Synthesis, characterization and photophysical properties of benzidine-based compounds

Article abstract of DOI:10.1016/j.tet.2008.04.058

The N-alkylation of the 3,3′-diaminobenzidine with innocent substituents leads to unusual properties. The emission of the benzidine core can be fine-tuned by subtle modifications, and the N-substitution with benzylic groups results in photoinduced exciplexes with distinct and increased emission. This compound constitutes the first example of intramolecular exciplex containing benzidine unit. We also show that these photoinduced processes could be modulated by proton input, and that the diprotonation of the benzidine core disrupted the intramolecular communication in the excited states with a concomitant inhibition of the ligand-centred fluorescence. Furthermore, upon photo-irradiation at 254 nm, semiquinone imine and quinone diimine systems are produced in CH₂Cl₂ of which the photolysis generates Cl^{{radical dot}} radicals, which rapidly oxidize the tetraamine compounds.

Full text of DOI:10.1016/j.tet.2008.04.058

Tetrahedron 64 (2008) 6522–6529
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis, characterization and photophysical properties of
benzidine-based compounds
,
Mohamad Hmadeh a, Hassan Traboulsi a, Mourad Elhabiri a, Pierre Braunstein b
,
*
,
c,
*
Anne-Marie Albrecht-Garya , Olivier Siri
*
a
´
Laboratoire de Physico-Chimie Bioinorganique, Institut de Chimie, UMR 7177 CNRSdUniversite Louis Pasteur, ECPM, 25 rue Becquerel, 67200 Strasbourg, France
b Laboratoire de Chimie de Coordination, Institut de Chimie, UMR 7177 CNRSdUniversite´ Louis Pasteur, 4 rue Blaise Pascal, 67070, Strasbourg Cedex, France
c Groupe de Chimie Organique et Mate´riaux Mole´culaires, UMR 6114 CNRS, Universite´ de la Me´diterrane´e, Faculte´ des Sciences de Luminy, case 901,
163 avenue de Luminy, 13288, Marseille Cedex 09, France
a r t i c l e i n f o
a b s t r a c t
The N-alkylation of the 3,30-diaminobenzidine with innocent substituents leads to unusual properties.
The emission of the benzidine core can be fine-tuned by subtle modifications, and the N-substitution
with benzylic groups results in photoinduced exciplexes with distinct and increased emission. This
compound constitutes the first example of intramolecular exciplex containing benzidine unit. We also
show that these photoinduced processes could be modulated by proton input, and that the diprotonation
of the benzidine core disrupted the intramolecular communication in the excited states with a con-
comitant inhibition of the ligand-centred fluorescence. Furthermore, upon photo-irradiation at 254 nm,
semiquinone imine and quinone diimine systems are produced in CH2Cl2 of which the photolysis gen-
erates Clꢀ radicals, which rapidly oxidize the tetraamine compounds.
Article history:
Received 6 March 2008
Received in revised form 10 April 2008
Accepted 14 April 2008
Available online 18 April 2008
Keywords:
Benzidine
N-Alkylation
Fluorescent probe
Exciplex
Ó 2008 Elsevier Ltd. All rights reserved.
Protonation constants
1. Introduction
successfully employed as key components of proton- or electron-
driven molecular machines. Supramolecular shuttles incorporating
4,40-Diamino-1,10-biphenyl (benzidine) 1 is a well known com-
pound, which has been widely used in the past in different areas of
science. (i) Molecules of this class were employed for a long time as
primary materials in the manufacture of azodyes. Being classified as
potentially hazardous and carcinogenic, the importation and in-
dustrial use of 1 are now prohibited in many countries except if
present in less than 1%.1
4,40-biphenol and benzidine units display reversible motion of a
p-
electron-deficient tetracationic cyclophane, serving as ring com-
ponent, which is triggered by oxido-reduction or protonation of the
benzidine moiety.5 (iv) Last, benzidines are relevant examples of
simple redox systems, which could find applications as OLEDs6 or
electroactive organic polymeric compounds.7
As part of our ongoing investigations on quinone diimines,8 we
describe herein the synthesis and full characterization of N-
substituted 3,30-diaminobenzidine systems containing either neo-
pentyl (noted 5) or benzyl (noted 6) groups.
