Tandem photoaffinity labeling of a target protein using a linker with biotin, alkyne and benzophenone groups and a bioactive small molecule with an azide group

Article abstract of DOI:10.1080/09168451.2015.1104240

A novel linker containing biotin, alkyne and benzophenone groups (1) was synthesized to identify target proteins using a small molecule probe. This small molecule probe contains an azide group (azide probe) that reacts with an alkyne in 1 via an azide- alkyne Huisgen cycloaddition. Cross-linking of benzophenone to the target protein formed a covalently bound complex consisting of the azide probe and the target protein via 1. The biotin was utilized via biotin-avidin binding to identify the cross-linked complex. To evaluate the effectiveness of 1, it was applied in a model system using an allene oxide synthase (AOS) from the model moss Physcomitrella patens (PpAOS1) and an AOS inhibitor that contained azide group (3). The cross-linked complex consisting of PpAOS1, 1 and 3 was resolved via SDS-PAGE and visualized using a chemiluminescent system. The method that was developed in this study enables the effective identification of target proteins.

Full text of DOI:10.1080/09168451.2015.1104240

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Tandem photoaffinity labeling of a target
protein using a linker with biotin, alkyne and
benzophenone groups and a bioactive small
molecule with an azide group
Tomoaki Anabuki, Miu Tsukahara, Hideyuki Matsuura & Kosaku Takahashi
To cite this article: Tomoaki Anabuki, Miu Tsukahara, Hideyuki Matsuura & Kosaku Takahashi
(2016) Tandem photoaffinity labeling of a target protein using a linker with biotin, alkyne
and benzophenone groups and a bioactive small molecule with an azide group, Bioscience,
Biotechnology, and Biochemistry, 80:3, 432-439, DOI: 10.1080/09168451.2015.1104240
To link to this article: http://dx.doi.org/10.1080/09168451.2015.1104240
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Date: 15 February 2016, At: 04:19

Bioscience, Biotechnology, and Biochemistry, 2016
Vol. 80, No. 3, 432–439
Tandem photoaffinity labeling of a target protein using a linker with biotin,
alkyne and benzophenone groups and a bioactive small molecule with an azide
group
Tomoaki Anabuki†, Miu Tsukahara†, Hideyuki Matsuura and Kosaku Takahashi*
Research Faculty of Agriculture, Division of Fundamental Agriscience Research, Hokkaido University, Sapporo,
Japan
Received August 21, 2015; accepted September 28, 2015
http://dx.doi.org/10.1080/09168451.2015.1104240
A novel linker containing biotin, alkyne and ben-
zophenone groups (1) was synthesized to identify
target proteins using a small molecule probe. This
small molecule probe contains an azide group (azide
probe) that reacts with an alkyne in 1 via an azide–
alkyne Huisgen cycloaddition. Cross-linking of ben-
zophenone to the target protein formed a covalently
bound complex consisting of the azide probe and
the target protein via 1. The biotin was utilized via
biotin–avidin binding to identify the cross-linked
complex. To evaluate the effectiveness of 1, it was
applied in a model system using an allene oxide syn-
thase (AOS) from the model moss Physcomitrella
patens (PpAOS1) and an AOS inhibitor that con-
tained azide group (3). The cross-linked complex
consisting of PpAOS1, 1 and 3 was resolved via
SDS–PAGE and visualized using a chemiluminescent
system. The method that was developed in this
study enables the effective identification of target
proteins.
small molecule, limiting the use of photoaffinity probes
for protein identification. In contrast, Budin et al. used
non-denaturing protein electrophoresis to determine that
a complex containing a non-cross-linked target protein
and an azide probe could be created through the Huis-
gen [3 + 2] cycloaddition known as click chemistry,
which is an important bioorthogonal reaction.7–10)
However, separation of proteins by non-denaturing gel
electrophoresis is less effective than separation by
SDS–PAGE. To overcome the limitations of photoaffin-
ity probes and the use of protein gel electrophoresis
under non-denaturing conditions, this study provides a
novel method to identify target proteins using an azide
probe, which is a small bioactive molecule; this probe
is created by introducing an azide group into a small
molecule, which minimizes the loss of both affinity for
the protein and the bioactivity of the original molecule.
A linker containing alkyne, biotin, and photophore
groups connects the azide probe with the target protein.
