Biotinylated quercetin as an intrinsic photoaffinity proteomics probe for the identification of quercetin target proteins

Article abstract of DOI:10.1016/j.bmc.2011.07.005

Quercetin is a flavonoid natural product, that is, found in many foods and has been found to have a wide range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has been no systematic approach to identifying a

Full text of DOI:10.1016/j.bmc.2011.07.005

Bioorganic & Medicinal Chemistry 19 (2011) 4710–4720
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Biotinylated quercetin as an intrinsic photoaffinity proteomics probe
for the identification of quercetin target proteins
Rongsheng E. Wang a, Clayton R. Hunt b, Jiawei Chen a,c, John-Stephen Taylor a,
⇑
a Department of Chemistry, Washington University, St. Louis, MO 63130, USA
b Department of Radiation Oncology, School of Medicine, Washington University, St. Louis, MO 63108, USA
c Center for Biomedical and Bioorganic Mass Spectrometry, Washington University, St. Louis, MO 63130, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Quercetin is a flavonoid natural product, that is, found in many foods and has been found to have a wide
range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has
been no systematic approach to identifying all potential targets of quercetin. We describe an O7-biotin-
ylated derivative of quercetin (BioQ) that can act as a photoaffinity proteomics reagent for capturing
quercetin binding proteins, which can then be identified by LC–MS/MS. BioQ was shown to inhibit heat
induction of HSP70 with almost the same efficiency as quercetin, and to both inhibit and photocrosslink
to CK2 kinase, a known target of quercetin involved in activation of the heat shock transcription factor.
BioQ was also able to pull down a number of proteins from unheated and heated Jurkat cells following UV
irradiation that could be detected by both silver staining and Western blot analysis with an anti-biotin
antibody. Analysis of the protein bands by trypsinization and LC–MS/MS led to the identification of heat
shock proteins HSP70 and HSP90 as possible quercetin target proteins, along with ubiquitin-activating
enzyme, a spliceosomal protein, RuvB-like 2 ATPases, and eukaryotic translation initiation factor 3. In
addition, a mitochondrial ATPase was identified that has been previously shown to be a target of quer-
cetin. Most of the proteins identified have also been previously suggested to be potential anticancer tar-
gets, suggesting that quercetin’s antitumor activity may be due to its ability to inhibit multiple target
proteins.
Received 5 May 2011
Revised 1 July 2011
Accepted 2 July 2011
Available online 13 July 2011
Keywords:
Quercetin
Photoaffinity probe
Biotinylated pull down probe
Heat shock proteins
ATP binding proteins
Casein kinase 2
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
anticancer,6–8 activities with almost no human toxicity.9 At the
molecular level, quercetin has been found to inhibit many ATP
Quercetin (Fig. 1A) is a natural product with multiple medicinal
properties which belongs to the flavonoid family.1 Initially
extracted from red wine, quercetin was subsequently found to exist
widely in leaves, fruits, and vegetables,2 and is available as a dietary
supplement. Quercetin has antioxidant,3,4 anti-inflammatory,5 and
binding enzymes and in particular kinases,10 suggesting that some
of its biological effects may be due to inhibition of signaling path-
ways. Thus quercetin is an interesting lead compound for further
pharmaceutical development,7 and has been discussed in over
6000 journal publications. In spite of this, very little is known about
the full range of proteins that it targets, and which targets are pre-
dominantly responsible for a particular biological effect.
Our interest in quercetin initially arose from its well known
ability to inhibit the heat induction of heat shock protein 70
(HSP70), one of many proteins induced by heat,11 that is, also
known as HSP70-1a, HSP70-1 and HSP72.12,13 One of us had previ-
ously found that HSP70 deficient mouse embryonic fibroblasts are
more sensitive to radiation following heat treatment,14 suggesting
that agents that suppress heat induction of HSP70 might also func-
tion as radiosensitizers. Despite the classification as a heat respon-
sive protein, HSP70 is also induced, along with other heat shock
proteins, in response to wide range of chemical and physiological
stresses, and is overexpressed in many cancers.15–17 Thus small
molecule inhibitors of HSP70 and its induction may have potential
Abbreviations: BSA, bovine serum albumin; CaMK2, Ca2+/calmodulin-dependent
protein kinase II; CK2, casein kinase II; COSY, correlation spectroscopy; DMSO,
dimethyl sulfoxide; DTT, dithiothreitol; EDC, 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide; EDTA, ethylenediaminetetraacetic acid; ESI, electrospray ioniza-
tion; HMBC, heteronuclear multiple bond coherence spectroscopy; HMQC,
heteronuclear multiple quantum coherence spectroscopy; HRESI, high resolution
electrospray ionization mass spectrometry; HSF1, heat shock factor 1; HSP, heat
shock protein; IC50, 50% inhibitory concentration; LC–MS/MS, combined high
performance liquid chromatography tandem mass spectrometry; LRESI, low
resolution electrospray ionization mass spectrometry; MES, morpholinoethane-
sulfonic acid; NMP, N-methyl-2-pyrrolidone; TFA, trifluoroacetic acid; TLC, thin
layer chromatography; Tris, tris(hydroxymethyl)aminomethane; UVA, ultraviolet
light of 290–320 nm.
⇑
Corresponding author. Tel.: +1 314 935 6721; fax: +1 314 935 4481.
E-mail address: taylor@wustl.edu (J.-S. Taylor).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.005

R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
4711
biotin must be attached to the compound at a position that does
not interfere with target protein binding.
OH
OH
A
B
Quercetin: R=H
7-MeQ:
7-CMQ:
7-BioQ:
O
R = CH3,
RO
O
O
To guide the synthesis of biotinylated quercetin derivatives, we
recently synthesized all of the mono-methyl and selected carbo-
methoxymethyl derivatives of quercetin (Fig. 1A) and determined
their ability to inhibit heat shock induction of HSP70.26 We found
that the C7 and C30 hydroxyls on quercetin can be derivatized with
a bulky carbomethoxymethyl group without affecting its ability to
inhibit HSP70 induction, and could therefore be used to attach bio-
tin. We also found that all quercetin derivatives capable of inhibit-
ing heat induction of HSP70, were also able to inhibit two protein
kinases known to activate heat shock transcription factor 1 (HSF1),
casein kinase II (CK2) and Ca2+/calmodulin-dependent protein ki-
nase II (CAMK2), suggesting that these may be targets of querce-
tin’s action. The Ki’s for these enzymes, and for most known
enzyme targets of quercetin are in the micromolar range, indicat-
ing that a photoaffinity approach would be required to pull down
the quercetin binding proteins. Quercetin is intrinsically photoac-
tive,27 however, and has previously been shown to photocrosslink
malate dehydrogenase to which it binds,28 suggesting that it could
serve as its own photoaffinity reagent. Herein, we report the syn-
thesis of the C7-biotinylated quercetin derivative, 7-BioQ
(Fig. 1A), and its use for pulling down potential quercetin target
proteins by the strategy shown in Figure 1B. We show that BioQ
can be photocrosslinked to CK2 in vitro, and to various proteins
in vivo following heat shock. Mass spectrometric analysis of tryp-
sinized fragments of the pulled down proteins led to the identifica-
tion of heat shock protein 70 and 90, two ATPases, an ubiquitin-
activating protein, and a translation initiation factor as possible
quercetin targets. One of the ATPases, mitochondrial ATP synthase,
has already been shown to be a quercetin binding protein,29 while
the others remain to be validated. Interestingly, most of these pro-
teins have been previously identified as potential therapeutic tar-
gets for cancer, which could potentially explain some of the
anticancer effects of quercetin.