H2N
NH2
1 Benzidine
R
R
HN
NH
(ii) Benzidines also play an important role in cell biology and
clinical work as valuable staining reagents.2 Due to its potential
risks, research has been carried out to develop analogues for bi-
ological and medical purposes. As a result, 3,30,5,50-tetrame-
HN
NH
R
R
5 R=neo-pentyl
6 R=benzyl
thylbenzidine (TMB) was found to be
a non-carcinogenic
compound, and is now widely used in routine assays.3,4 (iii) Be-
cause of their peculiar redox properties, benzidines have also been
If numerous substituted benzidine derivatives have been
reported in the literature, the possibility to introduce electroactive,
photoactive or binding units9 by N-alkylation has received less
attention.10 We show that N-benzylation of the benzidine backbone
confers new properties to these systems.
* Corresponding authors. Tel.: þ33 (0)3 90 24 26 38; fax: þ33 (0) 3 90 24 26 39.
E-mail address: amalbre@chimie.u-strasbg.fr (P. Braunstein).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.04.058

M. Hmadeh et al. / Tetrahedron 64 (2008) 6522–6529
6523
Table 1
2. Results and discussion
2.1. Synthesis
Protonation constants of 5 and 6 and of related compounds
Equilibrium
log KnHðꢅ3
s
Þ
112
5
6
713
813
Molecules 3 and 5 were prepared as described in the literature.11
The tetraphenyl 3,30-diaminobenzidine 6 was synthesized in two
steps (Scheme 1) from 3,30-diaminobenzidine 2. Its reaction first
with 4 equiv of pivaloyl chloride (PhC(O)Cl) afforded the corre-
sponding tetraamido derivative 4 as a grey-white solid, which was
then reduced with LiAlH4 in dry THF to yield the targeted com-
pound 6 as a yellow powder.
K1H
4.18a
4.95b
2.92a
3.84b
d
3.10(7)a
2.8(1)a
3.3(3)b
1.2(4)a
0.9(4)b
nd
4.05
3.33
L þ H % LH
K2H
LH þ H % LH2
K3H
1.6(1)a
2.83
2.62
LH2 þ H % LH3
nd
nd
d
d
d
d
K4H
LH3 þ H % LH4
d
nd
Solvent: CH3CN/H2O (9:1 v/v); I¼0.1 M (NEt4ClO4) for 5 and 6; T¼25.0(2) ꢃC; nd: not
determined under our experimental conditions. For the sake of clarity, charges have
been omitted.
s
¼standard deviation.
a
R
R
Absorption versus pH.
Emission versus pH.
b
O
O
HN
NH
RCOCl
H2N
H2N
NH2
NH2
HN
R
NH
R
H2N
O
NH2
NH2
O
O
7 3,3'-dimethoxybenzidine
8 3,3'-dimethylnaphthidine
2
O
3 R = t-Bu
4 R = Ph
LiAlH4
H2N
R
R
Scheme 2. Chemical structures of benzidine-based compounds 7 and 8.
HN
NH
40 of the biphenyl backbone. Values of log K3H and log K4H were
estimated to be significantly lower than 0.9, probably owing to
HN
R
NH
R
electrostatic interactions and electronic properties. Large
D
log K ¼
log K1Hꢁ log K2H values14 of about 1.3(2) were calculated for com-
pounds 1 and 5–8 (Table 1). This result reflects strong electrostatic
repulsions between the two molecular subunits.14 It is noteworthy
that the diimines derived from benzidine, which were obtained by
oxidation with K2Cr2O7, displayed two significantly higher pK
values of z8.95 and 5.05.15 The increase of the pK values with re-
spect to benzidine (4.95 and 3.84, Table 1) could be mainly
explained by inductive and mesomeric effects occurring in the
conjugated quinone diimine systems.8
5 R = t-Bu
6 R = Ph
Scheme 1. Synthesis of compounds 5 and 6.
2.2. Acido-basic properties
In order to determine the acido-basic properties of the chro-
mophoric benzidine-type units in 5 and 6, absorption spectro-
photometric titrations versus pH were carried out. The
spectrophotometric and potentiometric data of 5 between pH 1.5
and 9.5, and of 6 between pH 1.3 and 10.1 are given in Figures S1
and S2 in Supplementary data, respectively. Compounds 5 and 6
possess four protonable amino sites. The statistical treatment47–50
of the spectrophotometric data versus pH led to the determination
of two protonation constants (Eqs. 1 and 2). The reported errors on
Moreover, we report here the spectrophotometric properties of
5 and 6 under various acido-basic conditions. The electronic spectra
of the tetramino species 5 and 6 (Fig. 1) are characterized by the
presence of two main absorption bands (322 and 236 nm for 5; 324
and 240 nm for 6) in the UV region. These absorptions are attrib-
uted to short-axis (p2) and long-axis (p1) polarized
p
–
p transi-
*
tions of the biphenyl backbone, respectively.16 Interestingly, the
absorption spectrum of biphenyl in cyclohexane is constituted of
protonation constants are given as 3
s
(s
¼standard deviation).
