Exploration of the binding proteins of perfluorooctane sulfonate by a T7 phage display screen

Article abstract of DOI:10.1016/j.bmc.2012.05.016

Perfluorooctane sulfonate (PFOS) is a pollutant widely found throughout nature and is toxic to animals. We created a PFOS analogue on a polyethylene glycol polyacrylamide copolymer and isolated peptides that preferentially bound the PFOS analogue using a T7 phage display system. Bioinformatic analysis using the FASTAskan program on the RELIC bioinformatics server showed several human proteins that likely bound PFOS. Among them, we confirmed binding between PFOS and a recombinant soluble form of monocyte differentiation antigen CD14 (sCD14) by a surface plasmon biosensor. Furthermore, PFOS inhibited TNF-α production induced by the sCD14 in mouse macrophage RAW264.7 cells.

Full text of DOI:10.1016/j.bmc.2012.05.016

Bioorganic & Medicinal Chemistry 20 (2012) 3985–3990
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploration of the binding proteins of perfluorooctane sulfonate by a T7
phage display screen
Yuka Miyano a, Senko Tsukuda a, Ippei Sakimoto b, Ryo Takeuchi a, Satomi Shimura a, Noriyuki Takahashi a,
Tomoe Kusayanagi a, Yoichi Takakusagi a, Mami Okado a, Yuki Matsumoto a, Kaori Takakusagi a,
Toshifumi Takeuchi a, Shinji Kamisuki a, Atsuo Nakazaki c, Keisuke Ohta b, Masahiko Miura b,
Kouji Kuramochi d, , Yoshiyuki Mizushina e,f, Susumu Kobayashi c, Fumio Sugawara a, Kengo Sakaguchi a
⇑
a Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
b Oral Radiation Oncology, Department of Oral Restitution, Graduate School, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
c Department of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
d Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan
e Laboratory of Food & Nutritional Sciences, Department of Nutritional Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
f Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Perfluorooctane sulfonate (PFOS) is a pollutant widely found throughout nature and is toxic to animals.
We created a PFOS analogue on a polyethylene glycol polyacrylamide copolymer and isolated peptides
that preferentially bound the PFOS analogue using a T7 phage display system. Bioinformatic analysis
using the FASTAskan program on the RELIC bioinformatics server showed several human proteins that
likely bound PFOS. Among them, we confirmed binding between PFOS and a recombinant soluble form
of monocyte differentiation antigen CD14 (sCD14) by a surface plasmon biosensor. Furthermore, PFOS
Received 11 April 2012
Revised 9 May 2012
Accepted 9 May 2012
Available online 15 May 2012
Keywords:
inhibited TNF-
a
production induced by the sCD14 in mouse macrophage RAW264.7 cells.
Ó 2012 Elsevier Ltd. All rights reserved.
Perfluorooctane sulfonate
Solid-phase synthesis
Phage display
CD14
RELIC
1. Introduction
junction intercellular communication both in vitro and in vivo,12
and increases production of reactive oxygen species in cultured
cells including hepatoma Hep G2 cells13 and human umbilical vein
endothelial cells (HUVECs).14 In addition, PFOS substantially
inhibits 3b-hydroxysteroid dehydrogenase, 17b-hydroxysteroid
dehydrogenase 315 and 11b-hydroxysteroid dehydrogenase 2.16
Our group previously found that PFOS was a potent inhibitor of
Perfluorinated alkyl substances (PFAS) have been used in a vari-
ety of industrial products such as refrigerants, surfactants, compo-
nents of paper coatings, fire retardants, adhesives, cosmetics and
insecticides.1 While they have unique and useful properties such
as chemical inertness, thermal stability, low surface energy and
amphiphilic character, PFAS are difficult to break down in nature
and thus bioaccumulate.
Previous reports describe the wide distribution of perfluorooc-
tane sulfonate (PFOS) in the environment and its impact on a range
of biological events, although little is known about the detailed
mechanisms underlying its physiological activity (Fig. 1). PFOS
has been demonstrated to induce liver cancer in rats and mice by
mammalian DNA polymerases (pols)
a and b, and human terminal
deoxynucleotidyl transferase.17 Unlike classic fatty acid inhibitors
against mammalian DNA polymerases (many of which selectively
inhibit binding of the N-terminal 8 kDa domain of pol b to a DNA
template), PFOS inhibits primer extension catalyzed by the
31 kDa domain of pol b.
Phage display is a powerful system which can identify target
activation of the peroxisome proliferator-activated receptor
a
proteins of small molecules,18–23 and also determine the binding
(PPAR
a
) that leads to peroxisome proliferation.2–5 However, recent
reports show that PFOS-induced toxicity is independent of PPAR
a
activation.6–8 PFOS affects lipid metabolism,3,9–11 blocks gap
F
F F F F F F
F F F F F F
Figure 1. Structure of perfluorooctanesulfonoic acid.
F
SO3H
F
F
F
⇑
Corresponding author.
E-mail address: kuramoch@kpu.ac.jp (K. Kuramochi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.016

