Fluorescent Zn2+ chemosensors, functional in aqueous solution under environmentally relevant conditions

Article abstract of DOI:10.1016/j.tetlet.2009.12.069

The synthesis and evaluation of two new ratiometric chemosensors for the quantification of potentially toxic free Zn²⁺ ions in aqueous solutions are described. Both sensors show high selectivity for Zn²⁺ over other cations, and are functional at environmentally relevant pH with detection limits of 0.05 μM for free Zn²⁺.

Full text of DOI:10.1016/j.tetlet.2009.12.069

Tetrahedron Letters 51 (2010) 1161–1165
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fluorescent Zn2+ chemosensors, functional in aqueous solution under
environmentally relevant conditions
Amanda E. Lee a,b, Michael R. Grace a, Adam G. Meyer b, Kellie L. Tuck a,
*
a School of Chemistry, Monash University, Clayton, Vic 3800, Australia
b CSIRO Molecular and Health Technologies, Bag 10, Clayton South, Vic 3169, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and evaluation of two new ratiometric chemosensors for the quantification of potentially
toxic free Zn2+ ions in aqueous solutions are described. Both sensors show high selectivity for Zn2+ over
Received 3 November 2009
Accepted 11 December 2009
Available online 21 December 2009
other cations, and are functional at environmentally relevant pH with detection limits of 0.05
lM for free
Zn2+
.
Ó 2009 Elsevier Ltd. All rights reserved.
Owing to the fundamental and ubiquitous role Zn2+ and Cu2+
ions play in biological systems,1 the development of highly selec-
tive and sensitive chemosensors to detect such species in complex
biological mixtures or in environmental samples at low, but eco-
logically relevant concentrations, is paramount.2 Research into
ion recognition has expanded in recent decades to encompass
many areas of chemistry; from small molecule sensors to pep-
tide-based systems and elaborate supramolecular complexes.3
Regardless of the class of sensor, comparable design strategies ap-
ply: coupling a recognition site to a reporting component, where a
sensing event gives rise to a measurable signal. Currently, fluores-
cence-based systems show the greatest promise and sensitivity,
owing to the fact that the emission signal is proportional to the
substrate concentration.4
According to the widely accepted free ion activity model, free
metal ions generally have higher associated toxicity than their
respective colloidal or particulate forms.5 However, straightfor-
ward and direct measurement of free metal ions remains challeng-
ing.6 Current methodology relies on measurement of total metal
concentration through flame atomic absorption spectrometry,7,8
electrochemical or anodic stripping voltammetry (ASV) analysis,8
or ion specific electrodes.9 Despite the high sensitivity of tech-
niques such as ASV, it is impossible to distinguish between poten-
tially toxic, labile ion species (i.e., Zn2+) and less-toxic forms (i.e.,
coordinated Zn2+); species that coexist in environmental systems
yet have substantially different physicochemical and biological
properties.10 Additionally, ASV has difficulty in simultaneously dis-
tinguishing between copper and zinc, due to the formation of inter-
metallic compounds.10,11 This limitation is a notable disadvantage
in the measurement of zinc in complex environmental systems.
Herein, we report the development of two novel sensors for
detection of Zn2+ which are functional in aqueous media and at
environmentally relevant pH. These sensors exploit a photo-in-
duced electron transfer (PET) mechanism to give substantial
fluorescent enhancement upon Zn2+ binding,12 and rely on the
well-established dipicolylamine moiety as the binding unit.13
Mono- (1) and di-substituted (2) anthracene cores provide addi-
tional insight into binding modes and structural design in order
to achieve highly sensitive and selective metal ion recognition.
Chemosensors 1 and 2 were prepared from the corresponding
aldehydes and N1,N1-bis(pyridin-2-ylmethyl)ethane-1,2-diamine
314 via reductive amination, as outlined in Scheme 1. The dialde-
hyde 4 was synthesised by treatment of 9,10-dibromoanthracene15
with n-BuLi, and subsequent quenching of the resultant dianion
with DMF.
The fluorescence spectra of chemosensors 1 and 2 demon-
strated characteristic absorption and emission bands consistent
with the anthracene fluorophore. In all subsequent experiments,
the fluorescence spectra were measured using an excitation wave-
length of 375 nm, corresponding to a major absorption band of the
anthracene core. The optimal operating pH for chemosensors 1 and
2 was determined using a variable pH screen (see Supplementary
data, Fig. S1). Both chemosensors 1 and 2 were functional over
an environmentally relevant pH range (pH 5–8); this is important
if the sensors are to be used for real-time applications in environ-
mental and biological systems.
A fluorescence titration of Zn2+ with either chemosensor 1 or 2
(10
creased fluorescence was observed in a dose-dependent manner
until 1 mol equiv of Zn2+ (10
M) was added; after which, the fluo-
lM) in MES buffer (0.1 mM, pH 6.5) was performed. For 1, in-
l
rescence reached a saturated maximum (Fig. 1A). The addition of
Zn2+ to 2 resulted in a similar dose-dependent increase in fluores-
cence (Fig. 1B), however, despite two potential metal binding sites,
the fluorescence maximum was also reached after addition of
1 mol equiv of Zn2+ (10
lM). This system (2) demonstrated a
higher fluorescent intensity (ꢀ500 a.u.) than 1 (ꢀ300 a.u.), most
* Corresponding author. Tel.: +61 3 99054510; fax: +61 3 99054597.
E-mail address: kellie.tuck@sci.monash.edu.au (K.L. Tuck).
likely due to the PET contribution of two benzylic nitrogens in
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.069