KnH
a
broad and structureless band centred at 247 nm
LHðnnꢁꢁ11Þþ þ Hþ % LHnnþ with 1 ꢂ n ꢂ 4; L ¼ 5 or 6
(1)
(2)
(3
¼1.55ꢄ104 Mꢁ1 cmꢁ1).17 The substitution of the 4,40-positions of
the biphenyl chromophore by two amino functions (1) induces
a large bathochromic shift (
l
¼278 nm; 3278¼2.26ꢄ104 Mꢁ1 cmꢁ1 in
ꢀ
ꢁ
LHnnþ
water) of the long-axis-polarized transitions.12b Furthermore, in-
troduction of two additional amino groups in positions 3 and 30 (5
and 6) resulted in a bathochromic shift of the latter transitions
KnH
¼
h
i
ꢀ
ꢁ
ðnꢁ1Þþ
LH
Hþ
nꢁ1
(l
¼322 nm; 3322¼1.89ꢄ104 Mꢁ1 cmꢁ1 for 5;
l
¼324 nm; 3324¼1.57ꢄ
104 Mꢁ1 cmꢁ1 for 6, Fig. 1). The comparison of the electronic
spectrum of 6 with that of 5 evidenced a hyperchromic effect at
z250–300 nm due to the presence of the four benzylic arms
K1H and K2H values are given in Table 1 together with those de-
termined for closely related compounds benzidine12 1, 3,30-dime-
thoxybenzidine13 7 and 3,30-dimethylnaphthidine13 8 (Scheme 2).
Compounds 1, 7 and 8 were studied in water at 25 ꢃC by absorption
spectrophotometry versus pH.12b,13 Compound 1 was examined
using the same detection method in methanol/water (1:1 v/v).12a
Distribution diagrams of the protonated species of 5 and 6 as
a function of pH are given in Figures S3 and S4 in Supplementary
data. Despite differences in experimental conditions, comparison
with benzidine ligands 1, 7 and 8 allowed us to easily assign K1H and
K2H for 5 and 6 to the amino functions substituted in positions 4 and
(
lmax¼259 nm, 3259¼1.86ꢄ102 Mꢁ1 cmꢁ1 per benzyl unit).18,19
The first protonation of compounds 5 and 6 induces a hypo-
chromic effect and a concomitant broadening of the absorption
bands lying at lower energies (Fig.1). Besides, hypochromic (20–30%
of decrease) and bathochromic shifts (Dl¼10 nm for 5 and Dl¼6 nm
for 6) of the short-axis-polarized transitions could be also observed.
These spectrophotometric data are in agreement with those
reported for the monoprotonated benzidine 1, for which similar

6524
M. Hmadeh et al. / Tetrahedron 64 (2008) 6522–6529
characterized by a large Stokes shift of z80 nm. The absolute
(a)
fluorescence quantum yield of 5 is equal to 0.54(5)%. The fluores-
3
2
1
0
cence emission spectrum of biphenyl in cyclohexane displays a fine
em
max
vibrational structure (
l
¼ 326 nm with Dl¼1050 cmꢁ1). These
data are characteristic of a chromophore that is non-planar in the
ground state and more planar and linear in its excited singlet and
triplet states.16,24 By contrast, the absence of a structured profile for
the absorption and emission bands demonstrates that 5 possesses
a non-planar arrangement either in the ground state or in the ex-
cited state. Introduction of neo-pentylamino units in positions 3, 30,
4 and 40 induces a red shift of 74 nm of the emission band with
respect to biphenyl. Moreover, our spectrophotometric data are in
good agreement with those obtained for benzidine 1, for which an
emission signal centred at z380 nm, and attributed to the biphenyl
core was observed. A Stokes shift of z100 nm and a fluorescence
quantum yield of 0.3943 were measured for 1.
5
2+
5H2
5H+
250
300
350
(nm)
400
450
3
2
1
0
(b)
1.0
0.5
0.0
6
2+
6H2
6H+
250
300
350
(nm)
400
450
300
400
(nm)
500
Figure 1. Electronic spectra of the protonated species of 5 (a) and 6 (b). Solid line:
neutral species; dash line: monoprotonated species; dotted line: diprotonated species.
Solvent: CH3CN/H2O (9:1 v/v); I¼0.1 M (NEt4ClO4); T¼25.0(2) ꢃC.
