Design and synthesis of a photocleavable biotin-linker for the photoisolation of ligand-receptor complexes based on the photolysis of 8-quinolinyl sulfonates in aqueous solution

Article abstract of DOI:10.1016/j.bmc.2009.03.031

The ability of avidin (Avn) to form strong complex with biotin (Btn) is frequently used in the detection and isolation of biomolecules in biochemical, analytical, and medicinal research. The fact that the binding is nealy irreversible, however, constitutes a drawback in term of the isolation and purification of intact biomolecules. We recently found that 8-quinolinyl esters of aromatic or aliphatic sulfonic acids undergo photolysis when irradiated at 300-330 nm in aqueous solution at neutral pH. In this work, a biotin-dopamine (BD) conjugate containing a photocleavable 8-quinolinyl benzenesulfonate (QB) linker, BDQB, was designed and synthesized for use in the efficient recovery of dopamine-protein (e.g., antibody) complexes from an Avn-Btn system. The complexation of BDQB with a primary anti-dopamine antibody (anti-dopamine IgG₁ from mouse) on an Avn-coated plate was confirmed by an enzyme-linked immunosorbent assay (ELISA) utilizing a secondary antibody (anti-IgG₁ antibody) conjugated with horseradish peroxidase (HRP). Upon the photoirradiation (at 313 nm) of the BDQB-IgG₁ complex, the release of dopamine-IgG₁ complex was confirmed by ELISA. Characterization of the resulting photoreleased dopamine-anti-dopamine IgG₁ complex was performed by SDS-PAGE and Western blot.

Full text of DOI:10.1016/j.bmc.2009.03.031

Bioorganic & Medicinal Chemistry 17 (2009) 3405–3413
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of a photocleavable biotin-linker for the photoisolation
of ligand–receptor complexes based on the photolysis of 8-quinolinyl
sulfonates in aqueous solution
a
a
Shin Aoki a,b, , Nanako Matsuo , Kengo Hanaya , Yasuyuki Yamada a,b, Yoshiyuki Kageyama a
*
a Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan
b Center for Drug Delivery Research, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The ability of avidin (Avn) to form strong complex with biotin (Btn) is frequently used in the detection
and isolation of biomolecules in biochemical, analytical, and medicinal research. The fact that the binding
is nealy irreversible, however, constitutes a drawback in term of the isolation and purification of intact
biomolecules. We recently found that 8-quinolinyl esters of aromatic or aliphatic sulfonic acids undergo
photolysis when irradiated at 300–330 nm in aqueous solution at neutral pH. In this work, a biotin–dopa-
mine (BD) conjugate containing a photocleavable 8-quinolinyl benzenesulfonate (QB) linker, BDQB, was
designed and synthesized for use in the efficient recovery of dopamine–protein (e.g., antibody) com-
plexes from an Avn–Btn system. The complexation of BDQB with a primary anti-dopamine antibody
(anti-dopamine IgG1 from mouse) on an Avn-coated plate was confirmed by an enzyme-linked immuno-
sorbent assay (ELISA) utilizing a secondary antibody (anti-IgG1 antibody) conjugated with horseradish
peroxidase (HRP). Upon the photoirradiation (at 313 nm) of the BDQB–IgG1 complex, the release of dopa-
mine–IgG1 complex was confirmed by ELISA. Characterization of the resulting photoreleased dopamine–
anti-dopamine IgG1 complex was performed by SDS–PAGE and Western blot.
Received 9 February 2009
Revised 17 March 2009
Accepted 18 March 2009
Available online 24 March 2009
Keywords:
Antibody
Avidin
Biotin
Click chemistry
Dopamine
Photolysis
Quinolinol
Sulfonate
Ó 2009 Elsevier Ltd. All rights reserved.
Very stable Avn–Btn systems are in widespread use in the areas
of biological chemistry, cell biology, analytical chemistry, medici-
1. Introduction
nal chemistry, and therapeutic applications, mainly for the isola-
tion of specific receptors (affinity chromatography), the
localization of target molecules (affinity cytochemistry, cell cytom-
etry, and blotting technology), and medical diagnostics (e.g.,
immunoassay, histopathology, and gene probes).2 However, the
separation of biotinylated ligands or ligand–receptor complexes
from the avidin matrix requires harsh denaturing conditions such
as treatment with detergent, high or low pH, high salt concentra-
tions and other similar conditions.4
Biotin (Btn) is a water soluble vitamin found in egg yolk, beef
liver, and milk concentrates.1 Biotin forms stable complexes with
avidin (Avn, Mr = 67,000), a protein contained by egg white,2 neu-
travidin (Nevn, Mr = 60,000), a deglycosylated form of avidin, and
streptavidin (Stvn, Mr = 66,000–75,000), isolated from the bacte-
rium Streptomyces avidinii. Biotin forms extraordinary stable com-
plexes (the dissociation constant, Kd, for the Avn–Btn complex is
ca. 10ꢀ15 M), mainly due to van der Waals and hydrophobic inter-
actions at the binding site of avidin.3 The half-time for dissociation
of the Avn–Btn complex has been estimated to be 130–200 days.
As a result, this complexation is considered to be essentially
irreversible.
Past efforts to address and solve this problem can be classified
into following three categories. The first involves the development
of Btn derivatives which have
a lower affinity for Avn
a
(or Stvn) than that of Btn,5–7 such as a N ,B1-iminobiotinyl-linker
(dethiobiotin–X(spacer) linker)5,6 and a N ,B1-iminobiotinyl-linker
a
O
(iminobiotin).7
HN NH
The second approach is the development of an Avn mutant hav-
ing a reduced affinity for Btn,8 which requires tedious biological
manipulation.
The third approach is the design and synthesis of Btn linkers
that contain chemically cleavable groups.9–16 This category includes
biotinylated derivatives containing the disulfide linker,9,10 the
silyl-containing Btn linker,11 and the acid-labile Btn linker.12 The
CO2H
(+)-Biotin (Btn)
S
* Corresponding author. Tel./fax: +81 4 7121 3670.
E-mail address: shinaoki@rs.noda.tus.ac.jp (S. Aoki).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.031

3406
S. Aoki et al. / Bioorg. Med. Chem. 17 (2009) 3405–3413
cleavage of these three biotin-linkers requires excessive amounts
of reagents such as dithiothreitol (DTT),9,10 Fꢀ,11 and acid (H+),12
respectively, which might induce conformational changes and loss
of function of the target molecules.
OH
OH
H3N
Dopamine
O
HN NH
Meanwhile, light-induced cleavage reactions of chemically
masked groups have been a subject of considerable interest for
some time.13 Such procedures assume that photochemically cleav-
able biotin linkers would afford clean and useful methodologies for
the isolation of intact ligand–receptor complexes.14 In this context,
photocleavable biotin linkers having a 2-nitrobenzyl moiety were
developed previously.15–17 As of this writing, however, biological
applications have been limited.
O2
S
Me
Me
N
N
N
O2
S
OH
OH
S
3
O
O
O
N
H
N
H
N
H
N
N
N
(+)-Biotin
Photocleavable linker
Dopamine
1,2,3-triazole
(QB)
Left part
Right part
During our efforts to develop new fluorescent sensors for zin-
c(II) ions,18 we recently discovered that sulfonates of 5-N,N-dim-
ethylaminosulfonyl-8-hydroxyquinoline (general structure: 1)
undergo photolysis upon UV irradiation (at 300–330 nm) in
CH3CN/H2O (10 mM HEPES with I = 0.1 (NaNO3)) to yield the corre-
sponding alcohols 2 and sulfonates 3 (R2SO3ꢀ), as shown in Scheme
1.19 Our recent studies on mechanistic aspects of this reaction indi-
cated that this photolysis proceeds mainly via excited triplet state,
9 (BDQB)
Scheme 3.
