08:25-08:30, Yoshiki Chujo (a vice president of the CSJ)

Session 1
Chair: Yanyi Huang (Peking University)1）08:30-08:50, Kenjiro Hanaoka (The University of Tokyo）
Title: Development of Far-red to Near-Infrared Small-molecule
Fluorophores and their Applications to Multicolor Imaging2）08:50-09:10, Chunhai Fan (Shanghai Institute of Applied Physics, Chinese
Academy of Sciences)
Title: Creating dynamic organization with DNA nano structures3）09:10-09:30, Yousuke Takaoka (Tohoku University）
Title: Self-assembling turn-on nanoprobe for endogenous protein sensing4）09:30-09:50, Xing Chen (Peking University)
Title: Live-Cell bioorthogonal Raman imaging5）09:50-10:10, Hiroshi Abe ( Hokkaido University)
Title: DNA templated chemical reaction for detection of nucleic acids

10:10-10:30, Coffee break(10:28-10:30, Introduction of the CSJ journals by Shinichi Suzuki)

Session 2
Chair: Katsunori Tanaka (RIKEN)

6）10:30-10:50, Xingyu Jiang (National Center for NanoScience and Technology
of China)
Title: Live imaging of mitocondria in cells7）10:50-11:10, Tomoya Hirano (Tokyo Medical and Dental University (TMDU))
Title: Development of Various Fluorescent Sensors Based on theConstruction of Fluorescent Compound Library8）11:10-11:30, Zhi Zhu (Xiamen University)
Title: Nucleic Acid Aptamers for Bioanalysis and Biomedicine9）11:30-11:50, Shin Mizukami (Osaka University)
Title: Development of smart 19F MRI probes for in vivo imaging

12:00-13:30, Lunch(group photograph)Session 3
Chair: Zhi Zhu (Xiamen University)

10）13:40-14:00, Jing Zhao (Nanjing University)
Title: Inorganic Bioimaging: Cell and Synthetic Approaches

11）14:00-14:20, Katsunori Tanaka (RIKEN)
Title: Therapeutic In Vivo Synthetic Chemistry: Total Synthesis of Bioactive
Compounds in Live Animals

12）14:20-14:40, Peng Chen (Peking University)
Title: Protein chemistry in living cells

13）14:40-15:00, Mitsuru Hattori (The University of Tokyo)
Title: Development of Bioluminescent Probes for Analysis of Intracellular
Environment and Signaling

15:00-15:20, Coffee breakSession 4
Chair: Kenjiro Hanaoka (The University of Tokyo)

14）15:20-15:40, Qing Huang (Shanghai Institute of Applied Physics, Chinese
Academy of Sciences)
Title: Synchrotron radiation based X-ray imaging for cells

15）15:40-16:00, Toshitada Yoshihara (Gunma University)
Title: Phosphorescent molecular probes for imaging oxygen levels in living
cells and hypoxic tissues

16）16:00-16:20, Fuyou Li (Fudan University)
Title: Upconversion luminescent materials for bioimaging

17）16:20-16:40, Hiromu Kashida (Nagoya University)
Title: Highly-sensitive RNA detection by using In Stem Molecular Beacon

18）16:40-17:00, Yan He (Hunan University)
Title: Single molecule spectroscopy of plasmonic metal nanoparticles

18:00-20:00, Dinner (Quanjude Peking Duck)Development of Far-red to Near-Infrared Small-molecule
Fluorophores and their Applications to Multicolor Imaging
Kenjiro HANAOKA
Graduate School of Pharmaceutical Sciences, The University of Tokyo
Tokyo 113-0033, Japan

Fluorescence imaging is one of the most powerful
techniques for visualization of the temporal and spatial
biological events in living cells, and fluorescence probes
are very useful for this technique. For the development offluorescence probes, the fluorophores are important, forexample, fluorescein has been widely utilized as thefluorescent core for a large number of fluorescence probes.
On the other hand, fluorescence probes in green region
have been sufficiently developed, however, those in far-red
to near-infrared (NIR) color region are still needed.
Therefore, we set out to develop fluorescence probes in farred to NIR color region, since we expected that they would
be useful for multi-color imaging with various kinds of indicators having green
fluorescence such as GFP. So far, we have developed a novel fluorescein analogue,
TokyoMagenta (TM), in which the O atom at the 10 position of the xanthenechromophore of fluorescein is replaced with a Si atom. The absorption and emission
wavelengths of TM were about 90 nm longer than those of fluorescein. We further
developed a red fluorescence probe for β-galactosidase and Ca2+. We have also
developed a novel red fluorescent dye, 2Me SiR600, in which the O atom of Rhodamine
Green at the 10 position of the xanthene moiety is replaced with a Si atom, as a scaffold
for probes to detect protease activity. Furthermore, the NIR fluorescent dyes based on
Si-rhodamines have been developed, and could be utilized for the development offluorescence probes targeted to calcium ions and antibody labeling reagents.
In the talk, I will talk about hypoxia-sensitive fluorescence probes. We focused on the
fluorescence quenching property of azo compounds which are utilized for dark
quenchers and developed green and red fluorescence probes, MAR and MASR, fordetecting hypoxia on the basis of rhodamine green and SiR600 scaffolds. This kind of
azo compounds were also selectively reduced by reductases under hypoxic conditions.