Figure 2. Normalized absorption (solid line) and emission (dashed line) spectra of
compound 5. Solvent: CH2Cl2; T¼25.0(2) ꢃC. Compound 5: lexc¼276 nm; emission and
excitation slits¼7 nm. Compound 6: lexc¼269 nm; emission and excitation slits¼8 nm.
trends (
l
¼278 nm; 3278¼2.26ꢄ104 Mꢁ1 cmꢁ1 for 1;
l
¼273 nm;
3273¼1.76ꢄ104 Mꢁ1 cmꢁ1 for 1Hþ) were observed.12b The second
protonation leads to significant blue shifts of the long-axis-polar-
ized transitions (Dl¼24 nm for 5 and Dl¼14 nm for 6) simulta-
neously with a hypochromic shift of the short-axis-polarized
transitions. These data also agree with benzidine 1 spectrophoto-
The N-alkylation with benzyl groups significantly altered the
absorption and emission properties of the 3,30,4,40-tetraaminobi-
phenyl moiety. Upon excitation at 269 nm, compound 6 indeed
exhibits an intense, broad and non-structured fluorescence emis-
sion band at 486 nm together with a shoulder at z386 nm (Fig. 3).
metric data (
l
¼247 nm; 3247¼1.63ꢄ104 Mꢁ1 cmꢁ1 for 1H22þ).12b As it
was pointed out for biphenyl, the blurring of the absorption bands in
the ground state strongly indicates a distortion away from planarity.
In solution, the preferred twist angle for biphenyl was indeed esti-
mated to be z42ꢃ,20 while various X-ray diffraction studies pointed
to a planar structure for crystalline biphenyl.21 However, a recent
X-ray study22 revealed that benzidinium dichloride possesses
polymorphic properties (triclinic and orthorhombic structures).
Interestingly, the angle between the two benzene rings within the
two polymorphs is equal to 18.12(5)ꢃ and 45.60(5)ꢃ, respectively.
Density functional calculations supported these observations and
1.0
0.5
0.0
showed that the most stable conformation corresponds to
q
¼30ꢃ
rotated conformation about the C–C biaryl bond.23
2.3. Emission spectrophotometry
Figure 2 shows the normalized absorption and emission spectra
400
500
(nm)
600
700
of
(
5
in CH2Cl2. The corresponding electronic spectrum
lmax¼320 nm; 3320¼1.84ꢄ104 Mꢁ1 cmꢁ1) is similar to that pre-
viously determined for 5 in acetonitrile/water (9:1 v/v) mixture
(Fig. 1). Upon excitation into the transitions at 276 nm, compound 5
displays a broad and structureless band centred at 400 nm, and is
Figure 3. Normalized emission spectra of 6. Solvent: CH2Cl2; T¼25.0(2) ꢃC. Solid line:
lexc¼269 nm; dashed line: lexc¼318 nm; dotted line: lexc¼362 nm; emission and ex-
citation slits¼10 nm.

M. Hmadeh et al. / Tetrahedron 64 (2008) 6522–6529
6525
The fluorescence emission centred at 386 nm most likely originates
1.0
(a)
2
from the 3,30-diaminobenzidine backbone as shown by the similar
shape of the excitation (lana¼386 nm) and absorption spectra
(Fig. S5 in Supplementary data). By contrast, the excitation spec-
trum recorded with lana¼482 nm is strikingly different from the
absorption spectrum of 6, and is slightly red shifted (lmax¼332 nm)
as expected from a ground state association. These spectrophoto-
metric features suggest the formation of a photoinduced exciplex.
This exciplex type emission25,26 can thus be ascribed to intra-
3
0.5
molecular
p-stacking interactions in the excited state between
1
the benzylic units and the 3,30-diaminobenzidine moiety. This
intramolecular process, which follows the excitation of one of
the chromophores, leads to an excited species for which the two
aromatic residues are brought into proper superposition by
p
-stacking.27 Such properties were already reported for various
0.0
non-symmetric bichromophoric compounds such as fluorescent
chemosensors,28 switches,29 antibodies,30 dyads31 or chiral recog-
nition markers.32 Amine intramolecular exciplexes33 are unlikely to
be generated, since no red-shifted exciplex emission was observed
with compound 5. It should also be mentioned that dibenzylamine,
dibenzylether and other dibenzyl derivatives34 may undergo
intramolecular excimer formation with the appearance of a fluo-
rescence emission band with a maximum at about 335 nm in ad-
dition to the normal emission at 285 nm. Intramolecular excimer
solely involving benzyl units within compound 6 could be excluded
since no emission at z335 nm was observed.
At lexc¼368 nm, the absolute fluorescence quantum yield of 6
(3.9(4)%) was found to be more than seven times higher than that of
5 under the same experimental conditions.35 This result clearly
shows that the emission properties of the 3,30-diaminobenzidine
moiety can be tuned by subtle modification (e.g., N-alkylation).
Introduction of aromatic residues led to unexpected photoinduced
exciplex species with increased fluorescence efficiency. Impor-
tantly, compound 6 reveals to be the first example of intra-
molecular exciplex containing benzidine unit.
400
500
600
(nm)
800
(b)
λ = 395 nm
600
400
200
λ = 430 nm
1
2
3
4
5
6
pH
2.4. Effect of pH on emission
Figure 4. (a) Fluorescence spectra of 6 (5.25ꢄ10ꢁ6 M) versus pH. Solvent: acetonitrile/
water (9:1 v/v); T¼25.0(2) ꢃC; I¼0.1 M (NEt4ClO4); l¼1 cm, lexc¼250 nm. Spectra: (1)
pH¼0.85; (2) pH¼2.35; (3) pH¼4.97. Excitation and emission slits: 5 nm. (b) Fluores-
It was of interest to examine the effect of pH on the emission
properties of 6 in acetonitrile/water. It is noteworthy that when
3,30-diaminobenzidine is N-substituted by an alkyl group (R¼neo-
pentyl, 5), no emission signal from the biphenyl skeleton was ob-
served in acetonitrile/water (9:1 v/v). In contrast, 6 with benzylic
N-substituents exhibited interesting emission properties. A spec-
trofluorimetric titration of 6 versus pH was therefore undertaken.
Upon excitation at 250 nm (Fig. 1 and Figs. S1 and S2 in Supple-
mentary data), an emission maximum was observed at 392 nm at
pHz5 (Fig. 4a). Since exciplexes involve different fluorophores,
some degree of charge separation could be expected.
cence intensities versus pH at
l
¼395 nm and ¼430 nm.
l
exciplex species under acidic conditions.37 Therefore, the excited
unfolded diprotonated species 6H22þ behaves as compound 5 with
no emission properties. Interestingly, under these experimental
conditions, 6 possesses valuable fluorescent on/off switch proper-
ties, which can be easily modulated by proton input.
The processing of the spectrofluorimetric data versus pH led to
the determination of two protonation constants close to those
determined in the ground state by absorption spectrophotometry
(Table 1). The recalculated and normalized emission spectra of 6
and 6Hþ are given in Figure 5.
According to classical effects of solvent polarity,26,29 red-shifted
emission could be expected upon going from acetonitrile/water to
dichloromethane. In contrast, a blue shift was observed for 6 under
these conditions. Compound 6 is indeed an intricate system with
one to four benzyl units being able to interact with the benzidine
core in the excited state. Moreover, it has been suggested that
photoinduced folded structures could be favoured in less polar or
non-polar solvents, more open arrangements appearing in polar
solvents.36 The first protonation of one of the amine functions in-
duces a significant bathochromic shift of z40 nm of the ligand-
centred fluorescence signal, suggesting that the positive charge
borne by the 3,30-diaminobenzidine core may favour folded ar-
rangements within the corresponding excited species. The second
protonation process induces a quenching of the ligand-centred
fluorescence emission (Fig. 4b). Electrostatic repulsions most likely
disrupted the interactions between the two aromatic chromo-
phores in the excited state and prevent the formation of the
2.5. Photo-irradiation of 5 and 6 in CH2Cl2
Benzidines are relevant examples of simple redox systems,
which in non-aqueous media, undergo stepwise electrochemical
oxidation6,7 to form monoradical cations and diradical (diimine)
dications.38,39 These oxidation processes and the resulting products
were also observed from closely related compounds using free
radical initiators,40 oxidizing reagents41 and peroxidase/H2O2 sys-
tem.42 It was reported that photolysis of halomethanes, such as
CH2Cl2, leads to the formation of free radicals able to abstract
electrons from benzidine 1.43 Benzidine 1 was consequently
transformed to coloured mono- and diradical cations. Similarly,
photo-irradiation (lexc¼254 nm) of 5 (Fig. 6a and Fig. S6 in

6526
M. Hmadeh et al. / Tetrahedron 64 (2008) 6522–6529
6H+
800
(a)
1.0
0.5
0.0
2
6
600
400
200
1
400
500
600
0
(nm)
300
400
500
(nm)
Figure 5. Relative recalculated fluorescence spectra of 6 protonated species. Solvent:
acetonitrile/water (9:1 v/v); T¼25.0(2) ꢃC; I¼0.1 M (NEt4ClO4); 6H22þ was assumed to
be a non-emitting species in the statistical processing.
600
400
200
0
(b)
2
Supplementary data) and 6 (Fig. 6b and Fig. S7 in Supplementary
data) in CH2Cl2 is associated with significant spectral changes.
After photo-irradiation (z20 s), the intensity of the absorption
bands centred at z320 nm strongly decreases with concomitant
formation of weak absorption bands in the visible region
(labs¼547 nm for 5 and labs¼553 nm for 6). Prolonged irradiation
induces red shifts of these transitions (labs¼593 nm for 5 and
labs¼584 nm for 6). These spectrophotometric variations are
assigned to the formation of coloured monoradical and diradicals
(Figs. S6 and S7 in Supplementary data). These radical cations are
produced in solution according to Scheme 3 in which methylene
chloride is excited (lexc¼254 nm) and photolyzed to yield halogen
radicals.
1
Clꢀ radicals are hence able to abstract electrons from benzidine
with subsequent formation of the two benzidine radicals. It is
noteworthy that compounds 5 and 6 are photostable in tetrahy-
drofuran and acetonitrile/water. It is a good indication that the
oxidized products result from reactions with methylene chloride.
Emission spectra of 5 and 6 are also notably altered with large blue
shift and strong increase of the ligand-centred emission bands
(Fig. 6a and b). Prolonged irradiation in CH2Cl2 induces a photo-
bleaching of the samples, which results in intricate photo-degraded
products as described for benzidine 1 by mass spectrometric
methods (ESMS).44 These results agree well with the spectral
400
500
600
700
(nm)
Figure 6. Effect of photo-irradiation (lexc¼254 nm) on emission spectra of (a) 5 and (6)
in CH2Cl2. T¼25.0(2) ꢃC; (a) [5]tot¼5.53ꢄ10ꢁ6 M; lexc¼276 nm; excitation and emission
slits: 7 nm; the irradiation times are: 0 (1), 10, 20, 30, 40, 50, 60, 80, 110, 160, 220 (2) s;
(b) [6]tot¼3.5ꢄ10ꢁ6 M; lexc¼269 nm; excitation and emission slits: 8 nm; the irradia-
tion times are: 0 (1), 10, 20, 30, 40, 50, 60, 80 (2) s.
changes monitored for benzidine 1 in various halomethanes
(CHCl3, CH2Cl2, CCl4).43 Moreover, oxidation in acetonitrile/water
by tert-butyl-hydroperoxide under basic conditions results in slow
conversion of diamine to diimine as shown by the appearance of an
hν (254 nm)
CH2Cl2
+
CH2Cl
Cl
RHN
RHN
NHR
NHR
RHN
NHR
NHR
Monoradical cation
Cl-
+
+
Cl
RHN
RHN
RHN
NHR
NHR
RHN
RHN
NHR
NHR
Cl-
+
+
Cl
Diradical cation
5: R = neo-pentyl
6: R = benzyl
RHN
RHN
Scheme 3. Proposed oxidation scheme of benzidine-type compounds 5 and 6 following photo-irradiation of CH2Cl2 at 254 nm.
NHR
NHR

M. Hmadeh et al. / Tetrahedron 64 (2008) 6522–6529
6527
absorption band centred at z598 nm for 5 (Fig. S8 in Supple-
mentary data). Under these experimental conditions, no in-
termediate could be evidenced. It was reported42 that the
horseradish peroxidase (HRP) catalyzed oxidation of 3,30,5,50-tet-
ramethylbenzidine (TMB) in the presence of H2O2, and led to the
formation of the two-electron oxidation product (diimine), which
was characterized by an intense absorption band at 450 nm
insoluble grey-white solid 4 in suspension was then isolated by fil-
tration, washed with water and dried under air (88% yield). 1H NMR
(300 MHz, CDCl3)
d
: 7.04 (d, 3JHH¼9 Hz, 2H, aromatic C–H), 7.35–7.60
(m, 10H, aromatic C–H), 8.03 (d, 3JHH¼9 Hz, 4H, aromatic C–H), 8.29
(m, 6H, aromatic C–H), 10.44 (s, 2H, N–H), 10.52 (s, 2H, N–H). Anal.
Calcd for C40H30N4O4$H2O: C, 74.06; H, 4.97; N, 8.64. Found: C, 74.10;
H, 5.09; N, 8.52. MS (MALDI-TOF): m/z: 631.2 [Mþ1]þ.
(
3450¼5.9ꢄ104 Mꢁ1 cmꢁ1). Prior to the formation of this species,
a blue charge transfer complex of the parent diamine and the dii-
mine oxidation product was evidenced (lmax¼370 and 652 nm;
3652¼3.9ꢄ104 Mꢁ1 cmꢁ1). This charge transfer complex resulted
from dimerization of two 3,30,5,50-tetramethylbenzidine semi-
4.2.2. Synthesis of 6
The tetraamide 4 was dissolved in dry THFand an excess of LiAlH4
(10 equiv) was added to the solution. The mixture was then refluxed
for 12 h and the excess of LiAlH4 was quenched by addition of water.
After filtration of aluminium salts and evaporation to dryness, the
crudeproduct was taken up in CH2Cl2 and then extractedwith water.
The organic phase was dried with MgSO4 and evaporated to dryness
after filtration. Yellow product 4 was collected by filtration after
precipitation from a mixture of ether and petroleum ether (85%
ꢀ
quinone imine cation free radicals (TMBþ ) and subsequent dis-
proportionation within these species, but is, itself, in equilibrium
with the diamine and the diimine (log K¼5.45).42 With compounds
5 and 6, no charge transfer complexes could be evidenced either by
photo-irradiation in CH2Cl2 or by oxidation with H2O2 in tetrahy-
drofuran/water (9:1 v/v), probably due to the bulkiness of the
N-substituents.
yield). 1H NMR (300 MHz, CDCl3)
d: 3.65 (br s, 4H, N–H), 4.24 (s, 4H,
CH2), 4.27 (s, 4H, CH2), 6.66 (d, 3JHH¼9 Hz, 2H, aromatic C–H), 6.75 (s,
3
2H, aromatic C–H), 6.84 (d, JHH¼9 Hz, 2H, aromatic C–H), 7.30
3. Conclusion
(m, 20H, aromatic C–H). 13C NMR (75 MHz, CDCl3)
d: 49.00 (CH2),
49.03 (CH2), 110.93–112.24–117.58–127.28–127.86–127.95–128.64–
128.68–133.55–135.89–137.32–139.50 (aryl C). Anal. Calcd for
Our results show that N-alkylation of 3,30-diaminobenzidine
with innocent substituents leads to unusual properties. Indeed, the
emission properties of the benzidine core can be fine-tuned by
subtle modifications, and the introduction of benzylic residues led to
unexpected photoinduced exciplexes with distinct emission prop-
ertiesandimprovedfluorescence efficiency. Importantly, compound
6 constitutes the first example of intramolecular exciplex containing
benzidine unit. We could show that these photoinduced properties
could be modulated by proton input, and the diprotonation of the
benzidine unit disrupted the intramolecular communication in the
excited states with a concomitant inhibition of the ligand-centred
fluorescence emission. The semiquinone imine and quinone diimine
systems were generated in CH2Cl2 upon photo-irradiation at
254 nm. Photolysis of halomethane indeed generates Clꢀ radicals,
which were able to rapidly oxidize the tetraamine compounds. It
will be of particular interest to examine the dynamics of the exciplex
formation by time-resolved fluorescence spectroscopy. The poten-
tial of these new systems as chromogenic substrates for measuring
HRP activity using absorption and emission techniques will be also
examined. Furthermore, with two bischelating sites and owing to
their photophysical properties, compounds 5 and 6 will be also used
as ligands for the coordination of metal centres.45
C
40H38N4$1/2H2O: C, 82.30; H, 6.73; N, 9.60. Found: C, 82.52; H, 6.67;
N, 9.45. MS (MALDI-TOF): m/z: 575.3 [Mþ1]þ.
4.3. Physico-chemistry
4.3.1. Starting materials and solvents
For solubility reasons, the acido-basic properties of ligands 5
and 6 were examined in a mixture of acetonitrile/water (9:1 v/v).
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). The distilled
water, spectroscopic grade acetonitrile and CH2Cl2 (MERCK,
UvasolÒ, p.a.) were de-oxygenated using CO2- and O2-free argon
prior to use (SIGMA Oxiclear cartridge). All the stock solutions were
prepared by weighting solid products using an AG 245 METTLER
TOLEDO analytical balance (precision 0.01 mg). The complete
dissolution of the ligands was achieved with the help of ultrasonic
bath. The ionic strength was adjusted to 0.1 M with tetra-
ethylammonium perchlorate (NEt4ClO4, FLUKA, puriss).
CAUTION: perchlorate salts combined with organic ligands are
potentially explosive and should be handled in small quantity and with
the necessary precautions.46
4. Experimental section
4.1. General
4.3.2. Potentiometry
The free hydrogen ion concentrations were measured with an
Ag/AgCl combined glass electrode (METROHM 6.0234.500, Long
Life) filled with 0.1 M NaCl (FLUKA, p.a.) in water. Potential differ-
ences were given by a TACUSSEL Isis 20.000 millivoltmeter. Stan-
dardization of the millivoltmeter and the verification of the
linearity of the electrode response were performed using a set of
commercial MERCK buffered solutions (pH¼4.00, 7.00 and 9.00).
The experiments were carried out at 25.0(2) ꢃC maintained with
the help of HAAKE FJ thermostats.
1H NMR (300 MHz), 13C NMR (75 MHz) spectra were recorded
on a Bruker AC-300 instrument. FAB mass spectral analyses were
recorded on an autospec HF mass spectrometer and MALDI-TOF
mass spectral analyses were recorded on a Finnigan TSQ 700. Ele-
mental analyses were performed by the Spectropole center (Mar-
seille, France). Solvents were freshly distilled under nitrogen prior
to use. Molecule 5 was prepared according to the literature.11 All
reactions of air- or water-sensitive compounds were performed
using standard Schlenk techniques under dry argon atmosphere.
4.3.3. Protonated species of 5 and 6
Solutions of 5 (5.46ꢄ10ꢁ5 M) and 6 (5.25ꢄ10ꢁ5 M) were pre-
pared by quantitative dissolution of a solid sample in acetonitrile/
water (9:1 v/v). An aliquot of 40 mL was introduced into a jacketed
cell (METROHM 6.1414.150) maintained at 25.0(2) ꢃC by the flow
of a HAAKE FJ thermostat. The solution was continuously de-
oxygenated by bubbling with oxygen-free argon. The titration of 5
(1.47<pH<9.46) or 6 (1.34<pH<10.06) was carried out by addition
of known volumes of a 0.1 M standardized perchloric acid solution.
4.2. Syntheses
4.2.1. Synthesis of 4
To a solution of 3,30-diaminobenzidine (0.50 g, 2.33 mmol) in
Et2O (500 mL) and NEt3 (5 mL) was added PhC(O)Cl (9.33 mmol)
and the mixture was stirred for 18 h at room temperature. After
evaporation of Et2O, the crude product was taken up in water. The

6528
M. Hmadeh et al. / Tetrahedron 64 (2008) 6522–6529
Special care was taken to ensure that complete equilibration was
attained. Absorption spectra versus pH were recorded using a Var-
ian CARY 50 spectrophotometer fitted with HELLMA optical fibers
(HELLMA, 041.002-UV) and an immersion probe made of quartz
suprazil (HELLMA, 661.500-QX).
thank the French Ministry of Research and Education for granting
them a Ph.D. fellowship.
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2008.04.058.
4.4. Fluorescence titration
An aliquot of 40 mL of 6 (5.25ꢄ10ꢁ6 M) was introduced into
a jacketed cell (METROHM 6.1414.150) maintained at 25.0(2) ꢃC by
the flow of a HAAKE FJ thermostat. The solution was continuously
de-oxygenated by bubbling with oxygen-free argon. The initial pH
was adjusted at 0.85 with concentrated perchloric acid, and the
titration of 6 was carried out by addition of known volumes of
a tetrametylammonium hydroxide solution (0.1 M). An aliquot
(2 mL) was taken after each addition of base, and a fluorescence
emission spectrum (250–700 nm) was recorded versus pH with
1 cm quartz optical cell (HELLMA, 110-QS) on a PERKIN-ELMER LS-
50B spectrofluorimeter maintained at 25.0(2) ꢃC by the flow of
a HAAKE FJ thermostat. The excitation wavelength was 250ꢅ1 nm,
and the excitation and emission slit widths were set at 5 nm. The
absorbance at the excitation wavelength (lexcꢆ250 nm) was kept
lower than 0.1 to minimize reabsorption processes and to keep it
constant throughout the fluorescence titration. The light source
was a pulsed xenon flash lamp with a pulse width at half peak
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R
Isð
l
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Þ ꢄ Dr ꢄ n2s
Þ ꢄ Ds ꢄ n2r
R
4f ð Þ
4f ð Þ
s
¼
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Irð
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