes for bio-
logical applications require acid base properties within the
critical intracellular pH range (pH 5 8).[4] Molecule L2
which was found to be sensitive in the interesting pH range
between 4 and 7, gave rise to an unexpected large fluores-
cence decrease under acidic conditions (~80%). Therefore,
L2 can be regarded as a sensitive H+ chemosensor with
large Stokes shift (>120 nm) by comparison with the con-
ventional fluorophores, such as fluorescein (25 nm at
pH 9).[42] In conclusion, 2,5-diamino-1,4-benzoquinonedii-
mines bearing methylene groups could constitute stable and
tunable spectrophotometric probes with predetermined
physico-chemical properties in aqueous solution.
4.44 (s, 8H, CH2), 5.40 (s, 2H, Holefinic), 6.90 (brs, 2H, NH), 7.29 ppm (m,
20H, Harom); MS (40 eV, EI): m/z: 496 [M +]; elemental analysis calcd
(%) for C34H32N4: C 82.21, H 6.50, N 11.29; found: C 80.96, H 6.49, N
11.23.
(L2)H+ : Similarly to the procedure described for the synthesis of ana-
logues[10] but using acetone as solvent instead of Et2O during the work-
up, a few drops of diluted HCl were added to a solution of L2 dissolved
in THF until the colour changed from yellow to deep red. (L2)H+ was
1
obtained as a red solid (145 mg, 56%). H NMR (CDCl3): d = 4.51 (brs,
8H, CH2), 5.33 (brs, 2H, Holefinic), 7.20 7.26 (m, not integrated owing the
CDCl3 resonance, Harom), 7.30 7.35 ppm (m, 12H, Harom); elemental anal-
.
ysis calcd (%) for C34H33ClN4 H2O: C 74.10, H 6.40, N 10.17; found: C
72.93, H 6.05, N 10.06.
(L2)H22+ : Similarly to the procedure described for the synthesis of ana-
logues,[10] a large excess of HCl was added to a solution of L2 dissolved in
2+
THF. (L2)H2 was obtained as a green solid (118 mg, 67%). 1H NMR
(CDCl3): d = 4.63 (s, 8H, CH2), 5.33 (brs, 2H, Holefinic), 7.19 7.24 (m, 8
H, Harom), 7.30 7.35 ppm (m, 12H, Harom); elemental analysis calcd (%)
for C34H34Cl2N4: C 71.70, H 6.02, N 9.84; found: C 70.71, H 6.09, N 9.68.
Potentiometric and UV/Vis titrations: All solutions were prepared using
an AG 245 Mettler Toledo analytical balance (precision 0.01 mg). The
ionic strength was adjusted to 0.1m or 0.5m with tetraethylammonium
perchlorate (Fluka, purum) and tetrabutylammonium perchlorate (Fluka,
purum), respectively. Solutions of L1 (5.23î10ꢀ5 m and 5.76î10ꢀ5 m) and
L2 (4.23î10ꢀ5 m and 4.35î10ꢀ5 m) were prepared by quantitative dissolu-
tion of a solid sample in methanol/water (80/20 w/w). An aliquot of
40 mL was introduced into a jacketed cell (Metrohm) maintained at
25.0ꢁ0.28C by the flow of a Haake FJ thermostat. The free hydrogen
ion concentrations were measured with a combined glass electrode (Met-
rohm 6.0234.500, Long Life). The Ag/AgCl reference electrode was filled
with 0.1m NaCl (Fluka, p.a.) in MeOH/H2O (80/20 w/w). Potential differ-
ences were given by a Tacussel Isis 20.000 millivoltmeter. Standardisation
of the millivoltmeter and verification of the linearity (2.00<pH<13.60)
of the electrode were performed using buffers according to classical
methods.[45] The titration of L1 (2.66<pH<10.19, [L1]=5.76î10ꢀ5 m) and
L2 (2.86<pH<8.03, [L2]=4.23î10ꢀ5 m) were carried out by addition of
known volumes of 8.27î10ꢀ3 m tetrabutylammonium hydroxide solution.
The titration of L1 (1.31<pH<2.20, [L1]=5.23î10ꢀ5 m) and L2 (1.30<
pH<2.52, [L2]=4.35î10ꢀ5 m) were carried out by addition of known vol-
umes of a 9.98î10ꢀ1 m perchloric acid solution. Special care was taken to
ensure that complete equilibration was attained. Simultaneous pH and
UV/Vis measurements (230 600 nm) were recorded. Absorption spectra
were recorded using a Varian CARY 50 (Varian) probe UV/Vis spectro-
photometer fitted with Hellma optical fibres (Hellma, 041.002-UV) and
immersion probe made of quartz suprazil (Hellma, 661.500-QX).
