Detection of Zn(II) ions by fluorescent pyrene-derived molecular probes

Article abstract of DOI:10.1080/10610278.2013.835050

Two pyrene-based molecular probes have been synthesised from ortho-and meta-bis(azidodimethyl)benzene and their coordination with Fe(III), Al(III), Fe(II), Zn(II), Ni(II), Cu(II), Cd(II), Hg(II), Ca(II), Mg(II) and Na(I) cations is described. The greatest

Full text of DOI:10.1080/10610278.2013.835050

Supramolecular Chemistry, 2014
Vol. 26, Nos. 3–4, 141–150, http://dx.doi.org/10.1080/10610278.2013.835050
Detection of Zn(II) ions by fluorescent pyrene-derived molecular probes
a
Erendra Manandhar , Peter J. Cragg * and Karl J. Wallace *
b
a
a
Department of Chemistry and Biochemistry, Bobby Chain Technology Center (TEC), The University of Southern Mississippi,
Rm 441A, 118 College Drive, Box #5043, Hattiesburg, MS 39406, USA; School of Pharmacy and Biomolecular Sciences,
University of Brighton, Brighton, UK
b
(
Received 10 June 2013; accepted 12 August 2013)
Two pyrene-based molecular probes have been synthesised from ortho- and meta-bis(azidodimethyl)benzene and their
coordination with Fe(III), Al(III), Fe(II), Zn(II), Ni(II), Cu(II), Cd(II), Hg(II), Ca(II), Mg(II) and Na(I) cations is described.
The greatest spectral changes were observed with Zn(II) salts, which are important analytical targets in environmental and
2
2
2
2
2
2
biological chemistry, and a detailed investigation on the influence of the counter-ion (F , Cl , Br , I , ClO , NO ,
2
4
3
22
4
2
4
CH3CO , SO and BF ) was undertaken. Density functional theory studies suggest that Zn(II) is bound in the cleft of the
probes in a 1:1 host:guest ratio. A significant hypsochromic shift is observed in the excimer band in the presence of metal
2
salts and is greatest upon the addition of Zn(ClO ) .The limit of Zn(II) detection is in the nanomolar range for the meta-
4
2
isomer and the binding process is reversible allowing the system to be recycled several times. A protocol is proposed that
would allow detection of Zn(II) in aqueous samples.
Keywords: zinc; pyrene; excimers; fluorescence sensors; DFT
Introduction
cations, anions and neutral species (2a). Many fluorescent
Zn(II) sensors use an array of photophysical mechanisms,
for example internal charge transfer (10), fluorescence
resonance energy transfer (4k) or the heavy atom effect
(11) and, more recently, chelation-enhanced fluorescence
(CHEF) (12). The term CHEF was utilised in the early
1990s by Czarnik and co-workers (13) but has started to
appear in the literature more frequently to describe a
number of different mechanisms which include photo-
induced electron transfer as well as manipulation of
n–p * /p–p * frontier orbitals. For a detailed discussion
on these individual mechanisms, the reader is referred to
de Silva’s elegant review (9d).
The development of molecular podands and tripodal
molecular species to selectively bind guest species is an
area of continued interest (1), especially where it relates to
biologically and environmentally important analytes (2).
Molecular receptors that change their spectroscopic
signatures upon the addition of metal cations via changes
in fluorescence are attractive analytical tools as fluor-
escence signals are easily perturbed by changes in the
fluorophore’s local environment (3). The detection of Zn
(II) is of particular interest both in vitro and in vivo due to
its biological importance (4).
The human body contains ,2–3 g of zinc, mainly
found within the confines of proteins and enzymes, which
is essential to many biological functions (5); however,
there is also a dark side to Zn(II). For example, it has been
known since the mid-1980s that free zinc has an active role
in neuronal injury (6) and there have since been numerous
reports on the acute toxicity of free zinc and its link to
neurodegenerative effects in Alzheimer’s disease (4d, 7)
and amyotrophic lateral sclerosis (Lou Gehrig’s disease)
We have recently reported the synthesis of a simple
pyrene-based triazole receptor which was shown to self-
assemble in the presence of ZnCl by complexation-induced
2
dimerisation (14). This was an example of a molecular
dyad, whereby pyrene units are syn in orientation, and
both cation and anion aided in the templation of the
receptor. The fluorescent signal was generated by the self-
assembled induced excimer formation upon the addition of
Zn(II) ions.