R = CH2CO2Me
R =
7
OH
OH
H
H
N
N
H
N
O
H
H
O
O
N
S
O
H
1) incubate
2) h
ν
lyse
cells
streptavidin
beads
denaturing
wash
1) SDS
PAGE
1) trypsin
2) MS/MS
protein
identification
2) silver
stain
Figure 1. Strategy for pull down and identification of quercetin target proteins. (A)
Structure of quercetin and derivatives. BioQ was designed for pulling down protein
targets of quercetin. (B) Steps involved in pulling down and identifying quercetin
target proteins with BioQ.
2. Chemistry
therapeutic value for treating cancer. Unfortunately, quercetin’s
multiple biological effects and the high concentration required to
inhibit HSP70 induction diminish its potential as a therapeutic
agent or adjuvant.
2.1. Synthesis of the biotin quercetin conjugate, BioQ
The selective coupling of biotin to the 7-OH of quercetin was
carried out by two synthetic routes (Fig. 2) based on a previously
developed method for the selective methylation and benzylation
of the 7-OH.30 The first route was to alkylate the 7-OH with tert-
butylchloroacetate after which the ester would be converted to
an acid and coupled to an amine derivative of biotin. Thus, querce-
tin penta-acetate was refluxed in anhydrous acetone with excess
tert-butyl chloroacetate in the presence of potassium carbonate
and catalytic potassium iodide with TLC monitoring to minimize
over-alkylation. The tert-butyl ester 2 was converted to the acid
3 by treatment with 20% trifluoroacetic acid in methylene chloride
under anhydrous conditions to limit competing hydrolysis of the
remaining acetate protecting groups. Coupling of the amino biotin
derivative 631 with the tetra-acetate acid 3 was problematic. Both
reactants are very polar and only dissolved well in dimethyl form-
amide or water–acetonitrile mixtures. When the reaction was car-
ried out in the presence of N,N-diisopropylethylamine with HATU,
DCC, or ByBOP, extensive decomposition of the quercetin tetra-
acetate occurred, which may have been the result of deacetylation.
Similar results were observed with DCC or EDC in dimethyl form-
amide in the absence of base, suggesting the possible involvement
of the amino group of amino biotin 6 and hence the necessity of a
buffered solution in neutral to acidic pH. Final treatment with EDC
and N-hydroxysuccinamide at a slightly acidic pH of 6, in the
presence of MES buffer, afforded the desired coupled product 5
Identifying the protein target(s) responsible for quercetin’s inhi-
bition of HSP70 induction, however, could aid in the development
of more selective agents. One systematic approach to identifying
potential targets has involved screening chromatographic fractions
of cells extracts for quenching of protein fluorescence in the pres-
ence of quercetin, followed by SDS–PAGE of the active fractions,
and MASCOT analysis of the trypsinized fragments.18 Unfortu-
nately, without authentic protein samples, it is difficult to establish
which of the protein bands detected in the SDS–PAGE gel are the
quercetin binding proteins.
We decided to adopt a more promising approach for identifying
protein targets of a bioactive compound that involves derivatizing
the compound with biotin which serves as an affinity tag allowing
the bound proteins to be isolated with streptavidin.19,20 The iso-
lated proteins are then separated and identified by bottom up mass
spectrometry. There have been several reports in which com-
pounds with Kd’s of around 1 nM can be used to pull down target
proteins in this manner from cell lysates.21–23 To identify more
weakly binding proteins, phage display libraries have been used
in place of cell lysates to afford a higher concentration of protein,24
although this approach is limited by the contents of the library. An-
other approach is to increase the affinity of the compound by
attaching a photocrosslinking agent,19,20,25 or by making use of
intrinsic photoaffinity properties of the compound. In all cases,

4712
R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
O
OAc
OAc
OAc
Cl
O
O
O
O
K2CO3/acetone
reflux
AcO
O
O
O
OAc
OAc
OAc
40%
2
1
AcO
TFA/DCM
O
AcO
PhSH,
imidazole
DCM
90%
65%
OAc
OAc
OAc
O
H
O
O
O
HO
O
OAc
O
O
S
H
H
N
H
(CH2)4
OAc
N
OAc
H
3
H
AcO
4
AcO
O
N
NH2
O
2
O
6
R-I (7)
Cs2CO3
85%
EDC, NHS
DMF
OAc
OAc
15%
OH
OH
RO
O
RO
O
pH 7.4 phosphate buffer
O
OAc
H
OH
5
N
OH
74%
AcO
O
N
OH
O
8
9
O
S
H
N
N
R =
H
N
O
H
H
O
O
N
O
H
Figure 2. Two synthetic routes to BioQ.
in 10–15% yield. Attempts to improve the yield by further lowering
the pH, however, failed to give desired product. Complete deacet-
ylation was attempted under basic conditions, such as aqueous so-
dium hydroxide or sodium methoxide in methanol, but only
afforded the desired biotinylated quercetin 9 in low yield. Biotinyl-
ated quercetin could be obtained, however, in 74% yield upon
treatment with the hydroxamic acid 8 under near neutral condi-
tions in pH 7.4 phosphate buffer.
Because direct coupling of the quercetin acid 3 with biotin
amine 6 was so problematic we investigated whether we could
directly alkylate the 7-OH of quercetin with the commercially
available biotin alkylator, biotin PEO iodoacetamide 7. In princi-
ple, one could simply use the method described above in which
quercetin penta-acetate is alkylated selectively at O7 by refluxing
with the alkylating agent and potassium carbonate in acetone.
We were, however, concerned that the iodoacetamide 7 might
self-react under these conditions, so we decided instead to alkyl-
ate quercetin tetra-acetate 4 under milder conditions. Quercetin
tetra-acetate 4 has been previously prepared by treatment of
the penta-acetate 1 with lipase,32 or more recently, with imidaz-
ole and thiophenol in N-methyl-2-pyrrolidone (NMP).33 We found
that the latter method using dichloromethane in place of NMP
afforded quercetin tetra-acetate 4 in 65% yield (Fig. 2). Treatment
of 4 with the iodoacetamide 7 in anhydrous dimethyl formamide
with cesium carbonate in the dark afforded the desired biotinyl-
ated quercetin tetra-acetate 5 in 85% yield. Attempts to use
potassium carbonate, in place of cesium carbonate, failed to give
the desired product. Deacetylation of compound with N-
methyl-2-dimethylamino-acetohydroxamic acid again afforded
5
biotinylated quercetin 9.
2.2. Structural characterization of BioQ 9
To verify that the biotin was indeed coupled to the hydroxyl at
position 7 of quercetin, and had not been coupled to another hy-
droxyl that might have been produced by transesterification of
the acetate groups, we characterized the product by COSY, HMQC,
and HMBC NMR (Figs. 3, S1–S3). Aromatic protons H15 and 16
could be readily identified by their large ortho-coupling in the 1D
1H NMR and
a strong crossspeak B in the COSY spectrum
(Fig. S1). This allowed identification of proton 12 through a
crosspeak A with H16, leaving aromatic protons H6 and H8 which
showed a crosspeak C between them. The assignment of protons
H6 and H8 allowed identification of C7 in the HMBC spectrum
through crosspeaks J and H (Fig. S3). C7 in turn showed a crosspeak
F to the H17 protons of the acetamide group verifying the connec-
tion of the biotin derivative to O7. The assignment of the H17 pro-
tons was confirmed by a crosspeak E with C18 via HMBC, which in
turn showed a correlation K with the amide NH (d). The biotin
group could be easily identified by the crosspeak (D) between
H19 and H20. The rest of structure could be confirmed by key
proton–carbon crosspeaks in the HMBC spectrum, such as

R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
4713
O
S
Ha
Hb
H
N
N
19
20
Hc
N
H
D
O
O
Figure 4. Ability of BioQ to inhibit heat shock induction of HSP70 in Jurkat cells.