To demonstrate our methodology, we synthesized a dopamine
derivative that contains a photocleavable Btn linker 9 (a biotin–
dopamine (BD) conjugate containing a photocleavable 8-quinolinyl
benzenesulfonate (QB) linker, BDQB) (Scheme 3). Dopamine is an
important neurotransmitter found in the brain.21 Several dopa-
mine receptors (D1 to D5 receptors) have been identified21 and
are recognized as targets of therapeutic agents. In addition, anti-
bodies against dopamine and dopamine receptors are commer-
cially available. In this manuscript, we describe the synthesis of 9
and related model compounds, the complexation of 9 with an
anti-dopamine antibody, and the results of an enzyme-linked
immunosorbent assay (ELISA). The photorelease of the dopa-
mine–anti-dopamine antibody complex (corresponding to 8 in
Scheme 2), as verified by ELISA, sodium dodecylsulfonate–poly-
acrylamide gel electrophoresis (SDS–PAGE), and Western blot, will
be described.
in which the S–O bond of 1 is homolytically cleaved20
.
This finding prompted us to prepare a new photocleavable bio-
tin-linker. As summarized in Scheme 2, we synthesized a biotinyl-
ated ligand that contains a photocleavable 8-quinolinyl sulfonate
linker 4. It is likely that 4 would form an Avn–4 complex 5 in the
solid phase. A mixture of specific receptor candidates is added to
obtain ternary complexes 6, which are composed of Avn, 4, and
the ligand–specific receptor. We hypothesized that ligand–recep-
tor complexes 8 could be isolated upon the photoirradiation of 6
in aqueous solution, leaving Avn-Btn complex 7, without the need
for any other reagents.
SO2NMe2
SO2NMe2
2. Results and discussion
hν
R2SO3
+
in aqueous solution
mainly via triplet state
R1
N
R1
2.1. Synthesis of reference compounds 10 and 11 and
examination of their binding to anti-dopamine antibody (IgG1)
N
OH
O
R2
S
3
2
1
O2
Prior to the synthesis of 9, reference compounds 1022 and 11
(for their synthesis, see the Supplementary data) were synthesized
(Scheme 4). Using ELISA with an anti-dopamine antibody (IgG1), it
was found that 10 is not recognized by IgG1, while 11 is able to
readily bind to IgG1, suggesting that the ammonium cation form
of dopamine is important for its binding to IgG1, as previously sug-
gested for dopamine–dopamine-receptor interactions.23
Scheme 1.
Ligand
Ligand
Photocleavable
group
Avidin
(Avn)
2.2. Comparison of the photoreactivity of 12 and the reference
compound 13
(in the solid
phase)
Receptor candidates
Btn
The photoreactivity (photoirradiation at 328 nm) in CH3CN/
10 mM HEPES (pH 7.4 with I = 0.1 (NaNO3) (9/1)) of 12 and refer-
4
Avn-4-complex
(in the solid phase)
5
Ligand
O
HN NH
Specific
receptor
H
Photocleavage
OH
N
Ligand
S
hν
O
OH
Ligand-receptor
10
O
complex
8
HN NH
H2
N+
N
Avn-4-receptor
N
H
N
OH
OH
N
complex
Avn-Btn complex
Isolation
and
S
7
6
O
11
Scheme 4.
identification
Scheme 2.

S. Aoki et al. / Bioorg. Med. Chem. 17 (2009) 3405–3413
3407
Me2NO2S
N
O2
S
UV at 328 nm
O3S
1
2
O
(R = Me)
+
In CH3CN / 10 mM HEPES
O
O
N
H
Tol
+
(90/10)
N
N
N
N
H
N
H
Tol
N
pH 7.4 at 25 ºC
N
N
Φ = 2.8 x 10-4
12
14
UV at 328 nm
NH2
ON
O
O2N
O
O
+
Ph
byproducts
In CH3CN / 10 mM HEPES
O
O
N
H
N
H
Tol
N
Ph
N
Tol
N
(90/10)
N
H
N
N
N
pH 7.4 at 25 ºC
Φ = 7.6 x 10-4
15
13
16
Scheme 5.
Me
Me
NaN3
Br
NBS
Br
L
-Proline
N3
N3
AIBN
17
CuI
Benzene
58%
19
18
NaOH
MeOH/H2O
73%
OR3
OR3
OR3
OR3
19
H2N
N
nBu4NI
H
N3
Cs2CO3
Dopamine: R3 = H
TBDMSCl
DBU
CH3CN
41%
CHCl3/CH3CN
21
(Right part of 9)
20
(48% based on
)
20: R3 = TBDMS
Scheme 6.
SO2Cl
MeHN
N
NBoc
Me
O
O
OH
25
HN NH
HN NH
22
O2
(+)-Biotin
Me
N
Me
N
EDC·HCl
CH3CN/MeOH
96%
NR4
Me
S
N
Et3N
CH2Cl2
65%
S
S
N
3O
Me
3O
OH
23: R4 = Boc
26
TFA
CH2Cl2
78%
ClO2S
24: R4 = H
H2N
28
NCO
27
ClO2S
O
S
HN
NH
O
O2
S
Me
Me
N
N
H
N
H
N
O2
N
3
S
O
29
O
O
(not isolated)
N
H
N
H
30
9
(Left part of
)
Et3N
CH2Cl2/CH3CN
81%
CuSO4.5H2O
21
Sodium ascorbate
tBuOH/H2O
76%
O
HN
NH
O2
S
Me
Me
OR5
OR5
N
N
O2
S
N
S
3
O
O
O
N
H
N
H
N
H
N
N
N
: R5 = TBDMS
31
HF
CH2Cl2
35%
9 (as 3HF salt): R5 = H
Scheme 7.

3408
S. Aoki et al. / Bioorg. Med. Chem. 17 (2009) 3405–3413
ence compound 13, which contains commercially available o-
nitrophenyl-b-alanine as a photocleavable group (Scheme 5),24
was almost identical, as shown in Figure S1 in the Supplementary
data. It should be noted that the molar absorption coefficient of 12
Dopamine
Btn
Photocleavable
linker
328 = 1.1 ꢁ 103 Mꢀ1 cmꢀ1) is ca. three times as great as
Avn
at 328 nm (
that of 13 (
e
e
328 = 0.4 ꢁ 103 Mꢀ1 cmꢀ1) and the quantum yields for
= 2.8 ꢁ 10ꢀ4) is ca. one-third that of 13
9
Stvn- complex
the photolysis of 12 (
U
(in wells)
32
(U
= 7.6 ꢁ 10ꢀ4), resulting in a similar photolysis profile. More
importantly, the photolysis of 12 yielded 2 and 14 with almost
no detectable byproducts (Figs. S2 and S3 in the Supplementary
data), while the photoirradiation of 13 afforded several byproducts
as well as 15 and 16 (data not shown).
Anti-dopamine antibody
(1st IgG)
2.3. Synthesis of 9
Dopamine
The convergent synthesis of 9 is shown in Schemes 6 and 7. For
the dopamine part (the right part of 9 indicated in Scheme 3), the
CuI-catalyzed coupling reaction of 17 with sodium azide in the
presence of proline gave 18,25 which was then brominated to give
19.26 The two hydroxyl groups of dopamine were protected with t-
butyldimethylsilyl (TBDMS) groups to give 20.27 The monoalkyla-
tion of 20 with 19 in the presence of nBu4NI and Cs2CO3 gave 21,
the right part of 9, in 48% yield based on the used 20.
