A novel aggregation-induced emission based fluorescent probe for an angiotensin converting enzyme (ACE) assay and inhibitor screening

Article abstract of DOI:10.1039/c4cc07161a

A 'turn-on' fluorescent probe based on aggregation-induced emission (AIE) has been developed. It exhibits excellent selectivity and sensitivity for monitoring angiotensin converting enzyme (ACE) activity both in solutions and in living cells as well as fo

Full text of DOI:10.1039/c4cc07161a

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: H. Wang, Y.
Huang, X. Zhao, W. Gong, Y. Wang and Y. Cheng, Chem. Commun., 2014, DOI: 10.1039/C4CC07161A.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

View Article Online
DOI: 10.1039/C4CC07161A
Page 1 of 4
ChemComm
ChemComm
RSCPublishing
ARTICLE
A novel aggregation-induced emission based
fluorescent probe for angiotensin converting enzyme
Cite this: DOI: 10.1039/x0xx00000x
(
ACE) assay and inhibitor screening
a
a
b
b
a*
Haibo Wang , Yi Huang , Xiaoping Zhao , Wan Gong , Yi Wang , and Yiyu
Cheng
a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
A ‘turn-on’ fluorescent probe based on aggregation-induced class of organic fluorophores has been developed to exhibit
emission (AIE) has been developed. It exhibits excellent extraordinary aggregation-induced emission (AIE) behaviours,
selectivity and sensitivity to monitor angiotensin converting which are non-emissive fluorescent in solutions, but their
1
3,14
enzyme (ACE) activity both in solutions and in living cells fluorescent can be turned on in the aggregate state
as well as to screen ACE inhibitors in vitro based fluorophores have aroused great attention due to their
facile processability, high emission e ciency and contrast in
More importantly, AIE-active
. AIE-
.
ffi
Angiotensin converting enzyme (ACE), a zinc-dependent
peptidase known for hydrolyzing angiotensin I into the
vasoconstrictor angiotensin II, plays pivotal roles in the
1
5,16
the aggregated states
.
fluorogens have overcome the notorious ACQ problem.
Therefore, fluorescent probes with AIE characteristics have
provided a new method for biosensors and cell imaging .
However, to our knowledge, little AIE-based probe has been
exploited for inhibitor drug screening.
1
regulation of blood pressure, hematopoiesis , renal
1
7
2
3
development , immune response , and other physiological
4
processes . ACE inhibitors such as captopril and enalapril have
achieved dazzling successes in the treatment of cardiovascular
disorders, primarily hypertension and congestive heart
5
failure .To date, fluorescence resonance energy transfer (FRET)
was the most common method for determination of ACE
6
,7
activity and screening ACE inhibitors . The fluorescent probe
and the quencher acceptor were linked by a specific peptide
which can be recognized and cleaved by ACE. Peptide cleavage
can result in the loss of FRET and the ACE activity can be
8
evaluated via the changes in fluorescence intensity . However,
these fluorescent sensors often require more complicated
synthetic steps and still suffer from photo-bleaching and
9,10
background interference . Moreover, conventional dyes tend
to aggregate in aqueous medium, thus causing the ubiquitous
1
1,12
aggregation-caused quenching (ACQ) e
ff
ect
.
To address these issues, development of fluorescent sensors
Fig. 1 Illustration of TPE-SDKP for ACE Activities Study (A)
with high fluorescent efficiency and high signal-to-noise ratio is and cellular imaging (B).
still in urgent pursuit. Among the diverse candidates, a novel
In this contribution, we report a novel fluorescent probe
containing hydrophobic AIE-based fluorophore
tetraphenylethene (TPE)) and a hydrophilic Ser-Asp-Lys-
a
a
College of Pharmaceutical Sciences, Zhejiang University (Zijingang
(
Campus), Hangzhou, 310058, P. R. China.
b
Pro(SDKP)peptide sequence. The fluorescent probe was
soluble in water completely and non-emissive fluorescent in
aqueous medium. Because the circulating tetrapepetide N-
acetyl-Ser-Asp-Lys-Pro is a natural and specific substrate of the
College of Preclinical Medicine, Zhejiang Chinese Medical University,
Hangzhou 310053, PR China.
†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI:10.1039/b000000x/
1
8,19
N-terminal active site of ACE
, as shown in Fig.1, the
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