Kenjiro HANAOKA received his B.S. in 2000 from Faculty of Pharmaceutical Sciences, The Universityof Tokyo and then his Ph.D. in 2005 from Graduate School of Pharmaceutical Sciences, The Universityof Tokyo (Prof. Tetsuo Nagano). He was a JSPS research fellow (2005‒2007), and also worked with Prof.
Thomas Kodadek at University of Texas Southwestern Medical Center. In 2007, he was appointed as an
Assistant Professor at Graduate School of Pharmaceutical Sciences, The University of Tokyo. He is
Associate Professor in Graduate School of Pharmaceutical Sciences, The University of Tokyo. His
research interests are in the development of chemical biology tools which can visualize or regulate
biological phenomena, such as fluorescence probes and inhibitors.
Email: khanaoka@mol.f.u-tokyo.ac.jp

Creating Dynamic Organization with DNA Nanostructures
Chunhai Fan
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai
201800, China. E-mail: fchh@sinap.ac.cn
Living bodies are characteristic of dynamic organization. How to reconstruct
dynamic organization in vitro remains a grand challenge. DNA nanotechnology
provides a promising route to this long-term goal. Here I will present several examples
of DNA nanotechnology-enabled dynamic organization, ranging from molecular,
nanometer and macroscopic scale.
I will first demonstrate how we site specifically organize small molecules andbiomacromolecules with self-assembled DNA nanostructures. These nanoassemblies
have various applications for lasing, nanoreactors and nanoagents for intracellularimaging and delivery. We can also anchor metal nanoparticles to DNA nanostructures,endowing these soft materials with unique optoelectronic properties. And last, I willshow an example that we employ DNA nanostrcutres to engineering macroscopic thebiosensing interface. This strategy provides significantly enhanced spatial positioning
range and accessibility of the probes on surface over previously reported linear or stemloop probe structures. We demonstrate the use of this versatile DNA nanostructurebased platform for highly specific and sensitive electrochemical sensing of a wide rangeof biomolecules.

Self-assembling turn-on nanoprobe for
endogenous protein sensing
Yousuke TAKAOKA, Tatsuyuki YOSHII, Keigo MIZUSAWA, and Itaru HAMACHI
Graduate School of Science, Faculty of Chemistry
Tohoku University, Aramakiaza aoba, Sendai, JAPANWe developed a self-assembling turn-on fluorescent
nanoprobes that can be used for fluorescent imaging of protein
biomarkers under live cell contexts, as well as in test tube
settings.
Visualization of tumor-specific protein biomarkers on cell
membranes has the potential to contribute greatly to basic
biological research and therapeutic applications. We recently
reported a unique supramolecular strategy for specific protein
detection using self-assembling 19F-NMR or fluorescent
nanoprobes consisting a hydrophilic protein ligand and a hydrophobic detection
modality. This method is based on recognition-driven disassembly of the nanoprobes,
which induces a clear turn-on fluorescent signal. In the present study, we havesuccessfully extended the range of applicable fluorophores, by newly introducing ahydrophobic module to finely tune the aggregation properties of the probe. This greatly
enhanced the flexibility of the probe design. These probes allowed selective imaging of
membrane-bound endogenously expressed proteins under live cell conditions. Moreover,an extended nanoprobe was successfully used in a cell-based inhibitor assay for enzyme
on the surface of live cells. This highlights the supramolecular approach as reversible
sensing method for the target protein.

Yousuke TAKAOKA received his B.S. from Kyushu University and Ph.D (chemical biology) from
Kyoto University (supervisor: Prof. Hamachi) in 2005 and 2010, respectively. He worked at the
University of Tokyo with Prof. Kenzo Hirose (Postdoc in Neurobiology) and then moved to Kyoto
University with Prof. Itaru Hamachi (Assistant Professor in Chemical biology) from 2011 – 2014. He
joined Tohoku University with Prof. Minoru Ueda (Chemical biology for Natural Products) in July, 2014.
He is lector in Graduate school of Science. His current research interests are protein imaging, proteinlabeling and protein engineering in living cells.
Email: ytakaoka@m.tohoku.ac.jp

Live-Cell Bioorthogonal Raman Imaging
Senlian Hong1, Tao Chen2, Xiangdong Tian3, Liang Lin1, Ming Xiao1, Zhong-Qun Tian3,
Yanyi Huang2, Xing Chen1
1
College of Chemistry and Molecular Engineering, and 2College of Engineering
Peking University, Beijing 100871, China. 3College of Chemistry and Chemical
Engineering, Xiamen University, Xiamen, 361005, China

Live-cell imaging of biomolecules with high specificity and sensitivity as well as
minimal perturbation is essential for studying cellular processes. Here we report thedevelopment of a bioorthognal Raman imaging approach that exploits small Raman
reporters for visualizing various kinds of biomolecules including nucleic acids, proteins,glycans, and lipids. The Raman reproters including the azide, alkyne, nitrile, andcarbon-deuterium bond are small in size and spectroscopically bioorthogonal(background-free). We have demonstrated this strategy by using two Raman imaging
techniques, surface-enhanced Raman scattering (SERS) microscopy and stimulated
Raman scattering (SRS) microscopy. Our live-cell bioorthognal Raman imaging
strategy expands the capabilities of live-cell microscopy beyond the modalities offluorescence and label-free imaging.
Reference:
1.