Excess
Btn
Excess
Negligible
exchange
dopamine
Negligible
exchange
32-anti-dopamine antibody
complex
ELISA
UV (313 nm)
(32-1st IgG complex)
33
For the synthesis of the left part of 9 (Scheme 7), (+)-biotin was
reacted with N-Boc-N,N0-dimethylethylenediamine 2228 to give 23,
and the Boc group was deprotected by treatment with trifluoroace-
tic acid (TFA) to afford 24. Upon treatment of 24 with 25,18a,29 26
was obtained and the reaction of 26 with 29 (prepared from 27
and 28 and used without purification) yielded 30.
A Huisgen 1,3-dipolar [3+2] cycloaddition of the terminal al-
kyne group of 30 with 21 in the presence of a catalytic amount
of Cu2+ and sodium ascorbate according to procedures of click
chemistry developed by Hartmuth and Sharpless and co-workers30
gave 31 in moderate yields. Finally, deprotection of the TBDMS
groups of 31 by treatment with HF gave 9 as the 3HF salt.
2nd IgG-HRP
HRP
Secondary
antibody-HRP
conjugate
(2nd IgG-HRP)
Stvn-Btn complex
NH2
NH2
H2O2
2nd IgG
HRP
o-Phenylenediamine
(OPD)
Dopamine
1st IgG
N
N
NH2
NH2
Dopamine
Dopamine-1st IgG
complex
2,3-Diaminophenazine
2.4. Verification of the complexation of 9 with anti-dopamine
antibody (IgG1) by enzyme-linked immunosorbent assay
(ELISA)
35
(yellow)
Isolation
and
identification
The complexation of 9 with a primary anti-dopamine antibody
(mouse IgG1) was verified by means of an enzyme-linked immuno-
sorbent assay (ELISA) (Scheme 8).31 A primary anti-dopamine anti-
body (IgG1, 1st IgG) was added to a complex of Stvn and 9 in wells
of micro 96-wells plates (32?33 in Scheme 8). After washing with
phosphate buffered saline containing 0.05% (v/v) Tween 20 (PBST),
the 32–IgG1 complex 33 was treated with an anti-IgG1 antibody
conjugated with horseradish peroxidase (2nd IgG–HRP) to give
34, as indicated in the left half of Scheme 8. After washing, o-phen-
ylenediamine (OPD) and H2O2 were added as substrates of HRP,
and the increase in UV absorption at 450 nm (OD450) for 2,3-diami-
nophenazine formed by the HRP-catalyzed oxidative dimerization
of OPD32 was detected. Figure 1 shows that the OD450 values in-
creased with increasing amounts of 9, strongly suggesting the for-
mation of the quaternary complex 34 (Stvn–9–1st IgG–2nd IgG–
HRP). Addition of an excess amount of Btn or dopamine to 33
(and successive washing) had negligible effect on the results of ELI-
SA (data are not shown), indicating that the exchange reaction of
33 with Btn and dopamine hardly occurred.
32
-1st IgG-2nd IgG
quaternary complex
34
Scheme 8.
-1
μ
2.5. Evaluation of the complexation characteristics of 9 with
neutravidin (Nevn) and an anti-dopamine antibody (IgG1) using
a 27-MHz quartz-crystal microbalance
-1
μ
The complexation constant (Ks1) for 9 with Nevn was deter-
Figure 1. Results of ELISA of 9 with anti-dopamine antibody (1st IgG) and 2nd IgG–
mined by means of
a
27-MHz quarts-crystal microbalance
HRP.

S. Aoki et al. / Bioorg. Med. Chem. 17 (2009) 3405–3413
3409
(QCM).33 The log Ks1 value for Nevn and 9 was determined to be
8.2 0.2 (Kd = 6 3 nM) by analysis of typical titration curves
shown in Figure 2A. The log Ks value for Nevn and 30, which lacks
the dopamine part, was determined to be 8.1 0.2, indicating that
these modified biotin derivatives have a lower affinity than that of
biotin itself.34 In addition, IgG1 was added to the Nevn–9 complex
to obtain the titration curves displayed in Figure 2B, from which
the log Ks2 value for the complexation of the Nevn–9 complex with
IgG1 was estimated to be 7.5 0.2 (Kd = 20 ꢂ 50 nM).
Figure 4. Results of Western blot of 9–IgG1 complex after photoreaction. Lane 1:
The sample obtained by treatment of 9–IgG1 with SDS without photoirradiation.
Lane 2, 3, 4: photoproducts after photoirradiation (313 nm) for 0 min, 5 min and
30 min. Lane 5: Blank. Lane 6: Anti-dopamine antibody commercially purchased.
Neutravidin (Nevn) (1 mg/mL)
A
0
2.6. Photolysis of 9–IgG1 complex in streptavidin (Stvn)-coated
well, as followed by ELISA and detection of a dopamine–IgG1
complex by Western blot
-500
The photocleavage of the 9–IgG1 complex in Stvn-coated wells
was carried out by photoirradiation at 313 nm, as displayed in
the right half of Scheme 8. After irradiation of the ternary complex
33 (33?35 in Scheme 8), an ELISA of the photoreaction mixtures
was carried out. As shown in Figure 3, OD450 decreases with
increasing photoirradiation time, suggesting that the dopamine–
IgG1 complex is photochemically released from the wells.
The photoreaction product 35 (Scheme 8) was analyzed by
Western blot. In this experiment, avidin beads were used instead
of 96-wells for the solid phase. Lane 1 of Figure 4 shows the sample
obtained by the treatment of the 9–IgG1 complex on avidin beads
with SDS before photoirradiation. Lanes 2–4 show that the amount
of photoreleased IgG1–dopamine complex is dependent on the
photoirradiation time (lane 2, 0 min; lane 3, 5 min; lane 4,
30 min), which shows good agreement with the results of ELISA
(Fig. 3).
1800 200 Hz
-1000
-1500
-2000
-2500
9
1 μL each of a solution of
(4 μg/mL) was added.
~100 Hz
Time
Nevn-9 complex
B
0
-500
1200 200 Hz
3. Conclusion
1 μL each of a solution
-1000
In this study, we have reported on the design and synthesis of a
new biotinylated dopamine containing a photolabile 8-quinolinyl
benzenesulfonate moiety, 9 (BDQB). This photocleavable biotin-
linker can be easily incorporated into small molecules using a
Huisgen 1,3-dipolar [3+2] cycloaddition. We examined the com-
plexation of 9 with avidins and an anti-dopamine antibody (IgG1)
and the photorelease of dopamine–IgG1 complexes by ELISA, which
was also confirmed by Western blot. These methods may provide
effective strategies for the recovery of intact ligand–receptors com-
plexes under mild conditions, without the need for damaging
chemical reagents. Moreover, the photolysis of 9 proceeds cleanly
to give the corresponding quinolinols and sulfonates, which would
be advantage over the previous chemically cleavable biotin linkers.
We presume that this method should have merits in cases where
ligands and receptors are bound by covalent bonds. Efforts to im-
prove the efficiency of the photoreaction are currently underway.
of IgG1 was added.
~ 250 Hz
-1500
-2000
-2500
Time
Figure 2. Results of 27-MHz QCM of 9, Nevn, and IgG1. Time course of frequency
change of QCM. (A) A solution of 9 was added to Nevn; (B) a solution of IgG1 was
added to Nevn–9 complex.
2.0
1.5
1.0
0.5
0
4. Experimental
4.1. Materials
All reagents and solvents purchased were of the highest com-
mercial quality and were used without further purification. Anhy-
drous acetonitrile (CH3CN), THF, dichloromethane (CH2Cl2) were
obtained by distillation from calcium hydride. All aqueous solu-
tions were prepared using deionized and distilled water. The
Good’s buffer reagents (Dojindo) were commercially obtained:
HEPES (N-(2-hydroxyethyl)piperazine-N0-2-ethanesulfonic acid,
0
30
Photoirradiation time / min
10
40
20
Figure 3. The results of ELISA of 9–anti-dopamine antibody complex after
photoirradiation at 313 nm.

