A highly selective space-folded photo-induced electron transfer fluorescent probe for carbonic anhydrase isozymes IX and its applications for biological imaging

Article abstract of DOI:10.1039/c1cc12386f

The first highly selective and sensitive fluorescent probe Z1 for detection of carbonic anhydrase IX (CA IX) over isoforms CA I and CA II was developed. As demonstrated, Z1 worked effectively in both enzymatic systems and living hypoxia cells.

Full text of DOI:10.1039/c1cc12386f

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A highly selective space-folded photo-induced electron transfer
fluorescent probe for carbonic anhydrase isozymes IX and
its applications for biological imagingw
Shenyi Zhang, Chunmei Yang, Weiqiang Lu, Jin Huang, Weiping Zhu, Honglin Li,
Yufang Xu* and Xuhong Qian*
Received 24th April 2011, Accepted 31st May 2011
DOI: 10.1039/c1cc12386f
The first highly selective and sensitive fluorescent probe Z1 for
detection of carbonic anhydrase IX (CA IX) over isoforms CA I
and CA II was developed. As demonstrated, Z1 worked effectively
in both enzymatic systems and living hypoxia cells.
hypoxia inducible factor HIF-1 in tumors.6 Recent studies
have demonstrated that CA IX contributes to tumorigenesis
by manipulating cell proliferation, cell adhesion, malignant
cell invasion and pH regulation.7,8 Therefore, the isoform CA IX
has been recognized as a valuable target for cancer diagnostics
and treatment.9
Despite the fact that in the postgenomic and proteomic era a
vast array of genetic and proteomic information has been
revealed, opportunities and challenges exist for imaging to
play a significant role in basic, translational and clinical
research. Accordingly, the development of selective and sensitive
imaging probes for biomacromolecules has emerged as a new
frontier in biomedical research.1,2 Notably, small-molecule
fluorescent probes have been demonstrated as powerful means
for the study of the physiological roles of specific proteins or
enzymes and their correlations with disease development due
to their biocompatibility, stability, minimal interference, and
high sensitivity and specificity.3 However, a limited number of
such probes are available and the lack of selective imaging
probes is a major bottleneck in the study of these biomacro-
molecules. Toward this end, herein we wish to report a novel
highly selective space-folder photo-induced electron transfer
(SPET)-fluorescent probe for carbonic anhydrase isozymes IX
and demonstrate its utility in living cell imaging.
Although several fluorescent probes have been reported for
carbonic anhydrases,10,11 none of them could recognize CA IX
selectively. Supuran and colleagues12 described a nice fluores-
cent labeling agent for CA IX by taking advantage of its high
binding affinity against other isoforms of CA. Nevertheless
such a probe did not induce fluorescence signal change before
and after binding. Therefore, a selective fluorescent imaging
probe with ideal ‘‘off–on’’ intensity alternation is highly desirable.
Our design strategy is illustrated in Scheme 1. The fluores-
cent sensor Z1 consists of three parts: a recognition moiety, a
quencher and a fluorophore. First of all, achieving a high
specificity is a critical issue in any sensor design. We envisioned
that the employment of saccharin as a recognition motif
could lead to high specificity for CA IX because it has been
reported13 as a selective CA IX inhibitor over CA I and CA II.
In addition, it possesses favorable physical and chemical
properties, high stability, and excellent water solubility.
Carbonic anhydrases (CAs, EC 4.2.1.1), ubiquitous metallo-
enzymes present in prokaryotes and eukaryotes, catalyze an
important physiological reaction involving conversion of carbon
dioxide and water to bicarbonate and protons.2a Deregulation
of CAs has been correlated with a variety of disorders including
oedema, glaucoma, obesity, cancer, epilepsy and osteoporosis.
CA inhibitors (CAIs) have been subsequently developed for the
treatment of obesity, cancer and infectious diseases.4,5
Human carbonic anhydrase IX (hCA IX), a transmembrane
a-CA isoform, is significantly overexpressed in response to the
State Key Laboratory of Bioreactor Engineering,
Shanghai Key Laboratory of Chemical Biology,
School of Pharmacy East China University of Science and
Technology, Meilong Road 130, Shanghai, 200237, China.
E-mail: yfxu@ecust.edu.cn, xhqian@ecust.edu.cn;
Fax: +86 21-64252603
Scheme 1 Design of space-folded PET fluorescent probe Z1 for selective
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc12386f
detection of CA IX.
c
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Chem. Commun., 2011, 47, 8301–8303 8301