The linker thus performs the function of binding the
azide probe to the target protein and labeling the pro-
tein for detection, which are roles that conventional
photoaffinity probes have fulfilled until recently. A
summary of the method developed in this study is
shown in Fig. 1: (1) the azide probe is incubated with
the target protein; (2) the linker is added to a mixture
of the azide probe and the target protein, and a
conjugate between the linker and the azide probe is
formed via Huisgen cycloaddition; (3) UV irradiation
causes the complex to bind both the target protein and
the conjugate of the azide probe through the linker’s
benzophenone group; and (4) the ternary complex that
is formed between the target protein, the azide probe
and the linker is identified via Western blotting.
Therefore, the azide probe does not require a pho-
tophore and a tag that could prevent access to the bind-
ing site of the target protein. Thus, the method
proposed in this study can be used to effectively
identify target proteins.
Key words: Huisgen cycloaddition; linker; pho-
toaffinity labeling; target protein; Wes-
tern blotting
Photoaffinity probes have been utilized to identify
target proteins in combination with small bioactive
molecules with photophores and tags for detection.1)
Aryl azide, benzophenone and 3-aryldiazirine are
well-known photophores that are photoactivated by UV
irradiation and used to cross-link small molecules and
target proteins.1–3) Tags such as biotin or fluorescent
groups are used to detect proteins that are cross-linked
with photoaffinity probes.4–6) Photoaffinity probes are
useful for identifying proteins bound to small bioactive
molecules. However, the introduction of photophores
and tags into small molecules often decreases both their
affinity for the target protein and the bioactivity of the
*Corresponding author. Email: kosaku@chem.agr.hokudai.ac.jp
†These authors contributed equally to this study.
Abbreviations: AOS, allene oxide synthase; AOC, allene oxide cyclase; 12,13-EOT, (12,13S)-epoxy-(9Z,11E,15Z)-octadecatrienoic acid; 13-HPOT,
13(S)-hydroperoxylinolenic acid; IPTG, isopropyl-β-D-thiogalactopyranoside; OPDA, 12-oxo-phytodienoic acid.
© 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry

Tandem photoaffinity labeling of a target protein
433
COOH
COOH
α-Linolenic acid
Lipoxygenase
HOO
13-HPOT
Allene oxide synthase (AOS)
O
COOH
12,13-EOT
Allene oxide cyclase (AOC)
O
COOH
(+)-cis-OPDA
O
COOH
Jasmonic acid
Fig. 3. Biosynthesis of jasmonic acid.
(13-HPOT) into (12,13S)-epoxy-(9Z,11E,15Z)-octadeca-
trienoic acid (12,13-EOT),11) which is the first specific
reaction in the biosynthesis of jasmonic acid (Fig. 3).
AOS is classified as cytochrome P450 CYP74A and
contains a heme iron in its active center. The inhibitory
mechanism of cytochrome P450s has been studied.12)
Imidazole derivatives inhibit cytochrome P450s, appar-
ently due to the affinity of the electron pair on the
nitrogen in heterocyclic molecules for the heme iron in
the active center.13,14) Oh and Murofushi synthesized
imidazole derivatives with alkylcarboxylate moieties as
AOS inhibitors and introduced the alkylcarboxylate
moieties into the AOS inhibitors to mimic the partial
structure of 13-HPOT.15)
Fig. 1. Illustration of the method for identifying a target protein in
this study.
O
HN
NH
H
H
H
N
S
O
O
HN
O
O
O
O
1
This study shows that the method described above
enables the identification of target proteins using a
model system of PpAOS1 and an AOS inhibitor intro-
ducing only an azide group (azide probe).
O
Cl
O
R
O
N
2: R = H (AOS inhibitor)
3: R = N3
Cl
N
Experimental
General.
Fig. 2. Structures of linker 1, AOS inhibitor 2, and azide probe 3.
1H NMR spectra were recorded on a
JEOL EX-270 FT NMR (270 MHz) spectrometer
(JEOL Ltd., Tokyo, Japan), and CDCl3 (7.24 ppm) or
CD3OD (3.30 ppm) was used as an internal standard.
Linker 1 that was used in this study is shown in
Fig. 2. Biotin is commonly used as a tag for protein
detection via Western blotting. Benzophenone was
introduced into the linker. Polyether is commonly used
as a hydrophilic spacer, and an alkyne is necessary for
the Huisgen cycloaddition. To examine whether this
strategy is effective for identifying proteins targeted
with small molecules, an allene oxide synthase (AOS)
was selected as the target protein, and AOS inhibitor 2
and azide probe 3 were chosen as the small molecules
for use in this study (Fig. 2).