3410
S. Aoki et al. / Bioorg. Med. Chem. 17 (2009) 3405–3413
pKa = 7.5). Melting points were determined on a Yanaco Melting
Point Apparatus and are uncorrected. UV spectra were recorded
on a JASCO UV/vis spectrophotometer V-550 at 25 0.1 °C. IR spec-
tra were recorded on a Horiba FTIR-710 spectrophotometer at
room temperature. 1H (400 MHz) and 13C (100 MHz) NMR spectra
were recorded on a JEOL Lambda 400 spectrometer. 1H (300 MHz)
and 13C (75 MHz) NMR spectra were recorded on a JEOL Always
300 spectrometer. Elemental analyses were performed on a Per-
kin–Elmer CHN 2400 analyzer. Thin-layer (TLC) and silica gel col-
umn chromatographies were performed using a Merck 5554
(silica gel) TLC plates and Fuji Silysia Chemical FL-100D,
respectively.
purified by silica gel column chromatography (CH2Cl2/MeOH) to
yield 23 as a pale yellow amorphous solid (828 mg, 96% yield). IR
(KBr): 3284, 2972, 2930, 2866, 1697, 1458, 1397, 686 cmꢀ1 1H
;
NMR (400 MHz, CDCl3, TMS): d = 1.47 (br, 11H), 1.62–1.76 (m,
4H), 2.32–2.35 (m, 2H), 2.73 (m, 1H), 2.88–3.02 (m, 7H), 3.14–
3.21 (m, 1H), 3.34–3.53 (m, 4H), 4.31–4.35 (m, 1H), 4.53–
4.59 ppm (m, 1H); 13C NMR (75 MHz, CDCl3, TMS): d = 24.7, 28.2,
28.3, 28.4, 31.9, 33.0, 33.6, 34.7, 40.6, 47.3, 51.6, 55.4, 60.1, 61.8,
61.8, 61.9, 70.5, 163.5 ppm.
4.2.4. Synthesis of N-[2-(D
-Biotinylamino)-ethyl]-N,N0-
dimethylamine (24)
TFA (5.5 g, 47.7 mmol) was added dropwise over 5 min to a
solution of 23 (661 mg, 1.59 mmol) in CH2Cl2 (7.5 mL) at 0 °C and
the resulting solution was then stirred for 3 h at room temperature.
After concentrating the reaction mixture under reduced pressure,
the resulting residue was azetroped with toluene to remove TFA.
The residue was dissolved in CHCl3 and the solution was washed
with 10% aqueous NaOH. After drying the organic layers over anhy-
drous Na2SO4, the solvent was filtered and concentrated under re-
duced pressure to yield 24 as a colorless solid (468 mg, 94% yield).
4.2. Synthesis
4.2.1. Synthesis of 2-[3,4-bis(tert-butyldimethylsilyloxy)
phenyl]ethylamine (20)22
Dopamine hydrochloride (1 g, 5.27 mmol) was added to t-butyl-
dimethylsilyl chloride (1.75 g, 11.6 mmol) in CH3CN (20 mL) at
0 °C, and the reaction mixture was stirred for 10 min at 0 °C. After
the dropwise addition of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) (3.21 g, 21.08 mmol), the resulting solution was stirred at
0 °C for 4 h and then at room temperature for 20 h. The reaction
mixture was concentrated under reduced pressure and the result-
ing residue was purified by silica gel column chromatography
(hexane/CHCl3/MeOH) to yield 20 as a brown amorphous solid
(833 mg, 41% yield). IR (KBr): 3436, 2930, 2891, 2859, 1513,
IR (KBr): 3286, 2934, 2859, 1696, 1624, 1464, 1331, 685 cmꢀ1 1H
;
NMR (400 MHz, CDCl3, TMS): d = 1.44–1.51 (m, 2H), 1.64–1.80 (m,
4H), 2.34–2.46 (m, 5H), 2.72–3.04 (m, 7H), 3.15–3.18 (m, 1H),
3.38–3.53 (m, 2H), 4.30–4.33 (m, 1H), 4.48–4.53 ppm (m, 1H);
13C NMR (75 MHz, CDCl3, TMS): d = 24.8, 25.4, 28.2, 28.3, 32.5,
32.9, 33.4, 35.9, 36.2, 36.5, 40.5, 47.3, 49.3, 49.6, 49.7, 55.5, 55.6,
60.1, 61.8, 163.9, 173.3, 173.4 ppm; HRMS (FAB): m/z: calcd for
C14H26N4O2S: 315.1855 [M+H+]; found: 318.1852.
1472, 1254, 842, 781 cmꢀ1 1H NMR (400 MHz, CDCl3, TMS):
;
d = 0.18 (s, 12H), 0.96 (s, 9H), 0.97 (s, 9H), 2.97 (t, J = 8.8 Hz, 2H),
3.18 (t, J = 8.6 Hz, 2H), 6.67 (m, 2H), 6.75 ppm (d, J = 8.5 Hz, 1H);
13C NMR (100 MHz, CDCl3, TMS): d = ꢀ4.0, ꢀ4.1, 18.4, 18.4, 25.9,
33.3, 121.3, 121.6, 121.6, 129.2, 146.1, 147.0 ppm.
4.2.5. Synthesis of 8-hydoxy-5-{N-[2-(D-biotinylamino)-ethyl]-
(N,N0-dimethyl)sulfonamide}-2-methylquinoline (26)
4.2.2. Synthesis of N-(4-azidobenzyl)-N-(2-[3,4-bis(tert-butyldi
Distilled Et3N (134 mg, 1.32 mmol) was added to a solution of 24
in CH2Cl2 (26 mL) at 0 °C under an argon atmosphere, to which a
solution of 25 (170 mg, 0.66 mmol)18a,29 in CH2Cl2 (5 mL) was added
dropwise over 30 min. The reaction mixture was stirred at 0 °C for
30 min and at room temperature for 3 h. The solvent was removed
under reduced pressure and the remaining residue was purified by
silica gel column chromatography (CH2Cl2/MeOH) to yield 26 as a
yellow amorphous solid (196 mg, 56% yield). IR (KBr): 3293, 2929,
2859, 1699, 1631, 1462, 1326, 1149, 715 cmꢀ1; 1H NMR (300 MHz,
CDCl3, TMS): d = 1.43–1.47 (m, 2H), 1.59–1.80 (m, 2H), 2.23 (t,
J = 7.4 Hz, 2H), 2.71–3.02 (m, 11H), 3.27 (d, J = 5.8 Hz, 2H), 3.48–
3.60 (m, 2H), 4.32–4.36 (m, 1H), 4.48–4.52 (m, 1H), 7.15 (d,
J = 8.3 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H),
8.87 ppm (d, J = 9.0 Hz, 1H); 13C NMR (75 MHz, CD3OD): d = 24.8,
25.8, 26.2, 29.5, 29.8, 33.3, 33.9, 34.1, 34.6, 35.9, 36.5, 41.1, 46.2,
48.2, 56.9, 61.6, 63.2, 63.3, 110.0, 123.7, 124.3, 124.9, 125.4, 125.6,
132.7, 132.8, 134.9, 135.1, 139.3, 158.7, 158.9, 159.7, 159.8, 166.0,
175.5, 175.6 ppm; HRMS (FAB): m/z: calcd for C24H33N5O5S2:
536.2002 [M+H+]; found: 536.1998.
methylsilyloxy)phenyl]ethyl)amine (21)
To
a
mixture of tetrabutylammonium iodide (118 mg,
0.32 mmol), Cs2CO3 (609 mg, 1.87 mmol), and 20 (242 mg,
0.63 mmol) in CHCl3/CH3CN (7/3), 4-azidobenzylbromide 1926
(45 mg, 0.21 mmol) was added and the resulting solution was stir-
red for 1.5 h at room temperature. After removing the insoluble
salts by filtration, water was added to the filtrate and extracted
with CHCl3. After drying the combined organic layer over anhy-
drous Na2SO4, the solvent was filtered and concentrated under re-
duced pressure. The remaining residue was purified by silica gel
column chromatography (hexane/CHCl3/MeOH) to yield 21 as a
brown amorphous solid (52 mg, 48% yield based on the used 19).