Lin, L.; Tian, X.; Hong, S.; Dai, P.; You, Q.; Wang, R.; Feng, L.; Xie, C.; Tian, Z.; Chen, X. “A
Bioorthogonal Raman Reporter Strategy for SERS Detection of Glycans on Live Cells” Angew.
Chem. Int. Ed. 2013, 52, 7266-7271.

2.

Hong, S.; Chen, T.; Zhu, Y.; Li, A.; Huang, Y.; Chen, X. “Live-Cell Stimulated Raman Scattering
Imaging of Alkyne-Tagged Biomolecules” Angew. Chem. Int. Ed. 2014, 53, 5827-5831.

3.

Xiao, M.; Lin, L.; Li, Z.; Liu, J.; Hong, S.; Li, Y.; Zheng, M.; Duan, X.; Chen, X. “SERS Imaging of
Cell-Surface Biomolecules Metabolically Labeled With Bioorthogonal Raman Reporters” Chem.
Asian J. 2014, DOI: 10.1002/asia.201402151.

Xing CHEN completed his undergraduate degree in Chemistry from Tsinghua University in 2002. Dr.
Chen then obtained his Ph.D. in Chemistry from University of California, Berkeley in 2007, where his
research focused on chemical biology and bionanotechnology. After completing postdoctoral work at
Harvard Medical School in the field of structural biology and immunology, he joined the Peking
University faculty in September, 2010. The current research interest of Dr. Chen is focused on chemical
glycobiology.
Email: xingchen@pku.edu.cn

DNA templated chemical reaction for detection of nucleic
acids
Hiroshi Abe
Faculty of Pharmaceutical Sciences
Hokkaido University
Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan

Oligonucleotide-templated reactions are powerful tools forthe detection of nucleic acid sequences. One of the major
scientific challenges associated with this technique is therational design of non-enzyme-mediated catalytic templated
reactions capable of multiple turnovers that provide highlevels of signal amplification.
Herein, we report the development of a nucleophilicaromatic substitution reaction-triggered fluorescent probe.
The probe underwent a rapid tem-plated reaction without
any of the undesired background reactions. The fluorogenic
reaction conducted in the presence of a template provided a
223-fold increase in fluorescence after 30 s compared with
the non-templated reaction. The probe provided an efficientlevel of signal amplification that ultimately enabled particularly sensitive levels ofdetection. Assuming a simple model for the templated reactions, it was possible toestimate the rate constants of the chemical reaction in the presence and in the absence ofthe template. From these kinetic analyses, it was possible to confirm that an efficientturnover cycle had been achieved, based on the dramatic enhancement in the rate of thechemical reaction considered to be the rate-determining step. With maximized turnover
efficiency, it was demonstrated that the probe could offer a high turnover number of1500 times to enable sensitive levels of detection with a detection limit of 0.5 pM in the
catalytic templated reactions.

Hiroshi Abe received his B.S. (Pharmacy) and Ph. D. (Medicinal Chemistry) from Hokkaido University
in 1991 and 2001, respectively. He worked at MIT with JoAnne Stubbe (Postdoc in Biochemistry) andthen moved to Stanford with Eric Kool (Postdoc in Bioorganic Chemistry). He joined RIKEN as researchscientist in 2005 and became senior research scientist in 2009. He is Associate Professor in Faculty of
Pharmaceutical Sciences, Hokkaido University. His current research interests are bio-imaging and drug
medicinal chemistry.
Email: h-abe@pharm.hokudai.ac.jp

Imaging and Manipulation of
Mitoncondria in Live Cells using Molecular Aggregates
Wenwen Liu, Zhuo Wang and Xingyu Jiang
National Center for NanoScience and Technology, Beijing, 100190, China

We show here that an organic molecule that can both image
mitochondria and control its activity. This work shows that
aggregates of organic molecules in the nanoscale can both
image the mitochondria, and selectively inactivate
mitochondria to kill tumor cells. This molecule outperforms
existing dyes for mitochondrial imaging not only instability, but also in that it can monitor the mitochondrial
membrane potential (ΔΨm); it may also serve as highly
localized source of singlet oxygen in selective eradicationof cells (such as in cancer treatment) when irradiated withlight.

