Simple pyrazoline and pyrazole "turn on" fluorescent sensors selective for Cd2+ and Zn2+ in MeCN

Article abstract of DOI:10.1039/c2ob26608c

An efficient two-step synthesis of pyrazoline ligand 1 is described which is an effective "turn on" fluorescent sensor for Cd²⁺ in MeCN. Oxidation to the corresponding pyrazole ligand 2 creates a "turn on" fluorescent sensor now selective for Zn²⁺ and able to distinguish it from Cd²⁺.

Full text of DOI:10.1039/c2ob26608c

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Simple pyrazoline and pyrazole "turn on" fluorescent sensors selective
for Cd2+ and Zn2+ in MeCN
Alexander Ciupa,a Mary F. Mahon,b Paul A. De Bank*a and Lorenzo Caggiano*a
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
5
DOI: 10.1039/b000000x
An efficient two-step synthesis of pyrazoline ligand 1 is
described which is an effective “turn on” fluorescent sensor
for Cd2+ in MeCN. Oxidation to the corresponding pyrazole
ligand 2 creates a “turn on” fluorescent sensor now selective
10 for Zn2+ and able to distinguish it from Cd2+.
Cadmium is a toxic element widely distributed throughout the
environment originating from natural sources and as a pollutant
from industrial and agricultural use.1 Cadmium can accumulate in
the body over a period of years and has been linked to various
15 diseases including cancer, even at low-levels of exposure.1-2
Therefore the facile detection and monitoring of cadmium in
various media is of great importance.3
50
Scheme 1 Synthesis of pyrazoline 1 and pyrazole 2. i) PhCHO, 10%
NaOH(aq), 24h, ii) H2NNHMe, EtOH, 3h, iii) 10 mol% Pd/C, 200 C, 4h
pyrazole 2 in 80% yield. This simple synthesis is highly scalable
as it uses common commercially available starting materials and
all the products are highly crystalline.
Zinc is chemically similar to cadmium due to its position in the
periodic table, yet it has a vital role in many biological
20 processes.4 The detection of zinc in biological systems has
become a rapidly emerging field as excess free zinc is toxic.5
Many Cd2+ and Zn2+ probes are based on fluorescent sensors
which upon chelation to the cation result in a decrease ("turn off")
or increase ("turn on") in fluorescence intensity. A large variety
25 of complex and sophisticated Zn2+ fluorescent sensors have been
reported.5-6 Various Cd2+ sensors have also been reported,3, 6l, 7
some of which are able to distinguish cadmium from zinc ions.6l,
55
The pyrazoline ligand 1 has been previously reported
complexed with ruthenium, although no other metals were
described.9f Similarly, the synthesis of pyrazole 2 was recently
reported by an alternative route although no chelation properties
were reported.12 We now describe the investigation of pyrazoline
60 1 and pyrazole 2 with various cations‡ analysed by UV/Vis, NMR
and fluorescence spectroscopy.
7c-f
UV/Vis spectroscopy was performed in MeCN, as similar
ligands were investigated in this solvent,6a and in the presence of
Group 1 and 2 metals produced negligible results with both
65 ligands. Upon the addition of various transition metals, however,
both ligands 1 and 2 showed spectral changes consistent with
chelation. The effect of Zn2+ and Cd2+ with pyrazoline 1 is given
in Figure 1 and is representative of the various metals examined
with ligands 1 and 2 (the complete list is available in the
70 Electronic Supplementary Information). The absorbance at 320
nm (ε = 14800 M−1cm−1) is due to the pyrazoline ligand 1 only
and disappears upon the addition of Zn2+ or Cd2+. The formation
of a new band at 360 nm with Zn2+ (ε = 8650 M−1cm−1, Figure
1A) and 350 nm with Cd2+ (ε = 7650 M−1cm−1, Figure 1B) is
75 evident, which increased proportionally up to 1.0 equivalent of
the metal ion and levelled off thereafter, suggesting a 1:1 ratio of
the metal to ligand. Job plot analysis also suggests the formation
of a 1:1 complex with a 0.5 molar ratio of pyrazoline 1 and Zn2+
or Cd2+ (Figure 1).