IR (KBr): 2954, 2929, 2889, 2857, 1577, 1507, 1471, 1254, 839,
781 cmꢀ1 1H NMR (400 MHz, CDCl3, TMS): d = 0.17 (s, 6H), 0.18
;
(s, 6H), 0.97 (s, 9H), 0.98 (s, 9H), 2.69 (t, J = 6.4 Hz, 2H), 2.82 (t,
J = 6.6 Hz, 2H), 3.75 (s, 2H), 6.62 (dd, J = 2.0, 8.0 Hz, 1H), 6.66 (d,
J = 2.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.1 Hz, 2H),
7.25 ppm (d, J = 8.4 Hz, 2H); 13C NMR (75 MHz, CDCl3, TMS):
d = ꢀ4.1, 18.4, 25.9, 35.4, 50.5, 53.2, 118.9, 120.9, 121.5, 129.5,
132.9, 137.0, 138.6, 145.2, 146.6 ppm; HRMS (EI): m/z: calcd for
C27H44N4O2Si2: 512.3003 [M+]; found: 512.3000.
4.2.6. Synthesis of 2-methylquinoline-8-[p-(N0-2-propylureido)
benzenesulfonyloxy]-5-{N,N0-dimethyl-N0-[2-(
D-biotinylamino)
4.2.3. Synthesis of N-(tert-Butoxycarbonyl)-N0-[2-(
amino)-ethyl]-N,N0-dimethylamine (23)
D-biotinyl
ethyl]}sulfonamide (30)
A solution of propargylamine 28 (105 mg, 1.91 mmol) in CH2Cl2
(3 mL) was added dropwise over 1 h to a solution of 4-(chlorosul-
fonyl)phenyl isocyanate 27 (403 mg, 1.85 mmol) in CH2Cl2 (8 mL).
After stirring the resulting solution was stirred for 7 h at room tem-
perature, the solvent was removed under reduced pressure. The
remaining precipitate was resuspended in CHCl3, filtered, and the
filtrate concentrated under reduced pressure to yield 29 as a color-
less solid (314 mg), which was used without further purification
for the synthesis of 30. 1H NMR of 29 (300 MHz, CDCl3, TMS):
A solution of
ylamine 2228 (475 mg, 2.50 mmol) and 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide hydrochloride (518 mg,
D
-biotin (508 mg, 2.08 mmol), N-Boc-N,N0-dimeth-
(EDCꢃHCl)
2.70 mmol) in a 1/3 mixture of MeOH and CH3CN (20 mL) was stir-
red for 5 h at room temperature and the reaction mixture was con-
centrated under reduced pressure. The residue was resuspended in
MeOH and filtered through a Celite (No. 545). The filtrate was con-
centrated under reduced pressure and the resulting residue was

S. Aoki et al. / Bioorg. Med. Chem. 17 (2009) 3405–3413
3411
d = 2.30 (t, J = 2.5 Hz, 1H), 4.10 (d, J = 2.5 Hz, 2H), 6.97 (s, 1H), 7.62
(d, J = 9.0 Hz, 2H), 7.94 ppm (d, J = 9.0 Hz, 2H).
A solution of crude 29 (68 mg) in CH3CN (7 mL) was added
dropwise to a mixture of 26 (115 mg, 0.21 mmol) and Et3N
room temperature. After removing the solvent under reduced pres-
sure, the remaining residue was azetroped with CH3CN twice or
three times to remove HF to give a colorless amorphous solid.
THF was added to the amorphous solid and the insoluble solid
was collected by filtration and washed with THF to yield 9 as a col-
orless solid (13 mg, 35% yield). Mp 150–152 °C; IR (KBr): 3390,
2932, 2859, 1687, 1593, 1522, 1459, 1408, 1365, 1327, 1225,
(34 lL, 0.25 mmol) in CH2Cl2 (3 mL) at 0 °C. The solution was stir-
red at 0 °C for 30 min and at room temperature for 3 h. Water
(1 mL) was added to the reaction mixture and the resulting solu-
tion stirred for further 30 min. The reaction mixture was poured
into aq K2CO3, and extracted with CH2Cl2. The combined organic
layer was dried over K2CO3 and filtered, and then the filtrate was
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography (CH2Cl2/MeOH) to
yield 30 as a yellow solid (132 mg, 81% yield). Mp 120 °C; IR
(KBr): 3285, 2928, 2857, 1696, 1591, 1462, 1408, 1370, 1326,
1193, 1171, 795, 714 cmꢀ1 1H NMR (400 MHz, CD3OD, TSP):
;
d = 2.02–2.54 (m, 6H), 2.82 (t, J = 5.5 Hz, 2H), 3.31 (s, 3H), 3.42 (d,
J = 9.5 Hz, 1H), 3.56–3.60 (m, 2H), 3.59 (s, 3H), 3.63 (s, 3H), 3.86–
3.90 (m, 1H), 3.92 (t, J = 5.9 Hz, 2H), 4.05–4.10 (m, 2H), 4.20–4.31
(m, 2H), 4.97 (s, 2H), 5.05 (q, J = 2.6 Hz, 1H), 5.20 (q, J = 3.2 Hz,
1H), 5.26 (s, 2H), 7.30 (dd, J = 1.5, 4.6 Hz, 1H), 7.41 (d, J = 1.5 Hz,
1H), 7.46 (d, J = 6.1 Hz, 1H), 8.19–8.23 (m, 3H), 8.39 (d, J = 6.0 Hz,
2H), 8.45 (d, J = 6.6 Hz, 2H), 8.53 (dd, J = 4.2, 2.0 Hz, 1H), 8.68 (d,
J = 6.2 Hz, 2H), 8.86 (d, J = 6.2 Hz, 1H), 9.20 (s, 1H), 9.53 ppm (d,
J = 6.6 Hz, 1H); 13C NMR (100 MHz, CD3OD): d = 25.0, 25.9, 29.8,
33.6, 33.9, 34.7, 36.1, 36.1, 36.3, 41.1, 46.0, 46.1, 49.5, 49.7, 49.9,
50.4, 52.0, 56.9, 57.0, 61.6, 63.3, 116.7, 116.8, 118.3, 118.4, 120.9,
121.9, 122.3, 122.7, 125.3, 125.4, 128.3, 128.4, 129.6, 129.6,
129.7, 131.5, 132.3, 134.7, 138.7, 142.4, 145.5, 146.7, 147.3,
148.0, 156.9, 162.2, 175.8; HRMS (FAB): m/z: calcd for
C49H57N11O10S3: 1056.3530 [M+H+]; found: 1056.3530; elemental
Anal. Calcd for C49H60F3N11O10S3 (1116.26): C, 52.72; H, 5.42; N,
13.42. Found: C, 53.00; H, 5.17; N, 13.42.
1224, 1193, 1172, 715, 648 cmꢀ1 1H NMR (300 MHz, CD3OD,
;
TSP): d = 2.12–2.46 (m, 6H), 2.81 (t, J = 6.9 Hz, 2H), 3.27–3.34 (m,
4H), 3.39 (d, J = 12.9 Hz, 1H), 3.58–3.66 (m, 7H), 3.87–3.94 (m,
1H), 4.01 (q, J = 1.7 Hz, 2H), 4.21–4.29 (m, 2H), 4.66 (d, J = 2.7 Hz,
2H), 5.02 (m, 1H), 5.18 (m, 1H), 8.15–8.23 (m, 3H), 8.39–8.56 (m,
3H), 8.82 (d, J = 8.1 Hz, 1H), 9.50 ppm (d, J = 9.0 Hz, 1H); 13C NMR
(100 MHz, CD3OD): d = 25.0, 25.9, 29.5, 29.8, 30.1, 33.9, 34.8,
36.2, 36.4, 41.1, 46.0, 48.3, 57.0, 61.6, 63.2, 72.2, 81.3, 118.4,
122.7, 125.4, 125.4, 128.5, 129.6, 131.4, 134.7, 134.7, 142.4,
147.2, 149.8, 156.5, 162.2, 166.1, 175.8 ppm; HRMS (ESI): m/z:
calcd for C34H41N7O8S3: 772.2251 [M+H+]; found: 772.2251.