Mass spectra were collected on
a JEOL JMS-
T100GCV mass spectrometer (JEOL Ltd., Tokyo,
Japan). Specific rotation values were measured on a
JASCO DIP-310 polarimeter (JASCO Corporation,
Tokyo, Japan). Chemical reagents were purchased from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan),
unless otherwise noted.
tert-Butyl {2-[2-(2-hydroxyethoxy)ethoxy]ethyl}carba-
AOS, which is an enzyme involved in the biosyn-
thetic pathway of jasmonic acid (Fig. 3), a plant
hormone, converts 13(S)-hydroperoxylinolenic acid
mate (5).
2-[2-(2-Chloroethoxy)ethoxy]ethanol (4,
6.3 g, 37 mmol), NaI (1.8 g, 12 mmol), and NaN3 (3.9

434
T. Anabuki et al.
g, 60 mmol) were dissolved in 30 mL of deionized
water and then stirred at 60 °C for 16 h. The reaction
mixture was filtered to remove insoluble matter. The
filtered solution was extracted with 60 mL of EtOAc.
The organic layer was dried over Na2SO4 and evapo-
rated to yield the azide (6.5 g). The azide (6.5 g) was
dissolved in MeOH containing 0.5 g of 5% Pd–C and
was stirred under a hydrogen atmosphere at room tem-
perature for 12 h. The reaction mixture was filtered
using Celite and was evaporated to yield the corre-
sponding amine (6.2 g). The amine was dissolved in
150 mL of CH3CN. This was followed by the addition
of 30 mL of deionized water, 30 mL of 1 M NaOH,
and (Boc)2O (8.1 g, 37 mmol) at room temperature for
16 h. The reaction mixture was evaporated and the
resultant residue was added to 100 mL of water and
then extracted with an equal volume of EtOAc. The
organic layer was dried over Na2SO4. After evaporation
of the organic layer, the residue that was obtained was
purified by silica gel column chromatography (200 g,
MeOH-CHCl3 1/50) to yield 5 (6.2 g, 25 mmol) as a
colorless oil. The yield from the three steps was 68%.
1H NMR (270 MHz, CDCl3): 5.21 (1H, br. s), 3.68
(2H, t, J = 7.1 Hz), 3.61–3.52 (6H, m), 3.50 (2H, t,
J = 7.1 Hz), 3.24 (2H, t, J = 6.9 Hz), 2.88 (1H, br. s),
1.37 (9H, s); 13C NMR (67.8 MHz, CDCl3): 155.9,
79.1, 72.5, 72.4, 70.2, 70.1, 61.5, 40.3, 28.2; FD-MS
m/z (rel.int.): 250 (100, [M + H]+), 249 (13.0, [M]+),
57.0 (25.1), 31.0 (16.1); FD-HR-MS m/z: calcd. for
C11H24NO5, 250.16545, found 250.16572.
extracted with 60 mL of EtOAc. The organic layer was
dried over Na2SO4. The resultant matter was purified
by silica gel column chromatography (40 g, n-hexane-
EtOAc 3/2) to yield 7 (90 mg, 0.31 mmol, 20%) as a
1
yellow oil. H NMR (270 MHz, CDCl3): 5.00 (1H, br),
4.18 (2H, d, J = 3.3 Hz), 3.70–3.66 (4H, m), 3.64–
3.56 (4H, m), 3.50 (2H, t, J = 7.3 Hz), 3.28 (2H, t,
J = 7.2 Hz), 2.40 (1H, t, J = 3.3 Hz), 1.41 (s, 9H); 13C
NMR (67.8 MHz, CDCl3): 155.9, 79.5, 79.1, 74.5,
70.4, 70.3, 70.19, 70.17, 69.0, 58.3, 40.3, 28.3; FD-MS
m/z (rel.int.): 288 (100, [M + H]+), 287 (19.0, [M]+),
69.0 (95.7), 57.0 (91.3); FD-HR-MS m/z: calcd. for
C14H26NO5, 288.18110, found 288.18038.
2-[2-(2-Prop-2-ynyloxyethoxy)ethoxy]ethylamine (8).