Jurkat cells were treated with 145 M quercetin (Q), DMSO vehicle (V) or 145 lM
BioQ (BQ) for 2 h prior to a 30 min 43 °C heat shock (HS) and then allowed to
recover at 37 °C for 8 h before Western blot analysis of HSP70 and actin levels.
Unheated Jurkat cells (C) and heated Jurkat cells in the absence of any additive (ꢀ)
were used as controls. The results were reproducible.
B
l
J
H
15
O
H
OH
OH
F
O
H
8
A
O
16
K
C
O
17
18
N
7
6
12
H
H
probe for proteins involved in this and possibly other biological
process. In this regard, other derivatives of the 7-hydroxy group
of quercetin have been shown to retain antitumor activity.34
Hd
H
H
OH
OH
O
H
Figure 3. Structural characterization of BioQ by COSY, HMQC, and HMBC. Through
bond correlations A, B, C, and D were observed in 1H–1H COSY, one bond 1H–13
3.2. In vitro pull down of casein kinase 2 with BioQ
C
correlations were made by HMQC, and multiple through bond correlations F, H, J,
and K were by HMBC. See Figs. S1–S3 for the 2D spectra.
Our attention then focused on demonstrating that we could use
streptavidin to pull down CK2 with the BioQ probe. Initial experi-
ments indicated, however, that the streptavidin agarose beads
would also non-specifically bind and pull down proteins when
using the lysis buffer that would eventually be used for the
in vivo pull down experiments. Despite many attempts, we were
unsuccessful at finding a wash buffer that would remove non-spe-
cifically bound proteins from the streptavidin beads without also
removing BioQ bound proteins.
crosspeaks G (Hb–C21), Q (Ha–C21), I (H6–C5), L (H8–C9), M (H15–
C14), N (H16–C11), O (H12–C11) and P (H12–C14).
3. Results
3.1. Substrate activities of BioQ
Because quercetin has been reported to be photoreactive and
capable of photocrosslinking to malate dehydrogenase,28 we
thought that UV irradiation could be used to photocrosslink BioQ
to CK2 and other target proteins. Photocrosslinking would prevent
CK2 or other target proteins from dissociating during affinity puri-
fication with the streptavidin and allow more stringent washing
steps. Quercetin and BioQ show two absorption maxima at about
270 and 380 nm of about equal absorptivity (Fig. S4), the latter
of which is photoactive and ideal for in vivo photocrosslinking
experiments due to the lower toxicity of UVA light. Irradiation of
quercetin and BioQ in 10 mM pH 7.2 PBS buffer with a medium
pressure mercury arc lamp filtered with wood’s glass which trans-
mits light from 320–400 nm, with a peak intensity at 365 nm, led
to significant irreversible bleaching of the longer wavelength
absorption maximum within 30 min (Fig. S4). No bleaching was
observed, however, in Tris buffer, possibly due to quenching by
the buffer.35
In a model photocrosslinking study, an equimolar mixture of
two readily available peptides containing 13 of the 20 amino acids,
DRVYIHPFHL (angiotensin I) and RPKPQFFGLM (substance P), was
irradiated with 4 mM of BioQ. Analysis by LC–MS/MS (Figs. S5
and S6) showed the formation of an adduct between angiotensin
I and a photofragment of BioQ, demonstrating BioQ’s ability to
photocrosslink to a protein target. The molecular weight of the
BioQ photofragment detected is consistent with the known photo-
chemistry of quercetin.27,28
Previously, we had shown that quercetin and its 7-O-methyl
and carbomethoxymethyl derivatives could inhibit CK2 and
CAMK2, and that this inhibition was correlated to its ability to in-
hibit heat induction of HSP70.26 To determine whether derivatiza-
tion of quercetin at O7 with a much larger biotin-containing group
would interfere with binding to these enzymes, the ability of BioQ
to inhibit these enzymes was assayed by the same in vitro protein
kinase inhibition assay we used previously for the simple quercetin
derivatives (Table 1). The IC50s of both quercetin and BioQ for CK2
were similar and about 5
was about eightfold higher than for quercetin (26
to 3 M).
l
M, while the IC50 of BioQ for CAMK2
lM compared
l
To determine whether BioQ could inhibit heat induction of
HSP70, Jurkat cells, which have a very low basal expression of
HSP70, were heat shocked in the presence of 145 lM quercetin
or BioQ, and assayed by western blotting.26 BioQ showed substan-
tial inhibition of HSP70 induction compared to the controls, though
not as complete as quercetin (Fig. 4). The lower inhibition of HSP70
induction by BioQ is consistent with a higher in vitro IC50 for
CAMK2 than observed for quercetin, and our previous conclusion
that effective inhibitors had to be good inhibitors of both CK2
and CAMK2.26 It may also be that BioQ is not as permeable as quer-
cetin and unable to achieve a high enough concentration in the cell
to be completely effective against CAMK2, for which BioQ has an
in vitro IC50 of 26 lM compared to 3.0 for quercetin (Table 1).
To verify the ability of BioQ to photocrosslink to a target pro-
None-the-less, the ability of BioQ to cause substantial inhibition
tein, we incubated increasing concentrations of CK2 (from 1
lg
of heat shock induction of HSP70 validated its use as an affinity
to 4 g) with BioQ in 400 L 10 mM PBS buffer (pH 7.2) with and
l
l
without irradiation for 30 min with Wood’s glass filtered medium
pressure mercury arc lamp. The mixture was then incubated with
streptavidin beads, followed by centrifugation and washing of the
beads to remove non-specifically bound protein. CK2 is a tetramer
Table 1
In vitro IC50 values for casein kinase II (CK2) and calmodulin-dependent kinase II
(CAMK2) inhibition using the PKLight protein kinase assay
consisting of two 45 kDa
a-subunits and two 25 kDa b-subunits
Quercetin (
lM)
BioQ (lM)
which can be readily detected as two discreet bands on an SDS–
PAGE gel. The SDS–PAGE analysis, however, did not detect any
photocrosslinked CK2 by silver staining (Fig. 5a). The only bands
CK2
CAMK2
5.6 1.2
3.0 0.6
3.2 0.7
25.9 0.8

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R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
3.3. In vivo pull down of proteins from Jurkat cells with BioQ
Having verified that BioQ could be used to pull down a known
quercetin binding protein, we carried out pull down experiments
with cells that had been incubated with BioQ. In one set of exper-
iments the photocrosslinking step was carried out after cell lysis
(Fig. 6) and in a second set of experiments it was carried out before
cell lysis (Fig. 7). As can be seen from lanes 1–4 of the 12% SDS–
PAGE gel shown in Figure 6, a large number of proteins were pulled
down by the streptavidin beads in the absence of the denaturing
wash, whether or not the cells were incubated with BioQ and/or
heat shocked. With the exception of contaminating keratin pro-
teins, the high protein background was reduced to almost nothing
with the use of the denaturing wash as shown in lane 7 for cells
that were incubated with BioQ and heat shocked. When cell lysates
containing BioQ were UV irradiated for 30 min prior to pull down
and followed with the denaturing wash, discrete new bands A–F
were observed (lanes 5 & 6). Similar results were obtained when
the cells were irradiated prior to lysis (Fig. 7).