(
8). One of the major problems of Zn(II) ion detection is
that it is spectroscopically silent and thus difficult to detect
directly. Many fluorescence mechanisms have been used in
molecular sensing and their applications in the field of
supramolecular chemistry have been extensively reviewed
The number of papers appearing on zinc sensors attests
to the importance of this area of research and,
consequently, the field warrants further development.
Many groups have utilised different scaffolds, moieties
and mechanisms to detect zinc in both aqueous (15) and
non-aqueous systems (16). We now report a versatile
(9). Numerous recent examples have incorporated the
pyrene motif as a spectroscopic handle to detect ion pairs,
*Corresponding authors. Email: p.j.cragg@brighton.ac.uk; karl.wallace@usm.edu
q 2013 Taylor & Francis

1
42
E. Manandhar et al.
Scheme 1. Synthesis of molecular receptors 3a and 3b (ortho-isomer (a) meta-isomer (b)).
modular approach to a pyrene-derived molecular probe
in which two pyrene units are tethered to an aromatic
backbone.
Fluorescence studies
Fluorescence analysis of compounds 3a and 3b with
different metal ions Zn(II), Mg(II), Cd(II), Al(III), Hg(II),
Ni(II), Cu(II), Fe(II), Fe(III) and Na(I) as either their
chloride or perchlorate salts was initially tested with the
Results and discussion
addition of 10 equiv. of metal salts in CH CN, excited at
3
The molecular probes contain an amide functional group
and a triazole moiety attached to a benzene scaffold
325 nm. There were no significant differences in the
absorption spectra before or after the addition of metal ions
and, therefore, no colorimetric tests were investigated in
this study. The choice of either the chloride or perchlorate
salt was due to their commercial availability and solubility
in the chosen solvent system (Figure 1).
(ortho-derivative 3a and meta-derivative 3b). The functio-
nalised pyrene group was prepared by reacting pyrene
carboxylic acid with SOCl to form the acid chloride,
2
which was subsequently reacted with an equimolar amount
of propagylamine and then purified by silica column
chromatography to afford 1 in 60 % yield (17). Compound
The fluorescence signal of the free receptors showed
the typical monomer bands at 380 and 400 nm,
characteristic of pyrene-derived compounds, which are
assigned to the p–p * electron transitions (18). A very
broad band at 495 nm is also seen which was assigned to
1
2
was then reacted with bis(azidodimethyl)benzene (2a or
b) via an azide-alkyne Huisgen cycloaddition to afford
the target molecule in ,70% yield (Scheme 1).
Figure 1. (Colour online) Fluorescence intensity of 3a and 3b upon addition of 10 equiv. of metal chlorides and perchlorates at 400 nm
CH CN, l ¼ 325 nm).
(
3
ex

Supramolecular Chemistry
143
intersystem crossing from the excited state to the
corresponding excited triplet state, known as the heavy
atom effect, whereby the fluorescence is quenched by non-
radiative decay. Redox active species such as Fe(III) and
Cu(II) can quench the fluorescence by accepting an
electron into their d-orbitals which was donated by the
excited fluorophore (25). Therefore, it was not surprising
that many of the metal salts studied quenched the
fluorescence of our molecular probes. However, Zn(II) can
neither accept an electron into its d-orbitals as they are
already filled nor is it classified as a heavy metal. As a
consequence, quenching is not observed and a signal ‘turn-
on’ or fluorescence enhancement is seen.
It was clear, however, that the fluorescence intensity of
2
2
the ClO4 salt was more intense than that of the Cl salt.
We, therefore, explored the effect of different counter-ions
on Zn(II) binding. The binding affinities of 3a and 3b
2
2
2
2
2
2
towards Zn(II) salts (F , Cl , Br , I , NO , CH3CO ,
2
3
2
22
2
SO , BF and ClO ) in CH CN were investigated using
3
4
4
4
fluorescence spectroscopy at 298 K by excitation of a
5
mM solution of 3a or 3b at 325 nm (Figure 3).
There were significant spectroscopic changes upon the
incremental addition of both Zn(ClO ) (Figure 2(A)) and
4
2
Zn(BF ) (Figure 4). These large counter-ions seem to
4
2
dissociate significantly in CH CN in comparison with the
3
other anions studied. The excimer band at 495 nm shifted
hypochromically with a new hyperchromic band appear-
ing at 400 nm through an isoemissive point at 461 nm.