Compound 7 (90 mg, 0.31 mmol) was dissolved in 50%
TFA-CH2Cl2 (2 ml) and stirred at 0 °C for 2 h. The reac-
tion mixture was evaporated to yield 8 (58 mg,
1
0.31 mmol, 99%) as a yellow oil. H NMR (270 MHz,
CD3OD): 4.80 (2H, s), 4.20 (2H, d, J = 3.1 Hz), 3.68–
3.56 (8H, m), 3.30 (2H, t, J = 7.2 Hz), 2.95 (2H, t,
J = 7.4 Hz), 2.85 (1H, t, J = 3.1 Hz); 13C NMR
(67.8 MHz, CD3OD): 80.5, 76.0, 71.4, 71.25, 71.23,
70.5, 70.0, 59.0, 41.3; FD-MS m/z (rel.int.): 188 (100,
[M + H]+); FD-HR-MS m/z: calcd. for C9H18NO3,
188.12867, found 188.12836.
(+)-5-(2-Oxohexahydrothieno[3,4-d]imidazol-4-yl)-
pentanoic acid (2-(4-benzoylphenyl)-1-{2-[2-(2-prop-2-
ynyloxyethoxy)ethoxy]ethylcarbamoyl}ethyl)amide (1).
(+)-Biotin (9, 24.4 mg, 0.10 mmol), HOBT (13.5 mg,
0.10 mmol), and EDC (19.1 mg, 0.10 mmol) were dis-
solved in DMF (5 mL) and then stirred at room tempera-
ture for 1 h. 2-Amino-3-(4-benzoylphenyl)propanoic
acid (10, 20.0 mg, 0.090 mmol) was added to the reac-
tion mixture, and the mixture was stirred for 16 h. Then,
EDC (19.1 mg, 0.10 mmol) and 8 (15.0 mg, 0.08 mmol)
were added to the reaction mixture, which was then stir-
red for an additional 16 h. The reaction solution was
added to EtOAc (20 ml) and was then washed with
30 mL of 1 M HCl, followed by deionized water. The
organic layer was dried over Na2SO4 and was concen-
trated in vacuo. The resultant residue was purified by sil-
ica gel column chromatography (20 g, CHCl3-MeOH 9/
1) to give 1 (11 mg, 0.017 mmol, 21%) as a yellow oil.
1H NMR (270 MHz, CDCl3): 7.83 (2H, d, J = 8.0 Hz),
7.72 (2H, m), 7.60 (1H, m), 7.44 (2H, m), 7.21 (2H, m),
4.35 (1H, m), 4.15 (2H, m), 3.99 (2H, m), 3.63–3.46
(10H, m), 3.42 (2H, t, J = 7.3 Hz), 3.16 (1H, m), 2.87
(2H, m), 2.68 (2H, m), 2.14 (1H, m), 2.05 (2H, m), 1.72
(2H, m), 1.50 (2H, m), 1.16 (2H, t, J = 7.2 Hz); 13C
NMR (67.8 MHz, CDCl3): 196.4, 179.9, 171.4, 142.2,
138.3, 133.8, 132.6, 130.8, 130.3, 130.1, 129.9, 129.6,
128.6, 128.3, 127.9, 81.8, 77.2, 76.2, 72.7, 72.1, 71.4,
70.7, 70.3, 64.8, 57.9, 57.5, 56.8, 55.2, 51.5, 44.6, 42.7,
33.6, 28.3, 27.9, 24.7; FD-MS m/z (rel.int.): 665 (100,
[M+H]+), 664 (52.1, [M]+); FD-HR-MS m/z: calcd. for
tert-Butyl {2-[2-(2-bromoethoxy)ethoxy]ethyl}carba-
mate (6). Compound 5 (713 mg, 2.85 mmol), CBr4
(1.23 g, 3.70 mmol), and K2CO3 (510 mg, 3.70 mmol)
were dissolved in 5 mL of CH2Cl2. Ten mL of CH2Cl2
containing PPh3 (1.2 g, 4.56 mmol) was added by syr-
inge to the solution and stirred at room temperature for
24 h. After evaporation of the reaction mixture, the
resultant residue was added to n-hexane, and the insol-
uble material was filtered. The resultant material, which
was obtained by evaporation of the filtrate, was purified
by silica gel column chromatography (40 g, n-hexane-
EtOAc, 1/1) to yield 6 (745 mg, 2.40 mmol, 84%) as a
1
colorless oil. H NMR (270 MHz, CDCl3): 4.98 (1H,
br.s), 3.76 (2H, t, J = 7.0 Hz), 3.63–3.55 (4H, m), 3.50
(2H, t, J = 7.1 Hz), 3.43 (2H, t, J = 7.3 Hz), 3.25 (2H,
t, J = 7.1 Hz), 1.39 (9H, s); 13C NMR (67.8 MHz,
CDCl3): 155.8, 79.1, 71.0, 70.3, 70.1, 70.0, 40.2, 30.1,
28.3; FI-MS m/z (rel.int.): 314 (52.5), 313 (100), 312
(58.3, [M + H]+), 311 (94.8, [M]+), 57.0 (84.6); FI-
HR-MS m/z: calcd. for C11H22BrNO4, 311.07322,
found 311.07016.
