N’-acyl-N-methylphenylenediamine as a novel proximity labeling agent for signal amplification in immunohistochemistry

Article abstract of DOI:10.1016/j.bmc.2019.01.036

We synthesized novel phenylenediamine derivatives and evaluated them as labeling agents to label proteins in close proximity to a single electron transfer catalyst. We found that N’-acyl-N-methylphenylenediamine labels tyrosine effectively in a model expe

Full text of DOI:10.1016/j.bmc.2019.01.036

Accepted Manuscript
N ’-Acyl-N-methylphenylenediamine as a Novel Proximity Labeling Agent for
Signal Amplification in Immunohistochemistry
Shinichi Sato, Masaki Yoshida, Kensuke Hatano, Masaki Matsumura, Hiroyuki
Nakamura
PII:
S0968-0896(18)32130-8
https://doi.org/10.1016/j.bmc.2019.01.036
BMC 14728
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 December 2018
22 January 2019
29 January 2019
Please cite this article as: Sato, S., Yoshida, M., Hatano, K., Matsumura, M., Nakamura, H., N ’-Acyl-N-
methylphenylenediamine as a Novel Proximity Labeling Agent for Signal Amplification in Immunohistochemistry,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.036
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
N’-Acyl-N-methylphenylenediamine
Leave this area blank for abstract info.
as a Novel Proximity Labeling Agent
for Signal Amplification in Immunohistochemistry
Shinichi Sato,* Masaki Yoshida, Kensuke Hatano, Masaki Matsumura, and Hiroyuki Nakamura*

Bioorganic & Medicinal Chemistry
N’-Acyl-N-methylphenylenediamine as a Novel Proximity Labeling Agent for Signal
Amplification in Immunohistochemistry
a,
a,b
a
Shinichi Sato , Masaki Yoshida, Kensuke Hatano, Masaki Matsumura, a,b Hiroyuki Nakamura a,


a Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-
8503, Japan.
b School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan.
ARTICLE INFO
ABSTRACT
Article history:
Received
We synthesized novel phenylenediamine derivatives and evaluated them as labeling agents to
label proteins in close proximity to a single electron transfer catalyst. We found that N’-acyl-N-
Received in revised form
Accepted
Available online
methylphenylenediamine labels tyrosine effectively in
a
model experiment using
tris(bipyridine)ruthenium (Ru(bpy)32+) as the single electron transfer catalyst. By changing the
substituents on the nitrogen atom of the phenylenediamine derivatives, the electrochemical
properties of the labeling agent can be drastically changed. On the other hand, horseradish
peroxidase (HRP) also catalyzes the reaction with almost the same oxidation potential as
Keywords:
Protein labeling
Proximity labeling
Signal amplification
Horseradish peroxidase
Single electron transfer
2+
Ru(bpy)3 (~+1.1 V). HRP proximity labeling is applicable to signal amplification in
immunohistochemistry. We evaluated the phenylenediamine derivatives as labeling agents for
HRP proximity labeling and signal amplification, and found that N’-acyl-N-
methylphenylenediamine is a novel and efficient agent for signal amplification using HRP in
immunohistochemistry.
1. Introduction
([PPIX]· +FeIVO), which can abstract a single electron from a
substrate with ~+1.1 V redox potential11. On the other hand, we
Enzyme-linked signal amplification is a key technique to
enhance the immunohistochemical detection of protein, mRNA1,
and other biomolecules.2 This technique enables the high-
sensitivity detection of biomolecules, and is useful not only in
biochemical research but also in evaluation systems in medicinal
research and chemical biology.3 The signal amplification is based
on the covalent bond formation between biomolecules and small
reporter molecules in a local environment surrounding enzyme
molecules. Although several methods based on the avidin-biotin-
peroxidase complex4,5, enzyme-catalyzed nanoparticle assembly6,
quinone methide generation7, and ascorbate peroxidase8 have been
reported, the catalyzed reporter deposition (CARD) method using
horseradish peroxidase (HRP) is the most widely used. As the
covalent labeling agent, reporter-conjugated tyramide, a phenolic
compound (Figure 1), has been used. HRP catalyzes the oxidation
of tyramide by single electron transfer in the presence of hydrogen
peroxide (H2O2). The generated tyramide radical reacts with amino
acid residues, such as tyrosine, tryptophan, histidine, and
cysteine9, in close proximity to HRP10. Most of the signal
amplification reactions that involve peroxidase use tyramide as the
labeling agent, and therefore, the development of new labeling
agent remains important.
have developed a tyrosine covalent labeling reaction that uses
tris(bipyridine)ruthenium (Ru(bpy)32+) as the single electron
transfer
catalyst12,13
.
In
that
method,
N’-acyl-N,N-
dimethylphenylenediamine labels tyrosine through the activation
of ruthenium photocatalyst by visible light irradiation. The redox
2+
potential of Ru(bpy)3 activated by visible light irradiation
(Ru3+→Ru2+) under the physiological condition is almost the same
as that of HRP activated by H2O2 (~+1.1 V), and tyrosine residue,
a phenolic structure14, is oxidized (Figure 2). Therefore, we
hypothesized that the phenylenediamine derivatives would label
amino acid residues, such as a tyrosine residue, even with HRP
instead of ruthenium photocatalyst, and would emerge as a novel
labeling agent for signal amplification. We found that N’-acyl-N-
methylphenylenediamine is a novel agent for signal amplification
using HRP in immunohistochemistry.
HRP is activated by H2O2, and heme in the HRP molecule is
transformed into a highly reactive species called compound I
———
 Corresponding author. Tel.: +81-45-924-5245; fax: +81-45-924-5976; e-mail: shinichi.sato@res.titech.ac.jp
 Corresponding author. Tel.: +81-45-924-5244; fax: +81-45-924-5976; e-mail: hiro@res.titech.ac.jp

on the nitrogen atom increases. Morpholino derivative 4 shows a
relatively high Epa (0.72 V) and a moderate Ipc/Ipa (0.63). N-
isobutyl derivative 5 shows similar properties to N-methyl
derivative 2 (Epa: 0.65 V, Ipc/Ipa: 0.31). In the case of ortho-
substituted derivative 6, a high potential (Epa: 1.03 V) is required
for oxidation, and the generated radical species is unstable (Ipc/Ipa:
0).
Table 1. Electrochemical properties of labeling agents.