Xingyu JIANG obtained his B.S. at the University of Chicago (1999), followed by a PhD. (2004) from
Harvard University (Chemistry), working with Professor George Whitesides on microfluidics and cellpatterning. After a short postdoctoral fellowship with Professor George Whitesides, he joined the
National Center for Nanoscience and Technology in 2005 as a professor where he has remained since andis now also serving as the deputy director of the Key Laboratory for Biomedical Effects of Nanomaterials
and Nanosafety (Chinese Academy of Sciences). He is a “Hundred Talents Plan” Professor and Top
Young Talent of National High-level Talents Special Support Plan. He was awarded the National
Distinguished Young Scholars granted by NSFC. His current research interests are surface chemistry,
microfluidics, micro/nano-fabrication, cell biology, immunoassays, and nanomedicine.
Email: xingyujiang@nanoctr.cn

Development of Various Fluorescent Sensors Based on theConstruction of Fluorescent Compound Library
Tomoya Hirano
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University(TMDU), 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Fluorescent sensors, whose fluorescence properties arechanged by each analyte such as ion, small molecule andenvironment change, have been utilized in various fields ofscientific research. For development of such sensors, ourgroup has constructed a library of fluorescent compounds.
We chose coumarin as a core structure mainly due to thosevarious fluorescent properties by each substituent and its
drug-like structure that would be suitable for developmentof fluorescent bioactive compounds.
3-azido-and/or-6-bromocoumarins were set as the starting
materials, and Suzuki-Miyaura coupling with various arylboronic acid moieties and Huisgen 1,3-cycloadditon witharylacetylenes were performed to construct the library. From this library, several
fluorescent sensors like that for nitric oxide or sodium ion, and multi-analyte sensor, inwhich the modes of fluorescent changes induced by two analytes were different fromeach other, that is, one is the change of fluorescent intensity and another is the shift ofemission maximum wavelength, could be obtained. In addition, several environmentsensitive fluorescent molecules like viscosity sensor, whose fluorescent maximum
wavelength was sifted by the change of solvent viscosity, and pH sensor, which could
fluoresce selectively under neutral condition, were also obtained.
As well as those sensors, fluorescent bioactive compounds also could be developed
based on this library. For example, fluorescent ligands for progesterone receptor (PR),which is related to female reproductive function and some diseases, could be foundfrom the library. The fluorescence of this ligand was increased in the presence of PR,
which suggested that it would be utilized for the study of the physiological function of
PR. Thus, our methodology could be utilized as a facile method for development ofvarious functional fluorescent molecules.

Tomoya HIRANO received his B.S. in 1997 from the Faculty of Pharmaceutical Sciences, theUniversity of Tokyo, and M.S. in 1999 and Ph.D. in 2002 from Graduate School of Pharmaceutical
Sciences of the University of Tokyo (Supervisor: Professor Tetsuo Nagano). During 2002-2004, he was a
JSPS research fellow, and also worked with Professor Kevan M. Shokat at University of California, San
Francisco in 2003-2004. He became an Assistant Professor at Graduate School of Biomedical Science,
Tokyo Medical and Dental University in 2004, and became an Associate Professor at Institute of
Biomaterials and Bioengineering, Tokyo Medical and Dental University in 2009. His research interests
are in the development and applications of functional molecules for elucidating of physiological functions
and medical purpose.
Email: hira.chem@tmd.ac.jp

Nucleic Acid Aptamers for Bioanalysis and Biomedicine
Zhi ZHU, Yanling SONG, Xilan LI, Huimin ZHANG, Chaoyong YANG
Department of Chemical Biology, College of Chemistry and Chemical Engineering,
Xiamen University, Xiamen, 361005, China

Aptamers are single-stranded oligonucleotides that canbind different types of targets, such as small molecules,
proteins, cells, and even tissues, with high affinity andspecificity. In addition, their robustness, ease of synthesis
and modification, high stability, as well as low
immunogenesis are some of the defining properties thatmake aptamers desirable diagnostic and biosensing tools.
Therefore, the aptamer probes have found their wide
applications in bioanalysis and biomedicine, including
biosensing, biomarker discovery, drug screening, targeted
delivery, gene regulation and disease diagnosis. In thistalk, I will present some recent progress from our groupon nucleic acid aptamer selection, optimization, structure
modification and their applications in bioanalysis and biomedicine.

Zhi ZHU received her B.S. (Chemistry) from Peking University in 2006 and Sc.D. (Analytical Chemistry)from University of Florida in 2011. She joined Xiamen University faculty in 2011. She is Associate
Professor in Department of Chemical Biology, College of Chemistry and Chemical Engineering. Her
current research is particularly focused on molecular recognition, molecular engineering, point-of-care
testing, and microfluidics.
Email: zhuzhi@xmu.edu.cn

Development of smart 19F MRI probes for in vivo imaging
Shin Mizukami
Graduate School of Engineering, Osaka University, Suita 565-0871, Japan
Immunology Frontier Research Center (IFReC), Osaka University, Suita 565-0871,
Japan