tert-Butyl {2-[2-(2-prop-2-ylethoxy)ethoxy]ethyl}car-
bamate (7). 10 mL of dry THF was added to a flask
containing 2-propyn-1-ol (560 mg, 10 mmol) and NaH
(480 mg, 20 mmol) and was stirred on ice for 20 min.
After the addition of 6 (488 mg, 1.6 mmol) to the solu-
tion, it was stirred for an additional 2 h. An aqueous
solution of NH4Cl was added to the reaction mixture to
terminate the reaction, and the solution was then
C35H45N4O7S,
665.30089,
found
665.29875;
[α]D25 + 21.0° (c 0.40, MeOH)

Tandem photoaffinity labeling of a target protein
435
Synthesis of 2 and 3. The synthesis of 2 and 3 is
described in the Supplementary data.
1 h. Then, UV irradiation of the reaction mixture was
performed as described above.
For competition assays, AOS inhibitor 2 (300 μM)
was added to 60 μL of 50 mM sodium phosphate buf-
fer, pH 7.8, containing azide probe 3 (30 μM) and the
recombinant PpAOS1 (10 μM). Other procedures were
performed as described above.
AOS inhibitory assay. Recombinant PpAOS1 was
obtained according to the method of Bandara (2009).17)
An AOS inhibitory assay was performed according to
the method of Oh and Murofushi with minor modifica-
tions.15) The reaction mixture containing PpAOS1
(0.5 μM), 13-HPOT (10 μM), and each concentration
of AOS inhibitor in 100 μL of 50 mM phosphate buf-
fer at pH 7.0 was incubated at 25 °C for 30 min. A
portion of the reaction solution (10 μL) was loaded
onto an HPLC (column: Capcelpak C18 UG120,
4.6 mm i.d. × 150 mm, Shiseido, Japan; solvent: 50%
CH3CN aq. with 0.1% phosphoric acid; flow: 1.0 ml/
min; detection: UV, 235 nm). AOS inhibitory activity
was calculated from the decrease in the peak area of
13-HPOT as a substrate.
Evaluation of the dose-dependent PpAOS1 signal
increase by PpAOS1 and 3. To evaluate the concen-
tration dependence of PpAOS1 on chemiluminescence,
the PpAOS1 concentration was varied from 1.25 to
10 μM. Similarly, variable amounts of azide probe 3
from 3 to 3000 nM were added to the reaction mixture.
The other procedure for PpAOS1 detection was per-
formed as described above.
Detection of PpAOS1 in lysates of E. coli overex-
pressing PpAOS1. A single colony of E. coli trans-
formed with pET23a containing PpAOS1 was
incubated in 5 mL of LB medium containing 100 μg/
mL of ampicillin at 37 °C. After the culture had grown
to a cell density of OD600 = 0.6, PpAOS1 synthesis
was induced by the addition of IPTG at 0.2 mM. After
further incubation at 37 °C for 3 h, the cells were col-
lected by centrifugation at 5000 g for 5 min and then
resuspended with 50 mM sodium phosphate buffer at
pH 7.8 and disrupted by ultrasonication. The cell debris
was removed by centrifugation at 15,000 g for 30 min.
56 μL of the resultant supernatant (protein concentra-
tion; 500 μg/mL) was used for Western blotting. For
control experiment, the lysate of the E. coli strain was
prepared as described above except for IPTG treatment.
Detection of PpAOS1 by Western blotting. Azide
probe 3 (30 μM) and recombinant PpAOS1 (10 μM)
were pre-incubated in 60 μL of 50 mM sodium phos-
phate buffer at pH 7.8 for 1 h on ice prior to
bioorthogonal coupling. Azide alkyne Huisgen cycload-
dition was initiated by the addition of linker 1 (30 μM)
and CuI (0.5 μM) and was incubated for 1 h. After the
cycloaddition, the mixture was irradiated with UV
(365 nm) on ice for an additional 1 h. For control
experiments, PpAOC2 (10 μM)18) was added to the
reaction mixture instead of PpAOS1.