Similar hypsochromic shifts have been observed by
Nakahira et al. (19) and de Schryver et al. (20) in pyrene-
based polymeric materials and bis(pyrenyl)ether deriva-
tives, respectively. The binding affinities of 3a and 3b for
Zn(ClO ) , calculated using HypSpec (26), were
Figure 2. (Colour online) The fluorescence spectra of 3a (A)
and 3b (B) upon incremental additions (up to 10 equiv.) of Zn
(
ClO
4
)
2
(5 mM; CH
3
CN, lex ¼ 325 nm)
.
4
2
6
5
21
K11 ¼ 1.4 £ 10 and 3.8 £ 10 M , respectively. The
binding constants for all of the other zinc salts, including
salts of Mg(II), Ca(II) and Na(I), are shown in Table 1.
Interestingly, the binding affinity for Zn(ClO ) was a
intramolecular excimer formation, upon excitation at
25 nm. Intermolecular excimer formation was ruled out
3
as the effect was observed in the micromolar range,
whereas intermolecular excimer formation occurs at a
4
2
magnitude greater for 3a than for 3b, even though the
2
5
concentration .1 £ 10 M. On inspection of the
fluorescence spectrum, quenching was observed for all
the metals except Zn(II) (and to a lesser degree Cd(II) and
Mg(II)), and a new band was seen to increase at 400 nm.
Upon addition of 10 equiv. of metal salts, it was evident
that the Zn(II) salts showed significantly greater
fluorescence at 400 nm for 3b (Figure 2(B)) than for 3a,
for which a small increase in intensity was observed at
4
00 nm (Figure 2(A)).
The lack of fluorescence at 400 nm for 3a is a
consequence of the two pyrene units in 3a adopting an
anti-orientation which gives a greater pyrene–pyrene
separation than is available to 3b, in agreement with the
density functional theory (DFT) calculations (vide infra)
(
see Figures S2–S4 for complete spectra).
There are many ways in which metals can quench
fluorescence; transition metals such as Hg(II) enhance the
Figure 3. Fluorescence intensities of 3a and 3b with various Zn
(II) salts.

1
44
E. Manandhar et al.
metal probes and it is a major challenge to apply them to
real-world’ problems. Nevertheless, probes that can
monitor zinc in non-aqueous systems such as CH CN
‘
3
(
16a), THF (16b) and EtOH (16c) are often a good starting
point from which an understanding of the binding
properties of the molecular receptors may be gained.
Recent work has taken this further and reports zinc binding
either in mixed organic solvent–water or in 100% buffer
solution (15a). To show that 3b could, in principle, be used
to assay Zn(II) found in aqueous samples, we developed a
simple protocol. Solutions of 3b in CH CN were refluxed
3
with aqueous solutions of Zn(ClO ) between 0.0 and
4
2
0.5 mM for 24 h. The solvents were removed in vacuo and
the residues were redissolved in CH CN. Fluorescence at
3
4
21 nm increased linearly with increasing Zn(II) concen-
Figure 4. (Colour online) The fluorescence spectra of 3b upon
in
incremental 5 mM additions (up to 10 equiv.) of Zn(BF
CH CN (l ¼ 325 nm).
4
)
2
tration, demonstrating that receptor 3b could be used to
determine the zinc content of aqueous samples (Figures S6
and S7). We are currently preparing other molecular
probes to improve solubility.
3
ex
fluorescent change at 400 nm was not observed to be as
dramatic (Figure 1).
In fact this trend is seen for all the other analytes
studied, presumably due to the more favourable chelating
motif. However, the lack of a spectroscopic signal can be
justified due to the pyrene moieties adopting a trans
orientation, in which they are further apart, thus reducing
the magnitude of the electronic transition seen at 400 nm
for receptor 3a. These effects can be explained by the
binding cleft of 3a being more conducive to metal
coordination than 3b but its pyrene groups are further
apart, reducing its fluorescence signal compared to 3b.
We investigated the binding ability of 3b systems in a
mixed organic–water system, but only 1% water could be
added to the system before precipitation occurred.
