Cleavable linker for photo-cross-linked small-molecule affinity matrix

Article abstract of DOI:10.1021/bc900316q

The introduction of a cleavable site in a photoactivatable linker, which is used to immobilize small molecules on an affinity matrix via a site-nonselective carbene addition/insertion reaction, makes it possible to verify the presence of the immobilized small molecule on the affinity matrix. It also permits the efficient detection of proteins covalently bound to the immobilized small molecule.

Full text of DOI:10.1021/bc900316q

182
Bioconjugate Chem. 2010, 21, 182–186
TECHNICAL NOTES
Cleavable Linker for Photo-Cross-Linked Small-Molecule Affinity Matrix
Naoki Kanoh,*,†,‡ Hiroshi Takayama,† Kaori Honda,‡ Takashi Moriya,† Takayuki Teruya,‡,§ Siro Simizu,‡
Hiroyuki Osada,‡ and Yoshiharu Iwabuchi†
Graduate School of Pharmaceutical Sciences, Tohoku University, and Antibiotics Laboratory, Advanced Science Institute,
RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Received July 16, 2009; Revised Manuscript Received November 3, 2009
The introduction of a cleavable site in a photoactivatable linker, which is used to immobilize small molecules on
an affinity matrix via a site-nonselective carbene addition/insertion reaction, makes it possible to verify the presence
of the immobilized small molecule on the affinity matrix. It also permits the efficient detection of proteins covalently
bound to the immobilized small molecule.
linking efficiency between BASMs. We have already shown
with LC/MS/MS that a variety of small molecules may be photo-
cross-linked with the photoactivatable linker 1a (4, 5). However,
molecules having different functionalities and/or molecular sizes
are thought to react differently with the photogenerated carbenes,
resulting in different numbers of immobilized molecules. It was
even difficult to verify the presence of the immobilized small
molecule on the affinity matrix.
The second problem is the nature of covalent bond formation
between the BASM and the PALC affinity matrix. A growing
number of BASMs are known to bind covalently with their
protein targets (6), but such ligand-reacting proteins are
sometimes difficult to detach from the affinity matrix if the
ligands are immobilized covalently on the affinity matrix.
These problems led us to consider the introduction of a
cleavable site in the photoactivatable linker. Among the various
INTRODUCTION
The affinity purification of protein targets for biologically
active small molecules (BASMs) has traditionally been used
for forward chemical genetic studies (1). Such experiments have
provided not only key information on the mode of action of
BASMs, but also insights of relevance to other biological
processes. The traditional target discovery/identification process
consists of the following three steps: (1) the search for a position
on a BASM of interest that does not significantly abrogate the
intrinsic biological activity; (2) preparation of the BASM
conjugate with a biotin or an affinity matrix that is covalently
attached via a linker; and (3) biochemical pull-down experiments
using the synthesized conjugate. Although this traditional
approach has yielded many promising results in the past two
decades, it remains both time-consuming and challenging in
many cases.
In the course of our efforts to develop a rapid and widely
applicable approach to enhance the target discovery/identifica-
tion process, we recently reported a photo-cross-linked small-
molecule affinity matrix in which the BASM of interest is
immobilized on photoactivatable linker-coated (PALC) agarose
beads 1a via a site-nonselective carbene addition/insertion
reaction when treated with UV irradiation (Figure 1) (2). This
platform is very effective when the BASM of interest tolerates
UV irradiation (365 nm), and has been used to pull down cellular
targets for BASMs in our laboratory. Using this technology,
we recently identified a detoxification enzyme, namely, glyox-
alase I, as a molecular target of methyl gerfelin, a fungus-derived
osteoclastogenesis inhibitor (3).
Figure 1. Photo-cross-linked small-molecule affinity matrix.
Despite these successes, we identified serious problems with
the technology. First, there is a possible difference in the cross-
* To whom correspondence should be addressed. Naoki Kanoh,
Graduate School of Pharmaceutical Sciences, Tohoku Univer-
sity, Aobayama, Sendai 980-8578, Japan. E-mail: kanoh@
mail.pharm.tohoku.ac.jp; Fax: +81-22-795-6847; Tel: +81-22-795-
6847.