Fig 1. Catalyzed reporter deposition (CARD) using horseradish
peroxidase (HRP) and tyramide
On the other hand, we previously reported ruthenium-
photocatalyst-conjugated tyrosine derivative (7) in which
Ru(bpy)32+ is directly conjugated to the amine of L-tyrosine amide,
as a model substrate for the evaluation of tyrosine proximity
labeling using a single electron transfer catalyst15. Using 7 as a
substrate, the labeling properties of 1–6 were compared. We have
reported that 3 labels 7, and that a covalent bond is formed between
the ortho-carbon atom of the phenolic oxygen of the tyrosine
residue and the ortho-carbon atom of the dimethylamino group of
312,15. Compounds 1 and 2 labeled 7 with comparable efficiency to
3 (Figure 3). The labeling efficiency of compounds 4 and 5 was
lower than that of 1 or 2. Although 6 is a regioisomer of 3, 6
showed the property of radical species which is significantly
different from 3. Compound 6 did not label 7 (Figure S8). It was
suggested that para-substituted structure is essential for labeling
tyrosine. Interestingly, in the case of labeling with 2, multi adducts
(bis and tris adducts) were observed (Figure 3). As the oxidation
reaction and the formation of many types of adducts also
proceeded simultaneously,16 we could not identify the structure of
the crosslinked product. The tris adduct might be generated by the
covalent dimerization of 2 on the tyrosine residue. The ability of 2
to form multi adducts on a single amino acid residue is considered
useful in the reporter deposition technique for signal amplification.
As a result of the evaluation using the substrate Ru-(Pro)5-Tyr,
which was previously reported by us,17 the labeling efficiency of 2
was lower than that of 3 (Figure S10). Therefore, 2 is suggested to
be a proximity labeling agent with labeling radius shorter than 3.
Comparing the reactivities of 2 and 5, despite both exhibiting
Fig 2. Single electron transfer catalysts, horseradish peroxidase
2+
(HRP) and tris(bipyridine)ruthenium (Ru(bpy)3
)
2. Results and Discussion
We
have
reported
that
N’-acetyl-N,N-
dimethylphenylenediamine (3) is an efficient tyrosine
modification agent. As the mechanism of the labeling reaction, not
only the pathway via a tyrosyl radical, but also the pathway via a
radical species of a labeling agent is suggested (Figure 2). In both
pathways, the electrochemical properties that can be oxidized by
activated catalysts (~+1.1 V) are essential for efficient labeling
agents. The electron density on the phenylenediamine aromatic
ring, which is influenced by the substituent on the nitrogen atom,
is responsible for the radical properties of the labeling agent. We
prepared N’-acetyl- phenylenediamine (1), N’-acetyl-N-
methylphenylenediamine
dimethylphenylenediamine
(2),
(3),
and
and
N’-acetyl-N,N-
evaluated their
electrochemical properties by cyclic voltammetry (Table 1).
Increasing the number of substituents on the nitrogen atom
decreases Epa, which indicates the potential needed for radical
species generation (1: 0.71 V, 2: 0.66 V, 3: 0.63 V), and increases
Ipc/Ipa, which is an index of the stability of radical species (1: 0, 2:
0.38, 3: 0.94) (See Supporting Information Figures S1–S7 for
details). These results suggest that the radical species of the
labeling agent becomes more stable as the number of substituents

similar radical characteristics, the reactivity of 5 was low and no
multi adducts were detected. This low reactivity might be due to
the bulkiness of the N-alkyl group.
Fig 4. Model experiment using HRP bound to ELISA plate. (A)
Assay system of this experiment. (B) Structures of 8–13. (C)
Fluorescence intensities of samples treated with labeling agents 8–
13.
In the model experiment using BSA as a single kind of
substrate, labeling with 9-11 was less efficient than tyramide 8,
however, we expected that these labeling agents show high
efficiency in experiments using cells. In another model experiment
using HRP, proteins on cell membrane surface were used as the
substrate. HRP was specifically expressed on plasma membrane.
HEK293 cells were transfected with a plasmid for expressing a
protein in which HRP is bound to the N-terminus of the
transmembrane domain of PDGF receptor (HRP-TM)9. Using
HRP-TM expressing HEK293 cells, we evaluated 8–11. After
labeling, the cell lysate was treated with dibenzocyclooctyne-
biotin (DBCO-biotin) to conjugate biotin to the labeled proteins.
Labeling efficiencies were evaluated by detecting the biotinylated
proteins by western blot analysis using SAv-HRP (Figure 5A). The
housekeeping protein, alpha-tubulin, was also detected. Labeling
by 8–11 was confirmed. Even in the control sample that was not
treated with labeling agent, some signals were detected, which
were considered to be endogenous biotinylated proteins and
proteins nonspecifically labeled with DBCO-biotin via a thiol-yne
reaction between the cysteine residues of proteins and DBCO.
Compared to the control sample, labeling of various proteins on
the cell membrane was detected in all the samples treated with 9–
11. In particular, 10 exhibited better labeling efficiency than
tyramide derivative 8 (Figure 5B). Although we were unable to
identify the labeled proteins, labeling agent 10, which strongly
labeled cellular proteins, was thought to be a suitable candidate for
signal amplification using HRP.
Fig 3. Labeling of 7 using phenylenediamine-type labeling agents.
(A) Structure of 7. (B) Detection of labeled 7 with labeling agents
1–3 by mass spectrometry. Left figure: The vertical axis is
magnified 10 times in the area of bis and tris adducts.
Based on the results of model experiments using ruthenium
photocatalyst, the properties of each labeling agent were evaluated
using HRP. As our aim was to apply CARD using HRP, we
compared the labeling efficiencies of labeling agents in the model
experiment for proximity labeling to antigen-bound HRP. In the
model experiment, streptavidin-conjugated HRP (SAv-HRP) was
bound to an ELISA plate immobilized with biotin-modified BSA
and unmodified BSA (Figure 4A). We labeled proteins on the plate
using azide-conjugated labeling agents (8–13) (Figure 4B). Then,
the azide-labeled proteins were functionalized by the click reaction
using dibenzocyclooctyne-cyanine 3 (DBCO-Cy3). Labeling
efficiencies were evaluated by measuring the fluorescence
intensities of wells in the ELISA plate. As a control experiment,
the same protocol was carried out with BSA immobilization, but
without biotin-modified BSA (Figure 4C, background). In the case
of azide-tyramide (8) as positive control, a strong signal was
observed compared to the control (Figure 4C, signal). It was found
that 9–11 promoted labeling with HRP as the catalyst although the
labeling efficiencies in this model experiment were inferior to that
of tyramide 8. We also evaluated N-methyl luminol derivative
(12), a HRP-mediated tyrosine labeling agent18,19, and MAUra
(13), a ruthenium photocatalyst-proximity labeling agent15. These
labeling agents failed to label the protein in close proximity to
HRP. In a solution experiment that used HRP as the catalyst, we
found that 12 labeled BSA more efficiently than tyramide19. In our
model experiment, however, 12 failed to label BSA immobilized
on the plate using HRP bound to the solid phase. Considering the
previously reported reactivity of MAUra and N-methyl luminol
derivative,17 and the result of Figure S10, the effective distance of
radical labeling is presumed to be 11>10>12,13. The labeling
radius of the radical species of 12 and 13 might be too small for
signal amplification labeling using HRP bound to protein.