To confirm that the proteins appearing in the gel had been
photo-modified with BioQ, a western blot with a biotin-specific
antibody was carried out (Fig. 6, lanes 8–10, Fig. 7, lanes 9–14).
Samples for lanes 8–10 of Figure 6 were identically prepared from
heat shocked cells, except that the sample for lane 8 was not UV
irradiated and the sample for lane 10 lacked BioQ. Only the sam-
ples that contained BioQ and had been irradiated (lane 9) showed
bands with the anti-biotin antibody that matched the major bands
observed in the corresponding lane of the silver stained gel (lane
6). The same was true for the experiment in which the cells had
been irradiated prior to lysis (compare lanes 8 and 12 of Fig. 7).
Figure 5. Casein Kinase II pull down by BioQ. Increasing concentrations of CK2
(lanes 3–6: 17.5, 35, 52.5, 70 nM) in 400
incubated with 150 M BioQ and then irradiated at 365 nm for 30 min in the (a)
absence or (b) presence of 10 mM MgCl2 and 2 M ATP. The biotinylated proteins
lL, 10 mM PBS buffer (pH 7.2), were
l
l
were then pulled down by streptavidin beads and subjected to a denaturing wash
prior to electrophoresis. L is a Fermentas prestained protein ladder. Lane 1 in panel
(a) and lane 2 in panel (b) were controls that contained 0.05
panel (a) and lane 1 in panel (b) contained 0.1 g of CK2.
positions of the and subunits of CK2, and refers to the position of
contaminating keratin proteins.
l
g CK2, while lane 2 in
l
a
and b refer to the
a
b
k
detected corresponded to contaminating keratin proteins in the
45–70 kDa range.
As casein kinase II is an ATP dependent kinase, that is, known to
autophosphorylate,36 it was possible that autophosphorylation
was required for CK2 to bind to quercetin. Indeed, we found that
ATP was consumed by CK2 in the presence of quercetin and the
absence of substrate (Fig. S7). We therefore incubated CK2 and
3.4. Mass spectrometric identification of proteins pulled down
from the Jurkat cells
BioQ in the presence of 2 lM ATP in PBS buffer for 30 min, after
which the samples were irradiated for 30 min prior to analysis
by SDS–PAGE. After much experimentation, we found that a 2%
SDS Tris–HCl buffer was sufficient to remove non-specifically
bound proteins from the streptavidin agarose beads, without dena-
turing the streptavidin and causing the release of the biotinylated
Proteins contained in discrete bands A to F of lane 8 of the gel in
Figure 7 that were confirmed to be biotinylated (lane 12) were tryp-
sinized and analyzed by LC–MS/MS. Corresponding sections of lane
6 which had not been subjected to irradiation were similarly ana-
lyzed so that contaminating proteins such as keratins and albumins
could be excluded from consideration. MASCOT search37 of the
NCBI database led to the identification of the proteins shown in Ta-
ble 2 that did not appear in the control lane and that had high MAS-
COT scores (>100) and the expected molecular weight. In the case of
CK2. Once the concentration of CK2 reached 4
lg/400 lL (70 nM) a
significant amount of both and b subunits could be detected by
a
SDS–PAGE (Fig. 5b, lane 6). The identities of these proteins were
confirmed by trypsinization followed by LC–MS/MS and a MASCOT
search (Table 2).
Table 2
LC–MS/MS identification of proteins pulled down with BioQ using MASCOTa
Band
App. MW (kD)
Name
Gene ID
GI number
MW (Da)
MASCOT scoreb
Figure 5
a
45/40
25
Casein kinase II alpha subunit (CSNK2A1P)
Casein kinase II alpha isoform (CSNK2A1)
Casein kinase II beta subunit (CSNK2B)
283106
1457
1460
21464270
86156152
181155
45,261
45,423
25,242
369
186
188
b
Figure 7
A
110
Ubiquitin-activating enzyme E1 (UBA1)
Spliceosomal protein SAP 130 (SF3B3)
Heat shock protein HSP 90-alpha 2 (HSP90AA1)
Heat shock protein 70-2 (HSPA2)
RuvB-like 2 helicase ATPase (RUVBL2)
Mitochondrial ATP synthase beta subunit ATPase (ATP5B)
Mitochondrial ATP synthase alpha subunit precursor (ATP5A1)
Eukaryotic translation initiation factor 3, subunit 5 epsilon (EIF3F)
Mutant b actin (ACTB)c
7317
23450
3320
3306
10856
506
498
8665
60
23510338
6006515
61656603
23271312
5730023
32189394
4757810
4503519
28336
118,858
136,590
98,622
70,237
51,026
56,525
59,828
37,654
42,128
205
252
663
524
450
447
223
179
657
B
C
D
100
70
50
E
40
a
The target proteins were identified by comparison of the proteins detected in an unirradiated control sample band with an irradiated sample band so that contaminating
keratins and other proteins were excluded from consideration. Proteins were also excluded if they did not match the observed molecular weight range, or had MASCOT scores
less than 100.
b
The MASCOT score corresponds to ꢀ10 * log (P) where P is the probability that the match to the database is wrong.
c
Beta actin was also observed in the control lane, but because it is a known quercetin binding protein, it is included. Though MASCOT matched the protein to a mutant or
beta’ actin sequence, all the peptides identified also match the wild type sequence (GI: 46397333), except for one peptide that does not completely match either the wild type
or mutant sequence.

R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
4715
Figure 6. SDS–PAGE of proteins pulled down from Jurkat cells incubated with BioQ that were UV irradiated following lysis. Left: Silver stained 12% SDS–polyacrylamide
electrophoresis gel of proteins. Lanes 1 and 2: proteins pulled down from lysates of normal or heat shocked Jurkat cells by the streptavidin beads in the absence of BioQ and
UV irradiation. Lanes 3 and 4: proteins pulled down from lysates of normal or heat shocked cells that had been incubated with 150 lM BioQ in the absence of UV irradiation.
Lanes 5 and 6: proteins pulled down from lysates of normal or heat shocked cells that had been incubated with BioQ and UV irradiated for 30 min following lysis, and then
subjected to a denaturing wash. Lane 7: proteins pulled down under the same conditions as for lane 6, except that the lysate was not UV irradiated. Right: Western blot of
corresponding lanes with anti-biotin antibody. Legend: heat, heat shock applied after incubation with BioQ but before lysis; wash, washing beads with cell lysis buffer;
denaturing wash, washing beads with 2% SDS buffer.
L
1
2
3
4
5
6
7
8
9
10 11 12 13 14
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
BioQ
Heat
UV
Wash
Denat. wash
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
A/B
C
D
E
F
Figure 7. SDS–PAGE of proteins pulled down from Jurkat cells incubated with BioQ that were irradiated prior to lysis. Left: Proteins pulled down with streptavidin beads from
Jurkat cells under similar sets of conditions as in the gel in Figure 6. Right: Western blot with anti-biotin antibody. Legend the same as in Figure 6.
band A, two proteins, ubiquitin-activating enzyme E1 and spliceos-
omal protein SAP 130, were identified as potential quercetin target
proteins. For band B, heat shock protein 90 was identified and for
band C, heat shock protein 70 was identified. Three proteins were
detected in band D, RuvB-like 2 and both alpha and beta subunits
of mitochondrial ATP synthase. Band E contained a protein that
matched eukaryotic translation initiation factor 3. Band E also con-
tained beta actin, which may also have been pulled down by BioQ
despite the fact that it was present in the control lane, because it
is also a known target for quercetin.18,38
therapeutic targets.19,20,25 The general idea appears to have been
first described over 20 years ago for the isolation of ACTH recep-
tors,39 and successfully implemented a few years later for isolation
of a melanocyte-stimulating hormone receptor.40 In both cases,
biotin had been selected as the affinity purification agent because
of its high affinity for streptavidin, which in principal would enable
facile separation of a protein bearing a biotinylated ligand from
non-target proteins. In practice, however, it is difficult to find con-
ditions that reduce the non-specific binding of the proteins to the
streptavidin without dissociating the biotinylated ligand protein
complex, as we found this to be the case for BioQ. This led to the
idea of using a photoaffinity crosslinker to covalently link the bio-
tinylated ligand to the target protein, and permit the use of more
stringent wash conditions.