† Tohoku University.
‡ RIKEN.
§ Present address: Okinawa Laboratory, Lequio Pharma Co., Ltd.,
Uruma, Okinawa 904-2234, Japan.
Figure 2. Chemical structure of photoactivatable linker-coated affinity
matrix.
10.1021/bc900316q  2010 American Chemical Society
Published on Web 12/22/2009

Technical Notes
Bioconjugate Chem., Vol. 21, No. 1, 2010 183
2
options, we selected a disulfide bond because it was reported
to be stable in many biological applications and it can be cleaved
selectively using mild reducing agents such as glutathione or
dithiothreitol (DTT) (7). A cleavable photoactivatable hetero-
bifunctional reagent utilizing disulfide linkage was reported as
early as 1981 (8), but the utility in forward chemical genetic
study has not yet been fully assessed.
We therefore designed and prepared the cleavable photoac-
tivatable linker-coated (CPALC) affinity matrix 2a (Figure 2)
and compared it with 1a in terms of its ability to immobilize
BASM. We also demonstrate here that 2a could be used for
efficient detection of the BASM binding protein, especially in
the context of the protein-reacting ligand.
42.3, 40.6, 38.8, 37.5, 28.3 (q, JCF ) 40.1 Hz); HRMS (EI)
m/z 364.0641 [(M+H)+], calcd. for C13H15ON4F3S2 364.0639.
Cleavable Photoactivatable Linker-Coated (CPALC)
Agarose Beads 2a. Commercially available NHS-activated
Sepharose 4 fast flow beads (6) (GE Healthcare Bioscience,
Uppsala, Sweden; 160-230 nmol NHS/10 µL of swelled beads,
150 µL) were washed successively with 1 mM aq HCl (5 × 1
mL) and coupling solution (1:1 dioxane-0.1 M aq NaHCO3)
(2 × 1 mL) at rt. To swelled beads (150 µL) was added a
solution of 2b (6.3 mg, 17.3 mmol) in coupling solution (100
µL) and methanol (50 µL) at rt. The mixture was shaken by
using a CM-1000 mixer (EYELA, Tokyo, Japan) for 2 h. The
resultant beads were washed with the coupling solution (5 × 1
mL). To the beads was added a 1 mM solution of ethanolamine
in Tris-HCl buffer (0.1 M Tris-HCl, pH 8.0, 1 mL), and the
mixture was shaken for an another 1 h. The beads were washed
with Milli-Q (5 × 1 mL), then transferred into a spin column
using 1 mL of Milli-Q. The beads were washed with methanol
(3 × 400 µL), centrifuged, and dried in vacuo.
EXPERIMENTAL PROCEDURES
Materials and Instrumentation. All chemical reactions were
carried out under argon atmosphere with dehydrated solvents
under anhydrous conditions, unless otherwise noted. Dehydrated
THF and CH2Cl2 were purchased from Kanto Chemical Co.,
Inc. (Tokyo, Japan). Other solvents were dehydrated and/or
distilled according to standard protocols. Reagents were obtained
from commercial suppliers and used without further purification,
unless otherwise noted. Reactions were monitored by thin-layer
chromatography (TLC) carried out on Kieselgel 60 F254 silica
gel plates (Merck KGaA) or NH-silica gel plates (Fuji Silysia
Chemical Ltd., Aichi, Japan). Column chromatography was
performed on silica gel 60N (spherical, neutral, 63-210 µm;
Kanto Chemical Co., Inc., Osaka, Japan) or Chromatorex
NHDM 1020 (amine-coated silica gel, 100-200 mesh; Fuji
Silysia Chemical Ltd., Kasugai, Japan). IR spectra were recorded
on a JASCO FT/IR-410 Fourier transform infrared spectrometer.