Fig 5. Model experiment using cells expressing HRP on cell
membrane. (A) Labeling on cell membrane of HRT-TM
expressing HEK293 cells. (B) Western blots of biotin-labeled
protein and tubulin.
Finally, we examined whether this method can be applied to
experiments using HRP-conjugated secondary antibodies. The
signal amplification method is especially useful to confirm the
binding of an antibody that cannot produce a sufficient signal in a
conventional immunostaining method. Therefore, we performed
an experiment where we can amplify a weak signal by signal
amplification. We chose calbindin, which is specifically expressed
in cerebellar Purkinje cells (PCs), as the target of signal
amplification20,21. PCs localize in the PC layer found between the
molecular layer and the granule cell layer in the cerebellar cortex
(Figure 7A), and elaborated dendrites extend into the molecular
layer. These localizations are distinctive, and we considered that
calbindin on PCs is a suitable target for proof of our strategy
because the success of the signal amplification can be easily
judged by identifying the labeled area (Figure S11). In previous
model experiments (Figures 4 and 5), labeled proteins were
visualized by the click reaction between DBCO and azide
structures. However, non-specific protein labeling due to the thiol-
yne reaction had to be considered as well (Figure S12E)22. To
reduce noise signals, we considered conjugating biotin to the
labeling agent directly. Rhee and co-workers used a desthiobiotin
reporter instead of biotin, and accomplished the identification of
desthiobiotin-labeled proteins by mass spectrometry23. The
identification of labeled proteins by mass spectrometry is not
adopted in this study. Nevertheless, we recognize the importance
of desthiobiotin in future applications. Therefore, we synthesized
and used desthiobiotin-conjugated compounds 14–17 (Figure 6)24.
Proteins labeled with desthiobiotin molecules could be visualized
by streptavidin-Texas Red. Fluorescence images were acquired
with a fluorescence confocal microscope. In the detection of
calbindin using a biotin-modified secondary antibody, the signals
were very weak, and it was not possible to detect calbindin-
positive PCs (Figure 7B). On the other hand, clear fluorescence
images were obtained and PCs could be detected after signal
amplification bylabeling with 14 and 16. The efficiencies of signal
amplification by 14 and 16 were almost the same (Figures 7C and
7D). There was no significant enhancement effect even when 14
and 16 were used at the same time (data not shown). This result
suggested that the labeling sites of both agents are the same
(mainly tyrosine residues on proteins). A weak signal was
observed when the sample was treated with 17, and almost no
signal was detected when the sample was treated with 15 (Figure
S12).
Fig 7. Staining of PCs and signal amplification using anti-
calbindin and HRP-conjugated secondary antibodies. (A) Bright-
field image of cerebellum. Lower left side is granular layer, upper
right side is molecular layer, and between those two layers is the
Purkinje cell layer. Exposure time: 1.0 sec. (B) Staining with
biotin-conjugated secondary antibody. Exposure time: 0.5 sec. (C)
Imaging after signal amplification using tyramide 14. Exposure
time: 0.2 sec. (D) Imaging after signal amplification using 16.
Exposure time: 0.2 sec.
3. Conclusions
We found a novel proximity labeling agent for signal
amplification in immunohistochemistry. To identify a novel
compound functioning as a proximity labeling agent, we focused
on N’-acyl-N,N-dimethylphenylenediamine, which we previously
found as a tyrosine labeling agent. We developed the
phenylenediamine-type protein labeling agent in this study.
Experiments indicated that changing the substituent on the
nitrogen atom of N’-acetyl-N-methylphenylenediamine alters the
electrochemical properties of the radical species of the labeling
agents.
Among
the
derivatives,
N’-acyl-N-
methylphenylenediamine emerged as the most efficient proximity
labeling agent for signal amplification using horseradish
peroxidase. However, because oxygen addition and multimer
formation occurred simultaneously, it was not possible to identify
which amino acid residue in the protein is involved in the labeling
reactions. The model experiment using a ruthenium-photocatalyst-
conjugated tyrosine derivative suggested that N’-acyl-N-
methylphenylenediamine binds to tyrosine residues with
oligomerization. This property is required for the deposition of
reporter molecules in the proximity of HRP. These results
demonstrate that the chemical structures of proximity labeling
agents can be modified further for use in signal amplification, and
are not limited to tyramide. Research is ongoing to further improve
the signal amplification effect by the structural development of
labeling agents.
Fig 6. Structures of 14–17.

4. Experimental section
2.87 (d, J = 6.8 Hz, 2H), 2.06 (s, 3H), 1.87-1.80 (m, 2H), 0.95 (d,
J = 6.8 Hz, 6H); 13C NMR (125 MHz, CDCl3) δ 168.7, 145.7,
128.1, 122.5, 112.7, 52.1, 28.0, 24.1, 20.5; FT-IR (neat) 3296,
4.1. General methods
3139, 3068, 2955, 2928, 2869, 1656, 1519, 1473, 1314, 1262 cm-
NMR spectra were recorded on a Bruker biospin AVANCE III
1
;
Mp 78-80 °C; HRMS (ESI, positive): calcd. for
1
(500 MHz for H, 125 MHz for 13C) instrument in the indicated
[C12H18N2O+Na]+: 229.1311, found 229.1311.
solvent. Chemical shifts are reported in units parts per million
(ppm) relative to the signal (0.00 ppm) for internal
4.5. N-(2-(dimethylamino)phenyl)acetamide (6)
1
tetramethylsilane for solutions in CDCl3 (7.26 ppm for H, 77.0
ppm for 13C) or CD3OD (3.31 ppm for H, 49.0 ppm for 13C).
1
To a solution of 3’-aminoacetanilide (500 mg, 3.33 mmol) in
4.00 mL THF was added formaldehyde (5.16 mL). The reaction
mixture was cooled to 0 ºC and stirred for 5 min. Sodium
borohydride (629 mg, 16.7 mmol) was then added slowly to the
reaction mixture and the reaction was stirred at 0 ºC for 20 min.