4. Discussion
The strategy for coupling photoaffinity and affinity purification
agents to a biologically active ligand to facilitate isolation of a
target protein is a powerful approach to identifying potential
In our case, quercetin is known to have intrinsic photoreactivity,
and is able to crosslink to malate dehydrogenase,28 thereby only

4716
R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
necessitating the attachment of biotin to create a pull down probe.
We had already determined that the O7 position of quercetin did
not interfere with the ability of quercetin to inhibit HSP70 induc-
tion in vivo, or to inhibit CK2 and CAMK2, two of the presumed tar-
get proteins, and so this site was chosen for biotinylation.26 As we
had found for a simpler derivative, quercetin biotinylated at the O7
position retained its ability to penetrate cells and inhibit heat
shock induction of HSP70 (Fig. 4) as well as to inhibit CK2 and
CAMK2 (Table 1). Initial attempts to demonstrate photocrosslink-
ing of BioQ to malate dehydrogenase, or to BSA which quercetin
is also known to bind,41 failed. It is possible that derivatization of
the O7 position interfered with binding to these proteins, or that
there were no crosslinkable protein side chains at the binding site.
We did confirm, however, that the BioQ was photoreactive, and
could be photobleached by irradiation with a UVA light source
(Fig. S4).
In a very limited study of two commercially available peptides
used as standards for mass spectrometry, we found that irradiation
of one of them (angiotensin I) with BioQ resulted in the formation
of an adduct between a fragment of BioQ and the peptide (Figs. S5
and S6). The site or nature of the crosslink could not, however, be
rigorously established at this time. One can tentatively rule out
Arg, Pro, Lys, Gln, Phe, Gly, Leu, and Met as crosslinkable side
chains as no reaction was observed with substance P, suggesting
that either Asp or Tyr or His was involved in the crosslinking reac-
tion of angiotensin I.
found to be overexpressed in hepatocellular carcinoma and re-
quired for cell viability.44 Beta actin, is also a known target of quer-
cetin,18,38 and though it was identified in Band E, we cannot
unambiguously conclude that it was pulled down by BioQ because
it was also present in the control lane.
Both splicesomal protein (SAP130) and eukaryotic translation
initiation factor 3 were also identified as potential targets and
are known to be important in translation or post-translation pro-
tein synthesis steps.45,46 As such, these proteins may play a role
in the expression of inducible heat shock proteins 70 and 90,47
which quercetin is known to inhibit.48 The spliceosome is a known
target of some anticancer compounds,49 while the eukaryotic initi-
ation factor has been found to be upregulated in breast and pros-
tate cancer, and thus represents a new therapeutic target.45,50
The identification of HSP70-2 and HSP90 as possible quercetin tar-
gets could also explain the decreasing level of these proteins in
quercetin treated cells,51,52 which might result from the deactiva-
tion of these proteins and their subsequent degradation or aggre-
gation in the cell. Both HSP70 and HSP90 proteins are of current
interest as anticancer targets.53–56
Another potential target of quercetin that we identified was the
ubiquitin-activating enzyme E1 which initiates protein degrada-
tion through the E1–E2–E3 mediated proteosome pathway.57 Bind-
ing to this enzyme could explain the observation that quercetin
inhibits the ubiquitin-proteosome pathway for degradation of un-
folded proteins.58 Currently, ubiquitin-activating enzyme E1 is
being investigated as a therapeutic target for treatment of leuke-
mia and multiple myeloma.59 Inhibition of the proteosome path-
way, together with the heat shock protein pathway for repairing
unfolded proteins could induce cell apoptosis, which may be one
of the mechanisms behind the antitumor activity of quercetin.
We therefore turned to CK2 kinase as a model target protein,
which we had shown was inhibited by BioQ. Initial photocrosslink-
ing experiments with CK2 and BioQ in the absence of ATP and sub-
strate, however, did not result in any detectable photocrosslinking.
When ATP was added to the reaction, photocrosslinking took place,
suggesting that CK2, which is known to autophosphorylate,36 must
be in its phosphorylated state to bind quercetin. Further experi-
ments established that ATP was consumed by CK2 in the presence
of quercetin and in the absence of substrate (Fig. S7). In contrast to
many other enzymes that are inhibited by quercetin binding to an
ATP binding pocket, CK2 may be inhibited by another mechanism.
One possibility is that quercetin is binding to the allosteric cleft be-
5. Conclusion
We have shown that quercetin derivatized with biotin at the 7-
hydroxyl position, BioQ, can be used as a photoaffinity agent to pull
down possible quercetin target proteins from cells which can then
be identified by bottom up mass spectrometry. Many of the pro-
teins identified as possible targets of quercetin have also been
found to be potential therapeutic targets for cancer, and all might
play a role in the apoptotic and antitumor activities of quercetin. It
is conceivable that the anticancer activity of quercetin is due to
simultaneous but low level inhibition of multiple therapeutic tar-
gets, which might explain how a molecule with low specificity
and toxicity can achieve a positive therapeutic effect.
The O7 hydroxyl site for attaching biotin to quercetin was orig-
inally chosen because it did not substantially interfere with quer-
cetin’s ability to inhibit heat induced HSP70 induction or the
inhibition of CK2 and CAMK2 kinase activities, which are thought
to be involved in heat shock transcription factor activation. Sur-
prisingly, neither of these kinases was detected in the assay, possi-
bly because they were only present at much lower levels than the
other proteins. In this case, screening against a phage display li-
brary of proteins24 with a more uniform protein distribution might
enable one to identify quercetin binding proteins that are at too
low a level to be detected by BioQ in vivo. A phage display library
might also be able to provide the amount of protein needed for
more detailed analysis of the protein binding sites by mass spec-
trometric analysis.
tween a a, b subunits
/b surface of CK2,42 where the interaction of
is believed crucial to the function of CK2 kinase. In accord with this
idea, BioQ pulled down both subunits, though more efficiently for
the b subunit (Fig. 5).
BioQ was also examined for its ability to specifically pull down
target proteins from normal and heat shocked Jurkat cells as shown
in Figures 6 and 7. We found that discrete, biotin-containing protein
bands could be obtained by using a photocrosslinking step in com-
bination with a denaturing wash step. Protein identification was
only carried out on the heat shocked cells that were irradiated prior
to lysis (lane 8, Fig. 7), in order to avoid artifacts that may have
resulted as a consequence of lysing the cell. LC–MS/MS analysis of
trypsinized protein bands from lane 8 in comparison to the control
lane 6 led to the identification of a number of potential and known
target proteins of quercetin shown in Table 2. Five of these proteins
have known or putative ATP binding domains (UBA1, HSP90AA1,
HSPA2, RUVBL2 and ATP5B/ATP5A1), which is in accord with
quercetin’s known ability to inhibit ATPases and kinases.10 The
remaining proteins, SF3B3 and EIF3F are not associated with nucle-
otide binding domains, and may bind to quercetin through some
other site.
