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Project Details
Project Number :
706410
Design, Synthesis and Biological Applications of
Project Title(English) :
Fluorescent Probes for Peroxynitrite and
Hypochlorous Acid 
用于检测过氧亚硝酸根和次氯酸的荧光探针的设计﹑合
Project Title(Chinese) :
成及生物应用 
Principal Investigator(English) :
Prof Yang, Dan 
Principal Investigator(Chinese) :
Department :
Department of Chemistry
Institution :
The University of Hong Kong
E-mail Address :
[email address] 
Tel :
2859 2159 
Co - Investigator(s) :
Panel :
Physical Sciences
Subject Area :
Physical Sciences
Exercise Year :
2010 / 11
Fund Approved :
755,700
Project Status :
Completed
Completion Date :
30-9-2013
Project Objectives :
Develop red fluorescent and ratiometric probes for
detecting peroxynitrite
Develop new-generation green fluorescent and
ratiometric probes for detecting hypochlorous acid
Apply our fluorescent probes to investigate the roles
of peroxynitrite and/or hypochlorous acid in several
biological processes
Abstract as per original application
(English/Chinese):

Realisation of objectives:
We have developed a a rhodol-based probe named
HKYellow and its cell-permeable form HKYellow-AM
with fluorescence excitation and emission
wavelengths at 547 nm and 570 nm, respectively, for
sensitive and selective detection of peroxynitrite in
multiple cell lines and primary cells. An impressive
93-fold fluorescence turn-on response of HKYellow
was observed toward 1 equiv peroxynitrite treatment,
much higher response than that toward potentially
interfering hydroxyl radical or hypochlorous acid.
Moreover, a ratiometric fluorescent probe, FRET-1,
comprising a coumarin and rhodol as the fluorescence
donor and acceptor, respectively, has been found to
1 of 7

exhibit dual fluorescence responses at both 470 nm
and 535 nm upon peroxynitrite addition, and therefore
can be exploited for ratiometric detection and imaging
of peroxynitrite. Based on this design principle, we
then generated three new FRET probes (HJ-2-118,
HJ-2-124, HJ-3-163) which attempt to address
FRET-1’s limitations in cellular retention, photo-
transfer efficiency, and quenching of donor
fluorophore (coumarin). To our delight, in chemical
characterization, the probes showed improved
sensitivity over FRET-1, and ratiometric properties
that can be further validated in cellular systems. To
overcome the drawbacks of our previously published
BODIPY probe HKOCl-1, we have developed
new-generation green fluorescent probes, HKOCl-2
series. HKOCl-2 shows excellent sensitivity and
selectivity for detecting HOCl, and more importantly,
the fluorescent product of HKOCl-2 is much more
stable than that of HKOCl-1. Green fluorescent probe
HKOCl-2b has been reliably exploited in detection of
endogenous hypochlorous acid produced in human
and mouse macrophages stimulated with PMA or
yeast zymosan. More recently, we have discovered a
new switch motif for HOCl detection and developed
ultrasensitive green fluorescent probe HKOCl-3 and
yellow fluorescent probe HKOCl-yellow. Those new
probes are being validated in cellular systems. We
used HKGreen-4A, our fourth-generation peroxynitrite
probe, to establish methodologies for peroxynitrite
detection and imaging in live cells and tissues. The
roles of peroxynitrite in host-pathogen interactions in
immune defense and free radical-mediated tissue
injury in metabolic dysfunction were scrutinized in the
contexts of bacterial infection and atherosclerosis,
respectively. So far, in sensors literature, the kinetics
of peroxynitrite induction following bacterial infection
remains unknown. We address this by using a mouse
macrophage cellular model (RAW264.7 cells) of
Gram-negative bacterial infection to characterize the
time course of peroxynitrite formation in the immune
cells challenged with different pharmacological and
bacterial stimulants.
Summary of objectives addressed:
Percentage
Objectives
Addressed achieved
Develop red fluorescent
1. and ratiometric probes for Yes
100%
detecting peroxynitrite
Develop new-generation
green fluorescent and
2. ratiometric probes for
Yes
100%
detecting hypochlorous
acid
Apply our fluorescent
probes to investigate the
roles of peroxynitrite
3.
Yes
100%
and/or hypochlorous acid
in several biological
2 of 7