We report herein a simple two-step synthesis of a "turn on"
fluorescent sensor selective for Cd2+ which can be readily
30 oxidised in high yield to afford a "turn on" fluorescent sensor
selective for Zn2+ and able to differentiate it from Cd2+.
The ligands are based on the pyrazoline motif as it has been
previously reported to possess favourable photophysical
properties8 and chelate a variety of metals,6a-c, 9 including Zn2+.6a-
c, 9a, 9b
35
It is an attractive and versatile scaffold due to the ease of
large range of commercially available
synthesis from
a
acetophenones and benzaldehydes.
Pyrazoline 1 and related pyrazole ligand 2 were synthesised as
outlined in Scheme 1. Following previous literature precedent,10
40 2-acetylpyridine underwent Claisen-Schmidt condensation with
benzaldehyde in the presence of catalytic NaOH to afford the
corresponding chalcone in 97% yield. Treatment of the chalcone
with an excess of methylhydrazine gave the pyrazoline ligand 1
as the sole product in 72% yield. Similar transformations suggest
45 the reaction proceeds via 1,2-addition followed by cyclisation and
not initial 1,4-addition, which could generate a different isomer.8c
Following a procedure for the oxidation of a 1,2,3,4-tetrahydro--
carboline,11 pyrazoline 1 was readily oxidised with Pd/C to afford
80
This is consistent with a crystal structure of a similar
pyrazoline ligand in a 2:2 complex with Zn2+, with chloride atoms
acting as bridging ligands.9a Attempts to obtain a crystalline
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Figure 3 Partial 1H NMR spectra of i) pyrazole 2 (DMSO-d6, 63 mM)
with ii) 0.9, iii) 2.0 and iv) 3.0 equiv. Cd2+
30
spectroscopy in MeCN at 20 µM, as previously reported with
similar ligands.6a No change in fluorescence was observed with
various Group 1, 2 and transition metals, while exposure to Zn2+
or Cd2+ produced a large increase in fluorescence for both ligands
35 1 and 2 (Figure 4 and ESI). This result is particularly pleasing as
it demonstrates that although there is no selectivity in the
absorbance spectroscopy of either ligand with various metals,
they are however only fluorescent in the presence of either Zn2+
or Cd2+. Moreover, with pyrazole 2 they fluoresce at different
40 wavelengths providing a “turn on” fluorescent sensor that can
distinguish between Zn2+ and Cd2+ in MeCN. The effect of
different solvents on fluorescence was investigated with ligand 1
and 2 with Zn2+ and gave variable results, with complete
fluorescence quenching observed in the presence of water (ESI).
Figure 1 Absorbance spectra of pyrazoline 1 (MeCN, 500 µM) with the
addition of 0-1.5 equiv. (0.1 increments) of Zn2+ (A) and Cd2+ (B). Insets
at λem = 370 nm. Lower inset Job plot
45
The addition of 5 equivalents of Zn2+ to pyrazoline 1 resulted
in an 8 fold increase in fluorescence at 460 nm, whereas 5
equivalents of Cd2+ gave a 14 fold increase in fluorescence also at
460 nm with a Stokes shift of 100 nm (Figure 4A and 5). As
previously observed in the UV/Vis study, Job plot analysis is
5
complex of pyrazoline 1 with Zn2+ using the previously reported
conditions (EtOH/H2O, reflux, 24 h),9a instead resulted in a Zn2+
complex with the pyrazole ligand
2 (Figure 2). This is
presumably the result of aerobic and/or zinc mediated oxidation
during the harsh recystalisation process. Interestingly, the
50 consistent with a 1:1 ratio of metal to ligand for both cations
10 asymmetric unit was seen to contain four independent
Zn(pyrazole)Cl2 motifs. Three of these are 1:1 monomers but the
fourth, located proximate to a crystallographic inversion centre,
forms a 2:2 dimer, with bridging chloride atoms (ESI, Figure 2
only shows one of the monomeric 1:1 structures for clarity).