The sample solution was centrifuged to remove
insoluble CuI from the reaction mixture and was then
analyzed by SDS–PAGE. The gel was equilibrated with
a blotting buffer (25 mM Tris-HCl, pH 8.3, 192 mM
glycine, 10% MeOH) for 5 min. A PVDF membrane
(Millipore, USA), which was treated with MeOH and
water, was also equilibrated with the blotting buffer.
The protein was transferred from the gel onto the
PVDF membrane for 1 h using a Trans-blot semi dry
SD cell (Bio-Rad, USA). The electric current was set
at 0.8 mA/cm2. For blocking, the membrane was incu-
bated in 5% non-fat dry milk in PBS-T buffer (80 mM
Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl and 0.1%
Tween 20) overnight at 4 °C, and the membrane was
then washed twice using PBS–T buffer. The membrane
after blocking was incubated in PBS–T buffer contain-
ing streptavidin horseradish peroxidase conjugate
(50 ng/mL, Pierce, USA). After washing the mem-
brane, it was soaked in Amersham ECL Western blot-
ting detection reagents (GE Healthcare, USA) at room
temperature for 5 min. The chemiluminescence derived
from the ternary complex was detected using a Lumi-
VisionPro 400EX fluorescence imager (AISIN, Japan).
To examine the inefficiency of the bioorthogonal
coupling of linker 1 and azide probe 3 before incuba-
tion of 1 with PpAOS1 for PpAOS1 detection, linker 1
(1 mM), azide probe 3 (1 mM) and CuI (5 μM) were
mixed in 5 mL of MeOH, and the solution was incu-
bated at room temperature for 1 h. Two microliters of
the reaction mixture containing the Huisgen cycloaddi-
tion and 56 μL of PpAOS1 (10 μM) were incubated in
50 mM sodium phosphate buffer, pH 7.8, on ice for
Results and discussion
Synthesis of 1, 2, and 3
To synthesize linker 1, alcohol 5 was prepared from
2-[2-(2-chloroethoxy)ethoxy]ethanol 4 via three reac-
tions: introduction of the azide group, reduction of the
azide to an amine, and protection of the amino group
using a tert-butoxycarbonyl (Boc) group. Alcohol 5
was then converted to bromide 6. The reaction of bro-
mide 6 with 2-propyn-1-ol produced alkyne 7, and
deprotection of the Boc group formed polyether amine
8 (Scheme 1). The conjugation of biotin 9 and 2-
amino-2-[4-(phenylcarbonyl)phenyl]acetic acid 1016)
followed by amidation with biotin polyether amine 8
yielded the desired linker containing biotin, alkyne, and
benzophenone group 1 (Scheme 2).
The AOS inhibitor 2 and azide probe 3, which was
the azide group introduced into the structure of AOS
inhibitor 2, were synthesized as model compounds
according to the method of Oh and Murofushi with
some modifications (Supplementary data).15) The AOS
inhibitory activities of 2 and 3 were examined. The
AOS reaction was conducted by incubating purified
recombinant PpAOS117) from the model moss Physco-
mitrella patens with 13-HPOT as a substrate and vari-
ous concentrations of 2 or 3 at 25 °C for 30 min. The
activities of 2 and 3 were estimated by measuring the
decreased peak areas of 13-HPOT by HPLC analy-

436
T. Anabuki et al.
(a)
(b)
HO
O
HO
O
Br
O
O
Cl
O
NHBoc
O
NHBoc
4
5
6
(c)
(d)
O
O
O
O
O
NHBOc
O
NH2
7
8
Scheme 1. (a) (1) NaN3, NaI, H2O, 60 °C, 16 h. (2) Pd/C, H2, CH3OH, r.t., 12 h. (3) (Boc)2O, NaOH, CH3CN, r.t., 12 h, 68% (3 steps);
(b) CBr4, K2CO3, PPh3, CH2Cl2, r.t., 12 h, 84%; (c) 2-propyn-1-ol, NaH, THF, 0 °C, 2 h, 20%; (d) 50% TEA-CH2Cl2, 0 °C, 2 h, 99%.
