Both visual and ratiometric fluorescent sensor for Zn2+ based on spirobenzopyran platform

Article abstract of DOI:10.1016/j.tetlet.2012.02.031

A ratiometric fluorescent Zn²⁺ chemosensor, SPQH, based on spirobenzopyran platform, was synthesized. In aqueous HEPES 7.4 buffer solution, upon chelation with Zn(II), SPQH demonstrates high selectivity and subnanomolar sensitivity for zinc ion with 36-fold enhancement in the NIR fluorescence output.

Full text of DOI:10.1016/j.tetlet.2012.02.031

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Tetrahedron Letters 53 (2012) 2001–2004
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Both visual and ratiometric fluorescent sensor for Zn2+ based
on spirobenzopyran platform
⇑
Jian-Fa Zhu, Wing-Hong Chan , Albert W.M. Lee
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China
a r t i c l e i n f o
a b s t r a c t
A ratiometric fluorescent Zn2+ chemosensor, SPQH, based on spirobenzopyran platform, was synthesized.
In aqueous HEPES 7.4 buffer solution, upon chelation with Zn(II), SPQH demonstrates high selectivity and
subnanomolar sensitivity for zinc ion with 36-fold enhancement in the NIR fluorescence output.
Ó 2012 Elsevier Ltd. All rights reserved.
Article history:
Received 28 December 2011
Revised 27 January 2012
Accepted 7 February 2012
Available online 14 February 2012
Keywords:
Ratiometric
Fluorescent
Zn2+ chemosensor
Spirobenzopyran
Design and synthesis of fluorescent chemosensors with desir-
able properties is an active field of supramolecular chemistry ow-
ing to their potential applications in cell physiology, analytical,
environmental, and biological chemistry.1 Behind iron, zinc is the
second most abundant d-block metal ion in the human body, and
it plays diverse roles in biological processes.2–5 However, physio-
logical roles of Zn2+ in the human body are still far from well
understood.6
cell environment sensors capable of exhibiting near infrared
(NIR) emissive output signal are intensively sought after. To our
knowledge, only one ratiometric NIR Zn2+ sensor is known, no bio-
logical applications of this probe were or are reported as of yet.11
Recently, exploiting the unique optical properties of multi-
functional spiropyran derivatives,12 we have developed a water
soluble ratiometric probe for Cu2+ and Zn2+, respectively.13
To continue our quest for novel fluorescence Zn2+ probe, we
have defined the attributes of such probes as: (1) it should be read-
ily accessible synthetically; (2) it should be operative in aqueous
pH 7.4 buffer solution; (3) it must be a sensitive and selective
probe; (4) it should display fluorescence ratiometric response to
Zn2+ and (5) the output signal of the probe ideally can be in the
NIR region (i.e., >650 nm). To this end, here, by appending an elab-
orated metal binding motif onto the spirobenzopyran molecular
platform, we demonstrate that a NIR emission ratiometric Zn2+
probe, a spirobenzopyran quinolone hydrazine derivative, SPQH
(3) is able to detect the subnanomolar concentration of Zn2+ in
aqueous pH 7.4 HEPES buffer solution.
In particular, studies on various aspects of zinc in neurobiology
have recently emerged as a multi-disciplinary cutting edge re-
search field.7 The significance of zinc hemeostasis to neuro-physi-
ology and neuropathology has attracted much interest in devising
new ways to detect Zn2+ in biological samples. Owing to their high
sensitivity, fluorescent chemosensors have been extensively devel-
oped and recognized as one of the viable means for detecting
micromolar or subnanomolar concentration of Zn2+.8 Among these
recent publications of Zn2+chemosensors, some Zn2+ chemosensors
suffered from the cross interference of Cd2+ and/or Cu2+ 8e,f,k–m
It is
.
noteworthy that Lippard and coworkers have pioneered in this
field by utilizing fluorescein as the fluorophoric platform for
developing an array of Zn2+ chemosensors, allowing quantitative
measurements of intracellular changes in zinc metal ion concen-
tration.8l Ratiometric sensor in which the ratio between two emis-
sion intensities can be used to evaluate the analyte concentration
and provide a built-in correction for environmental effects is a
widely sought after sensing probe.9 To date, however, relatively
few small-molecule ratiometric Zn2+ probes are available.8f,g,10
Additionally, to eliminate the autofluorescence phenomenon in
We and others have shown that the novel design of spirobenz-
opyran-based specific metal binding optical probes can be realized
by incorporating metal ligating group on the C-8 position of the
spirobenzopyran scaffold.12b,c,13,14 Among metal ion chelates,
di(2-picolyl)amine (DPA) ligand was widely chosen for binding
Zn2+. Because the DPA moiety is well known for its binding affinity
to Mn2+, Fe2+, Co2+, Ni2+ and Cu2+ 8e,f,i–n,15
we deliberately avoided
,
the use of this ligand in the design of the probe. On this
background and our recent work,8g,13b as shown in Scheme 1, a
8-hydroxyquinoline-based multi-dentate ligand 2-(8-quinolin-
oxy)ethanohydrazide (1) was prepared and its subsequent
condensation with 8-carboaldehyde spirobenzopyran derivative 2
⇑
Corresponding author. Tel.: +852 34117076; fax: +852 34117348.
E-mail address: whchan@hkbu.edu.hk (W.-H. Chan).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.02.031