Nevertheless, a similar ratiometric response is seen, with
an excimer band decrease at 495 nm and increase at
Molecular modelling and IR studies
Numerous efforts were made to grow crystals of the host–
guest complex, to no avail. To gain insight into possible
ligand and complex geometries, an extensive compu-
tational approach was adopted (14). Molecular mechanics
calculations for 3a and 3b and their corresponding Zn
(ClO ) complexes were carried out (Figure 5). Both the
4 2
free receptors show interesting, but very different,
p-stacked interactions. The ortho-isomer, 3a, shows that
the aromatic scaffold of the molecular probes participates in
p-stacking and one of the pyrene arms seems to intercalate
and form an intramolecular sandwich interaction (Figure 5
(
A)). This intercalation is not observed in the meta-isomer,
˚
b, which shows 4.77 A p–p distance between the two
3
4
aqueous solubility is often encountered for pyrene-based
00 nm, upon the addition of Zn(ClO ) (Figure S5). Poor
4 2
pyrene units. The distance was measured between the
pyrene centroids (Figure 5(C)) and the CZH hydrogens
on the triazole rings orientated towards the centre of
the molecular probes. This is in excellent agreement
with the interactions expected to generate the excimer band
seen in the fluorescence spectrum. Both the Zn(II)
complexes show drastically different optimised geometries.
The Zn(II) ion is bound in a tetrahedral arrangement
coordinated to the oxygen atom of the carbonyl functional
group and a nitrogen atom in the triazole ring, forming a
seven-membered chelating ring. The distance between two
Table 1. Association constants (K11) between receptors 3a and
3b and ZnX in CH CN. (a) could not be refined; however, based
on the trend, the binding constant is predicted to be a magnitude
2 3
larger than 3a.
3
a 21
3b 21
11 (M
X
K
11 (M
)
K
)
2
3
3
F
Cl
1.4 £ 10
1.6 £ 10
1.1 £ 10
9.6 £ 10
5.0 £ 10
1.8 £ 10
3.1 £ 10
3.6 £ 10
1.4 £ 10
9.1 £ 10
8.4 £ 10
4.5 £ 10
3.5 £ 10
2.7 £ 10
1.2 £ 10
5.4 £ 10
5.6 £ 10
9.9 £ 10
5.7 £ 10
8.9 £ 10
3.8 £ 10
a)
2
3
4
3
3
3
4
5
6
3
2
2
3
2
4
4
3
2
3
4
5
2
þ
Br
˚
pyrene units in [Zn · 3a] is 10.8 A, whereas the distance
2
I
NO3
2þ
˚
between two pyrene units in [Zn · 3b] is 5.5 A, again
2
2
2
in excellent agreement with the fluorescence data in which
a broad excimer band is seen at 400 nm for the 3b complex;
the same band is significantly less intense for the 3a
complex.
CH3CO
SO4
BF4
ClO4
Mg(ClO
Ca(ClO4)2
Na(ClO
22
2
2
4 2
)
In order to verify the binding mode of Zn(ClO4)2,
2
5.3 £ 10
2
suggested by the molecular modelling calculations, an IR
study was conducted. The infrared spectrum of free
4
)
1.7 £ 10

Supramolecular Chemistry
145
4 2 4 2
Figure 5. (Colour online) DFT fully optimised structures of (A) 3a, (B) [Zn · 3a](ClO ) , (C) 3b and (D) [Zn · 3b](ClO ) .
receptor 3b was recorded as a solid using an ATR-IR and
compared with that of the metal complex, prepared by
grinding equimolar amounts of 3b and Zn(ClO ) in a
reflected in the IR wavelength shifts seen. Shifts of 46 and
2
1
16 cm in the red direction for amide I and amide II,
respectively, are seen. The amide A band is hypsochro-
4
2
2
1
pestle and mortar for 30 min to form a green solid. This
solid had been placed in the oven to dry for 2 h before the
ATR-IR was recorded. Compound 3b shows the
characteristic IR bands for the amide functional
mically shifted by 48 cm
as a consequence of the
hydrogen bonding interaction between the amide proton
2
and one of the oxygen atoms in the ClO ion (Figure S8).
The molecular models show that ClO4 participates in a
number of hydrogen bonding interactions, lowering the
4
2
2
1
group. The amide A stretch appears at 3333 cm for the
NH band. The amide I (CvO) stretch and the amide II
symmetry of the ion from a T to a C . As a consequence,
d 3v
(
NH in plane bending) stretch are seen at 1639 and
the degeneracy of the T (n ) ClZO stretching absorption
2 3
2
547 cm in 3b, respectively (Figure S8). It has been
1
21
1
at 1059 cm in the Zn(ClO ) salt is split into a doublet at
4 2
2
1
previously reported that significant infrared spectral
changes occur when perchlorate coordinates to a metal
ion through different modes (21). Determination of
1070 and 1043 cm assigned to A (n ) plus E (n ) (22),
1 1 4
which is in agreement with the binding present in the
modelling calculations.