processes
Research Outcome
Major findings and research outcome:
HKGreen-4A, our fourth-generation green
peroxynitrite probe, has recently been successfully
concluded as a manuscript submitted to J. Am. Chem.
Soc. It is currently under minor revisions and the
revised version will be submitted before July 5, 2014.
The current manuscript reports on our findings on the
probe’s high selectivity and sensitivity in chemical
characterization, the kinetics of endogenous
peroxynitrite formation induced by bacterial infection
(E. coli), and the enzymatic pathways involved. In
addition, we report the newly established methodology
of live tissue staining with HKGreen-4A in an
ApoE-KO mouse model of atherosclerosis. Using
HKGreen-4 as the probe, we have established a cell
based assay system for the detection of peroxynitrite
with flow cytometry and 96-well plates. Several
manuscripts on HKGreen-4 and its derivatives for
improved performance in cell imaging are in
preparation. HKOCl-2, a series of second-generation
BODIPY hypochlorous acid probes designed to
address the limitations of a previous probe HKOCl-1,
has been published Organic Letters (June 2014). In
this report, we demonstrated that HKOCl-2 probes are
superior to HKOCl-1 in three aspects of comparison:
selectivity, sensitivity and chemo-stability. More
recently, we have discovered a new switch motif for
HOCl detection and developed ultrasensitive green
fluorescent probe HKOCl-3 and yellow fluorescent
probe HKOCl-yellow. Manuscripts summarizing those
two new probes are in preparation. We have
collaborated with Prof. Hidehiko Nakagawa (Graduate
School of Pharmaceutical Sciences, Nagoya City
University, Japan) on the use of our fluorescent probe
HKGreen-3 in development of two peroxynitrite
donors. The results have been published in Chem.
Commun. (2011) and J. Am. Chem. Soc. (2012). We
have collaborated with Prof. Massimo Delledonne
(Dipartimento di Biotecnologie, Università degli Studi
di Verona, Italy) on the use of our fluorescent probe
HKGreen-2 in the first detection of peroxynitrite in
plants. The result has been published in Nitric Oxide
(2011). We have been collaborating with Dr.
Jian-Gang Shen (School of Chinese Medicine, The
University of Hong Kong) on the use of our probes
HKYellow-AM in detection of peroxynitrite in neuronal
cells under hypoxia condition. A manuscript on this
work is in preparation.
Potential for further development of the research Based on our success of developing green and red
and the proposed course of action:
fluorescent probes for detection of peroxynitrite and
hypochlorous acid in cells and tissues, we plan to
focus on the design and synthesis of fluorescent
probes that can be used for in vivo detection of
peroxynitrite and hypochlorous acid in living animals.
In addition, we will also explore further applications of
our probes in elucidating the biological roles of
peroxynitrite and hypochlorous acid in development
3 of 7