Magnetic resonance imaging (MRI) is one of themost promising technologies for in vivo studies, because
MRI can visualize deep regions in living animals. We
have focused on 19F MRI and developed the activatablesmall-molecule probes to detect protease activities on thebasis of a paramagnetic relaxation-based off/on switching
principle. However, the in vivo application of these small
molecule-based probes was not successful due to theweak signal intensity. Therefore, we improved thesensitivity of 19F MRI probes and succeeded to develop
core-shell nanoparticle PFCE@SiO2, which involves aliquid perfluorocarbon (PFCE: perfluoro crown ether)core and silica shell. The strong 19F MRI signals weresuccessfully detected from deep regions of the living mice bodies. Covering the surfaceof PFCE@SiO2 with polyethylene glycol (PEG) elongated the retention time in the blood.
When this PEGylated nanoparticle was injected to tumor-bearing mice, the 19F MRI
signals were observed from the tumor tissues.
Then, we tried to develop the off/on signal regulation of 19F MRI signals of PFC@SiO2
nanoparticles. To attenuate 19F MRI signal of PFCE@SiO2, paramagnetic species weremodified on the surface of the nanoparticle. Modification of Gd3+ complexes on thenanoparticle surface attenuated the 19F MRI signals in the Gd3+-density-dependent
manner. For further application, the paramagnetic species were conjugated to
PFC@SiO2 with an enzyme substrate peptide linker. By using this smart nanoparticle
probes, the enzyme reaction was detected by 19F MRI. This result indicates thepromising future of 19F MRI for visualization of enzyme activities in deep tissues ofliving animals.

Shin Mizukami received his B.S. (Pharmaceutical Sciences) and Ph.D. (Pharmaceutical Sciences) fromthe University of Tokyo in 1997 and 2002, respectively. He worked at National Institute of Advanced
Industrial Science and Technology (2002-2004, Postdoc in Materials Science) and then moved to
Stanford University (Prof. Eric T. Kool lab) (2004-2005, Postdoc in, Bioorganic Chemistry). He joined
Osaka University as Assistant Professor in 2005. He is Associate Professor in Graduate School ofEngineering, and Immunology Frontier Research Center (IFReC), Osaka University (Prof. Kazuya
Kikuchi lab). His current research interest is development of new methodologies of chemical biology,
such as imaging and regulation of biological signals with organic chemistry, materials science, andbiochemistry.
Email: smizukami@mls.eng.osaka-u.ac.jp

Inorganic Bioimaging: Cellular and Synthetic Approaches
Ling ZHANG, Wei WEI, and Jing ZHAO
School of Life Sciences
Nanjing University, Nanjing 210093, China

The inorganic species play important roles in physiological activity and diseases. We
have developed several approaches to detect the inorganic species.
Specifically, our work can be divided into three parts:(1) detecting heavy metal ions based on whole cells. Taking advantage of MerR family
metalloproteins, we developed a whole-cell gold biosensor that allows the selective
detection of gold ions by naked-eyes (Fig1).
(2) imaging the gasotransmitters by small molecule fluorescent dyes. We designed andsynthesized a series dual-function colorimetric and ratiometric fluorescent dyes for H2S
with facile structural manipulation.
(3) developing the first label-free fluorescence method that can selectively detect andimage inorganic drug cisplatin. Our method provides a starting point for fast
determination of cisplatin.
We hope that these biosensors will be powerful tools to study the roles of inorganic
species in physiological activity or diseases.

Fig. 1 Whole-cell based gold biosensor

Fig. 2 Ratiometric fluorescent probe for H2S

Jing ZHAO received his B.S. (Chemistry) from Nanjing University in 1998 and Ph.D. (Inorganic
Chemistry) from Yale University in 2005.. He worked at the University of California, Berkeley with
Dean Toste (postdoc in organometallic chemistry) and then moved to the University of Chicago with
Chuan He (postdoc in chemical biology). He joined Nanjing University faculty in 2008. He is Professor
in School of Life Sciences, Nanjing University. His current research interests are imaging inorganic
species with fluorescent dyes and cells based on metalloprotein structures.
Email: jingzhao@nju.edu.cn

Therapeutic In Vivo Synthetic Chemistry:
Total Synthesis of Bioactive Compounds in Live Animals
Katsunori TANAKA
Biofunctional Synthetic Chemistry Laboratory, RIKEN, Saitama 351-0198, Japan
Biofunctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan Federal
University, Kazan 420008, Russia

We are synthetically exploring the overlooked reactivityof the N-alkyl unsaturated imines, which are readily
derived from the various aldehydes and primary amines inbiosystems. The new reactivity of imines could then be
used to investigate the biological functions and in vivo
dynamics of the natural products, e.g., polyamines andglycans, and even to challenge the multi-step synthesis of
the biologically active molecules in live animals. Tworecent examples of are discussed.
1. New reactivity of N-alkyl unsaturated imines fromaminoalcohols and diamines: Synthetic application andtheir biological functions
We found that ,-unsaturated imines, obtained fromunsaturated aldehyde and aminoalcohols or diamines, could readily participate in the[4+4], [4+2], or [4+2+2] cycloadditions depending on the substrates. These reactions infact proceed in biosystems and regulate the various biofunctions. For instances, the[4+4] products derived from the polyamines and acrolein contribute new mechanisms
underlying acrolein-mediated oxidative stress and diseases.
2. In vivo glycan dynamics studies: application to natural products synthesis in live
animals
Efficient and general bioconjugation of various glycan molecules on the proteins andlive cells was established based on the rapid 6-azaelectrocyclization of the N-alkyl
unsaturated imines. Noninvasive whole body imaging and dissection experiments
revealed the organ- and cell-selective distribution of the neoglycoproteins depending on
their glycan structures. These glycan-based in vivo delivery systems were efficiently
applied to the natural products synthesis in live animal.