2
changing symmetry of the ClO4 ion upon binding, from
Td (ionic) to C3v (unidentate) and C2v (bidentate or
bridging), can help identify the binding mode of the
present system. The combination of the amide group in the
molecular probe and the distinctively intense ClZO
stretching frequencies observed in the IR spectrum of the
perchlorate anion makes these functional groups excellent
IR handles to show that the proposed model is consistent
with IR data. There is an increase in negative charge on the
metal ion as the Zn···O bond becomes stronger, and both
the amide I and amide II bands become weaker; this is
1
D and 2D NMR studies
There is a significant overlap of the pyrene signals in the
1
aromatic region of the H NMR spectrum, rendering the
exact assignment for receptor 3b difficult. This is
reasonable as there is free rotation around the methyl
groups in the molecule, which makes the individual signals
indistinguishable. Upon addition of 5 equiv. of Zn(ClO)2,
the aromatic pyrene protons shift to chemically different
environments, presumably due to the conformational

1
46
E. Manandhar et al.
1
Figure 6. (Colour online) H NMR complete spectrum (bottom) of receptor 3b without the addition of Zn(ClO ) and (top) upon the
4
2
4 2 3 3
addition of 5 equiv. of Zn(ClO ) in a mixture of CD Cl:CD CN (1:1).
Figure 7. (Colour online) ROESY spectrum of 3b (top) and after the addition of 5 equiv. of Zn(ClO
CD CN (1:1).
4 2 3
) (bottom) in a mixture of CD Cl:
3

Supramolecular Chemistry
147
pyrene) and CH adjacent to the NH functional group is
2
absent in the free receptor ligand. Upon addition of Zn
(ClO ) , a strong distinctive rOe signal was seen between
4 2
these same hydrogen atoms (labelled j in Figure 7, bottom).
Presumably, upon addition of Zn(II) ion, the two oxygen
atoms on the carbonyl coordinate to the metal centre
causing a rotation of the amide group such that both the
oxygen atoms participate in the coordination of Zn(II)
within the cleft. This contrasts with the free receptor where
the carbonyl functional groups point in opposite directions.
This change in geometry is in agreement with the molecular
modelling calculations.
Another two rOe signals of interest is between the
triazole proton and ortho-proton of the benzyl group
(
Figure 7, bottom, labelled h). In the free receptor there is
Figure 8. (Colour online) Calibration graph used to calculate
the LoD for Zn(II) (blue diamonds) and Ca(II) (red triangles)
with 3b (20 nM; CH CN, l ¼ 325 nm).
only one signal, whereas in the complex spectrum the
triazole proton has two distinct rOe signals consistent with
rotation of the benzyl functional group. Inspection of the
molecular model (Figure 5) shows that the benzyl group is
rotated by ,708. Unfortunately, the ortho (3a) isomer is
insoluble at the concentration required for NMR and
detailed 2D analysis was impossible.
3
ex
locking once Zn(II) ion has coordinated to the molecular
cleft (Figure 6). The NH signal is shifted downfield by over
1
ppm due to increasing acidity of the NH proton upon
coordination of the carbonyl oxygen atoms to the Zn(II)
metal centre. This is in excellent agreement with our
previously published work (14).
Sensitivity and reversibility
Upon addition of Zn(ClO ) , distinct NMR chemical
4
At low concentrations (0.05 mM), 3b showed an increase
in the fluorescence intensity at 380 nm (the wavelength
monitored in the detection studies at such low concen-
trations) upon the addition of Zn(ClO ) . This is a key
2
shifts were observed and careful 2D analysis (TOCSY,
HSBC and HSQC, see Supporting Information – Figures
S9–S21) allowed accurate assignments to be made. The
4
2
2
in the structural elucidation of the complex (Figure 7). The
D NMR spectroscopy, in particular ROESY, was helpful
requirement for the molecular receptor if it is to be used to
detect Zn(II) at biological concentrations: the same
experiment with 3a showed no spectroscopic change at
the same concentration. To determine the limit of detection
(LoD), the method of least squares was used to give a line
of regression. The confidence limit of the slope is defined
rOe spectrum recorded for 3b in a mixture of CD CN and
3
CDCl (1:1, v/v) showed one very weak and nine strong
3
rOe signals (Figure 7, top, see Supporting Information for
full assignments).
