A pyrene-based fluorescent sensor for Zn2+ ions: A molecular 'butterfly'

Article abstract of DOI:10.1039/c1cc13286e

A simple pyrene-based triazole receptor has been synthesised and shown to self-assemble in the presence of ZnCl₂ in an exclusively 2:1 ratio, whereas a mixture of 2:1 and 1:1 ratios are observed for other Zn²⁺ salts. The pyrene units are syn in orientation; this is supported by a strong excimer signal observed at 410 nm in the presence of ZnCl₂ in acetonitrile. DFT calculations and 2D NMR support the proposed structure.

Full text of DOI:10.1039/c1cc13286e

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A pyrene-based fluorescent sensor for Zn2+ ions: a molecular ‘butterfly’w
Erendra Manandhar,a J. Hugh Broome,a Jalin Myrick,a Whitney Lagrone,a Peter J. Cragg*b
and Karl J. Wallace*a
Received 2nd June 2011, Accepted 22nd June 2011
DOI: 10.1039/c1cc13286e
A simple pyrene-based triazole receptor has been synthesised
and shown to self-assemble in the presence of ZnCl2 in an
exclusively 2 : 1 ratio, whereas a mixture of 2 : 1 and 1 : 1 ratios
are observed for other Zn2+ salts. The pyrene units are syn in
orientation; this is supported by a strong excimer signal observed
at 410 nm in the presence of ZnCl2 in acetonitrile. DFT
calculations and 2D NMR support the proposed structure.
was subsequently reacted with an equimolar amount of
propargylamine, to produce compound 1.12 This was purified
by silica column chromatography to afford compound 1 in
60% yield. Compound 1 was then tethered to benzylazide via
azide-alkyne Huisgen cycloaddition to afford the target mole-
cule in 50% yield, (Scheme 1).
The binding affinity of compound 3 towards Zn2+ salts was
investigated by NMR and fluorescence spectroscopy. 1H-NMR
titrations were carried out in acetonitrile-d3 solution. There
were significant downfield 1H NMR chemical shifts for the
NH proton, the triazole proton and, to a lesser extent, for the
singlet of CH2 on the benzyl group. Upon the addition of
ZnCl2, for example, the changes in chemical shift for the NH,
CH (triazole) and CH2 (Bz) protons were 0.90 ppm, 0.25 ppm
and 0.1 ppm, respectively. The binding affinity (K21) of 3
for Zn2+ was calculated to be greater than 1.0 Â 105 MÀ1
(see ESIw Table S1*). Interestingly, the reaction of 3 with
ZnCl2 produced a 3 : Zn2+ complex that was exclusively in the
2 : 1 ratio, whereas non-linear curve fitting analysis can only be
successfully refined for the other Zn2+ salts if other ligand-to-
metal stoichiometries are taken into consideration. This was
further confirmed by Job’s Plot analysis (see ESI,w Fig. S11).
The NMR shifts can be rationalized through close contacts
between the triazole hydrogen and chloride atom on the
Zn2+ centre. Surprisingly, the NH proton is trans to the
metal centre, and does not participate in the binding event
(vide infra). The chemical shift seen for the amide proton can
be rationalised by the inductive effect. The amide oxygen atom
coordinates to the Zn2+ centre becoming more negatively
The design and synthesis of molecular receptors to target
metal analytes via changes in fluorescence are topics of current
interest as fluorescence signals are easily perturbed with any
change to the fluorophore’s local environment.1,2 Many fluores-
cence mechanisms have been employed in molecular sensing
and their applications in the field of supramolecular chemistry
have been elegantly reviewed.3 Numerous recent examples
have incorporated the pyrene motif as a spectroscopic handle
to detect ion pairs, cations,4,5 anions4 and neutral species.6
This is due to a broad strong excimer signal observed typically
in the 450 nm range when two pyrene units come into close
proximity. We have applied this fluorescent mechanism to the
problem of Zn2+ detection. The detection of Zn2+ is of
particular interest both in vitro and in vivo due to the biological
importance of Zn2+ 7,8
Zinc complexes (in particular ZnCl2)
.
also play an important role, as a moderate strength Lewis acid,
in Friedel–Crafts acylations.9,10 Unfortunately, Zn2+ is
spectroscopically silent making it difficult to detect Zn2+ ions
directly.
Here we report a pyrene-derived molecule (3) that contains
an amide functional group and a triazole moiety that self-
assembles around the metal and anion. The signal is generated
by the self-assembled induced excimer that forms upon the
addition of Zn2+. The Zn2+ ion coordinates to the oxygen
atom of the amide functional group rather than a nitrogen
atom in the triazole moiety, as seen in other Zn2+-binding
receptors.11 Compound 3 was prepared by reacting pyrene
carboxylic acid with SOCl2 to form the acid chloride, which
a Department of Chemistry and Biochemistry, 118 College Drive,
Hattiesburg, MS 39406, USA. E-mail: karl.wallace@usm.edu;
Fax: 1- 601- 266- 6075; Tel: 1-601-266-4715
b School of Pharmacy and Biomolecular Sciences,
University of Brighton, Brighton BN2 4GJ, UK.
E-mail: p.j.cragg@brighton.ac.uk; Fax: +441273679333;
Tel: +441273642037
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13286e
Scheme 1 Synthesis of molecular receptor 3.
c
8796 Chem. Commun., 2011, 47, 8796–8798
This journal is The Royal Society of Chemistry 2011