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Second, as mentioned above, an ideal fluorescent probe should
cause signal change upon binding. The use of conformational
change induced by an analyte upon binding to a receptor is a
fruitful strategy in sensor development. The pioneering work
by Koide et al.14,15 on PET fluorescent sensors for detecting
specific RNA has provided a cornerstone for our design. A
20,70-dichlorofluorescein (DCF) fluorophore coupled with a
piperazidine derivative as an electron donor (quencher) is
linked with the recognition moiety, saccharin. In a free state,
the designed probe Z1 is in a closed conformation where the
piperazine ring brings the aniline nitrogen atom proximal to
the xanthene ring of the fluorophore, leading to the efficient
quenching of fluorescence.15 However, upon binding of the
saccharin moiety to the CA IX, the piperazine ring adopts an
open conformation. The PET is shut down as a result of the
enlarged distance between the aniline nitrogen atom and the
xanthene ring, thus resulting in the recovery of fluorescence.
The space-folder photo-induced electron transfer (SPET) mecha-
nism has been verified by Sparano and Koide.15
Fig. 1 (A) The direct fluorescence response of Z1 to CA IX. Fluores-
cence responses of Z1 (0.1 mM) with 0–2 mM CA IX in enzyme buffer
(10 mM Tris-HCl, pH 7.1, containing 0.1 mM ZnCl2 and o1%
DMSO) at 37 1C. Fluorescence excitation was provided at 511 nm.
After adding CA IX to the Z1 buffer, the fluorescence data were
collected after certain time intervals (100 s) that are indicated in the
figure and marked with different colors. Inset: the plot represents
the corresponding emission intensity at 534 nm. (B) Variation in the
fluorescence intensity of Z1 (0.1 mM) at 534 nm with a series of
concentrations of CA IX (square-line), CA I (triangle-line) and CA II
(circle-line) in enzyme buffer (10 mM, Tris-HCl, pH 7.1, containing
0.1 mM ZnCl2 and o1% DMSO).
The designed fluorescent probe Z1 was synthesized (see ESIw)
and employed for the subsequent studies. First of all, the probe
should be selectively and tightly bound to CA IX. Therefore,
the binding experiments were carried out. A commonly used
CO2-stopped-flow method16 was performed to determine the
Ki values (Table 1). The results from the investigation showed
that the probe Z1 displayed high binding affinity with a Ki of
0.16 nM to CA IX. It is even more potent than those of known
acetazolamide (AZA) and ethoxzolamide (EZA) inhibitors in
comparison studies. Moreover, remarkably, it has an excellent
selectivity toward CA IX over CA I and CA II by as much as
at least 1000-fold.
was confirmed in our GST protein (26 kDa) study with Z1. It
was found that indeed the GST protein induced the decrease in
fluorescence intensity (Fig. S4w). However, at higher concen-
trations ranging from 0.1 to 0.3 mM, the fluorescence intensity
increased with these three CAs. This might attribute to non-
specific binding. When enzyme concentrations are in a range
of 0.3–2 mM, the behavior of fluorescence intensity change of
Z1 with CA IX is quite different from that with CA I or CA II.
It was found that significant enhancement in fluorescence
intensity (from 480 to 680) with CA IX was observed. The
reason, as mentioned above, is due to specific binding of the
Z1 probe to CA IX leading to its conformational change, thus
blocking the PET quenching efficiently. In contrast, CA I and
II quenched fluorescence. The intensity decreased from 470 to
420 and 530 to 420, respectively (Fig. S2 and S3w). As
expected, the ineffective binding of Z1 with CA I and II could
not induce conformational change of the piperazine ring,
thereby PET quenching is still in play. These studies clearly
indicated that the Z1 probe exhibited high specificity toward
CA IX and efficiently differentiated it from its isoforms CA I
and CA II.
With the encouraging data in hand, we carried out the
spectral property studies of probe Z1 afterwards. Absorption
and fluorescence spectral studies performed in an enzyme
buffer (10 mM Tris-HCl, pH = 7.1, containing 0.1 mM ZnCl2
and o1% DMSO) at 37 1C indicated that the absorption
and emission maxima were observed at 511 nm and 534 nm
(Fig. S1w), respectively. Therefore, all subsequent fluorescence
study experiments were performed at an excitation wavelength
of 511 nm.
We observed that the fluorescence intensities dropped by
some degree when Z1 was mixed with enzymes CA I, CA II
and CA IX at low concentrations. As shown in Fig. 1, when
0.1 mM of CA IX was added to a Z1 containing buffer, the
fluorescence intensity fell down from 480 to 420. A similar
trend was seen with CA I and CA II (Fig. S2 and S3w). This
may attribute to protein quenching of fluorescence,17 which
Most importantly, the fluorescence intensity of Z1 increased
in a linear fashion with the concentration of 0.3–1.6 mM of CA
IX (linearly dependent coefficient: R2 = 0.9729, Fig. S6w).
Therefore, Z1 could be employed to quantitate the concentration
of CA IX, an important biomarker in early stages of oncogenesis.
Finally, a fluorescence polarization assay, a powerful tool used
for the study of protein–protein and protein–ligand interactions,
membrane dynamics and protein–DNA and DNA–DNA
interactions,18 was also conducted to determine Z1–CA IX
interaction (Fig. S5w). We found that the DFP increase was
proportional to the concentration of CA IX in a Z1 (0.1 mM)
buffer, indicative of the probe binding to CA IX effectively.
To further demonstrate the practical application potential
of the Z1 probe, we also applied it for imaging CA IX in living
cells and studied the changes of its fluorescence intensity in
hypoxia cells. The SiHa cell line was chosen for these studies
owing to its relatively high endogenous expression of CA IX in
hypoxia.19 Two parallel SiHa cell lines were incubated in DMEM
Table 1 hCA I, II, IX inhibition data with acetazolamide (AZA),
ethoxzolamide (EZA) and Z1
Ki (nM)
Compound
hCA IXa
hCA Ib
hCA IIb
AZA
EZA
Z1
0.40 (25)c
0.19 (34)
0.16
26.80 (250)
1.22 (25)
4172.93
3.46 (12)
0.05 (8)
4172.93
a
Catalytic domain of human, with a GST-tag, recombinant enzyme.
c
Full length, from human erythrocytes. The number in the brackets
b
is the literature reported inhibition data.5b
c
8302 Chem. Commun., 2011, 47, 8301–8303
This journal is The Royal Society of Chemistry 2011