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
recorded on a JEOL JMN AL400 spectrometer. Mass spectra
were recorded on JEOL JMS-DX303, JEOL JMS-700, and
Waters Micromass ZQ2000 LC mass spectrometers.
Immobilization of Small Molecules on the PALC- and
CPALC-Agarose Beads. PALC- or CPALC-agarose beads (100
µL) were washed with Milli-Q (3 × 400 µL) using a spin
column and transferred to a glass sample vial. Solution of a
small molecule (10 mM in MeOH, 150 µL) was added to the
vial, and the mixture was concentrated using a rotary evaporator
and dried in vacuo. The beads were suspended in 2-propanol
(200 µL), residual water absorbed on the beads was removed
azeotropically using a rotary evaporator, and the beads were
dried again in vacuo. The beads were irradiated at 365 nm (4
J/cm2) by a CL-1000 L ultraviolet cross-linker (UVP Inc., San
Gabriel, CA), then washed with 50% aq MeOH, MeOH, DMSO,
and MeOH (3 × 400 µL each) to give small-molecule-
immobilized beads. The beads were suspended in PBS (8 mM
Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 3 mM KCl, pH
7.4) containing 1 mM sodium azide, and stored at 4 °C. The
beads were washed with PBS before use.
Synthesis of Cleavable Photoactivatable Linker-Coated
Agarose Beads.
Cleavage of Immobilized Small Molecules from the
CPALC Small Molecule Beads and MS Analysis. CPALC
small molecule beads (10 µL) were treated with a 100 mM DTT
solution in ethanol (100 µL) at 50 °C for 10 min. The
supernatant was analyzed by a Waters Micromass ZQ 2000 LC
mass spectrometer.
Disulfide 5. A solution of NHS ester 4 (72.2 mg, 0.221
mmol), N-(tert-butoxycarbonyl)cystamine (3) (87.5 mg, 0.347
mmol, 1.6 equiv), and Et3N (310 µL, 2.21 mmol, 10 equiv) in
CH3CN (3 mL) was stirred at rt for 5.5 h. After an addition of
water (10 mL), the mixture was extracted with ethyl acetate (2
× 70 mL). The combined organic layers were dried over
MgSO4, filtered, and concentrated in vacuo. The residue was
purified via silica gel column chromatography (ethyl acetate/
hexane ) 1:2) to afford 5 (96.6 mg, 0.208 mmol, 94%) as a
pale yellow oil. 5: IR (neat) 3323, 2978, 2928, 1694, 1644, 940
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J ) 8.2 Hz, 2H),
7.24 (d, J ) 8.3 Hz, 2H), 4.95 (s, 1H), 3.78 (q, J ) 6.1 Hz,
2H), 3.47 (q, J ) 6.1 Hz, 2H), 2.99 (t, J ) 5.5 Hz, 2H), 2.80
Preparation of Phoslactomycin D-Immobilized Beads
(PLM-D Beads). To NHS-activated Sepharose 4 fast flow beads
(50 µL), which were washed and swelled as described for the
synthesis of CPALC agarose beads (2a), was added phoslac-
tomycin D (1.1 mg, 1.7 µmol) in coupling solution (100 µL)
and MeOH (50 µL). The mixture was shaken for 2 h. The
resultant beads were washed with Milli-Q (2 × 1 mL) and
MeOH (5 × 1 mL). To the beads was added a 1 mM solution
of ethanolamine in Tris-HCl buffer (0.1 M Tris-HCl, pH 8.0,
1 mL), and the mixture was shaken for 1 h. The beads were
washed with Milli-Q (5 × 1 mL), then transferred into a spin
column using 1 mL of Milli-Q. The beads were washed
successively with 50% aq MeOH, MeOH, DMSO, and MeOH
(3 × 400 µL each) to give PLM-D beads. The beads were
suspended in PBS (8 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM
NaCl, 3 mM KCl, pH 7.4) containing 1 mM sodium azide, and
stored at 4 °C. The beads were washed with PBS before use.