4.2.7. Synthesis of 1-(4-{2-[3,4-bis(tert-butylsilyloxy)phenyl]
ethylaminomethyl}phenyl)-4-{2-methylquinoline-8-[p-(N0-2-
4.3. ELISA for 9, 10 and 11 with an anti-dopamine antibody
propynylureido)benzensulfonyloxy]}-5-{N,N0-dimethyl-N-[2-(
D
-
We used a monoclonal anti-dopamine antibody (IgG1 from
mice, Abcam) as a primary antibody and HRP (horseradish peroxi-
dase)-conjugated anti-IgG1 antibody (IgG from rabbit, MP Biomed-
icals) as a secondary antibody. A streptavidin immobilized 96-well
plate (purchased from Nunc Co. Ltd) was washed three times with
phosphate buffered saline containing 0.05% (v/v) Tween 20 (PBST)
prior to use. The biotinylated dopamine 9 diluted with PBST was
biotinylamino)]sulfamoyl}-1,2,3-triazole (31)
Sodium ascorbate (0.034 mmol, 34
lL of a 1 M solution in
water) and CuSO4ꢃ5H2O (3.4 mol, 34
l
lL of 100 mM solution in
water) were added to a suspension of 30 (132 mg, 0.17 mmol)
and 21 (94 mg, 0.18 mmol) in a 1/1 mixture of water and tert-
BuOH (12 mL). After the reaction mixture was stirred for 29 h at
room temperature, the solution was diluted with water and ex-
tracted with CHCl3. The organic layer was dried over anhydrous
Na2SO4, filtered, and the solvent was removed under reduced pres-
sure. The remaining residue was purified by silica gel column chro-
matography (CH2Cl2/MeOH) to yield 31 as a yellow amorphous
solid (162 mg, 76% yield). IR (KBr): 3329, 2929, 2857, 1697, 1592,
1541, 1463, 1409, 1362, 1254, 1225, 1193, 1171, 840, 784,
added at 100 lL/well, followed by incubation at room temperature
for 1 h. After washing, the 9 in each well was sequentially incu-
bated at room temperature for 1 h with anti-dopamine antibody
(1:2500 dilution) and the secondary antibody–HRP conjugate
(1:1000 dilution) and the plates were incubated for 1 h at room
temperature. After washing, o-phenylenediamine and H2O2 was
added to each well. After incubation at room temperature for
15 min, the absorbance at 450 nm was measured on a Bio-Rad
model 550 microplate reader (Bio-Rad, Hemel, Hempstead, UK).
714 cmꢀ1 1H NMR (400 MHz, CDCl3, TMS): d = 0.17 (s, 6H), 0.18
;
(s, 6H), 0.97 (s, 9H), 0.97 (s, 9H), 1.33–1.43 (m, 2H), 1.51–1.57
(m, 2H), 1.59–1.62 (m, 2H), 1.91–2.05 (m, 4H), 2.60 (s, 3H), 2.73
(s, 3H), 2.79 (d, J = 11.4 Hz, 1H), 2.88 (s, 3H), 2.90–2.95 (m, 1H),
3.02–3.05 (m, 2H), 3.13–3.15 (m, 1H), 3.24–3.27 (m, 2H), 3.37 (t,
J = 4.8 Hz, 2H), 3.49 (s, 2H), 3.51 (t, J = 4.8 Hz, 2H), 4.34–4.37 (m,
1H), 4.52–4.54 (m, 1H), 6.06 (s, 1H), 6.54 (s, 1H), 6.65 (d,
J = 6.0 Hz, 1H), 6.68 (s, 1H), 6.74 (d, J = 6.0 Hz, 1H), 7.29 (d,
J = 6.6 Hz, 1H), 7.39–7.60 (m, 8H), 7.72 (d, J = 6.3 Hz, 1H), 7.96 (s,
1H), 8.05 (d, J = 6.3 Hz, 1H), 8.72 ppm (d, J = 6.6 Hz, 1H); 13C NMR
(100 MHz, CDCl3, TMS): d = ꢀ4.2, ꢀ4.1, 18.3, 18.3, 24.3, 25.1,
25.9, 27.7, 27.9, 32.4, 33.8, 34.4, 35.0, 35.3, 35.7, 40.5, 45.0, 47.1,
50.7, 53.0, 55.4, 60.2, 61.7, 77.2, 117.1, 120.4, 120.9, 120.9, 121.4,
124.1, 124.1, 126.5, 128.1, 129.3, 130.1, 130.1, 130.1, 132.7,
132.7, 133.1, 135.5, 141.2, 145.1, 145.9, 146.0, 146.6, 148.6,
155.1, 160.6, 164.2, 173.2, 173.4 ppm; HRMS (ESI): m/z: calcd for
C61H85N11O10S3Si2: 1284.5254 [M+H+]; found: 1284.5254.
4.4. Determination of Ks values for 9 with Nevn and anti-
dopamine IgG1 by 27-MHz quartz-crystal microbalance (QCM)
analysis
QCM experiments were performed on an Affinix-Q4 apparatus
(Initium Inc., Japan). The clean Au (4.9 mm2) electrode equipped
on quartz crystal was incubated with an aqueous solution of 3,30-
dithiodipropionic acid (3 mM) at room temperature for 45 min.
The surfaces were activated by treatment with a mixture of
EDCꢃHCl (0.26 M) and N-hydroxysuccinimide (0.44 M) for 45 min.
The chip was allowed to stand to reach equilibrium at 25 °C in
500
tion (1 mg/mL) was injected and the frequency change of quartz
oscillator was recorded for specific time points. After adding 1
lL phosphate buffered saline (PBS). Then, 5 lL of Nevn solu-
lL
of an aqueous solution of ethanolamine (1 M) to block the remain-
ing activated groups, the PBS was replaced with PBST. For the
4.2.8. Synthesis of 1-{4-[2-(3,4-dihydroxyphenyl)ethylamino
methyl]phenyl}-4-{2-methylquinoline-8-[p-(N0-2-propyny
immobilization of Nevn–9, 1
(4 g/mL) was injected at several time points and the change in
frequency was recorded. Based on the fact that a frequency de-
crease ( F) of 1 Hz corresponds to a mass increase of 30 pg on
the electrode (0.049 cm2), Ks1 values were determined from the
lL of an aqueous solution of 9
lureido)benzensulfonyloxy]}-5-{N,N0-dimethyl-N-[2-(
D
-biotinyl
l
amino)]sulfamoyl}-1,2,3-triazole (9)
HF (15 drops) was added to
a
solution of 31 (43 mg,
D
0.033 mmol) in CH3CN (6 mL), the solution stirred for 30 min at

3412
S. Aoki et al. / Bioorg. Med. Chem. 17 (2009) 3405–3413
2. (a) Wilchek, M.; Bayer, E. A. Avidin–Biotin Technology (Methods in Enzymol.). In
Vol. 184; Academic Press: San Diego, 1990; (b) Meier, T.; Fahrenholz, F. A
Laboratory Guide to Biotin-Labeling in Biomolecule Analysis; Birkhauser:
Boston, 1996; (c) Biotin; Icon Group Intl: San Diego, 2004; (d) Hofmann, K.;
Kiso, Y. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3516; (e) Wilchek, M.; Bayer, E. A.