Of the proteins that were pulled down by BioQ, only mitochon-
drial ATP synthase has been previously shown to be a target of
Though we have identified a number of possible quercetin tar-
gets, further work will be needed to confirm them as actual targets,
with the exception of mitochondrial ATP synthase,29 and beta ac-
tin18,38 which have already been shown to be targets of quercetin.
It is also likely that a number of targets were not pulled down by
the BioQ probe, either because the position of the biotin interfered
quercetin, which binds between the a, b and c subunits and there-
by inhibits ATPase activity.29 ATP synthase has been found to be
upregulated in breast cancer, and the ATP synthase inhibitor
aurovertin B is being investigated as a therapeutic agent.43 RuvB-
like 2, is an ATPase as well as a putative helicase that has been

R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
4717
with binding, or because of an inefficient photocrosslinking reac-
tion. The mechanism and amino acid specificity of the photocross-
linking reaction will also need to be further investigated. To screen
for all the possible targets of quercetin, it might be necessary to at-
tach biotin along with an extrinsic photocrosslinking agent to
other positions of quercetin. In any case, our approach appears to
be a fruitful one for beginning to unravel the biomolecular basis
for the medicinal effects of a ubiquitous natural product in our diet.
(ethyl acetate/hexane/acetic acid 4.5:4.5:1) to afford 3 as a pale
white solid in 72% yield (142 mg, 0.27 mmol). 1H NMR (300 MHz,
(CD3)2CO) d 7.90 (dd, J = 8.0, 2.0 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H),
7.49 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 6.82 (d, J = 2.0 Hz,
1H), 4.95 (s, 2H), 2.34–2.30 (m, 12H). 13C NMR (151 MHz,
(CD3)2CO) d 169.9, 169.0, 168.4, 168.1, 168.0, 168.0 (6 C@O),
163.1, 158.5, 153.6, 151.4, 145.3, 143.2, 134.3, 128.5, 126.9,
124.8, 124.3, 111.7, 109.6, 100.5, 65.7 (OCH2–COO), 20.7, 20.2,
20.1, 20.0 (4 CH3CO). MS (C25H20O13), LRESI: [M+Na+] 551.1, HRESI:
[M+Na+] 551.0820, calcd 551.0802.
6. Experimental section
6.1. General
6.4. Quercetin-3,5,30,40-tetra-acetate 4
Imidazole (93.2 mg, 1.37 mmol) and 181 mg thiophenol
(1.2 equiv) were added to a solution of quercetin penta-acetate
(700 mg, 1.37 mmol) in anhydrous dichloromethane at 0 °C. After
1 h, TLC indicated that the reaction was near completion, and the
reaction was diluted with dichloromethane, and concentrated to
dryness under vacuum in a rotatory evaporator. The resulting res-
idue was dissolved in ethyl acetate washed with 1 N HCl and then
brine and concentrated to dryness. Flash column chromatography
of the residue (ethyl acetate/hexane: 4:3) afforded a yellow oil
from which 4 was obtained as a pale white solid in 65% yield
(418 mg, 0.89 mmol) following treatment with methanol. Com-
pound 4 gave spectroscopic data in accord with previously pub-
lished data.33
Anhydrous dimethyl formamide and acetone were from EMD
Chemicals Inc. Anhydrous dichloromethane was freshly distilled
from calcium hydride. All other reagents were directly purchased
from Omni Solvent or Sigma Aldrich unless otherwise specified.
Analytical thin layer chromatography was performed on Aldrich
Silica gel 60 F254 plates (0.25 mm) and visualized by UVG-54 min-
eral light UV lamp at 254 nm. Flash column chromatography was
conducted using E. Merck silica gel 60 (40–63 l
m). 1H NMR and
13C NMR spectra were carried out on Varian Mercury-300 MHz,
500 MHz or 600 MHz spectrometers. UV–vis spectra were obtained
on a Varian Cary 100 Bio spectrophotometer. High capacity strep-
tavidin agarose resin, silver SNAP stain kit, and avidin-conjugated
horseradish peroxidase were from Pierce Thermo Fisher Scientific
Inc., and a Western blot kit was from Bio-Rad Laboratories. ECL
staining reagents as well as mouse anti-actin antibody and horse-
radish peroxidase conjugated anti-mouse secondary antibody were
from GE Healthcare Systems. Silver staining was carried out by a
standard protocol60 or with the Pierce Silver Stain Kit (Pierce). Pro-
tease inhibitor cocktail tablets were from Roche Pharmaceuticals.
6.5. BioQ tetra-acetate 5
6.5.1. Method I
Compound 3 (20 mg, 0.038 mmol) was first incubated with EDC
(9.6 mg, 0.05 mmol) and N-hydroxysuccinimide (5.7 mg,
0.05 mmol) in 50% MES buffer/acetonitrile (0.1 M, pH 5.5) for
20 min. Compound 6 (20 mg, 0.053 mmol)31 was then added and
stirred under nitrogen for 12 h at room temperature after which
it was concentrated to dryness under vacuum. The resulting resi-
due was added to 50 mL of ethyl acetate and washed with satu-
rated ammonium chloride and brine three times and then
concentrated under vacuum. Purification by step gradient flash
chromatography (5–8% methanol in dichloromethane) afforded 5
in 15% yield (5 mg, 0.006 mmol).
6.2. 7-O-Carbo-tert-butoxymethyl-quercetin tetra-acetate 2
Anhydrous potassium carbonate (2.0 g, 15 mmol) and potas-
sium iodide (0.166 g, 1 mmol) were added to a solution of querce-
tin penta-acetate
1
(1.0 g, 1.95 mmol)61 and tert-butyl
chloroacetate (3.0 g, 19.5 mmol) in 50 mL of anhydrous acetone .
The suspension was refluxed for 2 h at which point TLC indicated
the reaction was near completion. The reaction mixture was then
filtered to give a yellowish clear solution, concentrated in a rota-
tory evaporator, and the residue purified by flash column chroma-
tography (ethyl acetate/hexane 4:3). The resulting solid was
recrystallized with acetone/hexane to give 2 as a white solid in
65 % yield (0.66 g, 1.27 mmol). 1H NMR (300 MHz, CDCl3): d 7.70
(dd, J = 8.0, 2.0 Hz, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 8.0 Hz,
1H), 6.80 (d, J = 2.0 Hz, 1H), 6.67 (d, J = 2.0 Hz, 1H), 4.60 (s, 1H),
2.40 (s, 3H), 2.32 (s, 9H), 1.49 (s, 9H). 13C NMR (75 MHz, CDCl3) d
170.2, 169.7, 168.3, 168.2, 168.1, 166.8 (6 C@O), 162.3, 158.2,
153.6, 151.2, 144.5, 142.4, 134.2, 128.3, 126.7, 124.2, 124.0,
112.0, 109.2, 100.1, 83.6 (C(CH3)3), 66.2 (OCH2–COO), 28.3 (3
CH3), 21.4(CH3CO), 21.0 (2CH3CO), 20.8 (CH3CO). MS (C29H28O13),
LRESI: [M+H+] 585.2, [M+Na+] 607.1, [M+K+] 623.1, [M+H+ꢀCH2CO]