Detection of PP2A Subunits Using Phoslactomycin
D-Immobilized Beads. HL-60 cells (RIKEN Cell Bank,
Ibaraki, Japan) were washed twice with PBS, then suspended
in binding buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM
EDTA, and 2.5 mM EGTA, pH 7.5) containing 1 mM DTT,
0.1 mM MgCl2, and Complete protease inhibitor cocktail
(Roche Diagnostics GmbH). Suspended cells were lysed by
sonication, and the resulting mixture was incubated at 4 °C
(t, J ) 6.7 Hz, 2H), 1.41 (s, 9H); 13C NMR (100 MHz, CDCl3)
1
δ 166.5, 156.0, 135.4, 132.2, 127.7, 126.5, 121.9 (q, JCF
)
)
2
274.4 Hz), 79.8, 39.6, 39.0, 38.8, 37.7, 28.3, 28.3 (q, JCF
40.9 Hz); HRMS (EI) m/z 464.1178 (M+), calcd. for
C18H23O3N4F3S2 464.1164.
Cleavable Photoactivatable Linker 2b. To a solution of 5
(69.7 mg, 0.15 mmol) in CH2Cl2 (3 mL) was added trifluoro-
acetic acid (330 µL, 4.5 mmol, 30 equiv) at rt. After stirring
for 3 h, the mixture was concentrated in vacuo, and the residue
was purified via amine-coated silica gel column chromatography
(MeOH-CHCl3 0:100 f 1:10 f 1:4) to afford 2b (47.6 mg,
0.131 mmol, 87%) as a pale yellow oil. 2b IR (neat) 3323, 1638,
1
1154 cm-1; H NMR (400 MHz, CDCl3) δ 7.81 (d, J ) 9.1
Hz, 2H), 7.25 (d, J ) 8.4 Hz, 1H), 6.71 (1H, brs), 3.82 (t, J )
6.1 Hz, 2H), 3.03 (t, J ) 6.1 Hz, 2H), 2.93 (d, J ) 6.1 Hz,
2H), 2.80 (t, J ) 6.1 Hz, 2H); 13C NMR (400 MHz, CDCl3) δ
1
166.3, 135.4, 132.4, 127.4, 126.6, 121.9 (q, JCF ) 275.3 Hz),

184 Bioconjugate Chem., Vol. 21, No. 1, 2010
Kanoh et al.
Scheme 1. Synthesis of Cleavable Photoactivatable Table Linker-Coated (CPALC) Agarose Beadsa
a Reagents and conditions: (a) 4, Et3N, CH3CN, 5.5 h, 94%; (b) trifluoroacetic acid, CH2Cl2, r.t., 3 h, 87%; (c) 6, 0.1 M aq NaHCO3-dioxane
(1:1), r.t., 2 h, then ethanolamine, r.t., 1 h.
Figure 3. Electrospray ionization of photo-cross-linked products cleaved
from the cleavable matrix by DTT reduction of disulfide bonds. (A)
Figure 4. Detection of CsA- and FK506-binding proteins using PALC-
and CPALC-agarose beads. (A) Detection of FKBP12 using FK506-
immobilized beads: PALC- (lanes 2-4) or CPALC- (lanes 5-7)
agarose beads were irradiated in the presence (lanes 3, 4, 6, and 7) or
absence (lanes 2 and 5) of FK506. The beads were incubated with cell
lysates of E. coli overexpressing (His)6-FKBP12 overnight. Beads in
lanes 4 and 7 were pretreated with DTT before being incubated with
cell lysate. The beads were washed and analyzed by 15% SDS-PAGE
followed by CBB staining. *protein unidentified. (B) Detection of
cyclophilin A (CpA) using cyclosporin A (CsA)-immobilized beads.