H2O (5.16 mL) was added and the pH was adjusted to pH 7 using
NaHSO4. The product was extracted into AcOEt. The organic
layers were combined and the solvent was removed in vacuo.
purified by silica gel column chromatography (Toluene : Acetone
Multiplicities are reported using the following abbreviations: s;
singlet, d; doublet, dd; doublet of doublets, t; triplet, q; quartet,
quint.; quintet m; multiplet, br; broad, J; coupling constants in
Hertz. IR spectra were recorded on a JASCO FT/IR-4100
spectrometer. IR spectrum was recorded on a JASCO Corporation
FT/IR-4100 FT-IR Spectrometer. ATR PRO ONE was attached to
the FT/IR-4100 in measuring solid IR spectroscopy by single
reflection attenuated total reflection. Only the strongest and/or
structurally important peaks were reported as the IR data given in
cm⁠−1. The absorption spectra were measured with JASCO V-670.
HRMS (ESI-TOF-MS) were measured with Bruker micrOTOF II.
Only the strongest and/or structurally important peaks are reported
as IR data given in cm-1. High-resolution mass spectra (HRMS)
were recorded on a Bruker ESI-TOF-MS (micrOTOF II).
Analytical thin layer chromatography (TLC) was performed on a
glass plate of silica gel 60 GF254 (Merck). Silica gel (Fuji Silysia,
CHROMATOREX PSQ 60B, 50-200 μm) was used for column
chromatography. Most commercially supplied chemicals were
used without further purification. Compound 1 was purchased
from TCI. Compounds 312, 715, 1112, 1218, 1315 were prepared
according to the previously reported procedures, and the observed
spectra were well consisted to the reported spectra.
1
= 4 : 1) afforded (404 mg, 68%). H NMR (400 MHz, CDCl3): δ
8.45 (s, 1H), 8.33 (d, J = 8.0, 1H), 7.16 (d, J = 8.0 Hz, 1H), 7.11
(t, J = 2.0 Hz, 1H), 7.04 (t, J = 4.0 Hz, 1H), 2.64 (s, 6H), 2.22 (s,
3H).
4.6. Azide-conjugated tyramide (8)
To a solution of tyramine (141.9 mg, 1.03 mmol), 5-
azidopentanoic acid (133.4 mg, 0.93 mmol), DMAP (184.1 mg,
1.51 mmol) and DIEA (0.26 mL, 1.51 mmol) in 7.0 mL of DMF
was added EDCI-HCl (214.2 mg, 1.12 mmol) at room temperature.
After stirring at room temperature for 27 h, AcOEt and H2O were
added to the reaction mixture. The organic layer was washed by
aqueous HCl solution (1 M) and saturated aqueous NaCl solution,
dried over Na2SO4, filtered and concentrated in vacuo. The residue
was purified by silica gel chromatography (Hexane : AcOEt = 1 :
4) to give 8 (162.9 mg, 67%) as a colorless oil. 1H NMR (400 MHz,
CDCl3) δ 7.04 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 5.46
(br, 1H), 3.50 (quint, J = 6.8 Hz, 2H), 3.26 (t, J = 6.8 Hz, 2H), 2.74
(t, J = 6.8 Hz, 2H), 2.16 (t, J = 7.2 Hz, 2H), 1.72-1.65 (m, 2H)
1.61-1.58 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 173.4, 155.3,
129.7, 115.7, 51.0, 41.0, 35.9, 34.6, 28.2, 22.9; FT-IR (neat) 3298,
3019, 2937, 2869, 2691, 2607, 2502, 2097, 1645, 1614, 1594,
1544, 1515, 1453, 1362, 1242, 1172, 1106, 1044 cm-1; HRMS
(ESI, positive): m/z calcd. For C13H18N4O2 [M+Na]+: 285.1322,
found 285.1320.
4.2. N’-acetyl-N-methylphenylenediamine (2)
A solution of 4'-aminoacetanilide (200 mg, 1.33 mmol), methyl
iodide (66.1 µL, 1.07 mmol) and potassium iodide (221 mg, 1.60
mmol) in 8.0 mL acetonitrile was heated at 90 °C for 6 h, and then
was cooled to room temperature and filtered. The filtrate was
concentrated and purified by silica gel column chromatography
(CH2Cl2 : methanol = 15 : 1) to afford a solid product (39.3 mg,
18%). 1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 8.8 Hz, 2H), 7.13
(br, 1H), 6.56 (d, J = 8.8 Hz, 2H), 2.81 (s, 3H), 2.12 (s, 3H); 13C
NMR (125 MHz, CDCl3) δ 168.3, 146.7, 128.2, 122.6, 112.7, 31.2,
24.4; FT-IR (neat) 3296, 3065, 2882, 2812, 1655, 1618, 1520,
1511 cm-1; Mp 83 °C ; HRMS (ESI, positive): calcd for
[C9H12N2O+Na]+: 187.0842, found 187.0842.
4.7. 5-Azido-N-(4-aminophenyl)pentanamide (9)
Isobutyl chloroformate (640 μL, 8.9 mmol) and N-
methylmorpholine (833 μL, 13.9 mmol) were added dropwise to a
solution of 5-azidopentanoic acid (1202 mg, 8.4 mmol) in THF (20
mL) at 0 °C. The resulting solution was stirred at 0 °C. After 1.5
h, a solution of N-(tert-Butoxycarbonyl)-1,4-phenylenediamine
(685 mg, 3.3 mmol) in THF (1 mL) was added and the reaction
mixture was stirred at room temperature for 12 h. The reaction
mixture was quenched with aqueous HCl solution (1 M) to acidify
and washed with AcOEt. A solution of aqueous NaOH solution (6
M) was added to the mixture to become basic. The product was
extracted with AcOEt and the solvent was removed under reduced
pressure. Purification by silica gel column chromatography
(AcOEt: hexane = 1:1) afforded intermediate (Boc derivative).