Katsunori TANAKA received his received his BS (1996) and Ph.D. (2002) from Kwansei Gakuin
University in Japan, under the direction of Professor Shigeo Katsumura. After a post-doc with Professors
Koji Nakanishi at Columbia University, New York (2002-2005), he was appointed as an AssistantProfessor in Osaka University. He moved to RIKEN (Biofunctional Synthetic Chemistry Laboratory) as
an Associate Chief Scientist in 2012. In 2014, he was also appointed as an international Professor,
Biofunctional Chemistry Laboratory, Kazan Federal University in Russia. His research interests include
exploring new synthetic methods for total synthesis (either in flask, microfluidics, live cells, or animals),
biological evaluation, molecular imaging, and molecular recognition of natural products.
Email: kotzenori@riken.jp

Protein Chemistry in Living Cells
Peng Chen1,2
Beijing National Laboratory for Molecular Sciences & Molecular Engineering
Synthetic and Functional Biomolecules Center, Peking University, Beijing 100871, China. 1
Peking-Tsinghua Center for Life Sciences, Beijing, China.2

Research in my laboratory has been centered on exploring and applying novel chemistrybased platform technologies to modulate protein-interactions and activities in living cells,
particularly with proteins involved in infectious diseases. In recent years, a new techniquetermed “Genetic-code Expansion” has become a highly powerful method for introducing
unnatual amino acids, beyond the canonical twenty, into a protein of interest (POI) in a
precisely controlled manner. Employing this strategy, we introduced diverse chemical
reactive groups into POIs inside living systems, with the applications ranging from protein
conjugation, protein activation to the study and imaging of protein interactions. Together, we
aim to develop novel chemical toolbox to study proteins within their native biological context,
which is advantageous because these biomolecules can be manipulated with anunprecedented precision.

Peng Chen obtained his BS degree in Chemistry from Peking
University in 2002 and Ph.D with Prof. Chuan He in Chemistry at The
University of Chicago in 2007. After a postdoctoral training with Prof.
Peter Schultz at The Scripps Research Institute between 2007 and2009, he started his independent career as an Investigator in
Chemical Biology at Peking University in July 2009. His Lab isinterested in developing and applying novel chemistry tools toinvestigate protein-based interactions and activities in living cells. He
received NSFC Distinguished Young Scholar Award in 2012, China
Young Scientists Award in 2013 and Chemical Society Review
Emerging Investigator lectureship in 2014.

Development of Bioluminescent Probes for Analysis ofIntracellular Environment and Signaling
Mitsuru HATTORI, Hideo TAKAKURA and Takeaki OZAWA
Department of Chemistry, School of Science, The University of Tokyo,
Tokyo 1130033, Japan

Bioluminescence technology based luciferase proteins has
been widely applied to a quantitative measurement and an
imaging for biomolecules. We have developed a new
bioluminescence technique based on complementation of
split luciferase fragments. It enables to detect the timing ofprotein-protein interaction by bioluminescence photon
counts. Using the technique, we can monitor several
protein dynamics, such as the reversible activity, structuralchanging and modification in living cells.
First, we introduce one of the applications for G-protein
coupled receptors (GPCRs) reaction. GPCRs arebiological sensors on plasma membrane. To monitoring
the activity with specific ligand, two of luciferase fragments were fused individuallywith GPCR and arrestin, a cytoplasmic protein binder to GPCR. We generated a stable
cell line expressing these GPCR and arrestin probes, and showed concentrationdependent bioluminescence responses for a specific ligand. Moreover, the highluminescence intensity of probes enabled meaningful imaging of GPCR signal range
from single cell to living subjects.
Second, we focus on an acidification of living tissue. Stress and disorder induce
acidification in tissue level. However, spatiotemporal analysis of the pH variation inliving tissue remains as an important challenge. We developed a bioluminescence pH
indicator based on combination of photo-reactive protein and luciferase-fragmentcomplementation. Bioluminescence of the fusion protein temporary diminished uponlight irradiation, and recovered gradually in the dark. We designated the fusion protein
as Photo-Inactivatable Luciferase (PI-Luc). The recovery time of bioluminescence (RT)was dependent to pH without subjecting to D-luciferin or ATP concentrations. PI-Luc
was induced living mice and tried to construct the RT images with low O2 condition.
Spots showing high RT values were generated in case of ischemic treatment, and the RT
values recovered to normal condition with reperfusion. Consequently, PI-Luc can beused to monitor such oxidative stress in living tissues.
Mitsuru HATTORI received his B.S. (Biology) and Sc.D. (Biology) from Nagoya University in 2003
and 2008, respectively. He worked at the University of Tokyo as a Research Fellow of the Japan Society
for the Promotion of Science and as a Project Researcher. His current research interest is the developmentof bioluminescence methods to monitor biomolecules in living organs.
Email: mitsuru@chem.s.u-tokyo.ac.jp