543.1. HRESI: [M+H+] 585.1606, calcd 585.1603.
6.5.2. Method II
Compound 4 (20 mg, 0.042 mmol) and compound 7 (30 mg,
0.055 mmol)31 were dissolved in 5 mL of anhydrous dimethyl
0
formamide under a nitrogen atmosphere in the presence of 4 ÅA
molecular sieves. Cesium carbonate (17 mg, 0.05 mmol) was then
added at 0 °C and the solution was stirred at room temperature
for 1.5 h during which time the flask was wrapped with aluminum
foil to protect the reactants from light. Hydrochloric acid (1 M) was
then added to a pH of about 6 to stop the reaction. The solution was
concentrated under vacuum and the resulting residue was purified
by flash column chromatography (5% methanol in dichlorometh-
ane) to afford compound 5 in 87% yield (33 mg, 0.037 mmol). 1H
NMR (CDCl3 600 MHz) d 7.73 (dd, J = 8.0, 2.0 Hz, 1H), 7.70 (d,
J = 2.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.07 (s, H–N in ethylene gly-
col, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.72 (d, J = 2.0 Hz, 1H), 6.45 (NH in
biotin, 1H), 6.31 (NH in biotin, 1H), 4.64 (s, CH2, 2H), 4.51 (m, 1H),
4.31 (m, 1H), 3.62–3.39 (m, 12H), 3.15 (m, 1H), 2.88 (dd, 12.0 Hz,
4.2 Hz, 1H), 2.68 (d, 13.8 Hz, 1H), 2.47–2.33 (m, 12H, 4 CH3CO),
2.21 (m, 2H), 2.09 (acetic acid), 1.67–1.61 (m, 4H), 1.43–1.41 (m,
2H). 13C NMR (151 MHz DMSO-d6) d 174.7, 173.6, 169.9, 169.7,
168.1, 167.9, 167.8, 167.2 (8 C@O), 164.5, 161.2, 157.9, 153.5,
151.0, 144.4, 142.3, 133.9, 127.8, 126.4, 124.0, 123.8, 112.0,
109.2, 100.0, 70.2, 70.1, 69.6, 69.5, 67.7, 62.0, 60.6, 55.0, 40.3,
39.2, 39.0, 35.6, 27.7, 25.2, 21.1, 20.7, 20.7, 20.6 (4 CH3). MS
6.3. 7-O-Carboxymethyl quercetin tetra-acetate 3
To 200 mg of 2 (0.38 mmol) in 4 mL anhydrous dichlorometh-
ane under nitrogen was slowly added 1 mL anhydrous TFA. After
stirring for 3 h at room temperature the solution was concentrated
by rotatory evaporation under vacuum. The residue was dissolved
in chloroform, washed with water and then brine until the aqueous
layer became neutral. The organic phase was concentrated in a ro-
tary evaporator and purified by flash column chromatography

4718
R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
(C41H48O16N4S)
LRESI:
[M+H+]
885.3,
[M+Na+]
907.3,
0.07 and 0.35
20 mM Tris–HCl, 50 mM KCl, 10 mM MgCl2 at 30 °C for 10 and
40 min, with or without 300 peptide substrate
(RRRADDSDDDDD).
lM) was assayed in 1 lM ATP, 10 lM quercetin,
[MꢀCH2CO+H+] 843.3; HRESI: [M+H+] 885.2827, calcd 885.2786,
[M+Na+] 907.2618, calcd 907.2686.
lM
6.6. BioQ 9
6.9. Assay for HSP70 heat induction inhibition in Jurkat cells
A solution of compound 5 (30 mg, 0.034 mmol) in 10 mL tetra-
hydrofuran/phosphate buffer/methanol (4.5:4.5:1, pH 7.4) was
treated with N-methyl-2-dimethylamino-acetohydroxamic acid
(4.5 equiv, 20 mg, 0.15 mmol) under a nitrogen atmosphere for
12 h. The solution was then acidified with hydrochloric acid
(1 M) to pH 5, concentrated under vacuum, extracted with 50 mL
ethyl acetate, and washed with saturated ammonium chloride.
The residue from the organic phase was purified by high perfor-
mance liquid chromatography on a Waters Xterra Prep MS C18
Assays were carried out as previously described for other quer-
cetin derivatives.26
6.10. Model peptide photocrosslinking study
Angiotensin I peptide (NRVYIHPFHL, 100
lM) and Substance P
(RPKPQFFGLM, 100 M) were mixed with 4 mM BioQ in 200 lL
l
of 10 mM PBS buffer, pH 7.2, and irradiated. The protein solution
was then irradiated for 30 min in a glass tube that was immersed
in ice 10 cm away from a 450 W UV medium pressure mercury
arc lamp in a water cooled immersion apparatus that was shielded
with Wood’s glass which transmits ultraviolet between 320 and
400 nm with a peak transmittance at 365 nm. After irradiation
the sample was diluted 50–100 fold and subjected to LC–MS/MS
analysis.
column (10
l
m, 7.8 ꢁ 300 mm) with a 30 min 0–50% and 10 min
50–100% linear gradient of solvent B (1% acetic acid in acetonitrile)
in solvent A (1% acetic acid in water) to afford compound 9 eluting
at 32 min in 74% yield (18 mg, 0.025 mmol). The purity was deter-
mined by analytical HPLC to be 98.3% when detected at 280 nm,
97.6% at 300 nm, and 100% at 254 nm. 1H NMR (DMSO-d6
300 MHz) d 8.20 (s, N–H), 7.82 (s, N–H), 7.72 (d, J = 2.0 Hz, 1H),
7.56 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.70 (d,
J = 2.0 Hz, 1H), 6.41 (d, J = 2.0 Hz, 1H), 6.40 (NH in biotin, 1H),
6.36 (NH in biotin, 1H), 4.63 (s, CH2, 2H), 4.29 (m, 1H), 4.11 (m,
1H), 3.49–3.07 (m, 13H), 2.80 (dd, 12.0 Hz, 4.2 Hz, 1H), 2.58 (d,
13.8 Hz, 1H), 2.05 (m, 2H), 1.91 (s, acetic acid), 1.58–1.04 (m,
6H). 13C NMR (151 MHz DMSO-d6) d 176.4, 172.5, 167.4, 163.5 (4
C@O), 163.1, 160.7, 156.2, 148.3, 147.8, 145.5, 136.5, 122.2,
120.4, 116.0, 115.6, 104.8, 98.4, 93.1, 69.9 (2C overlap), 69.6,
69.2, 67.6 (2C overlap), 61.4, 59.6, 55.8, 38.8, 38.7, 35.5, 28.6,
28.4, 25.6. MS (C33H40O12N4S) LRESI: [M+H+] 717.2, [M+Na+]
739.3, [M+K+] 755.2. HRESI: [M+H+] 717.2442, calcd 717.2363;
[M+Na+] 739.2256, calcd 739.2263; [M+K+] 755.1984, calcd
755.3343.
6.11. Casein kinase II pull down assay
The indicated concentrations of casein kinase II and150
BioQ in 400
0.01 M PBS buffer (pH 7.2)/protease cocktail containing 2
l
M
M
l
L of 0.01 M PBS buffer/protease cocktail (pH 7.2) or
l
ATP/Mg2+ were photocrosslinked for 30 min as described in proce-
dure 6.11 and then immediately incubated with 20 lL of streptavi-
din agarose beads for 1 h during which time the color of the beads
changed from white to yellow due to binding of BioQ or BioQ pho-
toreaction products. The beads were then centrifuged at
14000 rpm for 5 min and the supernatant removed. The beads
were then washed with lysis buffer (10 mM phosphate buffer, pH
7.2, 100 mM NaCl, 0.2% Triton X-100, and protease cocktail) or
denaturing buffer (20 mM TrisꢂHCl, 2% SDS, pH 7.4) and shaken
well for 3 min before each spin-down. The washing procedure
was repeated at least three times. The beads were then resus-
6.7. 2D NMR Spectroscopy of BioQ 9
1H and 13C NMR spectra were recorded on Mercury-300, Inova-
500 and Inova-600 (Varian Assoc., CA) NMR instruments while 2D
NMR spectra were recorded on an Inova-600 spectrometer. All the
data were processed with VNMR series software (Varian Assoc.,
CA) with standard functions and the chemical shifts were refer-
enced to TMS. Proton spectra were collected with an 8000 Hz spec-
tral width and 32 K data points while carbon spectra were
collected with a 30000 Hz spectral width and 64 K data points.