Then, to a solution of intermediate (907 mg, 27.2 mmol) in CH2Cl2
(40 mL) was added TFA (4 mL, 52.2 mmol) at room temperature
for 2.5 h. The reaction mixture was then diluted with saturated
aqueous NaHCO3 solution and extracted with CH2Cl2 and dried
over MgSO4.The CH2Cl2 solvent was removed under reduced
pressure. Purification by silica gel column chromatography
(AcOEt: hexane = 1:1) afforded compound 9 (2 steps, 503 mg,
25%). 1H NMR (400 MHz, CDCl3) δ 7.53 (s, 1H), 7.23 (d, J = 9.0
4.3. N-(4-morpholinophenyl)acetamide (4).
A solution of 4'-aminoacetanilide (100 mg, 0.665 mmol),
bis(bromoethyl)ether (635 µL, 0.505 mmol), potassium iodide
(188 mg, 1.13 mmol) and potassium carbonate (156 mg, 1.13
mmol) in 15.0 mL acetonitrile was heated at 90 °C for 24 h, and
then was cooled to room temperature and filtered. The filtrate was
concentrated and purified by silica gel column chromatography
(CH2Cl2 : methanol = 15: 1) to afford a solid product (130.4 mg,
89%). 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.8 Hz, 2H), 7.26
(br, 1H), 6.87 (d, J = 8.8 Hz, 2H), 3.85 (t, J = 4.8 Hz, 4H), 3.11 (t,
J = 4.8 Hz, 4H), 2.14 (s, 3H); 13C NMR (125MHz, CDCl3) δ 168.3,
148.4, 130.8, 121.7, 116.4, 67.0, 47.9, 24.5. ; FT-IR (neat) 3276,
3190, 3119, 2958, 2848, 2826, 1654, 1533, 1456, 1119 cm-1; Mp
208 °C ; HRMS (ESI, positive): calcd. for [C12H16N2O2+H]+:
221.1285, found 221.1286
4.4. N-[4-[(2-methylpropyl)amino]phenyl]acetamide (5).
This compound was prepared according to the similar
1
procedure with 2. H NMR (400 MHz, CDCl3) δ 7.83 (br, 1H),
7.24 (d, J = 8.8 Hz, 2H), 6.51 (d, J = 8.8 Hz, 2H), 3.68 (br, 1H),

Hz, 2H), 6.60 (d, J = 8.5 Hz, 1H), 3.42 (br 2H), 3.27 (t, J = 6.8 Hz,
2H), 2.29 (t, J = 7.5, 2H), 1.77-1.71 (m, 2H), 1.65-1.59 (m, 2H);
13C NMR (125 MHz, CDCl3) δ 170.8, 143.5, 129.2, 122.3, 115.4,
51.2, 36.6, 28.4, 22.9; FT-IR (neat) 3418, 3320, 3049, 2950, 2870,
2103, 1652, 1534, 1468 cm-1; Mp 73 °C; HRMS (ESI, positive) :
calcd. for [C11H15N5O+H]+: 234.1349, found 234.1349.
To a solution of N-Boc-p-phenylendiamine (53.7 mg, 0.258
mmol) in MeOH (5 mL), paraformaldehyde (37.4 mg) and sodium
methoxide (67.4mg, 1.248 mmol). The mixture was stirred for 11
h at reflux under an inert atmosphere. Then, the reaction mixture
was reacted with NaBH4 (14.5 mg, 0.383 mmol) at room
temperature and refluxed for an additional 4 h. The mixture was
concentrated in vacuo. The residue was dissolved in saturated
aqueous NH4Cl, was extracted with ethyl acetate and dried over
Na2SO4. The solvent was concentrated in vacuo and was purified
by column chromatography on silica gel with Hexane : ethyl
acetate (7 : 3) to give N’-Boc-N-methyl-p-phenylendiamine as a
yellow oil (45.7 mg, 80%). N’-Boc-N-methyl-p-phenylendiamine
was dissolved in 4 M HCl/dioxane (500 µL) and stirred at room
temperature for 2 hours. The solvent was concentrated in vacuo
and was dissolved in DMF (1 mL). This solution was added D-
desthiobiotin (45.1 mg, 0.210 mmol), EDCI-HCl (41.1 mg, 0.214
mmol), HOBt (28.2 mg, 0.209 mmol) and DIEA (103.4 µL, 0.6
mmol). The mixture was stirred at room temperature for 15 h and
was concentrated in vacuo. The residue was dissolved in saturated
aqueous NaHCO3, was extracted with ethyl acetate and dried over
Na2SO4. Then, the residue was purified by preparative thin layer
chromatography with CH2Cl2 : MeOH = 10 : 1 to give 16 as a white
solid (15.1 mg, 24%). 1H NMR (500 MHz, CD3OD) δ 7.29 (d, J =
8.9 Hz, 2H), 6.61 (d, J = 8.9 Hz, 2H), 3.86-3.80 (m, 1H), 3.74-3.70
(m, 1H), 3.37 (s, 1H), 2.76 (s, 3H), 2.35 (t, J = 7.5 Hz, 2H), 1.73
(quint, J = 7.4 Hz, 2H), 1.56-1.34 (m, 6H), 1.12 (d, J = 6.5 Hz,
3H); 13C NMR (125 MHz, CD3OD) δ 172.8, 164.8, 147.1, 128.2,
121.9, 112.3, 56.0, 51.3, 36.2, 29.7, 28.3, 28.9, 25.8, 25.5, 14.2;
FT-IR (neat) 3242, 3140, 3067, 2925, 2855, 1691, 1652, 1602,
1517, 1430, 1401, 1376, 1347, 1306, 1249, 1176, 1155, 1101,
4.8. 5-Azido-N-(4-(methylamino)phenyl)pentanamide (10)
A mixture of 9 (50 mg, 0.214 mmol), methyl iodide (60.6 mg,
0.428 mmol) and potassium carbonate (50.3 mg, 0.364 mmol) in
5.0 mL acetonitrile was heated at 90 °C for 6 h, then was cooled
to room temperature and filtered. The filtrate was concentrated and
purified by column chromatography on silica gel (AcOEt : hexane
1
= 1: 1) to afford a solid product (8.0 mg, 15%). H NMR (500
MHz, CDCl3) δ 7.29 (d, J = 9.0 Hz, 2H), 7.06 (br, 1H), 6.58 (d, J
= 8.0 Hz, 2H), 3.32 (t, J = 6.8 Hz, 2H), 2.82 (s, 3H), 2.34 (t, J =
7.3, 2H), 1.84-1.78 (m, 2H), 1.71-1.65 (m, 2H); 13C NMR (125
MHz, CDCl3) δ 170.4, 146.5, 128.3, 122.3, 112.9, 51.4, 36.8, 31.3,
28.5, 23.0; FT-IR (neat) 3292, 2929, 2871, 2818, 2096, 1653,
1520, 1457, 1466 cm-1; Mp 68-71 °C; HRMS (ESI, positive):
calcd. for [C12H17N5O+H]+: 248.1506, found 249.1506.