Synchrotron radiation based X-ray imaging for cells
Qing Huang
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai
201800, China. E-mail: huangqing@sinap.ac.cn
Synchrotron-based X-ray microscopy is a non-destructive imaging technique thatenables high resolution spatial mapping of metals with elemental level detection
methods. Here I will introduce some examples to show the potential usage of thisunique microscopy in cell imaging.
As we know, to detect and localize specific biomolecules in cells with highresolution may be the most important goals of cellular imaging. The resolution ofsynchrotron-based X-ray microscopy can easily extended to the nanoscale, offering
great promise for localization of specific intracellular proteins and organelles. On theother hand, Synchrotron-based X-ray microscopy is an advanced imaging technology
with excellent elemental specificity, provides a new platform for studying interactions
between nanomaterials and living systems. I will show how we use this platform to
study the mechanism of cytotoxicity of Qds and Carbon nanodiamonds.

Phosphorescent molecular probes for imaging oxygen levels in
living cells and hypoxic tissues
Toshitada YOSHIHARADivision of Molecular Science, Graduate School of Science and Technology,
Gunma University, Kiryu, Gunma 3768515, JapanThe oxygen levels in tumors are significantly lower thanthose of normal tissues because of abnormal and/or poorly
developed vasculature. Therefore, measurements of oxygen
levels of the tissues are of growing importance in detection oftumor and therapeutic treatment of tumor by radiation andphotodynamic therapy. Phosphorescence of molecule has been
applied to the observation of behavior of oxygen in cells andtissues, because its intensity and lifetime depend remarkably on
the concentration of oxygen. Thus, phosphorescent probes suchas -diimine complexes of Ru(II), and Pt(II) and Pd(II)complexes of decorated porphyrins have been developed for tumor imaging.
Iridium(III) complexes exhibit intense phosphorescence with moderately long
lifetimes. We used red-emitting iridium complex BTP (Fig. 1), because red emission
exhibits deeper tissue penetration [1]. In this study, we investigated the phosphorescent
properties of BTP in solutions, and performed the phosphorescence imaging experiment
for living cells and tumor-bearing nude mice.
The phosphorescence of BTP was remarkably
quenched by oxygen molecule in solutions andliving cells. The tumor images of nude mice
were obtained 1 hour after the injection of BTP.
The spectral and photophysical properties of
BTP can be easily modified by changing
aromatic ligands. We will also present the
BTP
BTPHSA
phosphorescent properties of the improved
iridium complexes (BTPHSA, Fig. 1) and the Fig. 1 Structures of BTP and BTPHSA
tumor imaging by using these complexes [2, 3].
[1] T.Yoshihara et al., Proc of SPIE, 7190, 71900X-1, 2009.
[2] S. Zhang et al., Cancer Res., 70, 4490, 2010.
[3] T. Yoshihara et al., Proc of SPIE, 8233, 82330A-1, 2012.
Toshitada YOSHIHARA received his M.S. (Physical Chemistry) and Sc.D. (Physical Chemistry) from
Gunma University in 1998 and 2001, respectively. He worked at Max-Planck-Institut fürbiophysikalische Chemie (Göttingen, Germany) with Prof. K. A. Zachariasse, and then moved to National
Institute of Advanced Industrial Science and Technology (Tsukuba, Japan). He joined department ofchemistry Gunma University in 2004. His current research interests are developments of fluorescent andphosphorescent probes for bioimaging, measurements of oxygen concentration in living cells, and opticalimaging of hypoxia in living animals.
Email: yoshihara@gunma-u.ac.jp

Upconversion luminescent materials for bioimaging
Wei Feng, Fuyou Li
Department of Chemistry, Fudan University, Shanghai, 200433, China* Corresponding author: fyli@fudan.edu.cn
Upconversion luminescence (UCL) is a unique process whereby continuous-wave low-energy
light is converted into higher-energy one through sequential multiple photon absorption or energy
transfer.1 Nowadays, the UCL emission can be achieved by f-f transition of the lanthanide ion andtriplet-triplet annihilation (TTA) process (Scheme 1). Lanthanide nanophosphors codoped with
Yb3+ (sensitizer) and Er3+ (activator) become popular to show visible and near-infrared UCL
emission under 980 nm excitation.2-5 Such Yb3+-sensitized upconversion nanoparticles have been
applied in bioimaging and sensing. Furthermore, by loading both sensitizer and annihilator into a
nanosystem, we have constructed water-soluble, high-effective upconversion nanosystems basedon TTA process.6-8 These upconversion nanosystems have been applied for high-contrast UCL
bioimaging in vivo under low power excitation.