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J.-F. Zhu et al. / Tetrahedron Letters 53 (2012) 2001–2004
1. K CO /BrCH CO Et (95%)
2
3
2
2
OH
N
O
N
O
HN NH2
O
N
2. NH NH (95%)
2
2
HN
N
1
CH OH, 12 h
3
O
CHO
O
N
OH
CHO
(60%)
N
O
N
SPQH (3)
(80%)
OHC
2
Scheme 1. Synthesis of SPQH (3).
Figure 1. Naked eye detection of Zn2+, Cu2+. (a) Color changes of 10
lM SPQH upon the addition of 0, 500 nM, 1
lM, 5
lM, 10 lM zinc ion in buffer solution (50 mM, HEPES,
40% methanol, pH 7.4) (b) Color changes of 10
lM SPQH upon the addition of 0, 500 nM, 1, 5, 10 lM copper ion in buffer solution (50 mM, HEPES, 40% methanol, pH 7.4).
665 nm
20
350000
300000
250000
200000
150000
100000
50000
0
300000
280000
260000
240000
220000
200000
180000
160000
140000
120000
100000
80000
60000
40000
20000
0
15
10
5
0
0.0
0.5
1.0
1.5
2.0
[Zn2+]/[SPQH]
560 nm
6
SPQH Zn2+ Hg2+ Pb2+ Cu2+ Co2+ Cd2+ Ni2+ Ca2+ Mg2+ Fe3+ Fe2+ Ag+ Na+ K+ Li+
550
600
650
700
750
800
850
Wavelength (nm)
Figure 3. Metal ion selectivity profiles of SPQH (10 lM) in the presence of various
metal ions in buffer solution (50 mM, HEPES, 40% methanol, pH 7.4): (a) (black bar)
fluorescence intensity at 665 nm of SPQH as the control; (b) (red bars) fluorescence
Figure 2. Emission spectra of SPQH (10 l
M) at increasing concentration of Zn2+ (0,
0.1, 0.4, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.5, 2.0 equiv) (50 mM HEPES buffer, 40%
ethanol, pH 7.4). Inset: emission ratio change of I665 nm/I560 nm with increasing
concentration of Zn2+. Excitation wavelength was 510 nm with 10 nm slit width.
intensity at 665 nm of the probe in interacting with 5 equiv of Zn2+, Hg2+, Pb2+, Cu2+
,
Co2+, Cd2+, Ni2+, Ca2+, Mg2+, Fe3+, Fe2+, Ag+, Na+, K+, Li+; (c) (blue bars) fluorescence
intensity of the probe in the mixture containing separately 5 equiv of Ag+, Hg2+
,
Pb2+, Ni2+, Co2+, Cd2+, Cu2+, Li+; 500 equiv of Na+, K+, Ca2+, Mg2+, Fe3+, Fe2+ in the
presence of 5 equiv of zinc ions.
afforded SPQH in
a 60% yield in a convergent manner
(Supplementary data). SPQH was fully characterized by NMR and
HRMS method (Figs. S1–S3).16,17
Figure 1, naked-eye selective and sensitive detection of these two
In aqueous ethanol solution (50 mM HEPES buffer, 40% ethanol,
pH 7.4), only Zn2+ and Cu2+ can mediate the ring opening of color-
less spiropyran form of SPQH triggering its formation of the pink
colored metal chelated merocyanine complex. As shown in
ions can be actualized. The absorption spectra of SPQH and Zn2+
–
SPQH (Fig. S4)16 show a dramatic change from the visible transpar-
ent pattern of the apo-ligand to the emergence of a broad peak at
550 nm of the metal–merocyanine complex. Additionally, the