15
Figure 2 Ortep-3 representation of one of the three 1:1 monomers
containing pyrazole ligand 2 and Zn2+ in the asymmetric unit of the
crystal structure, with the ellipsoids represented at 30% probability13
The interaction of the pyrazoline 1 and pyrazole 2 ligands with
1
20 Zn2+ and Cd2+ was also analysed by H NMR spectroscopy. The
addition of Cd2+ to pyrazole 2 is representative of the results
obtained and is shown in Figure 3 (complete study shown in ESI).
Significant chemical shifts of the pyridine and pyrazole protons
were observed upon the addition of the metal, broadening and
25 moving downfield as previously reported for other sensors upon
chelation to Zn2+ 6g-k, 7c and Cd2+ 6j, 6l, 7a-c (Figure 3).
Figure 4 Fluorescence spectra of pyrazoline 1 (A, λex=320 nm) and
pyrazole 2 (B, λex=285 nm, MeCN, 20 µM), upon addition of 5 equiv. of
metal. Metal screen at λem=460 nm (A) and 380 nm (B). Inset shows
Pyrazoline 1 and pyrazole 2 were examined by fluorescence
55
fluorescence with Zn2+ and Cd2+ at 313 nm (A) and 254 nm (B)
2
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Figure 6 Fluorescence spectra of pyrazole 2 (MeCN, 20 µM, λex=285 nm)
upon addition of 0-20 equiv. Zn2+ (A) and Cd2+ (B). Insets at λem=380 nm
(A) and 350 nm (B) upon addition of cation. Lower inset Job plot
Figure 5 Fluorescence spectra of pyrazoline 1 (MeCN, 20 µM, λex=320
nm) upon addition of 0-20 equiv. Zn2+ (A) and Cd2+ (B). Insets at λem=460
40
nm upon addition of cation. Lower inset Job plot
5
(Figure 5 insets). Similar pyrazolines have been previously
reported as ligands9a, 9b or sensors6a-c for Zn2+. The data presented
here however shows that the pyrazoline ligand 1 is more sensitive
towards Cd2+ than Zn2+ and could be a useful "turn on"
fluorescent sensor for cadmium.
10
Conversely, the related pyrazole ligand 2 displays increased
fluorescence in the presence of Zn2+ than Cd2+ (Figure 4B and 6).
A 13 fold increase in fluorescence at 380 nm is observed with
Zn2+ with a Stokes shift of 90 nm, whereas Cd2+ only exhibits a 5
fold increase in fluorescence at 350 nm with a Stokes shift of 60
15 nm. Titration with Zn2+ and Cd2+ is again consistent with the 1:1
stoichiometry previously observed by the UV/Vis analysis and X-
ray crystallography of pyrazole 2 with Zn2+ (Figure 6 insets).
Although it is challenging to selectively distinguish Zn2+ from
Cd2+ ions due to similar physical properties, the 30 nm difference
20 between the Zn2+ (380 nm) and Cd2+ (350 nm) emission maxima
enables the pyrazole ligand 2 to distinguish these cations. In
addition, the pyrazole ligand 2 exhibits increased sensitivity for
Zn2+ providing an effective "turn on" fluorescent sensor for Zn2+.
Figure 7 Competition experiments. The white bar represents pyrazoline 1
(MeCN, 20 µM, λex=350 nm, λem=460 nm) and 5 equiv. of the cation; the
black bar is the same plus 5 equiv. Cd2+ after equilibrating for 3 min.
45 as Pb2+ and Hg2+ is of particular interest.
Significant fluorescence quenching was observed upon the
addition of the paramagnetic metals such as Fe3+, Co2+ and Ni2+
as previously reported with other sensors.6c-f, 7c A similar trend
was observed for competition experiments with pyrazoline 1 with
50 Zn2+, although fluorescence quenching was more pronounced
(ESI). Combined with the increased sensitivity for Cd2+ over Zn2+
(Figure 3), these results suggest that the pyrazoline ligand 1 is
more suitable as a Cd2+ fluorescent sensor.