View Article Online
DOI: 10.1039/C4CC07161A
ChemComm
Page 2 of 4
ARTICLE
Journal Name
specific peptide sequence could be recognized and cleaved by
ACE. Then, the hydrophobic TPE residues could aggregate and
the fluorescence was switched on in aqueous solution. This
probe could be utilized for monitoring of ACE activity and
determinating of the efficiencies of ACE inhibitors both in
solution and in living cells. By monitoring the activity of ACE
in living cells, we further demonstrated this biosensor can be
applied in cellular imaging and in situ drug screening with high
fluorescence contrast.
As one of typicalAIE-based fluorophore, TPE has a widely
application in biosensor and cell imaging. The TPE-based
fluorescent probe was synthesized through two processes as
shown in Fig.1. Briefly, tetraphenylethylene with carboxylate
group was synthesized by McMurry reactions. Then, TPE-
1
SDKP was prepared by standard solid-phase synthesis. H NMR
and ESI-MS determined that we have obtained the fluorescent
probe successfully. HPLC analysis indicated that the purity of
TPE-SDKP was more than 98%(Fig. S1-S5).
Fig.3(A)Time-dependent PL spectra of TPE-SDKP upon
addition of ACE from 0 to 180 min. ACE = 18.75mM. (B) PL
intensity of TPE-SDKP when incubated with concentration-
dependent ACE, [TPE-SDKP]=200μM. (C) Concentration-
dependent captopril inhibited ACE addition upon TPE-SDKP.
(
D)Plot of (I−I )/I versus different enzyme/proteins, where I
0 0
and I represents the PL intensities of TPE-SDKP in Tris-buffer
0
with or without the protein (0.5mg/mL).
Further exploration of the relationship between ACE and
TPE-SDKP has been performed for optimizing the quantity of
ACE and TPE-SDKP used in the assays. TPE-SDKP with
varied concentrations from 0 to 100μM was incubated with
ACE (18.75 mM) at 37℃. The PL spectra were recorded from
400 to 600nm. As shown in Fig.2C, the PL intensity gradually
increased with the increasing concentration of TPE-SDKP.
When TPE-SDKP was incubated with ACE in the
concentration of 50μM, the PL intensity increased moderately.
Therefore, 50μM of TPE-SDKP was used in following study.
As shown in the Fig.2D, when the concentration of TPE-SDKP
was fixed to 50μM, the PL intensity increased with the
increasing concentration of ACE. Thus, the results suggested
that TPE-SDKP was sensitive to the concentration changes of
ACE, which could be an effective probe for detecting ACE.
The fluorescence detection of ACE hydrolyzing TPE-SDKP
have been explored and gained more possibility to explore its
kinetics by time. The increase of fluorescence could be
attributed to the catalytic kinetics of the peptide. It has been
proved Ser-Asp-Lys-Pro peptide sequence could be recognized
and cleaved in the presence of ACE. As shown in Fig.3A, the
Fig. 2 (A) Photoluminescence (PL) spectra of TPE-COOH and
TPE-SDKP in distilled water. Inset: photographs of TPE-
COOH and TPE-SDKP in water taken under illumination by an
UV lamp.(B) PL spectra of TPE-SDKP upon treatment with
ACE in the presence and absence of inhibitor captopril.(C) PL
spectra of varied concentrations of TPE-SDKP in the presence
of ACE. (D) PL spectra of TPE-SDKP in the presence of varied
concentrations of ACE. The incubation time is 3 h. [TPE-
COOH] = [TPE-SDKP] = 50 μ M; ACE = 18.75 mM; [inhibitor]
=
10 nM; λ = 320 nm.
ex
substrate without ACE sustained
a
low and stable
To explore whether the TPE-SDKP probe can be used in
fluorescence.However, when TPE-SDKP incubated with ACE,
the fluorescence rapidly increased within the first 100 min.
After 100 min, most of the peptide had been hydrolyzed and the
change rate of fluorescence became slower.Furthermore, when
a constant amount of TPE-SDKP incubated with different
concentration of ACE, the similar result was shown in Fig.3B.
The PL intensity became stronger with the concentration
increase of ACE. The intensity was rarely changed when the
concentration of ACE was above 2.2 mU/mL, suggesting most
of the peptides in TPE-SDKP have been enzymolyzed. To
study the potential possibility of the probe for inhibitor
screening, we tested the dose-dependent inhibition of captopril,
a well-known ACE inhibitor. As shown in Fig.3C, the
inhibitory activity was increased along with the concentration
detecting ACE activity, the fluorescence emission blocking
character of TPE-SDKP was compared with TPE (Fig.2A). It
was found that TPE-COOH showed intense fluorescence in
aqueous solution due to the amphiphilic property of TPE-
2
0
COOH . However, the TPE-SDKP was almost non-fluorescent
in the same medium, indicated that TPE-SDKP has a good
solubility in water. A following experiment has been performed
in vitro for ACE activity assay (Fig.2B). Mixtures of TPE-
SDKP and ACE were prepared and incubated with or without
captopril. The results showed that the fluorescence increased
sharply after TPE-SDKP treated with ACE, while impaired
activity of ACE caused by ACE inhibitor captopril could be
sensitively represented in fluorescence signal.
2
| Chemical Communications., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C4CC07161A
Page 3 of 4
Journal Name
ChemComm
ARTICLE
of captopril. The result showed that the IC50 of captopril was stimulated HUVEC show a strong fluorescent signal with
.84nM, which exerted a good accordance with previous approximate 3.7-fold (Fig.S8). Meanwhile, treatment of