Anal. Biochem. 1988, 171, 1; (f) Diamandis, E. P.; Christpoulos, T. K. Clin. Chem.
1991, 37, 625.
relationship between the decrease in frequency and the time for
equilibration.
4.5. Photoreaction for the release of dopamine–IgG complex
3. (a) Hendrickson, W. A.; Pahler, A.; Smith, J. L.; Satow, Y.; Merritt, E. A.;
Phizackerley, R. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2190; (b) Weber, P. C.;
Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85; (c)
Weber, P. C.; Wendoloski, J. J.; Pantoliano, M. W.; Salemme, F. R. J. Am. Chem.
Soc. 1992, 114, 3197; (d) Pugliese, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J.
Mol. Biol. 1993, 231, 698; (e) Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5076; (f) González, M.; Bagatolli, L. A.;
Echabe, I.; Arrondo, J. L. R.; Argarana, C. E.; Cantor, C. R.; Fidelio, G. D. J. Biol.
Chem. 1997, 272, 11288; (g) Freitag, S.; Le Trong, I.; Chilkoti, A.; Klumb, L. A.;
Stayton, P. S.; Stenkamp, R. E. J. Mol. Biol. 1998, 279, 211.
4. (a) Green, N. M. Adv. Protein Chem. 1975, 29, 85; (b) Finn, F. M.; Titus, G.;
Montibeller, J. A.; Hofmann, K. J. Biol. Chem. 1980, 255, 5742.
5. (a) Hofmann, K.; Titus, G.; Montibeller, J. A.; Finn, F. M. Biochemistry 1982, 21,
978; (b) Hofmann, K.; Zhang, W. J.; Romovacek, H.; Finn, F. M.; Bothner-By, A.
A.; Mishra, P. K. Biochemistry 1984, 23, 2547.
Streptavidin coated wells were treated with 9 and an anti-
dopamine antibody as described above and photoirradiated at
313 nm utilizing a USHIO Optical Module X equipped with a super
high pressure UV lamp (USHIO), UV transmitting and a visible
absorbing filter U-330 (Kenko, Co. Ltd, Japan), and an optical filter
HQBP313-UV (Asahi Spectra, Co. Ltd, Japan). After irradiation for
given periods, the wells were washed with PBST (100 lL) and
the secondary antibody (diluted 1:1000 with PBST) was added
to the wells. After incubation at room temperature and washing,
o-phenylenediamine and H2O2 were added and the absorbance
was measured at 450 nm on a Bio-Rad model 550 microplate
reader (Bio-Rad, Hemel, Hempstead, UK).
6. Hirsch, J. D.; Eslamizar, L.; Filanoski, B. J.; Malekzadeh, N.; Haugland, R. P.;
Beechem, J. M.; Haugland, R. P. Anal. Biochem. 2002, 308, 343.
7. Fudem-Goldin, B.; Orr, G. A. Methods Enzymol. 1990, 184, 167.
8. Reznik, G. O.; Vajda, S.; Sano, T.; Cantor, C. R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
13525.
4.6. Western blot
9. Haugland, R. P. Handbook of Fluorescent Probes and Research Products, 9th ed.;
Molecular probes: Eugene, OR, 2002.
10. (a) Shimkus, M.; Levy, J.; Herman, T. Proc. Natl. Acad. Sci. U.S.A. 1985, 93, 2593;
(b) Soukup, G. A.; Cerny, R. L.; Maher, L. J. Bioconjugate Chem. 1995, 6, 135; (c)
Kang, Y.-S.; Voigt, K.; Bickerl, U. J. Drug Targeting 2000, 8, 3425.
Avidin–agarose beads (purchased from Sigma–Aldrich) were re-
acted with 9 in PBST at room temperature for 2 h, washed three
times with PBST, and then reacted with the anti-dopamine anti-
body at room temperature for 1 h. After washing the resulting avi-
din beads three times with PBST, the beads were resuspended in
0.5 mM Tris–HCl buffer, and then transferred to 24-well plate.
These suspensions were photoirradiated at 313 nm as described
above to release dopamine-antibody complexes. The resulting
11. Lin, W.-C.; Morton, T. H. J. Org. Chem. 1991, 56, 6850.
12. van der Veken, P.; Dirksen, E. H. C.; Ruijter, E.; Elgersma, R. C.; Heck, A. J. R.;
Rjiker, D. T. S.; Slijper, M.; Liskamp, R. M. L. ChemBioChem 2005, 6, 2271.
13. (a)Caged Compounds (Methods in Enzymol.); Marriot, G., Ed.; Academic Press:
New York, 1998; Vol. 291, (b) Adams, S. R.; Tsien, R. Y. Ann. Rev. Physiol. 1993,
}
55, 755; (c) Banerjee, A.; Grewer, C.; Ramakrishnan, L.; Jurgen, J.; Gameiro, A.;
Breitinger, H.-G. A.; Gee, K. R.; Carpenter, B. K.; Hess, G. P. J. Org. Chem. 2003, 68,
8361; (d) Adams, S. R.; Kao, J. P. Y.; Grynkiewicz, G.; Minta, A.; Tsien, R. Y. J. Am.
Chem. Soc. 1988, 110, 3212.
mixture was filtered through DISMIC-13HP PTFE (0.45 lm,
ADVANTEC) to remove the beads, which were washed with
0.5 mM Tris–HCl buffer, and the combined filtrate was freeze-
dried. The resulting materials were dissolved in a mixture of 10%
14. Bochet, C. G. J. Chem. Soc., Perkin Trans. 1 2002, 125.
15. Thiele, C.; Fahrenholz, F. Anal. Biochem. 1994, 218, 330.
16. Olejnik, J.; Sonar, S.; Krzymanska-Olejnik, E.; Rothschild, K. J. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 7590.
17. (a) Pandori, M. W.; Hobson, D. A.; Olejnik, J.; Krzymanska-Olejnik, E.;
Rothschild, K. J.; Palmer, A. A.; Phillips, T. J.; Sano, T. Chem. Biol. 2002, 9, 567;
(b) Pandori, M. W.; Sano, T. Gene Ther. 2000, 7, 1999.
18. (a) Aoki, S.; Sakurama, K.; Matsuo, N.; Yamada, Y.; Takasawa, R.; Tanuma, S.;
Shiro, M.; Takeda, K.; Kimura, E. Chem. Eur. J. 2006, 12, 9066; (b) Kimura, E.;
Takasawa, R.; Tanuma, S.; Aoki, S. Science STKE 2004, pl 7; (c) Kimura, E.; Aoki,
S. BioMetals 2004, 104, 769; (d) Aoki, S.; Kimura, E. In Comprehensive
Coordination Chemistry II; Que, L., Jr., Tolman, W. B., Eds.; Elsevier:
Amsterdam, 2004; Vol. 8, p 601; (e) Aoki, S.; Kagata, D.; Shiro, M.; Takeda,
K.; Kimura, E. J. Am. Chem. Soc. 2004, 126, 13377; (f) Kimura, E.; Aoki, S.; Kikuta,
E.; Koike, T. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3731; (g) Aoki, S.; Kaido, S.;
Fujioka, H.; Kimura, E. Inorg. Chem. 2003, 42, 1023.