Competition assays were also performed with pyrazole 2 in the
55 presence of either Zn2+ or Cd2+ (ESI). In both cases large
variations were observed, although heavy metals such as Hg2+
and Pb2+ had little or no effect on fluorescence in competition
with Zn2+ in the presence of pyrazole 2.
14
Following previous reports,6c, the detection limits of Zn2+
25 and Cd2+ by the ligands 1 and 2 were calculated and show that the
pyrazoline 1 has a detection limit of Zn2+ 0.20 M and Cd2+ 0.12
M (ESI). Pyrazole 2 has a detection limit of Zn2+ 0.24 M and
Cd2+ 0.34 M, again highlighting that pyrazoline 1 is a more
effective fluorescent sensor for Cd2+ and pyrazole 2 a more
30 effective sensor for Zn2+.
Competition assays were performed with ligands 1 and 2 to
investigate the effect of detecting Zn2+ or Cd2+ in the presence of
other cations (Figure 7 and ESI). The addition of Mn2+, Pb2+,
Ru3+, Mg2+, Ca2+ and Hg2+ to a mixture of pyrazoline 1 and Cd2+
35 results in only minor decreases in fluorescence (Figure 7). The
ability to detect Cd2+ even in the presence of heavy metals such
In summary, pyrazoline 1 and pyrazole 2 were prepared in an
60 efficient synthesis and are effective “turn on” fluorescent sensors
in MeCN for Cd2+ and Zn2+ respectively. In addition, the pyrazole
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ligand 2 can distinguish between these ions in MeCN and
6. Selected examples include: (a) P. Wang, N. Onozawa-Komatsuzaki,
therefore complements other sensors which can differentiate
these ions in aqueous media.6j-l, 7c-f The ligands are also able to
detect Zn2+ or Cd2+ in the presence of other heavy metals, which
is of great importance for industrial applications. Although these
simple ligands do not operate in aqueous media and suffer from
competition with some biological ions, the modular design and
molecular framework of the ligands allows for further
functionalisation to fine-tune desirable physicochemical
60
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5
65
10 properties, which will be investigated and reported in due course.
Acknowledgements
70
We wish to thank Dr Timothy J. Woodman and Dr Anneke
Lubben (University of Bath) for their assistance with the NMR
and mass spectra, respectively. We are grateful to the University
15 of Bath for providing a studentship for AC. We also wish to
acknowledge RCUK and the University of Bath for the
fellowship to LC.
75
Notes and references
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a Medicinal Chemistry, Department of Pharmacy and Pharmacology,
20 University of Bath, Claverton Down, Bath, BA2 7AY, UK. Fax: +44 1225
386114; Tel: +44 1225 385709; E-mail: l.caggiano@bath.ac.uk
b X-ray Crystallography Unit, Department of Chemistry, University of
Bath, Claverton Down, Bath BA2 7AY, UK.
† Electronic Supplementary Information (ESI) available: Experimental
25 procedures, characterisation data, 1H NMR and 13C NMR spectra are
provided. Data from UV/Vis and fluorescence spectroscopy, NMR
titration experiments, competition experiments, X-ray crystallography,
calculations of detection limits and extintion co-efficients are also
provided. See DOI: 10.1039/b000000x/
30 ‡ The metal complexes used in this study were all metal chlorides (i.e.
MClx), except LiBr, CrO3, Pd(OAc)2, Hg(OAc)2 and Cd(OAc)2.2H2O.
The complete list is provided in the ESI.
§ Crystal data for pyrazole 2 complexed with ZnCl2, CCDC 885728.
Formula: C60H52Cl8N12Zn4.
80
85
90
M = 1486.22, monoclinic. Unit cell
35 parameters: a = 14.2600(2) Å, b = 26.1400(4) Å, c = 16.8220(2) Å.  =
90o,  = 101.476(1)o,  = 90o, V = 6145.15(15) Å3, T = 150(2) K, space
group P21/c, Z = 4, 99 814 reflections collected, 14032 independent
reflections [R(int) = 0.0737]. Final R indices [I>2(I)] R1 = 0.0452 and
wR2 = 0.1015. R indices (all data) R1 = 0.0782 and wR2 = 0.1156.
40
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