Upon addition of zinc salt, two distinct signals became
significant. The rOe between CH on the pyrene (C(2)
as b ^ t , where t is the t-value taken from the desired
sb
confidence and n 2 2 degrees of freedom. In our
Figure 9. (Colour online) The effect of sequential addition of 1,10-phenanthroline and Zn(II) to 3b (5.0 mM; CH CN, lex ¼ 325 nm).
3

1
48
E. Manandhar et al.
experiment, we chose a 95% confidence level (t-value
2
between the two different aromatic proton signals. UV–
vis experiments were carried out on a Beckman DU-70
spectrophotometer, Beckman Coulter, Inc., Brea, CA,
USA. Low resolution mass spectra was measured on a
Finnigan TSQ70 instrument Thermofisher Scientific,
Waltham, MA, USA. IR spectra were recorded on a
Nicolet Nexus 470 FT, Thermofisher Scientific, Waltham,
MA, USA paired with a Smart Orbit ATR attachment.
The characteristic functional groups are reported in wave
.23, df ¼ 10) (Figure 8). It is generally accepted that the
LoD is the analyte concentration giving a signal equal to
the blank signal plus three standard deviations from the
blank, i.e. y ¼ y þ 3S . The calculated LoD values for 3a
B
B
and 3b were 0.6 mM and 20 nM, respectively. Calcium
salts often interfere with zinc in fluorescence assays;
however, the Ca(II) complexes gave no spectroscopic
responses in the same nanomolar concentration (Figure 8).
Upon addition of 1,10-phenanthroline, a known Zn(II)
chelator, Zn(II), was ‘stripped’ from the molecular cleft. The
fluorescence spectrum returned to that of the receptor
although the intensity was ,50% of the original emission.
This could be repeated three times (Figure 9), and each time
2
1
numbers (cm ), and are described as weak (w), medium
(m), strong (s) and very strong (vs). Fluorescence
e
experiments were carried out on a QuantaMaster 40,
Photon Technology International, Birmingham, NJ, USA
intensity-based spectrofluorometer from PTI technol-
ogies in the steady state; slit widths 0.50 mm;
1,10-phenanthroline was removed, the intensity of the
original emission was reduced by a further 50%. It is
reasonable to assume that the quenching of fluorescence
intensity is due to the 1,10-phenanthroline electron clouds
being in proximity to the fluorophore of the molecular
receptor, thereby preventing emission (25). This reversibility
is essential if 3b is to be incorporated into a Zn(II) sensor; we
are currently investigating other chelators that strip Zn(II)
without affecting the receptor fluorescence intensity.
lex ¼ 325 nm, l ¼ 360–625 nm. Elemental analysis
em
was carried out at Atlantic Microlab, Inc., Atlanta, GA,
USA. The synthesis and characterisation of compound 1
was previously reported (17).
Synthesis
General procedure for preparation of bis(azidomethyl)
benzene
The desired (ortho or meta) bis(bromomethyl)benzene
(1.32 g, 5.0 mmol) and sodium azide (0.97 g, 15 mmol)
Conclusions
In summary, two pyrene-based triazole molecular
probes have been synthesised and shown to bind Zn(II)
were dissolvedin a3:1 mixture of acetone and water (30 ml)
and stirred at room temperature for 24 h (23). A mixture of
dichloromethane (25 ml) and water (25 ml) was then added
to the reaction mixture and stirred for 10 min. The organic
layer was separated and washed three times with water
2
very effectively. Molecular modelling of [Zn · 3a] and
þ
2
Zn · 3b] suggests that distances and angles between
þ
[
pyrene moieties in the two complexes are significantly
different. This is consistent with fluorescence data that
(50 ml), dried over magnesium sulphate, filtered and the
solvent was removed to produce a yellowish oil.
2
þ
support excimer formation for [Zn · 3b] . Receptor 3b
has a LoD in the sub-nM range and its Zn(II) binding
was shown to be reversible over several cycles.
A protocol for use with aqueous samples has been
developed and we are continuing our efforts to improve
this method.
Characterisation of compound 2a
1
Yield 752 mg, 4.0 mmol, 80%: H NMR (300 K,
DMSO, 400 MHz): d 7.38 (m, 4H, CH ), 4.45 (s, 4H,
Ar
1
CH2). C NMR (300 K, DMSO, 100 MHz): d 133.9,
3
1
2
30.0, 128.7, 51.0; IR (ATR solid); 2887 n
21
(w),
CZH
Experimental section
085nNuN (vs) cm
.