O
O
HN
NH
H
HN
NH
H
H
H2N
H
N
(a)
H
+
O
S
COOH
10
O
OH
S
O
HN
O
O
O
O
9
O
1
Scheme 2. (a) (1) EDC, HOBT, DMF, r.t., 18 h; (2) 8, EDC, DMF, r.t., 16 h, 21% (2 steps).
sis,15) showing that the AOS inhibitory activity of an
azide probe 3 was higher than that of AOS inhibitor 2
(Fig. S1). The azide group, which was attached to the
terminal alkyl group in 3, increased AOS inhibitory
activity. The data indicated that azide probe 3 also
interacts with AOS and provides access to the AOS for
chemical labeling. Therefore, azide probe 3 was used
in the next step.
Detection of a recombinant PpAOS1 as a model
system
Detection of PpAOS1 was attempted using azide
probe 3 and linker 1. Azide probe 3 was incubated
with PpAOS1 in 50 mM phosphate buffer at pH 7.8.
Linker 1 was conjugated with 3 via the click reaction
using Cu (I) as a catalyst (Fig. 4). The PpAOS1 and
the conjugate 11 (Fig. 4) were cross-linked by UV irra-
diation to a benzophenone group. The ternary complex
Fig. 4. Structure of conjugate of linker 1 and azide probe 3 (11).

Tandem photoaffinity labeling of a target protein
437
Fig. 7. Examination of the effectiveness of azide probe 3 for detect-
ing the chemiluminescence of the PpAOS1–azide probe–linker com-
plex.
Notes: Lane 1: PpAOS1 incubated with azide probe 3 prior to
Huisgen cycloaddition of linker 1; lane 2: PpAOS1 incubated with
the conjugate 11.
action. Accordingly, AOS inhibitor 2 and azide probe 3
bound to the same binding site of PpAOS1, as
expected.
To examine whether azide probe 3 specifically binds
PpAOS1, PpAOS1 was replaced with PpAOC2, which
is another JA biosynthetic enzyme.18) As a result, no
signal corresponding to PpAOC2 was detected, con-
firming the specific binding of 3 with PpAOS1. There-
fore, the method could specifically detect a target
protein bound to an azide probe.
Fig. 5. Identification of the PpAOS1–azide probe–linker complex
by Western blotting.
Notes: Azide probe 3 was incubated with PpAOS1 and was then
bound to linker 1 via a Huisgen cycloaddition. The conjugate 11 was
covalently bound to PpAOS1 by UV irradiation (365 nm). The tern-
ary complex was subjected to SDS–PAGE and was detected by Wes-
tern blotting using streptavidin conjugated with horseradish
peroxidase. For the competitive inhibition assay, PpAOS1 was incu-
bated with azide probe 3 and a 10-fold excess of AOS inhibitor 2
(competitor) prior to Huisgen cycloaddition with linker 1. To examine
the specificity of this method, PpAOS1 was replaced by PpAOC2
and chemiluminescent detection was attempted as described above.
Dose dependence of azide probe 3 and PpAOS1 for
PpAOS1 chemiluminescent signal intensity
To investigate whether the intensity of the chemilu-
minescent signal varied with the PpAOS1 concentra-
tion, the concentration of PpAOS1 was varied from
1.25 to 10 μM. The results indicated that the intensity
of the chemiluminescent signal increased dose depen-
dently from 1.25 to 10 μM PpAOS1 (Fig. 6(A)). The
concentration dependence of the chemiluminescent sig-
nal intensity on azide probe 3 was also examined. The
concentration of 3 ranged from 3 to 3000 nM, and a
dose-dependent increase in the chemiluminescent signal
of azide probe 3 was also observed (Fig. 6(B)). These
data confirm that the developed method for identifying
target proteins bound to small molecules yielded dose-
dependent increases in the chemiluminescent signal
intensity with the target protein and the azide probe.
containing PpAOS1, 1 and 3 was analyzed by SDS–
PAGE. To detect the biotinylated components, the tern-
ary complex was electrophoretically transferred onto a
membrane surface and the blot was visualized via
chemiluminescence using streptavidin and horseradish
peroxidase. Chemiluminescence arising from the tern-
ary complex was observed (Fig. 5).