Scheme 1. Schematic illustration of the upconversion nanocapsules for bioimaging in vivo
References(1) Auzel, F. Chem. Rev. 2004, 104, 139.
(2) Zhou, J.; Liu, Z.; Li, F. Y. Chem. Soc. Rev., 2012, 41, 1323.
(3) Liu, Y.; Chen, M.; Cao, T. Y.; Sun, Y.; Li, C. Y.; Liu, Q.; Yang, T. S.; Yao, L. M.; Feng, W.; Li,
F. Y. J. Am. Chem. Soc., 2013, 135, 9869.
(4) Liu, J. L.; Liu, Y.; Liu, Q.; Li, C. Y.; Sun, L. N.; Li, F. Y. J. Am. Chem. Soc., 2011, 133,
15276.
(5) Liu, Q.; Sun, Y.; Yang, T. S.; Feng, W.; Li, C. G.; Li, F. Y. J. Am. Chem. Soc., 2011, 133,
17122.
(6) Liu, Q.; Feng, W.; Yang, T. S.; Yi, T.; Li, F. Y. Nat. Protoc., 2013, 8, 2033.
(7) Liu, Q.; Yin, B. R.; Yang, T. S.; Yang, Y. C.; Shen, Z.; Yao, P.; Li, F. Y. J. Am. Chem. Soc.,
2013, 135, 5029.
(8) Liu, Q.; Yang, T. S.; Feng, W.; Li, F. Y. J. Am. Chem. Soc., 2012, 134, 5390.

Highly-sensitive RNA detection by using In Stem Molecular
BeaconHiromu Kashida
Graduate School of Engineering, Nagoya University, Nagoya 4648603, JapanWe develop In-Stem Molecular Beacons (ISMBs) detecting
target DNA or RNA with high sensitivity. These probes canbe used for visualization of mRNA in cells.
Recently, localization of mRNA has been paid muchattention due to its relevance to spatial and temporal protein
expression. To clarify roles of mRNA localization in living
cells, monitoring tools with high sensitivity are strongly
required. We have developed In-Stem Molecular Beacons(ISMB) by incorporating fluorophores (perylene) andquenchers (anthraquinone) into stem portion of molecularbeacon. ISMB could detect target DNA and RNA with higher sensitivity thanconventional molecular beacons. Its detection sensitivity was further improved by theaddition of cationic polymer. We also synthesized ISMB tethering multiple Cy3
fluorophores and quenchers (Nitro Methyl Red), which showed high detection ability
both with DNA and RNA. ISMB could detect endogenous mRNA in fixed cells.
We developed Serinol Nucleic Acid (SNA), which can hybridize natural DNA andRNA with high affinity. Molecular beacon composed of SNA (SNA-MB) showed
extremely high sensitivity for RNA detection; its emission increased by up to 1000
times after the addition of target RNA. In addition, SNA-MB exhibited high resistanceto nucleases. We believe ISMB and SNA-MB can be versatile tools for the visualizationof various kinds of RNA in living cells.

Hiromu Kashida is an associate professor at the Graduate School of Engineering, Nagoya University. He
received his Ph.D. (Engineering) in 2006 from the University of Tokyo under the supervision of Prof.
Makoto Komiyama. He became an Assistant Professor at Nagoya University in 2007. He was promoted
to Associate Professor in 2011. He received the PCCP prize from the Royal Society of Chemistry in 2011
and The Chemical Society of Japan Award for Young Chemists in 2014. His research interests include
the design of artificial nucleic acids and their applications as probes and nano-materials.
Email: kashida@nubio.nagoya-u.ac.jp

Single molecule spectroscopy of plasmonic metal
nanoparticles
Dong Xu, Yinhe Peng, Xiaodong Cheng, Edward S. Yeung and Yan He
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
and Chemical Engineering, Hunan University, Changsha, 410082, China

Optical microscopy is a convenient yet powerfulstrategy for noninvasive studying in complex
surrounding with single event resolution. Observing
molecules one by one can effectively remove
ensemble averaging. Statistical distributions, timedependent fluctuations and rare events could be
revealed from the undisturbed raw data. So far, theoptical imaging method for single molecule detection
is dominated by fluorescence microscopy techniques.
But the inherent photo-instability of dye molecules
prevents long-time, dynamic tracking of individual
fluorescent probes. A promising alternate for this issue
is to use the noble metal plasmon resonance nanoparticles (PRPs) as the optical contrast
agent due to their unique size, shape and composition dependent optical features. One
attractive optical property of PRPs is their resonance frequency could be finely
manipulated via adjusting their size, shape and composition. Another one is theirabsorption and scattering cross-sections are several orders of magnitude larger than dyes.
Moreover, compared with fluorescent molecules, PRPs exhibit no photobleaching,photoblinking, and optical saturation. Here, based on our previous studies on single
molecule PRP imaging, we present improved apparatus and techniques that enable us todetect small PRPs and track their translational and rotational motions inside noisy living
cells. Our methods could be widely applied to study vital biological events with PRPs as
efficient optical contrast agents, and assist the investigation and regulation of the
kinetics of nanomachines to achieve highly efficient gene and drug delivery.
Isx

Isy

Yan HE received his B.S. from Peking University in 1995. He received his Ph.D. (Chemistry) in The
University of Iowa in 2001. He worked as a Postdoc in Iowa State University from 2002 to 2005. He
joined Hunan University faculty in 2005. He is Professor in College of Chemistry and Chemical
Engineering. His current research interests are plasmonic imaging, single molecule, single particle andsingle cell analysis, and self-assembly and long range interactions.
Email: yanhe2021@hun.edu.cn