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A. E. Lee et al. / Tetrahedron Letters 51 (2010) 1161–1165
Scheme 1. Synthesis of the mono- (1) and di-substituted (2) chemosensors. Reagents and conditions: (a) Boc2O, EtOH, 0 °C?rt, 95%; (b) 2-chloromethylpyridine, Na2CO3,
EtOH, , 4 Å MS; (ii) NaBH4, MeOH,
D
, 72%; (c) TFA/CH2Cl2, 0 °C?rt, quant.; (d) Br2, CH2Cl2, 0 °C, 70%; (e) (i) n-BuLi, 0 °C?rt; (ii) À78 °C, DMF, 58%; (f) (i) CH2Cl2/MeOH,
D
0 °C?rt; 1: 92%, 2: 85%.
Figure 1. Fluorescence spectra of chemosensor: (A) 1 (10
kem 420 nm versus [Zn2+].
lM) and (B) 2 (10 l lM; kex 375 nm. Inset: fluorescence intensity
M); in MES buffer (0.1 mM, pH 6.5), [Zn2+] = 0–100
chemosensor 2 compared with only one in chemosensor 1. PET ef-
fects are known to result in substantial fluorescent enhancement
on metal-binding.12
immediate qualitative feedback regarding the nature of random
samples, and could therefore find application in critical, real-time,
field-based studies.
A fluorescence titration of Cu2+ with either chemosensor 1 or 2
Both chemosensors, 1 and 2, were evaluated in a competitive
metal screen to determine the relative selectivity and tolerance
for Zn2+ over other relevant cations. Both chemosensors 1 and 2
showed no significant response when treated with 1.0 equiv
(10 lM) in MES buffer (0.1 mM, pH 6.5) resulted in fluorescence
quenching. Cu(II) is a recognised fluorescence quencher.16 The
step-wise addition of Cu2+ to 1 resulted in a dose-dependent de-
crease in fluorescence (Fig. 2A), until saturation was reached upon
(10
Pb2+, Sr2+, Al3+, K+, Na+, Li+, Cr3+, Ba2+ and Cd2+). However, the co-
addition of Zn2+ (10
M) resulted in a significant increase in fluo-
l
M) of metal ions (Cu2+, Mn2+, Ca2+, Mg2+, Ni2+, Fe3+, Ag+,
addition of 1 mol equiv of Cu2+ (10
lM). Likewise, a ratiometric
dose-dependent decrease in fluorescence was observed when
Cu2+ was added to 2 (Fig. 2B) with the fluorescent minima compa-
rable to that achieved with chemosensor 1.
Qualitative differences in fluorescent intensity of the sensors in
the absence and presence of Zn2+ can be observed by the naked eye
(Fig. 3). As a result, these sensors have the potential to provide
l
rescence (see Fig. 4A and B and Supplementary data, Figs. S2 and
S3). Importantly, for both chemosensors 1 and 2, the presence of
Na+, K+, Ca2+ and Mg2+, highly prevalent species in biological and
environmental systems, did not induce any distinct increase in
fluorescence, and the presence of these and other cations did