Cleaved product from the cyclosporin A-immobilized beads; (B) from
the FK506-immobilized beads. (C) Structures and calculated masses
for the cleaved products.
for 2 h. After the insoluble material was removed by
centrifugation (13 000 × g, 4 °C, 30 min), the supernatant
was collected as cell lysate. The total protein concentration
of the cell lysate was determined by the Bradford method to
be 5.7 mg/mL. The lysate was diluted with binding buffer
containing 0.1 mM DTT and 0.1 mM MgCl2, and the diluted
cell lysate (800 µL, 2 mg protein) was incubated with 10 µL
of PLM-D beads or 20 µL of CPALC PLM-D beads at 4 °C
for 1 h. The reacted beads were washed with binding buffer
containing 0.2% NP-40 (5 × 700 µL). The bound proteins
were eluted from the beads by boiling with 20 µL of SDS
sample buffer (140 mM Tris-HCl, 22.4% glycerol, 6% SDS,
10% 2-mercaptoethanol, 0.02% bromophenol blue, pH 6.8)
at 110 °C for 10 min. The total volume of sample eluted
from CPALC PLM-D beads and 1/10 of the sample eluted
from PLM-D beads were subjected to SDS-PAGE.
Tween 20, pH 8.0) containing 6% CS (#SH30072.03; Hyclone
Laboratories Inc., Logan, UT) at rt for 1 h, the membrane was
incubated with rabbit anti-PP2A/a antibody (#07-250; Milli-
pore) in the same buffer at a dilution of 1:1000 at 4 °C overnight.
After washing with TBS-T, the membrane was incubated with
peroxidase-labeled antirabbit IgG antibody (#NA934 V; GE
Healthcare) at a dilution of 1:1000 at rt for 1 h. PP2A/a protein
was detected with SuperSignal West Pico chemiluminescent
substrate (Pierce, Rockford, IL) according to the manufacturer’s
instructions. After the detection, the membrane was treated with
stripping buffer (25 mM glycine-HCl, 1% SDS, pH 2) at rt
for 45 min. The stripped membrane was incubated with anti-
PP2A/cR antibody (#610555; BD Biosciences, San Jose, CA)
in TBS-T containing 5% skim milk at a dilution of 1:1000 at 4
Western Blot Analysis. Eluted proteins from beads were
separated on SDS-polyacrylamide gel (10%), and then trans-
ferred to PVDF membrane (Millipore, Bedford, MA). After
blocking with TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.05%

Technical Notes
Bioconjugate Chem., Vol. 21, No. 1, 2010 185
Figure 5. Detection of PP2A subunits using phoslactomycin D-immobilized beads. (A) Structure of phoslactomycin D (PLM-D). (B) Western
blotting analysis of PP2A subunits coprecipitated with PLM-D beads and CPALC PLM-D beads. (C) Schematic presentation of PLM-D beads,
CPALC PLM-D beads, and coprecipitated PP2A subunits.
°C overnight. PP2A/cR protein was detected with SuperSignal
To validate the ability of the cleavable beads to capture and
purify the binding protein in the context of the immobilized
ligand, cleavable affinity beads were treated with a protein
mixture containing their target proteins. The CsA molecule is
known to bind to cyclophilin A (CpA) (10), a peptidyl prolyl
cis-trans isomerase, with a KD of 36.8 nM (11), whereas FK506
binds to another peptidyl cis-trans isomerase FKBP12 with a
KD of 0.4 nM (12). Cleavable CsA and FK506 beads were mixed
with the cell lysates of an E. coli BL21(DE3)pLysS strain
overexpressing GST-cyclophilin A conjugate (GST-CpA) and
(His)6-tagged FKBP12, respectively. Proteins that coprecipitated
with the respective beads were then subjected to SDS gel
electrophoresis.
As can be seen in Figure 4, CPALC CsA and FK506 beads
can be used to purify their binding proteins (lanes 6 and 13 in
Figure 4A and B, respectively), although we identified some
difference between the cleavable and noncleavable versions,
especially in the amount of nonspecific binding protein (denoted
by the asterisks). However, when the cleavable beads were
pretreated with DTT (100 mM in ethanol, 50 °C, 10 min) before
being incubated with cell lysates, the beads largely lost their
ability to isolate binding proteins because of the absence of the
ligand.