4.9. Desthiobiotin-conjugated tyramide (14).
To a solution of D-desthiobiotin (28.9 mg, 0.135 mmol),
tyramine (21.2 mg, 0.162 mmol) and HOBt・H2O (24.8 mg, 0.162
mmol) were added EDCI-HCl (31.1 mg, 0.162 mmol) at room
temperature. After stirring at room temperature for 32 h, the
reaction mixture was concentrated in vacuo. The residue was
purified by silica gel chromatography (CH2Cl2 : MeOH = 10 : 1 →
1
7 : 1) to give 14 (20.1 mg, 47%) as a white solid. H NMR (400
1062 cm-1; Mp 85-87 °C HRMS (ESI, positive): m/z calcd. for
;
MHz, CD3OD) d 7.02 (d, J = 8.4 Hz, 2H), 6.92 (br, 1H), 6.70 (d,
J = 8.4 Hz, 2H), 3.80 (q, J = 7.2 Hz, 1H), 3.69 (quint, J = 7.2 Hz,
2H), 3.37-3.34 (m, 2H), 2.69 (t, J = 6.8 Hz, 2H), 2.15 (t, J = 7.2
Hz, 2H), 1.60-1.56 (m, 2H), 1.48-1.44 (m, 2H) 1.34-1.25 (m, 4H),
1.10 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 176.1,
166.2, 156.9, 139.1, 131.2, 130.7, 129.6, 126.1, 116.2, 57.4, 52.7,
42.1, 37.0, 35.6, 35.4, 30.9, 30.7, 30.1, 27.1, 26.8, 21.3, 15.6; FT-
IR (neat) 3639, 3243, 3085, 2921, 2854, 1685, 1636, 1556, 1542,
1514, 1431, 1360, 1229, 1149, 1103, 1026 cm-1; Mp 64-67 °C;
HRMS (ESI, positive): m/z calcd. for C18H27N3O3 [M+Na]+:
356.1945, found 356.1950..
C17H26N4O2 [M+Na]+: 341.1948, found 341.1945.
4.12. N’-Desthiobiotin-N,N-dimethylphenylenediamine (17)
A solution of N, N-dimethyl-p-phenylenediamine in DMF
(500µL) was added D-desthiobiotin (23.6 mg, 0.110 mmol),
EDCI-HCl (20.5 mg, 0.106 mmol), HOBt (14.3 mg, 0.106 mmol)
and DIEA (26.0 µL, 0.100 mmol). The mixture was stirred at room
temperature for 22 h and was concentrated in vacuo. The residue
was dissolved in H2O, extracted with ethyl acetate and dried over
Na2SO4. The solvent was concentrated in vacuo and was purified
by column chromatography on silica gel with CH2Cl2 : MeOH (10
: 1) to give 17 as a black solid (8.8 mg, 24%). 1H NMR (500 MHz,
CD3OD) δ 7.81 (s, 1H), 7.39 (d, J = 9.0 Hz, 2H), 6.69 (d, J = 9.0
Hz, 2H), 5.80 (s, 1H), 4.69 (s, 1H), 3.85-3.80 (m, 1H), 3.71-3.68
(m, 1H), 2.90 (s, 6H), 2.33 (t, J = 7.3 Hz, 2H), 1.74 (quint, J=6.9
Hz, 2H), 1.53-1.21 (m, 6H), 1.10 (d, J = 6.5 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 171.1, 163.8, 147.9, 128.2, 121.7, 113.1,
56.0, 51.4, 41.0, 36.7, 29.5, 28.4, 25.7, 25.3, 15.8; FT-IR (neat)
3231, 3109, 3057, 2929, 2852, 2789, 1699, 1653, 1596, 1518,
1457, 1419, 1375, 1340, 1315, 1253, 1161, 1129, 1103, 1057 cm-
1; Mp 138-139 °C ; HRMS (ESI, positive): m/z calcd. for
C18H28N4O2 [M+Na]+: 355.2104, found 355.2103.
4.10. N’-Desthiobiotin-phenylenediamine (15)
Isobutyl chloroformate (364 µL, 2.8 mmol) and 4-
methylmorpholine (308 µL, 2.8 mmol) were added to a solution of
D-desthiobiotin (501.6 mg, 2.3 mmol) in DMF (10 mL). This
solution was stirred at room temperature for 1.5 hour. Then, N-
Boc-p-phenylendiamine (585.5 mg, 2.8 mmol) was added in
resultant mixture and it was stirred at room temperature overnight.
The solvent was removed in vacuo and the residue was purified by
column chromatography on silica gel with CH2Cl2 : CH3OH (10 :
1). Next, the resultant mixture was dissolved in CH2Cl2 (2.7 mL)
and TFA (0.3 mL, 10%) was added. It was stirred at room
temperature for 1.5 hour. The solvent was removed in vacuo to
4.13. Electrochemical measurement.
1
give compound 15 (9.5 mg, 3 steps 1 %) as a yellow solid. H
Electrochemical measurements were made with a Hokuto-
Denko HZ-5000 analyzer (observed in CH3CN; [compound] = ca.
1 mM; [NBu4PF6] = 0.1 M; working electrode: Pt, counter
electrode: Pt, reference electrode: Ag/AgCl; scan rates were 100
mV/s). After the measurement, ferrocene (Cp2Fe) was added to the
mixture and the potentials were calibrated with respect to the
Cp2Fe/Cp2Fe+ redox couple25.
NMR (400 MHz, CD3OD) δ 7.74 (d, J = 8.9 Hz, 2H), 7.33 (d, J =
8.9 Hz, 2H), 3.86-3.79 (m, 1H), 3.73-3.68 (m, 1H), 3.06 (s, 1H),
2.93 (s, 1H), 2.40 (t, J = 7.4 Hz, 2H), 1.73 (quint, J = 7.2 Hz, 2H),
1.51-1.40 (m, 6H), 1.11 (d, J = 6.4 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 137.9, 129.0, 128.2, 125.3, 56.3, 51.7, 35.6, 33.0, 29.2,
29.0, 26.1, 24.7, 21.4, 15.4; FT-IR (neat) 3269, 3060, 2925, 2850,
2606, 1663, 1542, 1512, 1457, 1420, 1378, 1310, 1257, 1177,
1132 cm-1; Mp 84-86 °C; HRMS (ESI, positive): m/z calcd. for
C16H24N4O2 [M+Na]+: 327.1791, found 327.1796.
4.14. Evaluation of labeling efficiency using 7
4.11. N’-Desthiobiotin-N-methylphenylenediamine (16)

4.17. Staining of PC and signal amplification using anti-
calbindin and HRP-conjugated secondary antibody
To a solution of 7 (final concentration 10 μM) in MES buffer
(10 mM, pH 6.0), labeling reagent (a 100 mM stock solution in
DMSO, final concentration 1 mM) were added, and the mixture
was briefly vortexed and incubated at room temperature for 5 min.