General
1
13
H, C and 2D NMR spectra were recorded on a Bruker
Characterisation of 2b
1
Yield 790 mg, 4.2 mmol, 84%: H NMR (300 K, DMSO,
Ultrashield Plus 400 MHz spectrometer, Bruker Biospin
Corporation, Billerica, MA, USA in the appropriate
deuterated solvents. Chemical shifts are reported in parts
per million (ppm) downfield from tetramethylsilane
400 MHz): d 7.38 (t, 1H, J ¼ 7 Hz, CH ), 7.28–7.30 (m,
Ar
1
3H, CH ), 4.37 (s, 4H, CH ). C NMR (300 K, DMSO,
3
Ar
2
(
(
0 ppm) as the internal standard and coupling constants
J) were recorded in Hertz (Hz). The multiplicities in the
100 MHz): d 136.1, 129.2, 128.0, 53.4; IR (ATR solid);
2
1
2929 nCZH (w), 2086 nNuN (vs) cm
.
1
H NMR spectra are reported as (br) broad, (s) singlet, (d)
doublet, (dd) doublet of doublets, (ddd) doublet of
doublet of doublets, (t) triplet, (sp) septet and (m)
multiplet. All spectra were recorded at ambient
temperature, unless stated otherwise, the abbreviations
py(pyrene) and Ar(aromatic) are used to distinguish
General procedure for preparation of compounds 3a
and 3b
N-(prop-2-ynyl)pyrene-1-carboxamide (17) (75 mg,
0.26 mmol) and the desired ortho- or meta-bis(azido-

Supramolecular Chemistry
149
2
5
methyl)benzene (25 mg, 0.13 mmol), copper(II) sulphate
3.25 mg, 0.013 mmol) and sodium ascorbate (5.15 mg,
.026 mmol) were dissolved in a mixture of acetone and
solution (1.32 £ 10 M) of compound 3a was prepared
(
by dissolving 0.5 mg in 50 ml of CH CN. Compound 3b
3
0
was prepared by dissolving 0.8 mg in 50 ml CH CN (2.12
3
2
5
water (15 ml, 4:1) and stirred at room temperature for 48 h.
The reaction mixture was then poured into ice-cold water
£ 10 M) and then 1 ml was transferred to the quartz
cuvette. From the stock solution, eight successive dilutions
were carried out, Table S1 shows the molar absorptivity
calculated for each new compound.
(20 ml) to obtain a solid which was filtered, washed with
water (200 ml) and dried to give the desired isomer.
Characterisation of compound 3a
1
Yield 75 mg, 0.1 mmol, 77%; m.p. 2448C: H NMR (300 K,
Fluorescence experiments
A stock solution of compound 3 was prepared in CH CN.
3
DMSO-d , 400 MHz): d 9.24 (t, 2H, J ¼ 5 Hz, NH), 8.47 (d,
6
The solution was excited at l ¼ 325 nm and scanned
from l 360 to 625 nm with slit widths set to 0.5 mm.
A 100 times more concentrated solution of the Zn(II) salt
2
H, J ¼ 10 Hz, CH ), 8.35 (s, 1H, CH ), 8.34 (s, 2H,
py py py
py
py
CH ), 8.32 (d, 2H, J ¼ 5 Hz, CH ), 8.29 (s, 1H, CH ),
8
CH
.24 (d, 2H, J ¼ 10 Hz, CH ), 8.18–8.21 (m, 6H, CH and
triazole py
py
py
was prepared in CH CN and 10 ml (10 ml ¼ 0.5 equiv. of
3
), 8.09–8.15 (m, 4H, CH ), 7.37–7.39 (dd, 2H,
metal salt) aliquots was added to compound 3;
fluorescence spectra were recorded after each addition.
Dilution factors were taken into consideration upon
binding study determination. The binding constants were
determined from fluorescence titrations using HypSpec
J ¼ 3.5, 5.6 Hz, CH ), 7.20–7.22 (dd, 2H, J ¼ 3.5, 5.6 Hz,
Ar
CH ), 5.89 (s, 4H, CH
Ar
), 4.69 (d, 4H, J ¼ 5 Hz,
3
2trizole
1
3
CH Namide). C NMR (300 K, CDCl , 100 MHz): d 169.3,
2
1
1
1
34.8, 132.1, 131.9, 131.1, 130.6, 129.6, 129.3, 128.8,
28.7, 128.3, 127.6, 127.0, 126.3, 126.1, 125.7, 125.0,
24.8, 124.2, 124.1, 123.9, 50.4 and 35.5. ESI-MS m/z
2
006 (26).