The COSY experiments were obtained with 512 real points in the
F1 dimension and 2000 complex points in the F2 dimension. HMBC
(heteronuclear multiple bond correlation) spectra were collected
pended in 20
l
L of 2ꢁ SDS–PAGE gel loading buffer (50 mM
TrisꢂHCl, 100 mM dithiothreitol, 8 M urea, 2% SDS, 10% glycerol)
and boiled for 10 min to denature the streptavidin and release
the biotin conjugates. Samples were loaded onto an SDS polyacryl-
amide gel and electrophoresed at 125 V for about one hour at
which point the 10 kD protein marker would reach the bottom of
the gel. The SDS polyacrylamide gel was then incubated overnight
in a 100 mL solution of ethanol/glacial acetic acid/water (30:10:60)
with gentle shaking to fix the proteins. After washing with deion-
ized water, the gel was silver stained either by a published proce-
dure,60 or using the Pierce Silver Stain Kit (Pierce).
with 10.2 l l
s 90° 1H pulse width and a 14.0 s 90° 13C pulse width.
HMQC (heteronuclear multiple quantum correlation) spectra were
recorded using a 0.3 s 1H–13C nulling period.
6.12. Pull down assay from Jurkat cells
6.8. Kinase inhibition and autophosphorylation assays
Jurkat cells were exponentially grown in RPMI media with 10%
fetal bovine serum, until about 15 ꢁ 106 cells were obtained, after
which the cells were harvested and treated with 3% aqueous DMSO
The kinase inhibition and autophosphorylation assays were car-
ried out with the PKLight assay kit for ATP (Lonza Rockland, Rock-
land ME), following a previously reported procedure.26 To test for
or 150 lM BioQ in 3% aqueous DMSO for 2 h. Then the cells were
subjected a 43 °C heat shock for 30 min and allowed to recover
at 37 °C for 1 h when the level of activated heat shock factor in cells
typically reaches a maximum. The Jurkat cells were then cooled to
0 °C while being transported to the location of the UV lamp
(10 min), where they were irradiated at 4 °C as described in proce-
dure 6.11 immediately before or after cell lysis. The Jurkat cells
were lysed by sonication in a cold room in 2 mL of cell lysis buffer
(10 mM phosphate buffer, pH 7.2, 100 mM NaCl, 0.2% Triton X-100,
and protease inhibitors) and centrifuged at 5000 rpm for 10 min.
kinase inhibition of CK2, CK2 was incubated with 300
lM peptide
substrate (RRRADDSDDDDD), 100 M ATP in 20 mM Tris–HCl,
l
50 mM KCl, and 10 mM MgCl2 at 30 °C for 30 min, with increasing
concentrations of BioQ. To test for kinase inhibition of CAMK2, pre-
activated CAMK2 was mixed with increasing concentrations of
BioQ, 300 lM autocamtide-2 (KKALRRQETVDAL), 100 lM ATP in
pH 7.5 50 mM Tris–HCl, 10 mM MgCl2, 2 mM DTT, and 0.1 mM
Na2EDTA at 30 °C for 30 min. Autophosphorylation of CK2 (0.035,

R. E. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4710–4720
4719
The supernatant was then incubated with the streptavidin beads
and processed as described in procedure 6.12. For the detection
of biotin, proteins were transferred from SDS–polyacrylamide gels
to nitro-cellulose membranes which were then immersed in 3%
BSA overnight at 4 °C to block non-specific binding. After washing
with PBS Tween buffer, the blots were incubated with horseradish
peroxidase (HRP) conjugated anti-biotin antibody for 1 h. The blots
were visualized with a 50 mL solution containing 30 mg of 4-
chloro-1-napthol and 0.01% hydrogen peroxide which produces a
purple color at sites of horseradish peroxidase (HRP) activity.
(New Objective, Woburn, MA). A full mass spectrum of eluting pep-
tides was obtained with the FT mass spectrometer component at
high mass resolving power (100,000 for ions of m/z 400). Ions sub-
mitted for MS/MS were placed in a dynamic exclusion list for 8 sec-
onds. The MS/MS collections for the six most abundant eluting ions
were performed with wide-band activation, in the LTQ mass spec-
trometer at a normalized collision energy of 35%, with a 2 Da iso-
lation width. Raw data was uploaded to MASCOT licensed
version (Matrix Science, MA) and submitted to identity search
through NCBInr 20090707 database, with 9,252,587 sequences
(226,521 from Homo sapiens). Carbamidomethyl cysteine was
treated as a fixed modification, and methionine single oxidation
as variable modification, and up to one missed trypsin cleavage
was allowed. Monoisotopic mass was used, with a peptide mass
tolerance of 20 ppm, and fragment mass tolerance of 0.6 Da,
using an ESI-FTICR instrument type. MS/MS results on the model
peptide were manually analyzed with Xcalibur software (Thermo
scientific, USA).
6.13. In-gel trypsin digestion
All equipment was first thoroughly washed with 70% ethanol
followed by deionized water using non-latex gloves and a face
mask. Silver stained bands were immediately excised with a gel
cutter or scalpel blade, taking only the silver stained gel area.
The excised bands from (Lane 8, Fig. 7), as well as corresponding
sections from the control lane without irradiation (Lane 6, Fig. 7)
were further cut into small pieces and placed into microfuge tubes,
into which destaining buffers supplied with the silver SNAP stain
kit were added. After destaining, gel pieces were washed at least
three times with 100 mM sodium bicarbonate until the gel color
turned a pale white. Acetonitrile (200 lL) was added to each tube
for 5 min to dehydrate the slices, and then removed, after which
the samples were dried by centrifugal evaporation in a Speedvac.
Oxidized cysteines were reduced by adding 50 lL of 10 mM DTT/
Acknowledgment
We thank Jeffrey L. Kao of the Washington University NMR
facility for help in acquiring the 2D spectra. This research was sup-
ported by NIH PO1 CA 104457 and the Washington University NIH
Mass Spectrometry Resource (Grant No. P41 RR000954) and the
Washington University NMR facility (Grant No. RR1571501).
50 mM ammonium bicarbonate to the dried slices for 30 min at
room temperature. After decanting the DTT solution, the gel slices
were incubated with 50 mM iodoacetamide solution in 50 mM
ammonium bicarbonate for 30 min at room temperature in the
dark to cap the freshly reduced cysteines. The gel slices were then
washed three times with 100 mM ammonium bicarbonate and two
times with 50 mM ammonium bicarbonate in 50% acetonitrile. The
Supplementary data
Supplementary data (two dimensional NMR spectra of BioQ,
irradiation studies of quercetin and BioQ, and autophosphorylation
studies of CK2) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmc.2011.07.005. These data include
MOL files and InChiKeys of the most important compounds de-
scribed in this article.
gel slices were dehydrated with 200
lL of acetonitrile for 5 min
and then 500 L acetonitrile for 20 min. After removal of the ace-
l
tonitrile, the samples were completely dried in a centrifugal evap-
orator and subjected to trypsin digestion. Promega sequencing
grade trypsin (10 ng) was dissolved in 1 mL ice-cold 50 mM
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