SDS (8
l
L), glycerol (4
l
L), and water (8
lL), loaded on a 15%
SDS–polyacrylamide gel for electrophoresis (20
l
L/lane), and then
transferred to a PVDF membrane (Immobilon-P Transfer Mem-
brane, Millipore) under wet conditions. The membrane was
blocked with 3% bovine serum albumin (BSA, Sigma) and incubated
with HRP–conjugated anti-IgG1 antibody diluted with PBST for 1 h
at room temperature. The membrane was rinsed with TBST
(5 min ꢁ 3) and visualized using ECL Plus Western blotting detec-
tion reagents on a LAS 3000 lumino-image analyzer (FUJIFILM).
Acknowledgments
19. Aoki, S.; Sakurama, K.; Ohshima, R.; Matsuo, N.; Yamada, Y.; Takasawa, R.;
Tanuma, S.; Takeda, K. Inorg. Chem. 2008, 47, 2747–2754.
20. It has been confirmed that 8-quinolinyl sulfonate derivatives that have no
Zn2+-chelation groups also undergo photolysis. Detail about mechanistic study
of this photocleavage reaction will be shortly reported (Kageyama, Y.;
Ohshima, R.; Fujiwara, Y.; Tanimoto, Y.; Yamada, Y.; Aoki, S., submitted for
publication).
We wish to thank Professor Yasuhiko Masuho, Dr. Junya Kohr-
oki, Prof. Fumio Fukai, and Dr. Toshiyuki Owaki (Faculty of Pharma-
ceutical Sciences, Tokyo University of Science) for helpful
discussions and assistance. We thank Dr. Mikihiko Nakamura and
Ms. Yukiko Suzuki (ULVAC, Inc., Japan), for helpful assistance on
QCM experiments. This work was supported by grants-in-aid from
the Ministry of Education, Culture, Sports, Science, and Technology
(MEXT) of Japan (Nos. 18390009, and 19659026) and the High-
Tech Research Center Project for Private Universities (matching
fund subsidy from MEXT). S.A. is grateful for grants from the Sumi-
tomo Foundation (Tokyo), the Naito Foundation (Tokyo), and the
Tokuyama Science Foundation (Tokyo).
21. (a) Cooper, J. R.; Bloom, F. E.; Roth, R. H. The Biochemical Basis of
Neuropharmacology, 8th ed.; Oxford University Press: New York, 2002; (b)
Bloom, F. E.; Nelson, C. A.; Lazersohn, A. Brain, Mind, and Behavior, 3rd ed.;
Worth: New York, 2002; (c) Hall, Z. W. An Introduction to Molecular
Neurobiology; Sinauer Associates: Sunderland, Massachusetts, 1992; (d)
Hardie, D. G. Biochemical Messengers; Chapman & Hall: London, 1991; (e)
Strange, P. G. Pharmacol. Rev. 2001, 53, 119; (f) Sealfon, S. C.; Olanow, C. W.
Trends Neurosci. 2000, 23, S34; (g) Huang, X.; Lawler, C. P.; Lewis, M. M.;
Nichols, D. E.; Mailman, R. B. Int. Rev. Neurobiol. 2001, 48, 65; (h) de Almeida, R.
M. M.; Ferrari, P. F.; Parmigiani, S.; Miczek, K. A. Eur. J. Pharmacol. 2005, 526, 51.
22. Sugawara, K.; Tanaka, S.; Nakamura, H. J. Electroanal. Chem. 1999, 466, 117.
23. (a) Simpson, M. M.; Ballesteros, J. A.; Chiappa, V.; Chen, J.; Suhiro, M.; Hartman,
D. S.; Godel, T.; Snyder, L. A.; Sakmar, T. P.; Javitch, J. A. Mol. Pharmacol. 1999,
56, 1116; (b) Appell, M.; Berfield, J. L.; Wang, L. C.; Dunn, W. J., III; Chen, N.;
Reith, M. E. A. Biochem. Pharmacol. 2004, 67, 293.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.03.031.
24. Weiboldt, R.; Ramesh, D.; Jabri, E.; Karplus, P. A.; Carpenter, B. K.; Hess, G. P.
J. Org. Chem. 2002, 67, 8827–8831.
25. Zhu, W.; Ma, D. J. Chem. Soc., Chem. Commun. 2004, 7, 888.
26. Mornet, R.; Leonard, N. J.; Theiler, J. B.; Doree, M. J. Chem. Soc., Perkin Trans. 1
1984, 879.
27. (a) Gerlach, M.; Jutzi, P.; Johannes, P.; Przuntek, H. Anorg. Chem., Org. Chem.
1983, 38B, 237; (b) Denora, N.; Laquintana, V.; Lopedota, A.; Serra, M.; Dazzi, L.;
References and notes
1. Clercq, P. D. J. Chem. Rev. 1997, 97, 1755.

S. Aoki et al. / Bioorg. Med. Chem. 17 (2009) 3405–3413
3413
Biggio, G.; Pal, D.; Mitra, A.; Latrofa, A.; Trapani, G.; Liso, G. Pharm. Res. 2007, 24,
1309; (c) Nakonieczna, K.; Przychodzen, W.; Chimiak, A. Amino Acids 1995, 8,
109.
Hockfield, S.; Carlson, S.; Evans, C.; Levitt, P.; Pintar, J.; Silberstein, L. In Selected
Methods for Antibody and Nucleic Acid Probes (Moleculr Probes of the Nervous
System); Cold Spring Harbor laboratory Press: New York, 1993; Vol. 1; (d)
Crowther, J. R. The ELISA Guidebook (Methods in Moleuclar Biology). In Vol. 149;
Humana Press, 2000.
28. (a) Eiseführ, A.; Arora, S. P.; Sengle, G.; Takaoka, R. L.; Nowick, S. J.; Famulok, M.
Bioorg. Med. Chem. 2003, 11, 235; (b) Amir, R. J.; Pessah, N.; Shamits, M.; Shabat,
D. Angew. Chem., Int. Ed. 2003, 42, 4494.
29. Pearce, D. A.; Jotterand, N.; Carrico, I. S.; Imperiali, B. J. Am. Chem. Soc. 2001,
123, 5160.
30. (a) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 24, 1128; (b) Wang,
Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem.
Soc. 2003, 125, 3192; (c) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem.,
32. (a) Tarche, P. J.; Chu, V. P.; Whittern, D. Anal. Biochem. 1987, 165, 230; (b) Liu,
H.; Wang, Z.; Liu, Y.; Xiao, J.; Wang, C. Thermochim. Acta 2006, 443, 173.
33. (a) Okahata, Y.; Matsunobu, Y.; Ijiro, K.; Mukai, M.; Murakami, A.; Makino, K.
J. Am. Chem. Soc. 1992, 114, 8299; (b) Okahata, Y.; Niikura, K.; Sugiura, Y.;
Sawada, M.; Morii, T. Biochemistry 1998, 37, 5666; (c) Okahata, Y.; Kawase, M.;
Niikura, K.; Ohtake, F.; Furusawa, H.; Ebara, Y. Anal. Chem. 1998, 70, 1288–
1296; (d) Nishino, H.; Nihira, T.; Mori, T.; Okahata, Y. J. Am. Chem. Soc. 2004,
126, 2264.
34. Recently, we have synthesized biotin–dopamine conjugates having longer
photocleavable linkers than 9. Preliminary experiments have indicated that its
affinity with Nevn and its photoreactivity were almost identical to that of 9.
Detail will be described elsewhere.
´
Int. Ed. 2001, 40, 2004; (d) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.;
Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002,
41, 1053; (e) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596.
31. (a) Engvall, E. Methods Enzymol. 1980, 70, 419; (b) Goding, J. W. Monoclonal
Antibodies: Principles and Practice, 3rd ed.; Academic Press: London, 1996; (c)