þ
þ
[
solid); 3339 n (m), 3115 n (w), 3037 n (w), 1633
M þ H] ¼ 755.4 and [M þ Na] ¼ 777.5; IR (ATR
NH
CH
CH
1
2
nCO amide I (s), 1600 nCO amide II (m), cm . Anal. Calcd
Computational methods
for C H N O : H 4.54%; N 14.84%; C 76.38%%; Anal.
48 34 8 2
Equilibrium geometries for the ligands (3a and 3b) and
their zinc complexes ([Zn·3a](ClO
Recalcd for C H N O ·9H O·10(CH ) CO: H 4.43%; N
2
) and [Zn·3b]
4 2
4
8
34
8
2
3 2
1
4.32%; C 74.50%; found for C H N O ·9H O·10
2
(ClO ) ) were determined by full conformational
4 2
4
8
34
8
2
(
CH ) CO: H 4.72%; N 14.26%; C 74.47%.
3
searches of the 676 possible structures (identified
through the number of rotatable bonds) followed by
molecular mechanics energy minimisation methods
2
Characterisation of compound 3b
Yield 68 mg, 0.1 mmol, 69%; m.p. 1958C: H NMR
(
using the merck molecular force field). No formal
1
bonds between zinc and ligands or anions were
introduced. Initial refinement was followed by geometry
optimisation at semi-empirical PM3d level with the
resulting structures used as input coordinates for density
functional calculations (B3LYP/6-31G * ). Calculations
were made using Spartan’10 installed on a desktop
computer equipped with Intel Xenon Dual Quad Core
CPUs running at 2.33 GHz (24).
(
300 K, DMSO, 400 MHz): d 9.22 (t, 2H, J ¼ 5 Hz, NH),
8
.49 (d, 2H, J ¼ 10 Hz, CH ), 8.32–8.36 (m, 5H, CH ),
py py
py
py
8
CHpy and CHtriazole), 8.09–8.13 (m, 4H, CHpyrene), 7.40–
.30 (s, 1H, CH ), 8.26 (s, 1H, CH ), 8.20–8.24 (m, 7H,
7
.43 (m, 2H, CH ), 7.30–7.32 (d, 2H, J ¼ 8 Hz, CH ),
Ar
Ar
5
.64 (s, 4H, CH
), 4.67 (d, 4H, J ¼ 5 Hz, CH N
).
C NMR (300 K, CDCl , 100 MHz): d 169.3, 137.2,
2
trizole
2
amide
1
3
3
1
1
1
32.1, 131.9, 131.2, 130.6, 129.7, 128.8, 128.6, 128.3,
28.2, 128.1, 127.6, 127.0, 126.3, 126.1, 125.7, 125.1,
24.8, 124.2, 124.1, 123.7, 53.0, 35.5. ESI-MS m/z
Acknowledgements
þ
þ
[
solid); 3333 n (m), 3120 n (w), 3036 n (w), 1639
M þ H] ¼ 755.1 and [M þ Na] ¼ 777.5; IR (ATR
KJW thanks the organisers of 2013 8-IMSIC (Profs Jeffery
Davis, Amar Flood and Lyle Isaacs) held in Arlington for the
invitation to write a manuscript for the special edition of
Supramolecular Chemistry in honour of this event.
NH
CH
CH
2
1
nCO amide I (s), 1547 nCO amide II (m), cm Anal. Calcd
for C H N O : H 4.54%; N 14.84%; C 76.38%; Anal.
4
8 34 8 2
Calcd for C H N O ·10H O·35(CH ) CO: H 4.28%; N
4
8
34
8
2
2
3 2
1
4.01%; C 72.08%; found for C H N O ·10H O·35
48 34 8 2 2
Funding
(
CH ) CO: H 4.91%; N 13.95%; C 71.87%.
3 2
KJW thanks the NSF [OCE-0963064] for funding the ongoing
research in the Wallace Laboratory.
UV–vis experiments
Supplemental data
Supplemental data for this article can be accessed here:
http://dx.doi.org/10.1080/10610278.2013.835050.
The general procedure for the molar absorptivity of
compounds 3a and 3b was carried out as follows. A stock

1
50
E. Manandhar et al.
(9) (a) Formica, M.; Fusi, V.; Giorgi, L.; Micheloni, M.
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