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charged, the carbonyl carbon is affected (+ve) as is the amide
nitrogen (Àve). On closer inspection of the NMR spectra,
specifically the aromatic region around 7.0 ppm, compound 3
shows two sets of multiplets assigned to the benzyl protons.
Upon the addition of ZnCl2, only one set of equivalent signals
is observed (ESI,w Fig. S10). This can be explained by the
prevention of free rotation of the benzyl group and the
resultant symmetry of 32ZnCl2. We can rule out the possibility
of the second ligand binding in the anti orientation, as an excimer
band is seen in the fluorescence experiments (see below).
PET quench the pyrene moiety, but to a lesser degree. The
intensity of the excimer signal is dependent on the proximity of
the two pyrene units. In absence of the Zn2+ ion, the excimer
peak is not observed, as was confirmed by the analogous
spectroscopic studies with tetrabutylammonium salts. This
suggest that the Zn2+ halide is templating the pyrene units.
The geometry around the Zn2+ ion is tetrahedral and the
pyrene moieties adopt a syn configuration. The fluorescence
titrations are in good agreement with the NMR studies showing
a 2 : 1 non-linear fitting predominated for the ZnCl2 complex
with a calculated binding constant (K21) of 1.8 Â 106 MÀ1
(ESI,w Table S1). In the later stage of the titration
(43 equivalents) it is clear from the fluorescence spectra
(Fig. 1, black lines) that there is a loss of defined spectroscopic
transitions, giving a broad and featureless band. This is
entirely consistent with a second Zn2+ coordination event of
a much weaker affinity, such as the formation of complexes
with higher metal to ligand ratios.
The binding behaviour of compound 3 was also studied by
investigating the photophysical properties of 3 towards various
Zn2+ salts in acetonitrile solution at 298 K. A 6.5 Â 10À4
M
solution of compound 3 was excited at 355 nm; the monomer
showed two distinct emission bands at 382 and 402 nm with a
broad shoulder at 420 nm, typical of pyrene-derived monomers,
assigned to the p–p* electron transitions.13 Upon the addition
of sub-equimolar amounts of Zn2+salts (FÀ, ClÀ, BrÀ, IÀ,
BF4À, CH3CO2À SO42À, H2PO4À, ClO4À, CNÀ, NO3À) different
fluorescence changes were observed. Full titrations were
carried out for those Zn2+ salts that showed excimer formation
only. For example, the excimer baÀnd at 410 nm was observed
for FÀ, ClÀ, (Fig. 1) BrÀ, IÀ, NO3 , whereas no excimer band
was seen for the other anions. However, an increase in the
fluorescence intensity of the monomer at 382 and 402 nm was
Zn2+ is versatile with respect to the number of ligands it can
adopt in its coordination sphere. Zn2+ is known to be tetra-
hedrally coordinated with halides in the inner coordination
sphere, whereas octahedral Zn2+ is most common in aqueous
solutions.14 However, there are many tetrahedral biological
Zn2+ complexes, and some of these systems are supported by
extensive DFT studies to show the preference for tetrahedral
2À
seen for the remaining anions CNÀ, CH3CO2À, SO4 and
Zn2+ 15
. Therefore it is reasonable to assume that compound 3
H2PO4À. It is well known that many organic functional
groups, such as amines, effectively PET quench the fluores-
cence of pyrene units.1 The amide functional group can also
would self-assemble around the Zn2+ salt in a tetrahedral
fashion for the halides. Nitrate, on the other hand, can
coordinate to the metal centre via two oxygen atoms and form
an octahedral geometry. The fluorescence experiments clearly
show the formation of an excimer, meaning that the pyrene
units had to be in close proximity for the p–p* transition and
in the syn orientation. To investigate this further we turned to
molecular modelling calculations.16 Molecular mechaniÀcs
calculations for 32ZnX2 (where X = FÀ, ClÀ or NO3
)
showed that complexes of the form syn-32Zn(NO3)2 should
be the most stable geometries. This is evident for the
32Zn(NO3)2 complex where there is a difference of approxi-
mately 16 kJ molÀ1 between the anti and syn isomers, in
excellent agreement with the fluorescence and NMR studies.
DFT calculations revealed that only two structures, anti-
32Zn(NO3)2 and syn-32Zn(NO3)2, appeared to remain viable
once bonding constraints had been removed. These were also
two of the three lowest energy structures identified by
Monte Carlo molecular dynamics and subsequent geometry
refinement by molecular mechanics. The pyrene–pyrene inter-
actions suggested that, of these structures, the more stable
syn-32Zn(NO3)2 was an excimer, with the two pyrene groups
separated by 6.8 A at an angle of 361. When the same protocol
was applied to the 32ZnF2 and 32ZnCl2 complexes, the pyrene
groups in the resulting 32ZnF2 structure were a similar
distance apart, however, the angle between the pyrene moieties
was now 761, explaining the lack of excimer signal in the
fluorescence study (ESI,w Fig. S12 and Table S2). In the
analogous 32ZnCl2 complex, the interplane distance was
calculated to be 4.4 A at an angle of 4.91 (Fig. 2). This explains
why the excimer signal observed in the fluorescence studies is
more intense for the chloride than the other Zn2+ salts
studied. The result can also be attributed to the ionic size of
Fig. 1 Fluorescence titration of compound 3 and ZnCl2 in CH3CN
(6.5 Â 10À4, M), lEx = 355 nm. (A) Fluorescence spectra and
(B) binding isotherm.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 8796–8798 8797