The solution was vortexed and the reaction was performed with
the irradiation of the light (RELYON, Twin LED light, 455 nm)
on ice 0.5 cm from the light source for 5 min irradiated with light
on ice for 5 min. The reaction mixture (0.5 µL) with 0.1% TFA 0.5
μL was mixed with 0.5 μL of CHCA solution (0.5 mg/mL solution
in acetonitrile : 0.1% TFA = 1 : 1) on MALDI-TOF plate and dried
at room temperature. The modified protein peaks were detected by
MALDI-TOF MS (Bruker, UltrafleXtreme).
The tissue sections of mouse brain were deparaffinized by
washing these sections in lemosol for 5 minutes, three times, and
in EtOH for 5 minutes, three times. Then, these sections were
washed sequentially with 90% EtOH, 80% EtOH, 70% EtOH,
PBS. These were washed again in PBS twice after treated with 3%
H2O2/MeOH for 30 minutes. Sections were treated with 5%
BSA/PBS and were washed by TTBS three times. The sections
were treated with anti-calbindin rabbit IgG antibody (Cell
Signaling Technology, #2173, 200 times dilution with 1% BSA
PBS) for 2 h followed by secondary antibody (anti-rabbit IgG
biotinylated antibody (Cell Signaling Technology, #14708, 1000
times dilution with PBS) or goat anti-rabbit IgG-HRP (Santa Cruz
Biotechnology, sc-2004, 1000 times dilution with PBS)) for 45
min. After washing by TTBS three times, labeling reactions (4 µM
labeling reagent, 200 µM H2O2, 10 mM Tris buffer) were
performed on sections for 10 min, and sections were washed by
TTBS three times, treated with streptavidin-Texas Red (Vector
Laboratories) for 30 min. After washing by TTBS three times and
PBS once, samples were treated with Hoechst 33342 solution
(Dojindo) for 15 min, washed by TTBS twice, and enclosed with
VECTASHIELD mounting medium (VECTOR Laboratories).
Fluorescence images were observed by fluorescence microscope.
4.15. Evaluation of labeling efficiency using HRP bound to
ELISA plate.
A solution of maleimide-biotin (Aldrich) in DMSO (100 µM)
was added to a solution of BSA (10 µM) in 1×PBS, then incubated
at room temperature for 1 h. After removing excess amount of
maleimide-biotin by Sephadex G-25 column (GE), biotin-
conjugated BSA was obtained. A solution of biotin-BSA (1 µg/mL
in 1×PBS) 100 µL was added to each well on 96 well ELISA plate
(flat-bottom, black, high binding) and incubated at 4 °C for 12 h,
then the ELISA plate was washed three times using 1×PBS 200
µL. A solution of BSA (3% (w/v) in 1×PBS) 200 µL was added to
each same well on ELISA plate and incubated at 37 °C for 4 h,
then the ELISA plate was washed three times using 1×PBS 200
µL. A solution of SAv-HRP (final conc. 1 µg/mL in 1×PBS) 100
µL was added to each same well on ELISA plate and incubated at
room temperature for 1 h, then the ELISA plate was washed three
times using 1×TTBS 200 µL. A solution of azide-conjugated
labeling agents (4 µM) and H2O2 (1 mM) in 1×TBS 100 μL was
added to each well on ELISA plate. The ELISA plate incubated at
room temperature for 10 min, then the ELISA plate was washed
one time using 1×TTBS 200 µL. A solution of DBCO-Cy3
(Aldrich, 10 mM in DMF, final conc. 4 µM in 1×PBS) 100 µL was
added to each well on ELISA plate and incubated at 37 °C for 30
min, then the ELISA plate was washed three times using 1×TTBS
200 µL. Fluorescence intensity of each well on ELISA plate was
measured by microplate reader (Infinite F200).
Acknowledgement
This work was partially supported by Grants-in-Aid for “Grant-
in-Aid for Young Scientists (A) (15H05490 to S. Sato)”,
“Homeostatic regulation by various types of cell death (15H01372
to S. Sato)”, and “Chemistry for Multimolecular Crowding
Biosystems (18H04542 to H. Nakamura)” from MEXT, Japan. We
would like to thank Prof. Hyun-Woo Rhee (Seoul National
University) for donation of HRP-TM plasmid, and Prof. Nobuhiro
Nishiyama and Dr. Makoto Matsui (Tokyo Institute of
Technology) for donation of tissue sections of mouse brain.
Supplementary Material
4.16. Evaluation of labeling efficiency using cells expressing
HRP on cell membrane.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.xxxx.xx.xxx.
HEK293 cells (2 x 105 cells/well) were seeded on 24 well plate
in Dulbecco's Modified Eagle's medium (DMEM) supplemented
with 10% FBS at 37 °C in an atmosphere of 5% CO2 and 100%
humidity for 24 h. Transfections were performed using
Lipofectamine 2000 (Thermo Fisher Scientific, 2.5 µL/well) and
pCAG HRP-TM9 (500 ng/well, the plasmid was donated by Prof.
Hyun-Woo Rhee), and cells were cultured for 24 h. Cells were
treated with each compound (final 500 µM in medium) for 30 min
at 37ºC. H2O2 (final 1 mM) is added to each well. After 1 min
incubation, quencher (the mixture of 50 mM Trolox, 100 mM
Ascorbate and 100 mM NaN3) was added. Medium was removed
and cells were lysed in lysis buffer (20 mM Tris, 150 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1% TritonX). Lysate was treated with
DBCO-biotin (Aldrich final 100 µM) for 1 h after the size
exclusion column using Micro Bio-Spin 6 column (Bio Rad). The
proteins were separated by SDS-PAGE with 4-20% acrylamide
gels (Bio Rad) and were transferred to PVDF membrane. It was
incubated with Immuno Block (DS Pharma) for 1 h. and washed
by TTBS buffer for 15 min in 3 times. HRP-streptavidin was
treated at 4 ºC for 16 h. the membrane was treated with
Immobilon® Forte Western Substrates (Millipore). The
chemiluminescence images were obtained with Fusion Solo 4S
(Vilber Lourmat).
References and notes
1.
Andras SC, Power JB, Cocking EC, Davey MR.
Strategies for Signal Amplification in Nucleic Acid
Detection. 2001;19.
2.
Stack EC, Wang C, Roman KA, Hoyt CC.