This platform was then applied to detect a protein that forms
a covalent bond with its ligand. We recently reported that the
antifungal antibiotics known as phoslactomycins (13) inhibit
protein phosphatase 2A (PP2A) through the covalent modifica-
tion of cysteine-269 of the catalytic subunit of PP2A (PP2A/c)
(14). However, when we prepared affinity beads on which
phoslactomycin D (PLM-D: 3, Figure 5A) was immobilized
covalently through an amide linkage (Figure 5C, left) and used
them for a pull-down experiment, only a very small amount of
PP2A/c was detected by Western blot analysis (Figure 5B, lane
3, upper section) as compared to the amount of PP2Aa, a
scaffolding subunit of PP2A that is known to bind to PP2A/c
West Pico chemiluminescent substrate.
RESULTS AND DISCUSSION
The cleavable photoactivatable linker 2b was synthesized in
two steps using known N-(tert-butoxycarbonyl)cystamine and
4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-benzoic acid N-hydroxy-
succinimidyl ester (Scheme 1). Conjugation of 2b on agarose
beads was also quite straightforward, yielding the CPALC
affinity matrix 2a. The amount of the cleavable linker 2b
introduced on the matrix 2a was estimated to be 269 ( 41 nmol
in 1 mL of swollen beads using a spectroscopic method (9).
This result is similar to the amount of 1a introduced on the
PALC agarose matrix (241 ( 5 nmol mL-1).
First, we immobilized cyclosporin A (CsA) and FK506 onto
2a by using the same procedure that was used for the
immobilization of these compounds onto 1a (2). Specifically, a
solution of the respective small molecules was independently
mixed with the CPALC beads 2a, and the mixtures were
concentrated and dried in vacuo. The dried beads covered with
the respective small molecules were exposed to UV light (365
nm, 4 J/cm2) and then washed thoroughly with methanol,
DMSO, and PBS. Control beads were prepared by the photolysis
of the CPALC beads 2a in the absence of small molecules.
To verify the extent of compound immobilization, both the
resulting CPALC CsA and FK506 beads were treated indepen-
dently under the cleavage conditions (100 mM DTT in ethanol,
50 °C, 10 min). The MS analysis of the supernatant solution
clearly confirmed the presence of cleaved photo-cross-linked
products, as shown in Figure 3. No ion peaks from residual
compounds (m/z 1202 and 803 for CsA and FK506, respectively)
were observed. The MS analyses also confirmed that the above
condition almost completely (>95%) cleaved the disulfide
linkage, which was not affected in the absence of reducing agent
(data not shown).

186 Bioconjugate Chem., Vol. 21, No. 1, 2010
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through hydrogen bonding networks (15, 16) (lane 3, lower
section). Although there are examples in which ligand-binding
proteins were detected by using similar protocols (17), this was
an obvious example in which the primary target molecule for a
protein-reacting ligand was not detected sufficiently using an
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immobilized. PALC PLM-D beads also gave similar results (data
not shown). On the other hand, using the CPALC affinity matrix
developed in the present study, the same analysis successfully
detected both PP2A/c and PP2A/a (lane 5).
In summary, we have shown here that the cleavable small-
molecule affinity matrix enables confirmation of small-molecule
immobilization. We also confirmed its use in the detection of
target proteins in the context of a protein-reacting ligand.
The combined use of this platform with LC/MS/MS may
make it possible to quantitatively determine the amount of
immobilized small molecule and/or small molecule-protein
conjugate by comparison with an internal standard. Furthermore,
it may be possible to determine what and how many conjugates
are formed on the photo-cross-linked affinity matrix by com-
bining this method with LC/MS/MS analysis. Efforts toward
this goal are ongoing in our laboratory.
ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Young
Scientists (A) (No. 19681023; to NK) from the MEXT, Japan,
and by the RIKEN Chemical Biology Project. We thank Dr.
Takemichi Nakamura (RIKEN) for his helpful input. We also
thank Prof. Takayuki Doi and Dr. Masahito Yoshida for
assistance with the LC/MS analysis.
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