7
2
1
reported literature . TPE-SKDP based assay was also applied captopril in PMA-stimulated HUVEC induced a significant
to screen active components from Tongmaiyangxin Pills, a decrease in fluorescent signal. Additionally, we also performed
botanical drug for treating cardiovascular diseases and found real-time imaging experiments in HT-29 human colorectal
two components with ACE inhibitory activity (Fig.S6).
cancer cells and human mesenchymal stem cells (hMSC). As
Moreover, cell system could be much complicated compared shown in Fig.S7, HT-29 showed rare ACE expression, while
with the simple buffer solution, which promoted us to inspect hMSC showed a relatively high expression of ACE, which was
whether the characters of TPE-SDKP could be affected by in accordance to previous report that local rennin angiotension
different enzymes or proteins. TPE-SDKP was incubated with expression participated in the differentiation of hMSC to
2
4
ACE, bovine serum albumin (BSA), trypsin, collagenase I/II, adipocytes . This result suggested that our probe might be used
human serum albumin (HSA), and cytochrome C (CYC). in track the phenotype of hMSC during its differentiation to
Fig.3D showed that compared to ACE, other enzymes/proteins adipocytes.
presented relatively weaker changes in (I-I )/I , which proved
In summary, we have developed a fluorescent probe that
0
0
the proposed biosensor could be used as a specific probe for contained a hydrophobic AIE-based fluorophore (TPE) and a
track ACE related cellular events in vitro. Slight increase of hydrophilic peptide sequence. The fluorescent probe was
fluorescence signal in the presence of BSA and HSA might be soluble in water and non-emissive fluorescent in aqueous
attributed to the electrostatic induced assembly between ε- medium. However, when the specific peptide sequence could
amino-group of lysine in the SDKP peptide and carboxyl anion be recognized and cleaved by ACE, the hydrophobic TPE
groups of BSA, which was in accordance to the reports that residues could aggregate and the fluorescence was switched on
2
2
BSA–TPE derivative conjugates has AIE characteristics .
in aqueous solution. This probe could be utilized for monitoring
ACE activity and evaluating the activities of ACE inhibitors in
solution. The outstanding properties of this probe also highlight
its potential use as a novel living cell imaging probe for
determinating of ACE activity and ACE inhibitors screening.
Acknowledgements
This study was supported by National Key Scientific and
Technological Project of China (no. 2012ZX09304-007).
Notes and references
1
2
X. Z. Shen and K. E. Bernstein, Cell Cycle, 2011, 10, 1363.
J. F. Giani, T. Janjoulia, S. Fuchs, J. Peti-Peterdi, K. E. Bernstein and R.
A. Gonzalez-Villalobos, Faseb J, 2013, 27.
3
4
K. E. Bernstein, R. A. Gonzalez-Villalobos, J. F. Giani, K. Shah, E.
Bernstein, T. Janjulia, Y. Koronyo, P. D. Shi, M. Koronyo-
Hamaoui, S. Fuchs and X. Z. Shen, Biol Chem, 2014.
K. E. Bernstein, F. S. Ong, W. L. B. Blackwell, K. H. Shah, J. F. Giani,
R. A. Gonzalez-Villalobos, X. Z. Shen and S. Fuchs, Pharmacol
Rev, 2013, 65, 1.
5
6
C. G. Smith and J. R. Vane, Faseb J, 2003, 17, 788.
M. C. Araujo, R. L. Melo, M. H. Cesari, M. A. Juliano, L. Juliano and A.
K. Carmona, Biochemistry, 2000, 39, 8519.
Fig. 4 Bright-field (BF), fluorescence (FL), and overlay images
of normal HUVEC,HUVEC treated with PMA, and HUVEC
treated with PMA plus captopril. The images were acquired
using a fluorescence microscope (ZEISS) equipped with DAPI
filter. All images share the same scale bar (50μm).
7
A. K. Carmona, S. L. Schwager, M. A. Juliano, L. Juliano and E. D.
Sturrock, Nat Protoc, 2006, 1, 1971.
8
9
M. A. Sentandreu and F. Toldra, Nat Protoc, 2006, 1, 2423.
K. Li, J. Pan, S. S. Feng, A. W. Wu, K. Y. Pu, Y. T. Liu and B. Liu, Adv
Funct Mater, 2009, 19, 3535.
We further explored the characters of TPE-SDKP for live-
cell imaging of human umbilical vein endothelial cell
1
1
1
0
R. N. Day and M. W. Davidson, Chem Soc Rev, 2009, 38, 2887.
1 Y. Hong, J. W. Lam and B. Z. Tang, Chem Commun (Camb), 2009, 4332.
(
HUVEC). After cultivated for 24 h, HUVEC cells were
2
Z. J. Ning, Z. Chen, Q. Zhang, Y. L. Yan, S. X. Qian, Y. Cao and H.
separately incubated with normal medium, 250nM
phorbolmyristate acetate (PMA) which is a strong stimulus for
Tian, Adv Funct Mater, 2007, 17, 3799.
up-regulation of ACE expression, and PMA plus captopril 13 W. Z. Yuan, P. Lu, S. Chen, J. W. Lam, Z. Wang, Y. Liu, H. S. Kwok, Y.
(
100nM). After 36 h incubation, TPE-SDKP (50μM) was added
Ma and B. Z. Tang, Adv Mater, 2010, 22, 2159.
Y. Hong, J. W. Lam and B. Z. Tang, Chem Soc Rev, 2011, 40, 5361.
A. Qin, J. W. Y. Lam and B. Z. Tang, Prog Polym Sci, 2012, 37, 182.
X. Zhang, X. Zhang, L. Tao, Z. Chi, J. Xu and Y. Wei, J Mater Chem B,
2014, 2, 4398.
into each sample. The bright field and fluorescent images were
captured after 3 hours incubation. As shown in Fig.4, normal
HUVEC show a relatively low fluorescent signal, as previous
literature has reported that normal expression of enzymatically
active ACE in the HUVEC , especially located in the
membrane (Fig.S7). In comparison of normal cells, PMA
1
1
1
4
5
6
2
3
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