Multiplexed immunohistochemistry , imaging , and
quantitationꢀ: A review , with an assessment of
Tyramide signal amplification , multispectral imaging
and multiplex analysis. Methods. 2014;70(1):46-58.
doi:10.1016/j.ymeth.2014.08.016
3.
4.
Li P, Li J, Wang L, Di L. Proximity Labeling of
Interacting Proteinsꢀ: Application of BioID as a
Discovery Tool. 2017;1700002:1-10.
doi:10.1002/pmic.201700002
Hsu S-M, Raine L FH. Use of Avidin-Biotin-
Peroxidase Immunoperoxidase Techniquesꢀ: A
Comparison Complex ( ABC ) in between Unlabeled

Antibody ( PAP ). J Histochem Histochem Cytochem.
1981;29:577-580.
15.
16.
Sato S, Hatano K, Tsushima M, Nakamura H. 1-
Methyl-4-aryl-urazole (MAUra) labels tyrosine in
proximity to ruthenium photocatalysts. Chem
Commun. 2018;54(46):5871-5874.
5.
Toda Y, Kono K, Abiru H, et al. Application of
tyramide signal amplification system to
immunohistochemistry: A potent method to localize
antigens that are not detectable by ordinary method.
Pathol Int. 1999;49(5):479-483. doi:10.1046/j.1440-
1827.1999.00875.x
doi:10.1039/C8CC02891E
These oxidations are observed only when the
phenylenediamine derivatives were used as
labeling agents, and they are not observed with
other labeling agents such as MAUra. The +On
peaks are also observed when Ru(bpy)3Cl2 was
used, which suggests that it is an oxidation in the
ruthenium complex moiety (See Supporting
Information Figure S9).
6.
7.
Pham XH, Hahm E, Kim TH, et al. Enzyme-catalyzed
Ag Growth on Au Nanoparticle-assembled Structure
for Highly Sensitive Colorimetric Immunoassay. Sci
Rep. 2018;8(1):1-7. doi:10.1038/s41598-018-24664-w
Polaske NW, Kelly BD, Ashworth-Sharpe J, Bieniarz
C. Quinone Methide Signal Amplification: Covalent
Reporter Labeling of Cancer Epitopes using Alkaline
Phosphatase Substrates. Bioconjug Chem.
2016;27(3):660-666.
17.
18.
Sato S, Hatano K, Tsushima M, Nakamura H. 1-
Methyl-4-aryl-urazole (MAUra) labels tyrosine in
proximity to ruthenium photocatalysts. Chem
Commun. 2018;54(46):5871-5874.
doi:10.1039/c8cc02891e
doi:10.1021/acs.bioconjchem.5b00652
Sato S, Nakamura K, Nakamura H. Tyrosine-Specific
Chemical Modification with in Situ Hemin-Activated
Luminol Derivatives. ACS Chem Biol.
2015;10(11):2633-2640.
8.
Lee J, Song EK, Bae Y, et al. An enhanced ascorbate
peroxidase 2/antibody-binding domain fusion protein
(APEX2-ABD) as a recombinant target-specific
signal amplifier. Chem Commun. 2015;51(54):10945-
10948. doi:10.1039/c5cc02409a
doi:10.1021/acschembio.5b00440
19.
20.
Sato S, Nakamura K, Nakamura H. Horseradish-
Peroxidase-Catalyzed Tyrosine Click Reaction.
ChemBioChem. 2017;18(5):475-478.
doi:10.1002/cbic.201600649
9.
Rhee HW, Zou P, Udeshi ND, et al. Proteomic
mapping of mitochondria in living cells via spatially
restricted enzymatic tagging. Science (80- ).
2013;339(6125):1328-1331.
doi:10.1126/science.1230593
Whitney ER, Kemper TL, Rosene DL, Bauman ML,
Blatt GJ. Calbindin-D28k is a more reliable marker of
human Purkinje cells than standard Nissl stains: A
stereological experiment. J Neurosci Methods.
2008;168(1):42-47.
10.
Rees JS, Li XW, Perrett S, Lilley KS, Jackson AP.
Selective proteomic proximity labeling assay using
tyramide (SPPLAT): A quantitative method for the
proteomic analysis of localized membrane-bound
protein clusters. Curr Protoc Protein Sci.
2015;2015(April):19.27.1-19.27.18.
doi:10.1016/j.jneumeth.2007.09.009
21.
22.
23.
24.
Kim BJ, Lee SY, Kim HW, et al. Optimized
immunohistochemical analysis of cerebellar Purkinje
cells using a specific biomarker, calbindin D28k.
Korean J Physiol Pharmacol. 2009;13(5):373-378.
doi:10.4196/kjpp.2009.13.5.373
doi:10.1002/0471140864.ps1927s80
11.
Kersten PJ, Kalyanaraman B, Hammel KE,
Reinhammar B, Kirk TK. Comparison of lignin
peroxidase, horseradish peroxidase and laccase in the
oxidation of methoxybenzenes. Biochem J.
1990;268(2):475-480. doi:10.1042/bj2680475
Van Geel R, Pruijn GJM, Van Delft FL, Boelens WC.
Preventing thiol-yne addition improves the specificity
of strain-promoted azide-alkyne cycloaddition.
Bioconjug Chem. 2012;23(3):392-398.
12.
13.
14.
Sato S, Nakamura H. Ligand-directed selective
protein modification based on local single-electron-
transfer catalysis. Angew Chemie - Int Ed.
doi:10.1021/bc200365k
2013;52(33):8681-8684. doi:10.1002/anie.201303831
Lee SY, Kang MG, Shin S, et al. Architecture
Mapping of the Inner Mitochondrial Membrane
Proteome by Chemical Tools in Live Cells. J Am
Chem Soc. 2017;139(10):3651-3662.
Sato S, Morita K, Nakamura H. Regulation of target
protein knockdown and labeling using ligand-directed
Ru(bpy)3 photocatalyst. Bioconjug Chem.
doi:10.1021/jacs.6b10418
2015;26(2):250-256. doi:10.1021/bc500518t
Compounds 1, 2, 3, 14 and 16 showed no cytotoxicity
to living cells. In MTT assay using A549 cells (48 h
incubation), no cytotoxicity was observed even when
a 1 mM solution was treated for 48 h.
Fancy DA, Kodadek T. Chemistry for the analysis of
protein-protein interactions: Rapid and efficient cross-
linking triggered by long wavelength light. Proc Natl
Acad Sci. 1999;96(11):6020-6024.
doi:10.1073/pnas.96.11.6020

25.
Connelly NG, Geiger WE. Chemical Redox Agents
for Organometallic Chemistry. Chem Rev.
1996;96(2):877-910. doi:10.1021/cr940053x