Experimental Section
General: Analytical-grade reagents were obtained from commercial sup-
pliers and were used as received. Azophenine (5) is a long known 2,5-dia-
mino-1,4-benzoquinonediimine which can be obtained in various ways[43]
from aniline. Solvents were distilled under argon prior to use and dried
by standard methods. Distilled water was further purified by passing
through a mixed bed of ion-exchanger (Bioblock Scientific R3-83002,
M3-83006) and activated carbon (Bioblock Scientific ORC-83005) col-
umns and was boiled for two hours and saturated with argon before use.
Spectrophotometric grade methanol (Merck, SeccoSolv, p.a.) was also sa-
turated with free-CO2 and O2 argon prior to use (Sigma Oxiclear car-
tridge).
Potentiometric and fluorescence titrations: A stock solution of L2 (7.58î
10ꢀ6 m, I=0.1m NEt4ClO4, Fluka, puriss) was prepared by dilution of a
mother solution (3.76î10ꢀ6 m) and an aliquot of 40 mL was introduced
into a jacketed cell (Metrohm) maintained at 25.0ꢁ0.28C by the flow of
a Haake FJ thermostat. The initial pH was adjusted at 3.34 with perchlor-
ic acid (1.15m), and the titration of L2 was carried out by addition of
known volumes of a 4.05î10ꢀ2 m tetrabutylammonium hydroxide solu-
tion. An aliquot (2mL) was taken after each addition of base, and fluo-
rescence emission spectra (350 700 nm) were recorded versus pH with
1 cm quartz optical cell (Hellma, 110-QS) on a Perkin Elmer LS-50B
maintained at 25.0ꢁ0.28C by the flow of a Haake FJ thermostat. The ex-
citation wavelength was 358ꢁ1 nm and the slit widths were set at 3 nm
for both excitation and emission. The light source was a pulsed xenon
flash lamp with a pulse width at half peak height < 10 ms and power
equivalent to 20 kW. Absorption spectra were also measured along the
fluorescence titration by using a Varian CARY 300 spectrophotometer.
CAUTION: Perchlorate salts combined with organic ligands are potential-
ly explosive and should be handled in small quantity and with the necessa-
ry precautions.[44]
1H NMR spectra were recorded in CDCl3 and [D6]DMSO on a AC300
1
Bruker spectrometer, operating at 300 MHz for H NMR spectra. Chemi-
cal shifts are reported in d units, in parts per million (ppm) relative to
the singlet at d=7.26 for CDCl3. Splitting patterns are designed as s, sin-
glet; m, multiplet; br, broad. In the 1H NMR spectra of the protonated
forms of L2, the NH resonance could not be identified. This could result
from rapid proton exchange with the other protonated forms present in
small amount. Elemental analyses were performed by the ™Service de mi-
croanalyses de L×Institut de Chimie∫, Strasbourg. EI mass spectral analy-
ses were recorded on a Finnigan TSQ 700. The EHT calculations have
been carried out as detailed in ref. [10].
1,2,4,5-Tetrabenzamidobenzene (4): Similarly to the procedure described
for the synthesis of analogues,[10] tetraaminobenzene tetrahydrochloride
(500 mg, 1.76 mmol) was reacted with benzoyl chloride (990 mg,
Refinement of the data: The spectrophotometric data were processed
with both the Specfit[24] and Letagrop-Spefo[25] programs, which adjust
the stability constants and the corresponding extinction coefficients of
the species formed at equilibrium. Letagrop-Spefo[25] uses the Newton
Raphson algorithm to solve mass balance equations and a pit-mapping
method to minimise the errors and determine the best values of the pa-
rameters. Specfit[24] uses factor analysis to reduce the absorbance matrix
and to extract the eigenvalues prior to the multiwavelength fit of the re-
duced data set according to the Marquardt algorithm.[46]
7.04 mmol) to afford
4 as a
white solid (720 mg, 74%). 1H NMR
([D6]DMSO): d = 7.5 (m, 12H, Harom), 7.95 (m, 8H, Harom), 8.01 (s, 2H,
Harom), 10.15 ppm (s, 4H, NH); MS (40 eV, EI): m/z: 554 [M +]: elemental
analysis calcd (%) for C34H26N4O4: C 73.63, H 4.73, N 10.10; found: C
72.53, H 4.67, N 10.03.
L2: Similarly to the procedure described for the synthesis of analogues,[10]
compound 4 (720 mg, 1.30 mmol) was reduced with LiAlH4 and L2 was
obtained as a deep orange solid (458 mg, 71%). 1H NMR (CDCl3): d =
140
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 134 141

Acid Base Sensors
134 141
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Acknowledgements
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We thank the Ministõre de la Recherche et Nouvelles Technologies and
the CNRS for financial support and Dr. M. Bÿnard (Strasbourg) for the
theoretical calculations and fruitful discussions. A.S.T. thanks the ERAS-
MUS program for a scholarship.
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Received: June 5, 2003 [F5206]
141
Chem. Eur. J. 2004, 10, 134 141
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim