Fluorescent molecular sensing of amino acids bearing an aromatic residue

Article abstract of DOI:10.1039/b105480p

The tetra-amino tripodal ligand, 3, containing two anthracene subunits, has been prepared through a multi-step synthesis and its solution properties have been studied by potentiometric and spectrofluorimetric techniques. The zinc(II) complex, [Zn^II

Full text of DOI:10.1039/b105480p

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Fluorescent molecular sensing of amino acids bearing an aromatic
residue
Luigi Fabbrizzi,* Maurizio Licchelli, Angelo Perotti, Antonio Poggi, Giuliano Rabaioli,
Donatella Sacchi and Angelo Taglietti
2
Dipartimento di Chimica Generale, Università di Pavia, via Taramelli 12, I-27100 Pavia, Italy
Received (in Cambridge, UK) 21st June 2001, Accepted 1st August 2001
First published as an Advance Article on the web 20th September 2001
The tetra-amino tripodal ligand, 3, containing two anthracene subunits, has been prepared through a multi-step
synthesis and its solution properties have been studied by potentiometric and spectrofluorimetric techniques. The
zinc() complex, [ZnII(3)]2ϩ, which displays the typical emission of anthracene derivatives, interacts with natural
amino acids, showing a particular affinity towards phenylalanine and tryptophan. This selective behaviour of the
complex derives from its ability to establish two kinds of interaction: (i) a metal–ligand interaction between the
zinc() ion and the amino acid’s carboxylate group; (ii) a π-stacking interaction involving the aromatic moieties
positioned on the complex and on the amino acid, inducing an extra stability of the adducts with tryptophan and
phenylalanine (7–8 kJ molϪ1). The formation of the 1 : 1 adduct with tryptophan is signalled by a strong fluorescence
quenching while no effect on the emission intensity has been observed in all other cases.
receptors to build fluorescent molecular sensors for a variety of
Introduction
anions.10
Fluorescent molecular probes are currently and profitably used
We have recently demonstrated that a type of interaction
other than hydrogen bonding can be conveniently used for
to detect a variety of analytes in biological fluids.1 The field has
been opened by Tsien and co-workers, who in the early 80’s
anion recognition and fluorescent sensing: the metal–ligand
designed a photoactive molecule suitable for signalling the
interaction.11 Actually, metal–ligand interactions may have
activity of Ca2ϩ within the cell,2 in real time and in real space,
some significant advantages with respect to electrostatic
interactions (which include hydrogen bonding): they are
more energetic (a useful feature for compensating the strongly
endothermic desolvation of the negatively charged analyte) and
by making use of the fluorescence microscopy technique.3
Tsien’s approach consisted of covalently linking through a
spacer a selective receptor for Ca2ϩ (e.g. a derivative of the
multidentate ligand ethylenebis(oxyethylenenitrilo)tetraacetic
acid, EGTA) to a powerful fluorophore: ion binding drastically
altered the emissive properties of the fluorophore (either an
display a definite directional character.
In this connection, we showed that the [ZnII(tren)]2ϩ com-
plex, 1 (tren: tris(2-aminoethyl)amine) is a useful platform for
anion recognition: the metal complex exhibits a trigonal-
enhancement of the fluorescent intensity or a substantial shift
of the emission band).4 The Ca2ϩ cation plays a fundamental
bipyramidal stereochemistry and maintains a vacant axial
role in many functions at the cellular level, controlling
position, available for the coordination of a further ligand,
(i) muscle contraction; (ii) nerve cell communication; (iii)
either a solvent molecule or an anion (Scheme 1). System 2, in
hormone secretion; (iv) immune cell activation,5 by increasing
its concentration from 10Ϫ7 M, when in the resting state, to
10Ϫ6—10Ϫ5 M, in the active state.4,6 The fluorescent molecular
probes introduced by Tsien (Indo-1, Fura-2, Fluo-3) are today
routinely used in laboratories of cell physiology all over the
world.1a,d The fluorophore–spacer–receptor synthetic approach
was later extended by de Silva and co-workers to the design of
molecular fluorescent sensors for alkali metal ions.7 These
systems were all based on a photoinduced electron transfer
mechanism (PET): in the absence of the analyte (e.g. Kϩ), the
emission of the fluorophore (an anthracene fragment) was
quenched through an intra-molecular electron transfer process,
which was interrupted when the metal ion fell down into the
receptor subunit (e.g. an NO5 crown).8 Thus, metal recognition
could be signalled through a sharp fluorescence revival.
Scheme 1
which the [ZnII(tren)]2ϩ platform has been equipped with an
anthracene fragment, displays a special affinity towards the
carboxylate group (e.g. of a benzoate anion, PhCOOϪ): when
the PhCOOϪ ion bears an electron-donor or -acceptor substitu-
ent (e.g., –N(CH3)2 or –NO2, respectively, in the 4-position),
carboxylate binding to ZnII induces the occurrence of an
efficient electron transfer process between the photoexcited
fluorophore and the substituent on PhCOOϪ. As a con-
sequence, benzoate recognition is signalled by a substantial
quenching of the anthracene emission.11a
PET fluorescent sensors for anions were first designed by
Czarnik and co-workers, using a similar synthetic approach: in
this case the receptor was a polyammonium ion and the
covalently linked fluorophore was anthracene. In the present
case, the hydrogen bonding interaction between the poly-
Ϫ
ammonium subunit and the anion (e.g. H2PO4 ) interrupted an
We considered that systems like 1 could be used for recog-
nition of amino acids, which can bind the ZnII centre
through their –COOϪ residue. However, 1 itself is not a good
receptor for amino acids, for two main reasons: (i) due to the
operating PET mechanism, thus inducing an enhancement of
the anthracene emission.9 More recently, Lehn and co-workers
used more sophisticated cyclic and polycyclic polyammonium
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This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b105480p

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electrostatic repulsions between Zn2ϩ and the –NH3ϩ fragment
of the zwitterion, the affinity of the amino acid towards the
[ZnII(tren)]2ϩ platform is drastically reduced with respect to
CH3COOϪ and PhCOOϪ (K decreasing to values lower than
103); (ii) in any case, the simple ZnII–carboxylate interaction
cannot provide any selectivity among amino acids. Thus, we
considered equipping the [ZnII(tren)]2ϩ platform with further
substituents, capable of establishing additional interactions
ϩ
with a given NH3 –CH(R)–COOϪ amino acid. In particular,
this extra-interaction has to be established with the R moiety,
thus increasing affinity and, hopefully, providing selectivity.
Fig. 1 Right vertical axis, distribution curves of the species present at
equilibrium for ligand 3 (L) in 4 : 1 dioxane–water solution at 25 ЊC: (a)
LH33ϩ; (b) LH22ϩ; (c) LHϩ; (d) L. Left vertical axis (full triangles),
dependence of the fluorescence intensity of the solution, IF, on [Hϩ].
In this connection, we synthesised derivative 3, which con-
tains two anthracenyl and one benzyl substituent. The corre-
sponding ZnII complex is expected to display selective affinity
ϩ
towards NH3 –CH(R)–COOϪ amino acids, whose R residue
has a π-nature and is therefore prone to establish π-donor–
acceptor interactions. In this sense, the [ZnII(3)]2ϩ complex
appears as a reasonable candidate for fluorosensing phenyl-
alanine and tryptophan, provided that recognition induces
a drastic modification of the emission of the photoexcited
anthracene fragment. This article describes the investigation of
the solution equilibria involving [ZnII(3)]2ϩ and natural amino
acids and the study of the photophysical effects induced by
the recognition process. It will be shown that [ZnII(3)]2ϩ is an
efficient fluorescent molecular sensor of tryptophan, whose
recognition is signalled through a substantial quenching of
the anthracene emission.
Results and discussion
Design of the [ZnII(tetramine)]2؉ platform and characterisation
of its photophysical behaviour in solution
The selectively substituted tripodal tetramine 3 was prepared
through the synthetic procedure illustrated in Scheme 2.
In particular, two anthracene subunits were appended at
the terminal atoms of the triamine 2,2Ј-diaminodiethylamine,
through Schiff base condensation with anthracene-9-carb-
aldehyde, to give 4. Nucleophilic addition of 4 to the bromo-
imine fragment 5, which supplies the benzyl substituent, gave
Scheme 2
which, at such a high [Hϩ] value (10Ϫ1 M) is the predominant
species in solution.
the unsaturated derivative 6, whose C᎐N double bonds were
᎐
hydrogenated with sodium borohydride.
The emission properties of system 3 at varying pH were
investigated through a spectrofluorimetric acid–base titration
experiment. In particular, a solution of 3 containing excess acid
was titrated with standard sodium hydroxide and the emission
spectra were recorded after each addition of base. Fluorescence
measurements within the pH range 1–2, in which the electrode
response is not reliable, were carried out on solutions contain-
ing known amounts of acid. The profile of the emission
intensity, IF (at λmax = 413 nm) has been superimposed on the
distribution diagram in Fig. 1. The highest emission intensity
The protonation state of 3 (indicated as L in the following) in
a dioxane–water solution (4 : 1, v/v), at 25 ЊC, was determined
through potentiometric titration experiments. In particular, the
following species were found to be present at the equilibrium,
3ϩ
2ϩ
over the investigated pH range: LH3 (pKA1 = 2.24), LH2
(pKA2 = 7.19), LHϩ (pKA3 = 8.54), and L (see Experimental
section). The % concentrations of the different species are
shown in the distribution diagram in Fig. 1. Although the
determination has been carried out over the 2–12 pH range, the
species distribution plots have been extrapolated to Ϫlog [Hϩ] =
1 in order to follow the abundance increase of the LH33ϩ species
(IF = 100) is observed at the beginning of the titration (Ϫlog
3ϩ
[Hϩ] = 1), where the dominating species is LH3
.
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It is suggested that in this species the three protons are bound
to the terminal groups of the tripodal tetramine (see formula
a in Scheme 3). Even if the tertiary amine group itself is more
Fig. 2 Right vertical axis, distribution curves of the species present at
equilibrium for the system 3 (L, 1 equiv.)–ZnII (1 equiv.) in 4 : 1
dioxane–water solution at 25 ЊC: (a) LH33ϩ; (b) LH22ϩ; (c) LHϩ; (d)
[Zn(L)]2ϩ; (e) [Zn(L)(OH)]ϩ. Left vertical axis (᭞), dependence of the
fluorescence intensity of the solution, IF, on [Hϩ].
2ϩ
that the relative emission intensity at the flex (100% of LH2
present) is about 50% of the original one.
On further deprotonation, which produces the LHϩ species,
a further drastic decrease of the emission intensity is observed.
This is an expected behaviour, because in the LHϩ species
(formula c in Scheme 3) both anthracenylammonium groups
have been deprotonated and, no matter which of the two
anthracene fragments has been excited, its emission will be
killed by the adjacent amine group, through an electron transfer
mechanism.
The last deprotonation step, LHϩ
L, occurring at pH > 8,
induces a slight, yet detectable, decrease of the fluorescent
emission. This would also indicate that the benzylamine group,
now deprotonated, is in some way capable of transferring an
electron to a distant photoexcited anthracene fragment. This
process is expected to take place according to a “through space”
mode, i.e. following the occasional contact of the dangling
ethylamine arms of L (formula d in Scheme 3). Thermal
motions of the molecular framework can take place easily in L,
which is no longer stiffened by electrostatic repulsions.
Analogous titration studies, both potentiometric and spec-
trofluorimetric, were carried out on system 3, in the presence of
an equivalent amount of Zn2ϩ. In particular, curve fitting
analysis of the potentiometric titration profile by a non-linear
least-squares method indicated that the following coordination
complexes are present at the equilibrium: [ZnII(L)]2ϩ, which
reaches its maximum concentration (75%) at pH ≅ 7, and
[ZnII(L)(OH)]ϩ, which at pH у 9 is present at 100%. It is
Scheme 3
basic than a primary one, in a polar medium, the situation
illustrated by a minimises the electrostatic repulsions between
the three ammonium groups. On decreasing [Hϩ], LH33ϩ depro-
tonates to give LH22ϩ, while the emission intensity decreases to
reach a short plateau at a pH value slightly higher than 4.
Noticeably, the plateau (or the pronounced flex) corresponds to
the formation of 100% of the doubly protonated species LH2
In this connection, it is suggested that the LH3
deprotonation step involves one of the two anthracenyl-
ammonium groups, which, due to the presence of an extended
aromatic substituent, are expected to be more acidic than the
benzylammonium group. At this stage, an intramolecular
hypothesised that both complexes exhibit
a
trigonal-
bipyramidal stereochemistry, such as that shown in Scheme 1.
In the [ZnII(L)]2ϩ species, the vacant axial position is occupied
by a water molecule. With increasing pH, the coordinatively
bound water molecule deprotonates, and the [ZnII(L)(OH)]ϩ
complex forms.
2ϩ
.
3ϩ
2ϩ
LH2
In Fig. 2, the profile of the spectrofluorimetric titration has
been superimposed on the distribution diagram: the highest
emission is observed at the highest investigated [Hϩ] value, 10Ϫ1,
and its intensity decreases with [Hϩ]. However, at pH = 5, IF
stops decreasing and starts to increase, to reach a maximum at
pH ≅ 7. At higher pH values, the fluorescent emission decreases
again, its quenching being complete at pH ≥ 9. The spectro-
fluorimetric behaviour can be interpreted in terms of pH con-
trolled formation of the ZnII complexes of 3. Until pH = 5, no
metal complex is formed: Zn2ϩ is present as an aqua ion and
does not interact with the various protonated forms of 3. Thus,
the photophysical behaviour at pH values <5 is the same as
observed in the titration experiment in the absence of metal, as
illustrated in Fig. 1. At pH = 5, the [ZnII(L)]2ϩ complex begins
to form: in these circumstances, all the amine groups of 3 are
engaged in the coordination of the metal and are no longer
2ϩ
electron transfer process takes place within the LH2 species
(formula b in Scheme 3) from the amine group to the nearby
photoexcited anthracene fragment, quenching its emission. A
photo-induced electron transfer process from an amine group
to a proximate anthracene subunit is a documented phenom-
enon, which has allowed the development of a class of fluor-
escent pH indicators.12 Under the irradiation conditions in the
spectrofluorimetric cuvette, excitation may involve either the
anthracene fragment bound to the amine group (electron trans-
fer takes place) or that bound to the ammonium group (electron
transfer does not take place): this situation accounts for the fact
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Fig. 4 Molecular model for the [Zn(L)(trp)]2ϩ adduct as obtained by
using the HyperChem software package (MMϩ force field). Hydrogens
are omitted for clarity. Zinc() ion is in a trigonal-bipyramidal
coordinative arrangement; the indole moiety of trp and one of the
anthracene subunits lie in parallel planes at a distance of 3.5–3.6 Å.
Fig. 3 Variation of the relative fluorescence intensity, IF, of a 10Ϫ5
M
[Zn(L)]2ϩ solution with tryptophan (circle), phenylalanine (full triangle)
and glycine (triangle). n = number of equivalents of the added amino
acid.
demonstrated by the fact that in an ethanolic solution of
[ZnII(L)(trp)]2ϩ, vitrified at 77 K, the fluorescence is fully
restored (i.e. its intensity is equal to that of a solution of plain
[ZnII(L)]2ϩ, under the same conditions). In this connection, it
has to be considered that an electron transfer process causes the
formation of an ion pair (AϪ ∼ Dϩ), a process which is accom-
panied by a drastic rearrangement of the solvent molecules,
which have to solvate the ion pair. Immobilisation of the sol-
vent molecules in the glass at 77 K strongly disfavours ion pair
formation and prevents the occurrence of the eT mechanism,
thus allowing the radiative decay of the photoexcited fragment
to take place and reviving the fluorescence.14 With the exception
of a few examples of ultrafast eT processes,15 the reviving of
fluorescence in frozen solution can therefore be considered as
evidence that an eT rather than an energy transfer process is
active.
Thus, spectrofluorimetric studies have shown that the
[ZnII(3)]2ϩ complex, in a solution adjusted to pH ≅ 7, is an
efficient sensor of tryptophan, operating through an electron
transfer mechanism. However, this investigation does not say
anything about selectivity with respect to other amino acids.
What can be concluded is that, whatever the affinity of the
other amino acids for the [ZnII(3)]2ϩ receptor, no mechanism
exists that perturbs the emission of the anthracene substituent.
Therefore, the affinity of [ZnII(3)]2ϩ towards amino acids other
than tryptophan has to be assessed by looking at a different
property than fluorescence. A convenient property to investi-
gate is the absorbance in the UV region. In particular, the
[ZnII(3)]2ϩ complex presents intense absorption bands in the
range 350–400 nm, to be assigned to π–π* transitions pertinent
to the aromatic substituents on the tetramine. The absorbance
of these bands is significantly altered on titration with a given
amino acid.
available to transfer electrons to the photoexcited anthracene
fragment. As a consequence, any electron transfer mechanism is
interrupted and fluorescence is restored. Interestingly, the high-
est emission is reached at pH ≅ 7, corresponding to the highest
concentration of the [ZnII(L)]2ϩ complex. With increasing pH,
the concentration of [ZnII(L)]2ϩ decreases, to the advantage of
the [ZnII(L)(OH)]ϩ species. Accordingly, the fluorescence inten-
sity IF decreases to fully quenched, corresponding to 100%
formation of the hydroxide-containing metal complex (pH ≥ 9).
Quenching is ascribed to the occurrence of an intra-complex
electron transfer process from the electron rich OHϪ anion to
the nearby photoexcited anthracene fragment. Photoinduced
electron transfer processes from metal-bound hydroxide to a
proximate fluorophore have been observed in analogous zinc()
containing systems.11b,13 These investigations have shown that a
solution containing equimolar amounts of 3 and Zn2ϩ, as the
triflate salt, adjusted to pH ≅ 7, is appropriate for carrying out
recognition studies on amino acids. In fact, at this pH, the
dominating species is [ZnII(L)]2ϩ, whose recognition site, one of
the axial positions of the trigonal-bipyramid, is occupied by a
labile and weakly bound water molecule, which can be replaced
by the carboxylate group of the amino acid. Moreover, quite
interestingly for sensing purposes, the [ZnII(L)]2ϩ receptor dis-
plays the most intense fluorescent signal.
Fluorosensing of tryptophan
A neutral solution 10Ϫ4 M both in 3 and in Zn2ϩ buffered at pH
6.8 with lutidine was titrated with a standard solution of a
given amino acid. Among all the natural amino acids, only
tryptophan (trp) induced a modification of the fluorescent
emission of the anthracene fragment of [ZnII(L)]2ϩ, in particu-
lar causing substantial quenching.
Fig. 5 shows how the absorbance at 388 nm varies with the
addition of a standard tryptophan solution to a solution 10Ϫ4
M in both 3 and Zn2ϩ, adjusted to pH = 6.8. The absorbance vs.
equiv. profile is consistent with the formation of a 1 : 1 adduct,
whose association constant log K is 4.21 0.02 (in good agree-
ment with the value obtained by spectrofluorimetry). Analo-
gous titration experiments were carried out with other natural
amino acids, AA: the absorbance vs. equiv. profiles corre-
sponded in any case to the formation of a 1 : 1 association
complex [ZnII(3)(AA)]2ϩ and the constants for the pertinent
The profile of the spectrofluorimetric titration (relative fluor-
escence intensity at 413 nm, IF, vs. equiv. of amino acid) is
shown in Fig. 3. Least-squares analysis of the titration profile
was consistent with the formation of a 1 : 1 receptor–amino
acid adduct and the constant for the association equilibrium
([ZnII(L)]2ϩ ϩ trp
[ZnII(L)(trp)]2ϩ) was 4.28 0.04 log units.
Molecular modelling studies performed on the [ZnII(L)-
(trp)]2ϩ adduct in a vacuum suggest that the amino acid is
bound to the zinc() polyamine receptor not only through the
metal–carboxylate coordinative interaction, but also through a
donor–acceptor interaction of π nature, involving the indole
residue of the amino acid and at least one of the anthracene
substituents on the tetramine (see Fig. 4). Therefore, the
substantial quenching of the fluorescence is ascribed to the
occurrence of an electron transfer (eT) process from the indole
subunit (donor, D) to the photoexcited anthracene fragment
(acceptor, A*). The electron transfer nature of the process is
association equilibrium ([ZnII(3)]2ϩ ϩ AA
[ZnII(3)(AA)]2ϩ)
were in all cases around 3 log units, i.e. more than one order of
magnitude lower than for trp. There was however one excep-
tion: phenylalanine (phe), whose log K value for the association
equilibrium was found to be 4.48 0.05. It is hypothesised that
the extra stability shown by the [ZnII(3)(phe)]2ϩ adduct is due to
the establishing of π interactions between the phenyl residue
of the amino acid and the aromatic substituent(s) on the
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Table 1 Summary of experimental parameters for the system zinc()–
N-anthracen-9-ylmethyl-NЈ-[2-(anthracen-9-ylmethylamino)ethyl]-NЈ-
[2-(benzylamino)ethyl]ethane-1,2-diamine
Solution composition
[TL]range/mol dmϪ3
[TM]range/mol dmϪ3
I/mol dmϪ3, electrolyte
pH range
0.001–0.003
0.001–0.003
0.1, KNO3
2.0–11.8
Experimental method
pH titration,
calibrated in
concentration
25
T /ЊC
Total number of data points
Protonation
132 (2 titrations)
144 (2 titrations)
HYPERQUAD17
Complexation
Method of calculation
Protonation and stability constants
(errors as ꢀ)
Fig. 5 Spectrophotometric titration of a 10Ϫ4 M [Zn(L)]2ϩ solution
with tryptophan. The solid line indicates the best fit curve. n = number
of equivalents of the added tryptophan.
log βLH
8.54 0.04
14.73 0.07
17.97 0.10
6.55 0.12
log βLH
2
log βLH
3
log βZnL
polyamine framework of the receptor, in a similar way to that
observed in the case of trp. However, the phenyl substituent on
phe displays lower electron donor tendencies than the indole
subunit of trp and is not able to transfer one electron to the
facing photoexcited anthracene subunit, whose fluorescent
emission is not altered. In conclusion, [ZnII(3)]2ϩ selectively
interacts with natural amino acids bearing an aromatic residue,
trp and phe, but it signals, through a drastic decrease of the
fluorescent emission, only the recognition of trp.
Spectrofluorimetric titration experiments with tryptophan
on a [ZnII(3)]2ϩ solution containing phenylalanine confirmed
that the latter amino acid interacts with the receptor and
competes with the former for the fluorescent sensor, therefore
behaving as an interferent. Moreover, some empirical con-
siderations can be made on the energetics of the interactions
within the [ZnII(3)(AA)]2ϩ association complex. The log K for
the HyperQuad software package.17 Electrode calibration was
carried out according to Gran’s method.18 The experimental
details are summarized in Table 1.
Spectrofluorimetric titrations
pH titrations were performed on 10Ϫ4 M dioxane–water 4 : 1
solutions of 3 (or 3 and Zn(ClO4)2) adjusted to pH ∼2 by adding
small amounts of a standard solution of HClO4. Then, addi-
tions of a standard 0.1 M NaOH solution were made until a
basic pH (>10) was obtained. Emission spectra (excitation
wavelength 366 nm) were recorded after each addition of base.
Fluorescence measurements at [Hϩ] higher than 10Ϫ2 M were
perfomed on 104 M solutions containing known amounts of
HClO4.
Titrations at neutral pH were carried out on dioxane–water
4 : 1 solutions (10Ϫ4 M) of equimolecular mixtures of 3 and
Zn(ClO4)2, buffered with lutidine (0.025 M, pH 6.8). Small
amounts of aqueous solution (0.01 M) of the selected amino
acid were added and spectra recorded after every addition. Very
similar experimental conditions were used for the spectro-
photometric titrations.
Association constant values were determined by non-linear
regression analyses of absorbance or fluorescence intensity data
versus amino acid concentration.17
the association equilibrium involving glycine is 3.06
log units, whereas for the negatively charged N-acetylglycine
derivative a value of 3.51 0.03 has been found. The ∼0.5
0.06
log units difference, which corresponds to ∼3 kJ molϪ1, can
be explained in terms of electrostatic repulsion between the
ammonium group of the amino acid and the Zn2ϩ centre.
Thus, the energy associated with the simple metal–carb-
oxylate interaction, in the absence of electrostatic repulsive
effects, can be estimated as around 20 kJ molϪ1. The extra
stability due to π-interactions, as observed for trp and phe,
ranges from 7 (trp) and 8 (phe) kJ molϪ1 (∆log K with respect to
glycine being 1.2 and 1.4, respectively). Thus, π-interactions
contribute to the formation of the association complex to a
distinctly lesser extent than metal–ligand interactions. Yet, this
π-contribution is essential in determining a selectivity pattern in
favour of trp and phe.
N-Anthracen-9-ylmethylene-NЈ-[2-(anthracen-9-ylmethylene-
amino)ethyl]ethane-1,2-diamine (4)
A solution of 2,2Ј-diaminodiethylamine (dien, 1 mmol) in
MeCN (20 ml) is slowly added to a solution of anthracene-9-
carbaldehyde (2 mmol) in MeCN (30 ml) over a period of 1 h
under magnetic stirring at room temperature. Stirring is con-
tinued for 24 h and the yellow precipitate is collected by vacuum
filtration (69%). Found: C, 84.98; H 6.07; N, 8.52%. C34H29N3
requires: C, 85.14, H 6.09, N 8.76%.
Experimental
UV-vis spectra were recorded on a Hewlett-Packard 8452A
diode array spectrophotometer. All fluorescence measurements
were carried out on a Perkin-Elmer LS-50B luminescence
spectrometer. Emission spectra at 77 K were measured in dry
ethanol (10Ϫ5 M), by using quartz sample tubes and the same
luminescence spectrometer equipped with a Perkin-Elmer
low temperature luminescence accessory. Mass spectra were
obtained with a Finnigan TQS 700 mass spectrometer. NMR
data were obtained on a Bruker AMX 400 MHz.
Benzylidene-(2-bromoethyl)amine (5)
2-Bromoethylamine hydrobromide (2.5 mmol) was dissolved in
1 M NaOH solution (30 ml) and extracted with CH2Cl2 (5 × 30
ml). The combined organic extracts were concentrated (30 ml)
and slowly added to a solution of benzaldehyde (1 mmol) in
MeCN (25 ml) at room temperature. The resulting solution was
stirred for an hour. The oil obtained after removing the solvent
(yield 78%) was used directly in the following step.
Potentiometric titrations
N-Anthracen-9-ylmethylene-NЈ-[2-(anthracen-9-ylmethylene-
amino)ethyl]-NЈ-[2-(benzylideneamino)ethyl]ethane-1,2-diamine
(6)
Potentiometric determinations were performed in dioxane–
water 4 : 1 v/v (0.1 M KNO3, 25 ЊC) according to the instru-
mental and general procedures reported elsewhere.16 Proton-
ation and complexation constants were determined by using
Diimine 4 (0.5 mmol) was dissolved in a suspension of Cs2CO3
2112
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Br., 1995, 31, 216; (i) B. Valeur and I. Leray, Coord. Chem. Rev.,
(0.5 mmol) in toluene (30 ml). The mixture was heated at 50 ЊC
and stirred under a nitrogen atmosphere. A solution of 5
(0.6 mmol) in toluene (20 ml) was added dropwise, then the
mixture was refluxed for 24 hours. After removing the solvent,
the solid obtained was treated with MeCN and the unreacted
compound 4 was filtered off. MeCN was evaporated in vacuo
and the product was used without further purification (yield
60%).
2000, 205, 3.
2 (a) R. Y. Tsien, Biochemistry, 1980, 19, 2396; (b) G. Grynkiewicz,
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N-Anthracen-9-ylmethyl-NЈ-[2-(anthracen-9-ylmethylamino)-
ethyl]-NЈ-[2-(benzylamino)ethyl]ethane-1,2-diamine (3)
NaBH4 (0.7 g) was added in small portions to a solution of 6
(2 mmol) in methanol (100 ml) at 40 ЊC, then the solution was
refluxed under stirring overnight. The solvent was removed
with a rotary evaporator, giving a solid which was dissolved
in 5% HCl solution. The resulting solution was heated at
50–60 ЊC for 30 minutes, made alkaline by addition of NaOH
solution and extracted with CH2Cl2 (10 × 30 ml). The combined
organic phases were dried over Na2SO4 and the solvent was
1
removed in vacuo giving an oily product (yeld 90%). H-NMR
(400 MHz; CDCl3; Me4Si) 8.30 (2H, s, aromatic-H of anthra-
cene), 8.20 (4H, d, aromatic-H of anthracene), 7.90 (4H, d,
aromatic-H of anthracene), 7.30 (8H, m, aromatic-H of
anthracene), 7.20 (5H, m, aromatic-H of phenyl), 4.60 (4H, s),
4.50 (2H, s), 2.80–2.90 (6H, m), 2.60–2.70 (6H, m). MS (ESI):
617 ([M ϩ H]ϩ).
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12 A. P. de Silva and R. A. D. D. Rupasinghe, J. Chem. Soc., Chem.
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Acknowledgements
We thank the Consiglio Nazionale delle Ricerche, “Progetto
Finalizzato Biotecnologie”, for financial support. Thanks are
also due to Dr Laura Linati, Dr Luca Gianelli and Centro
Grandi Strumenti (Università di Pavia) for NMR and mass
spectra.
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