Azide probe 3 was a derivative of 2 with the azide
group bound to the terminal alkyl group. To determine
if 3 binds to the same PpAOS1 site as 2, a 10-fold
excess of AOS inhibitor 2 as a competitor was incu-
bated with 3 in a PpAOS1 solution. The results indi-
cated that the addition of 2 caused the PpAOS1
chemiluminescent signal to disappear (Fig. 5). A com-
petition assay was performed with an excess of 2 to
confirm that labeling was the result of a specific inter-
Fig. 8. Detection of PpAOS1 in lysates of E. coli overexpressing
PpAOS1.
Notes: Lysates of a strain of E. coli with an expression vector con-
taining PpAOS1 with or without IPTG induction were used for the
detection of PpAOS1. After each lysate was incubated with 3,
bioorthogonal coupling of 1 with 3 was performed; then, the conju-
gate 11 with PpAOS1 was irradiated by UV (365 nm). The lysates
were analyzed by SDS–PAGE and then visualized by Western blot-
ting and CBB staining.
Fig. 6. Dose-dependent increase in chemiluminescence with both
PpAOS1 and azide probe 3.
Notes: (A) PpAOS1 (1.25–10 μM), linker 1 (30 μM), and azide probe
3 (30 μM) in 50 mM phosphate buffer, pH 7.8. (B) Azide probe 3
(3–3000 nM), PpAOS1 (10 μM), and linker 1 (30 μM) in 50 mM
phosphate buffer, pH 7.8.

438
T. Anabuki et al.
Efficacy of the developed method using linker 1 and
detection of target proteins in a mixture. This strategy
azide probe 3
facilitates the detection of proteins bound to small
bioactive molecules.
In the previously described experiments to detect
PpAOS1 as a target protein, a Huisgen cycloaddition was
performed after incubating 3 with PpAOS1. Azide probe
3 detected PpAOS1 without requiring a photophore in the
structure of 3. To test the efficacy of the azide probe for
detecting a target protein, the conjugate 11 was formed via
Huisgen cycloaddition and was then incubated with
PpAOS1. As a result, no chemiluminescent signal was
detected via Western blotting (Fig. 7). Although the
detailed structure of PpAOS1 and 3 as a complex has not
been examined, previous studies on cytochrome P450
inhibitors indicated that the benzene and imidazole moi-
eties are located near the active site.13,14) The azide group
in 3 appear to reside on the surface of PpAOS1. Linker 1
could provide access to the azide group of 3 on the surface
of PpAOS1, and the resultant conjugate 11 still provides a
stable interaction with PpAOS1. On the other hand, the
bulky moiety in conjugate 11, which is preliminarily
formed before incubating 3 with PpAOS1, should inhibit
its approach to the binding site of PpAOS1. Therefore,
these data showed the efficacy of the method for
identifying the binding protein using an azide probe that
was minimally modified by an azide group.
Authors contributions
Study concept and design: Kosaku Takahashi. Acqui-
sition of data: Tomoaki Anabuki, Miu Tsukahara, and
Kosaku Takahashi. Analysis and interpretation of data:
Tomoaki Anabuki, Miu Tsukahara, Hideyuki Matsuura,
Kosaku Takahashi. Drafting of the manuscript: Hide-
yuki Matsuura and Kosaku Takahashi. All authors
reviewed and approved the final manuscript.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Supplemental material
Supplemental material for this article can be accessed
at http://dx.doi.org/10.1080/09168451.2015.1104240.
Acknowledgments
Detection of PpAOS1 in a crude protein extract
As described above, the developed method to identify
a target protein with small bioactive compounds was
shown to work in a model system using recombinant
PpAOS1 and azide probe 3. However, it is possible that
the method in this study detects non-specific protein sig-
nals in a crude protein solution, which can obscure a
signal derived from a target protein. To examine
whether the method in this study can effectively detect
the target protein of a small bioactive compound in a
protein mixture, linker 1 and azide probe 3 were incu-
bated in a lysate of E. coli transformed with an overex-
pressing vector containing PpAOS1; then, detection of
PpAOS1 was attempted by Western blotting (Fig. 8). A
chemiluminescent signal derived from PpAOS1 in a
lysate of the E. coli strain, whose protein expression
was induced by isopropyl-β-D-thiogalactopyranoside
(IPTG), was clearly observed. Conversely, a PpAOS1-
derived signal was not detected in a lysate of the E. coli
strain without IPTG treatment. These data show that the
method developed in this study can be applied to the
detection of a target protein of a bioactive small mole-
cule in a crude protein extract.
We thank Dr. Eri Fukushi and Mr. Yusuke Takata for
collecting the mass spectral data.
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