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Research and Development Program of China (2011AA10A207),
the National Natural Science Foundation of China (21076077),
the China 111 Project (B07023), the Shanghai Leading Academic
Discipline Project (B507) and the Fundamental Research Funds
for the Central Universities.
Notes and references
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Fig. 2 (A) Fluorescence microphotographs of SiHa cells incubated
with Z1 (10 mM) without serum for 15 min at 37 1C after incubation in
normoxia and hypoxia for 48 h. (a) and (c) show bright-field transmission
images of SiHa cells after normoxia and hypoxia incubation. (b) and
(d) show fluorescent images of SiHa cells after normoxia and hypoxia
incubation; (B) mean fluorescence intensity change of SiHa cells
incubated with Z1 (10 mM) without serum for 0 to 25 min at 37 1C after
incubation in normoxia and hypoxia for 48 h, respectively. ImageJ
software gave an average emission value of fluorescent images.
for 48 h in normoxia and hypoxia at 37 1C, respectively. Then
Z1 was added to SiHa cells with additional 15 min incubation
in the absence of serum at 37 1C, and washed with a PBS buffer
(containing 1% DMSO, pH 7.4) to remove extracellular
probes. The images were taken by a fluorescence microscope.
Due to the overexpression of CA IX in hypoxia, as expected,
SiHa cells exhibited stronger fluorescence emission than normoxia
cells (Fig. 2A). These outcomes clearly showed that the probe
Z1 displayed high selectivity to CA IX over other CA enzymes
or proteins. Subsequently, we performed the dynamic change
studies of fluorescence emission in SiHa cells (Fig. S7w), the
fluorescent images were respectively taken in 1 to 22 min after
SiHa cells incubation with Z1 in DMEM without serum at
37 1C. A dynamic binding of Z1 to CA IX process in living
hypoxia cells was observed, while SiHa cells did not exhibit a
similar trend in normoxia (Fig. S7). Additionally, we collected
12 fluorescent images from 0 to 22 min and found a notable
fluorescence change (Fig. S7w). The use of ImageJ software
gave an average emission value of fluorescent images at 534 nm.
As shown in Fig. 2B, a rapid increase in mean fluorescence
intensity for hypoxia SiHa cells from 0–3 min was observed,
but slight enhancement for normoxia SiHa cells. The observation
was also consistent with the above results. Therefore, Z1 could be
used as a useful fluorescent probe for measurement of CA IX
in vitro and in living cells.
In summary, we have developed a novel fluorescent probe
Z1 for direct detection of carbonic anhydrase IX (CA IX)
against CA I and CA II with high sensitivity and selectivity. To
our knowledge, this is the first example of an ‘‘off–on’’ selective
fluorescent probe for CA IX. The design strategy described here
relying on the unique space-folder photo-induced electron-transfer
(SPET) mechanism serves as a general approach for the develop-
ment of new imaging probes to selectively detect a variety of bio-
markers simply by rational manipulation of recognition moieties.
We thank Prof. Seppo Parkkila (University of Tampere,
Finland) for providing the plasmid of carbonic anhydrase IX.
This work is financially supported by the National Basic Research
Program of China (010CB126100), the National High Technology
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c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 8301–8303 8303