using zebrafish models.
Layman's Summary of
Peroxynitrite (ONOO–) is a potent oxidizing and
Completion Report:
nitrating species implicated in numerous diseases
including diabetes, stroke, cancer, and
neurodegenerative disorders. Another important ROS
(reactive oxygen species), hypochlorous acid (HOCl),
figures prominently in innate immune response
against microbial infections, but potentially contributes
to pathological conditions such as atherosclerosis and
Alzheimer's disease. Rapid and accurate detection of
ONOO– and HOCl has been challenging due to their
transient nature and difficulty of achieving high
selectivity and sensitivity for single ROS in probe
design. Thus far, many existing probes for these
species are either employing green-emission
fluorophores or possessing insufficient selectivity
and/or sensitivity for single ROS detection in live cell
or tissues. We have developed the highly selective
and sensitive HKRed-1 and HKYellow as red and
yellow probes for cellular ONOO– detection,
respectively. The ratiometric probe FRET-1 and
analogs have been successfully developed for
exploration in quantitative ONOO– detection. A
HKOCl-2 series of green HOCl probes has been
designed to address practical limitations of our
previously published HKOCl-1. In biological
applications, we used a green ONOO– probe
HKGreen-4A to define the kinetics of ONOO–
formation in mouse macrophages during bacterial
infection, and establish a method for live tissue
ONOO– staining in atherosclerotic mice
Research Output
Peer-reviewed journal publication(s)
Year of
arising directly from this research project :
Author(s)
Title and Journal/Book
Publication
(* denotes the corresponding author)
HKGreen-3: A
Rhodol-Based
T. Peng, D.
2010
Fluorescent Probe for
Yang* 
Peroxynitrite. Org. Lett.,
2010, 12, 4932–4935. 
N. Ieda, H.
Nakagawa*,
Peroxynitrite Generation
T. Horinouchi, from NO-releasing
T. Peng, D.
Nitrobenzene Derivative
2011
Yang, H.
in Response to
Tsumoto, T.
Photoirradiation. Chem.
Suzuki, K.
Comm. 2011, 47,
Fukuhara, N. 6449–6451. 
Miyata*. 
Detection of
Peroxynitrite
F. Gaupels, E. Accumulation in
Spiazzi-
Arabidopsis thaliana
2011
Vandelle, D.
During the
Yang, M.
Hypersensitive Defense
Delledonne* 
Response. Nitric Oxide
2011, 25, 222–228. 
4 of 7

Photo-controllable
N. Ieda, H.
Peroxynitrite Generator
Nakagawa*,
Based on N-Methyl-
T. Peng, D.
2012
N-nitrosoaminophenol
Yang, T.
for Cellular Application.
Suzuki, N.
J. Am. Chem. Soc.
Miyata* 
2012, 134, 2563–2568. 
HKOCl-2 Series of
Green BODIPY-based
Jun Jacob
Fluorescent Probes for
Hu, Nai-Kei
Hypochlorous Acid
Wong,
Detection and Imaging
2014
Qiangshuai
in Live Cells. Organic
Gu, Xiaoyu
Letters. ASAP Articles
Bai, Sen Ye,
Publication Date (Web):
Dan Yang* 
June 20, 2014 (Letter)
DOI:
10.1021/ol501496n 
Tao Peng,
Nai-Kei
Wong,
Xingmiao
Molecular Imaging of
Chen,
Peroxynitrite with
Yee-Kwan
HKGreen-4 in Live Cells
Chan, Derek
and Tissues. Journal of
Hoi-Hang Ho, The American Chemical
Zhenning
Society. Under minor
Sun,
revision. 
Jiangang
Shen, Hani El
Nezami, and
Dan Yang* 
Recognized international conference(s)
Conference
in which paper(s) related to this research
Month/Year/City
Title
Name
project was/were delivered :
Using Synthetic the 1st Korea
Organic
06/2011/Seoul,
Forum on
Chemistry to
South Korea
Organic
Probe Biological Chemistry 
Mechanisms 
Developing
Pacifichem 2010
Highly Selective symposium
Fluorescent
12/2010
“Molecular
Sensors for the
/Honolulu,
Probes and
Detection of
Hawaii, USA
Fluorophores for
Peroxynitrite and Cellular
Hypochlorite in
Imaging” 
Cells 
Using Synthetic
Organic
Asian Chemical
02/2012/Hanoi, Chemistry to
Biology Initiative,
Vietnam
Probe Biological Hanoi Meeting 
Mechanisms 
Inspiration from
TWAS 22nd
09/2011/Trieste, Triptolide: New
GENERAL
Italy
Synthetic
Methods and
MEETING 
5 of 7