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J.-F. Zhu et al. / Tetrahedron Letters 53 (2012) 2001–2004
2003
O
N
Zn2+
HN
N
N
O
O
O
NH
O
N
EDTA
O
N
Zn
N
SPQH - Zn2+
SPQH
Figure 4. Possible binding model of SPQH–Zn2+
.
brightness of the metal complex at 550 nm was found to be
215.6 MÀ1 cmÀ1 (0.0069 Â 31335 MÀ1 cmÀ1).
scopic information, the binding mode of SPQH–Zn2+ is proposed
in Figure 4. Finally, the reversibility of binding between the probe
and Zn2+ was established by successive treatment with EDTA and
Zn2+ (Fig. S10).16
In summary, we developed a novel spirobenzopyran-based
ratiometric NIR Zn2+ chemosensor SPQH that effectively and selec-
tively recognizes Zn2+ under aqueous conditions. On the basis of
the low dissociation constant of SPQH–Zn2+ complex, the probe
can detect zinc ion in the subnanomolar concentration range.
Due to the visible excitation, great fluorescence enhancement of
the emission at the NIR region and the large Stokes shift, the probe
will be useful for chemical and biochemical researches.
The binding characteristics of SPQH toward Zn2+ in aqueous
HEPES buffer solutions were examined by fluorescent titrations.
Figure 2 revealed that SPQH can be developed as a ratiometric sen-
sor for Zn2+. Upon gradual addition of Zn2+ into 10
lM SPQH mea-
suring solution, excited with 510 nm, the relatively weak emission
peak of the sensor at 560 nm decreased with the concomitant for-
mation of new peak at 665 nm. A large Stokes shift of 110 nm,
which is rarely observed in the sensor, is apparent. On further addi-
tion of the Zn2+ probe, the ratiometric change of the fluorescence
spectra became evident with a clear isoemission point at 585 nm.
The observed bathochromatic shift of the probe after binding with
Zn2+ is conceivably induced by intramolecular charge transfer
(ICT).18 The emission ratio of I665 nm/I560 nm records a 36-fold signal
enhancement and remained at a plateau in the presence of more
than 1 equiv of Zn2+ (inset, Fig. 2). The Job’s plot confirms the 1:1
binding stoichiometry between 3 and Zn2+, consistent with the
fluorescent titration results (Fig. S5).16 On the basis of non-linear
fitting of the titration curve of 1:1 binding model, the dissociation
Acknowledgements
The financial supports from the Research Grant Council of Hong
Kong (HKBU 200407) and Hong Kong Baptist University (FRGI/10-
11/026) are acknowledged.
Supplementary data
constant of Zn2+–SPQH was computed to be (1.34 0.70) Â 10À7
M
(R2 = 0.9888), indicating that the probe can detect Zn2+ in the subn-
anomolar range.
Supplementary data (synthesis, experimental details, 1H, 13C
NMR and HRMS spectra of SPQH, naked-eye detection of Zn2+
and Cu2+ by SPQH, NMR titration data and HRMS of metal com-
plex) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.tetlet.2012.02.031.
The fluorescence of the metal–ligand complex attains a con-
stant value in the biological relevant pH range (i.e., 6.5–8.0,
Fig. S6).16 To define the application scope of the probe in metal
sensing, interference studies were undertaken. Figure 3 shows that
all biological relevant cations (i.e., Na+, K+, Ca2+, and Mg2+) even in
500 equiv excess are non-responsive to the probe. Among common
transition metals, moderate quenching on the fluorescence of the
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