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sensor in a practical application, we carried out a Friedel–
Crafts acylation reaction by preparing 1-(2,4-dihydroxyphenyl)-
ethanone (ESI,w Scheme S1), which uses ZnCl2 in the synthetic
procedure, to determine how much residual ZnCl2 remained
after the chemical work up. Initial results (ESI,w Fig. S18)
show that we do observe excimer formation. However, the
amount of water in the system competes with the self-assembly
process, preventing qualitative detection limits; this is currently
been investigated and will be reported in due course.
In summary a pyrene-based triazole receptor has been
synthesised and shown to self-assemble in the presence of
ZnCl2; due to the size of ZnCl2, this occurs in an exclusively
2 : 1 ratio. The calculated binding constants show a high
affinity for the Zn2+ salt and 2D NMR and DFT calculations
support the proposed structure.
All new compounds were characterised by 1H NMR,
13C NMR, 2D NMR (COSY, ROSEY, HMBC) ESI-MS
and elemental analysis. Binding studies were determined from
1H NMR and fluorescence titrations using HypNMR 2008
and HypSpec 2008, respectively. Financial support for this
work was provided by the NSF grant OCE-0963064, additional
acknowledgments in ESI.w
Fig. 2 DFT fully optimised structure of 32ZnCl2. Calculated pyrene
distance 4.4 A and angle 4.91.
À
FÀ (1.33 A), ClÀ (1.81 A) and NO3 (1.96 A). The larger size
of the chloride forces the pyrene units closer together. Another
factor is the electronegativity of the fluorine and oxygen
atoms, which spreads the pyrene units further apart. The
observation is supported by the calculated bond distances
between the anion and CH (triazole), CH (methyl amide)
and CH (benzyl) protons. The distance is shorter for anions
that are more electronegative than chloride (ESI, Table S2).
This is in agreementÀwith the less intense excimer behaviour
observed in the NO3 and FÀ fluorescence studies.
Notes and references
Further evidence to support the tetrahedral arrangement of
32ZnCl2 was determined by 2D NMR spectroscopy. An rOe
spectrum recorded for compound 3 in CD3CN showed five
strong rOe signals (ESI,w Fig. S16). The signal that is of
greatest significance is the rOe between the NH proton and
the hydrogen atom attached to C(10) on the pyrene moiety.
Upon the addition of ZnCl2, this signal disappears and an rOe
is observed between the NH and C(2) proton. This clearly
demonstrates that rotation about the C(1) pyrene has
occurred. This correlation is very distinctive, the calculated
bond distance between the NH group and C(2)H is greater
than 4 A for compound 3, too far to feel an rOe effect.
However, the calculated bond distance between the NH and
C(2)H is 2.386 A for the complex, a reasonable distance for an
rOe signal. The proposed geometry is also supported by an
additional weak rOe signal between the triazole proton and the
ortho proton of the benzyl group (ESI,w Fig. S17), absent in
the free ligand. This clearly supports that ZnCl2 acts as a
template for two equivalents of compound 3 in the proposed
geometry seen in Fig. 2.
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c
8798 Chem. Commun., 2011, 47, 8796–8798
This journal is The Royal Society of Chemistry 2011