Probes 
A Highly
Selective
The 1st
Fluorescent
International
Sensor for
Chinese
Peroxynitrite
Symposium on
08/2012
Detection in a
Free Radical
/Lanzhou, China Mouse
Biology and
Macrophage
Free Radical
Model of
Medicine
Bacterial
Research 
Infection 
Design,
Synthesis and
Applications of
Fluorescent
The 17th
Probes for
Biennial Meeting
03/2014/Kyoto, Molecular
of Society for
Japan
Imaging of
Free Radical
Superoxide,
Research
Peroxynitrite,
International 
and
Hypochlorous
Acid 
Fluorescent
Croucher
Probes for
Advanced Study
12/2013/Hong
Molecular
Institute on
Kong
Imaging of ROS Chemical
and RNS 
Biology 
Inspiration from
Trends in
Natural Products Organic
— New
Chemistry
03/2014/Umeå, Synthetic
Symposium on
Sweden
Methods,
Natural Product
Probes and
Inspired
Protein Targets  Chemistry 
Detection of
Hypochlorous
the 17th Biennial
Acid in Activated Meeting of
Phagocytes with
03/2014/Kyoto,
Society for Free
a Highly
Japan
Radical
Selective and
Research
Sensitive
International 
Fluorescent
Probe 
A Highly
Selective
The 17th
Fluorescent
Biennial Meeting
Probe for
03/2014/Kyoto,
of Society for
Peroxynitrite
Japan
Free Radical
Detection and
Research
Imaging in Live
International 
Cells and
Tissues 
Other impact
Several patents on our fluorescent probes have been
(e.g. award of patents or prizes,
awarded: 1. D. Yang, H.-L. Wang, Z.-N. Sun, J.-G.
collaboration with other research institutions,
Shen. Reagents for Highly Specific Detection of
6 of 7


technology transfer, etc.):
Peroxynitrite. U.S. Patent 7,705,040, 2010. 2. D.
Yang, Z.-N. Sun, Y. Chen, F.-Q. Liu. Reagents for
Detection of Hypochlorous Acid. U.S. Patent
7,858,598, 2010. 3. D. Yang, Z.-N. Sun, Y. Chen, F.-Q.
Liu. Reagents for Detection of Hypochlorous Acid.
European Patent EP2134724B1, 2011. 4. T. Peng, D.
Yang. Luminescence Quenchers and Fluorogenic
Probes for Detection of Reactive Species. U.S. Patent
No. 8,114,904, 2012. 5. T. Peng, D. Yang. Fluorophore
Compounds. U.S. Patent No. 8,148,423 B2, 2012. 6.
D. Yang, H.-L. Wang, Z.-N. Sun, J.-G. Shen.
Reagents for Highly Specific Detection of
Peroxynitrite. Chinese Patent No. ZL200680045462.9,
2012. 7. T. Peng, D. Yang. Fluorophore Compounds.
China Patent Application No. 200980112496.9 filed on
30 September 2010 was approved on 28 May, 2014.
Several patent applications on our new fluorescent
probes have been filed: T. Peng, D. Yang.
Diarylamine-Based Fluorogenic Probes for Detection
of Peroxynitrite. U.S. Provisional Application (No.
61/592,122), filed on Jan. 30, 2012. T. Peng, D. Yang,
J.-G. Shen, X.-M. Chen. Diarylamine-Based
Fluorogenic Probes for Detection of Peroxynitrite. U.S.
Application (No. 13/754,499), filed on 30 January
2013. T. Peng, D. Yang, J.-G. Shen, X.-M. Chen.
Diarylamine-Based Fluorogenic Probes for Detection
of Peroxynitrite. PCT Application, filed on 30 January
2013. In addition, the patent on HKGreen-4 has been
licensed to Invitrogen for commercialization. Dan Yang
has won the following awards: Chinese Young Women
in Science Fellowship, 2011 TWAS Prize in Chemistry,
2010 Dan Yang has been invited to deliver lectures at
the following international conferences or institutions
for the work on fluorescent probes: Keynote lecture at
the 2012 Dorothy Crowfoot-Hodgkin (DCH)
Symposium in University of Zurich (Oct., 2012; Zurich,
Switzerland) Invited lecture at the 8th Pong Ding Yuen
International Symposium on Traditional Chinese
Medicine cum The 2nd International Chinese
Symposium on Free Radical Research & The 6th
Symposium for Three Districts of Cross-straits on
Free Radical Research (Nov. 2014; Hong Kong)
Keynote lecture at the IUPAC 2015 Pusan (Aug. 2015;
Pusan, South Korea) Invited lecture at University of
Toronto (Dec. 2013; Toronto, Canada) Invited lecture
at Fudan University (Oct. 2013; Shanghai, China)
Invited lecture at the ABCI Seminar Series of Hong
Kong Polytechnic University (Jan. 2013; Hong Kong)
Nankai University Lectureship in Organic Chemistry
(Jun. 2013; Tianjin, China)
SCREEN ID: SCRRM00542
7 of 7