A. E. Lee et al. / Tetrahedron Letters 51 (2010) 1161–1165
1163
Figure 2. Fluorescence spectra of chemosensor: (A) 1 (10
kem 420 nm versus [Cu2+].
l
M) and (B) 2 (10
l
M); in MES buffer (0.1 mM, pH 6.5), [Cu2+] = 0–100
lM; kex 375 nm. Inset: fluorescence intensity
Figure 3. Chemosensors: (A) 1 (10
lamp, kex 365 nm.
lM) and (B) 2 (10 lM) in the absence (0 l lM); MES buffer (0.1 mM, pH 6.5); illuminated with a hand-held UV
M) and presence of Zn2+ (10
not interfere with the ratiometric response to Zn2+. While Cd2+
did generate a measurable response in both 1 and 2, it is gen-
erally present at concentrations P100-fold less than Zn2+ in
environmental systems, thus Cd2+-derived interference would
therefore be negligible (See Supplementary data, ST1 and ST2).17
Due to the greater fluorescent response of 2 than 1 in the pres-
ence of Zn2+, further analysis was completed with chemosensor 2.
The detection limit of 2 was determined using the relative chemo-
sensor/Zn2+ concentration that gave an instrumental signal which
was significantly different from the background signal (limit of
detection = yB + 3sB; where yB is the signal associated with the
blank, and 3sB is equal to three standard deviations of the blank).18
Using this approach, the detection limit of 2, in the presence of
current, cumbersome methodologies. Particulate and colloidal
forms of Zn2+ were emulated by addition of Na2S to standard
Zn2+ solutions, an effective protocol for precipitation of metal
ions.19 The presence of high concentrations of Na2S (P10
lM),
had no effect on the basal fluorescence of 2. The addition of Na2S
to the highly fluorescent [2-Zn2+] complex caused an immediate
decrease in fluorescence intensity, indicating that only free Zn2+
ions are detected by 2 (Fig. 5). This analogy was extended by the
addition of humic acids, complex heterogeneous mixtures of
small-size and poly-aromatic acids, known to chelate metal ions.20
In the presence of environmentally relevant concentrations of hu-
mic acids, the high fluorescence associated with the [2-Zn2+] com-
plex was significantly decreased (Fig. 6), which further confirms
the selectivity of 2 for free Zn2+ ions, rather than equivalent partic-
ulate or colloidal forms. Unfortunately, the presence of high con-
centrations of humic acids (P10 mg/L), suppressed the basal
Zn2+, was found to be 0.05
lM (Supplementary data, Fig. S4).
High specificity for free Zn2+ ions in solution is essential if these
small molecule chemosensors are to be viable replacements for

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A. E. Lee et al. / Tetrahedron Letters 51 (2010) 1161–1165
Figure 4. Alternate metal screen with chemosensors: (A) 1 and (B) 2; kem 420 nm in MES buffer (0.1 mM, pH 6.5), Grey bars: 1 or 2 (10
l
M) with addition of alternate metal
ions (10 M). Black bars: 1 or 2 (10 M) with addition of alternate metal ions (10 M). White bar: basal fluorescence of 1 or 2 (10 l
l
l
l
M) and Zn2+ (10
l
M).
Figure 5. Chemosensor 2 (10
(10 M); in MES buffer (0.1 mM, pH 6.5); kex 375 nm, kem 420 nm.
lM) in the presence of Na2S (10–100 l
M) and Zn2+
Figure 6. Chemosensor 2 (10
lM) in the presence of humic acids (1 lV to 5 lM)
l
and Zn2+ (10
l
M); in MES buffer (0.1 mM, pH 6.5); kex 375 nm, kem 420 nm.
fluorescence associated with 2.21 ASV, the current state-of-the-art
in zinc sensing, also suffers from similar quenching in the presence
of humic acids, due to adsorption of organic matter on the mercury
electrode.10,22
In conclusion, the synthesis and evaluation of two novel, ratio-
metric chemosensors with a high selectivity and sensitivity for
Zn2+ (Zn2+ detection limit of 0.05
lM) have been reported. Impor-
tantly, these chemosensors operate in aqueous solution over an
environmentally relevant pH range. Due to the higher sensitivity
observed for Zn2+, and relative ratios of [Zn2+]>>[Cu2+] in environ-
mental samples; these novel chemosensors are an exacting means
of measuring potentially toxic free Zn2+. Importantly, these chemo-
sensors rely on different competitive equilibria to the standard ASV
methodology, and thus provide an interesting comparison and
alternative insight into the binding of Zn2+ in complex environ-
mental systems. Additionally, due to their high water solubility,
they are potentially applicable as real-time quantitative sensors
for Zn2+ in vivo.
The function of chemosensor 2 was evaluated under mock-envi-
ronmental conditions utilising a competitive Zn2+/Cu2+ titration. As
zinc is generally more prevalent (0.06–2.9
fresh-water systems than copper (0.03–0.16
0.07
M),17 environmentally relevant ratios of [Zn2+]:[Cu2+] rang-
l
M, median 0.4
lM) in
lM, median
l
ing from 3:1 to 15:1 (Fig. 7 and Supplementary data, ST1) were
investigated. Despite the presence of a basal level of Cu2+, chemo-
sensor 2 still showed a measurable, dose-dependent response to
Zn2+. Thus, the problematic Cu2+ interference associated with
ASV methodology is not a concern with the use of a small molecule
chemosensor such as 2.10,11

A. E. Lee et al. / Tetrahedron Letters 51 (2010) 1161–1165
1165
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Acknowledgements
The authors acknowledge the support of the School of Chemis-
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.12.069.