View Article Online
DOI: 10.1039/C4CC07161A
ChemComm
Page 4 of 4
ARTICLE
Journal Name
1
1
1
7
8
9
K. Dhara, Y. Hori, R. Baba and K. Kikuchi, Chem Commun (Camb),
012, 48, 11534.
2
A. Rousseau, A. Michaud, M. T. Chauvet, M. Lenfant and P. Corvol, J
Biol Chem, 1995, 270, 3656.
M. Azizi, A. Rousseau, E. Ezan, T. T. Guyene, S. Michelet, J. M.
Grognet, M. Lenfant, P. Corvol and J. Menard, J Clin Invest, 1996,
9
7, 839.
2
2
0
1
X. Xue, Y. Zhao, L. Dai, X. Zhang, X. Hao, C. Zhang, S. Huo, J. Liu, C.
Liu, A. Kumar, W. Q. Chen, G. Zou and X. J. Liang, Adv Mater,
2
014, 26, 712.
R. Kapel, A. Chabeau, J. Lesage, G. Riviere, R. Ravallec-Ple, D.
Lecouturier, M. Wartelle, D. Guillochon and P. Dhulster, Food
Chem, 2006, 98, 120.
2
2
2
2
3
4
J. P. Xu, Y. Fang, Z. G. Song, J. Mei, L. Jia, A. J. Qin, J. Z. Sun, J. Ji
and B. Z. Tang, Analyst, 2011, 136, 2315.
I. V. Balyasnikova, S. M. Danilov, V. R. Muzykantov and A. B. Fisher,
In Vitro Cell Dev Biol Anim, 1998, 34, 545.
K. Matsushita, Y. Wu, Y. Okamoto, R. E. Pratt and V. J. Dzau,
Hypertension, 2006, 48, 1095.
4
| Chemical Communications., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012