Abstract of 706410
As two kinds of highly reactive species generated in biological systems, peroxynitrite and 
hypochlorous acid are important mediators of a variety of physiological and pathological events. 
The abnormal production of peroxynitrite and hypochlorous acid have been implicated in a series 
of human diseases such as vascular diseases, ischemia–reperfusion injury, circulatory shock, 
inflammation, cancer, and neurodegenerative disorders. Unfortunately, further investigations of 
the biological actions of peroxynitrite and hypochlorous acid have been greatly hampered by the 
difficulty of direct and unambiguous detection of them. To facilitate the direct studies of these 
species in biological systems, it is of ultimate importance to develop methods, especially 
fluorescent probes providing high sensitivity, real-time detection and spatial imaging, for the 
detection of peroxynitrite and hypochlorous acid.  
In our previous research funded by RGC, we have developed several efficient green 
fluorescent probes, the HKGreen series and HKOCl-1, for the sensitive and selective detection 
of peroxynitrite and hypochlorous acid, respectively. In this proposal, we plan to further expand 
the repository of our probe family by developing preferable probes, such as red fluorescent 
probes avoiding the interference from the auto-fluorescence of cells, and ratiometric probes 
allowing for quantification of peroxynitrite and hypochlorous acid. Another objective of this 
project is to address certain biological questions related to peroxynitrite and/or hypochlorous 
acid by utilizing our new probes.  
This project is of great significance as it will provide powerful tools to directly visualize 
and quantify peroxynitrite and hypochlorous acid produced in biological systems, which will be 
extremely helpful in studying the pathological roles and mechanisms of these two species in 
related human diseases. This project will also find its merits in elucidating the actions of 
peroxynitrite and hypochlorous acid in immune response, inflammatory diseases and  neuron 
degeneration.  
过氧亚硝酸根和次氯酸是两种在生物体内产生的高活性反应物,它们在很多的生理
和病理过程中都起着非常重要的作用。过氧亚硝酸根和次氯酸的非正常水平产生已经跟很
多的人类疾病联系在一起,包括心血管疾病,缺血-再灌注损伤,循环休克,炎症,癌症
以及神经退行性疾病等等。但不幸的是,对过氧亚硝酸根和次氯酸在生物体内作用的进一
步研究却由于难以直接确定地检测它们而受到极大的阻碍。因此,为了促进对它们生物体
内作用的直接研究,发展对过氧亚硝酸根和次氯酸的检测方法,尤其是具备高灵敏度,实
时检测以及空间成像特点的荧光探针检测方法是非常重要的。
在我们之前获香港研究资助局资助的研究中,我们已经成功地研发出了几种绿色荧
光探针HKGreen系列和HKOCl-1,它们分别实现了对过氧亚硝酸根和次氯酸的高灵敏高
选择性检测。在这份研究计划里,我们打算进一步发展更优越的荧光探针成员来丰富我们
的探针“家族”,比如说红色荧光探针以及比例检测荧光探针。前者可以有效地避免检测
过程中来自细胞自发绿色荧光的干扰,而后者可以实现对过氧亚硝酸根和次氯酸的定量检
测。此外,这份研究计划的另一个目的是利用我们的新探针来解决一些与过氧亚硝酸根或
者次氯酸相关的生物问题。
这份研究计划的重要性在于它将提供一些强有力的工具来直接地观察和定量检测过
氧亚硝酸根和次氯酸在生物体内的产生,这对于研究过氧亚硝酸根和次氯酸在人类疾病中

的病理作用和机制是有很大帮助的。同时,这份研究对于阐明过氧亚硝酸根和次氯酸在免
疫反应,炎症以及神经退化中的作用也有相当大的意义。