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Y. Miyano et al. / Bioorg. Med. Chem. 20 (2012) 3985–3990
sites of target proteins for small compounds.24–26 Analysis of affin-
ity-selected peptides from phage libraries by RELIC, a bioinformat-
ics server for combinatorial peptide analysis, is anticipated to
become a powerful tool for identifying both binding proteins of
small molecules and protein–ligand interaction sites.27–29 We pre-
viously illustrated the potential of polyethylene glycol polyacryl-
amide copolymer (PEGA) resin as an affinity matrix for phage
display screening.30 PEGA resin, which is generally used for solid-
phase synthesis, has several attractive features including chemical
stability, high coupling capacity and favorable swelling in both or-
ganic solvents and aqueous media.31 Thus, both the synthesis of li-
gand and the subsequent ligand affinity selection can be achieved
on the same PEGA resin.
p-nitrophenyl ester 5 for immobilization on amino-PEGA resin.
After immobilization of 5 onto the resin, the residual amino groups
were blocked with acetic anhydride. The removal of the p-
methoxybenzyl group on resin 6 with DDQ in dichloromethane–
water (10:1) gave PEGA-supported 1H,1H-perfluornonanol 7. Fi-
nally, treatment of 7 with SO3ꢀpyridine, followed by ion exchange
with saturated NaCl solution gave a PEGA-supported PFOS ana-
logue 8. In addition, a control resin 9 by acetylation of amino-PEGA
resin was prepared (Scheme 1B).
A library displaying random peptides on phage particles was
incubated with the PEGA-supported PFOS analogue 8 or the control
resin 9. After removal of unbound phage clones, the bound phage
particles were eluted and amplified in Escherichia coli. The recov-
ered phages were subjected to the next round of selection. In the
third and fourth rounds of selection, approximately 3- to 4-fold
more phage particles were recovered from 8 than from 9 (Fig. 2).
Among phages eluted from 8 in the fourth round of selection, 32
randomly picked clones were sequenced (Table 1). We next sought
to explore PFOS-binding proteins by bioinformatically analyzing a
pool of amino acid sequences displayed by phage particles that
bound to 8. The FASTAskan program developed by the RELIC bioin-
formatics ranked proteins in descending order of score that is cal-
culated from sequence similarity between queried peptides and
human gene products.27,32 Among nine proteins with the highest
score (Table 2), we focus on monocyte differentiation antigen
CD14.
In this study, we synthesized a PFOS-analogue-immobilized
PEGA resin and screened for peptides that bind to PFOS using a
T7 phage display screen.
2. Results
Peptides with high affinity to PFOS were searched using T7
phage display with a PEGA-supported PFOS analogue. Since it is
difficult to immobilize PFOS on affinity supports directly and con-
struct the sulfonate moiety of PFOS on PEGA resin, 1H,1H-perfluor-
ononyl sulfate moiety was synthesized on the surface of PEGA
resin (Scheme 1A). The synthesis was started from 2,2,3,3,4,4,5,5,
6,6,7,7,8,8,9,9-hexadecafluoro-1,10-decanediol (1). One hydroxyl
group was protected as its p-methoxybenzyl ether. Treatment of
2 with ethyl bromoacetate in the presence of potassium carbonate
gave 3. Hydrolysis of the ethyl ester with lithium hydroxide affor-
ded carboxylic acid 4, which was subsequently converted into
A physical interaction between PFOS and CD14 was analyzed
using a surface plasmon resonance biosensor instrument, Bia-
core3000 (Fig. 3). A soluble form of recombinant CD14 that lacked
the C-terminal 21 amino acids (sCD14 [1–335]) was immobilized
O
(A)
Br
PMBCl
K2CO3
EtO
K2CO3
O
F
F F F F F F F
F
F F F F F F
F
F F F F F F F F
HO
HO
O
OPMB
OH
EtO
OPMB
acetone
reflux
55%
acetone
reflux
77%
F
F F F F F F F
F
F F F F F F
F
F
F F F F F F F
2
1
3
O2N
p-nitrophenol
DCC, DMAP
O
O
O
F
F F F F F F
F
F
F F F F F F
F
LiOH
O
O
HO
OPMB
OPMB
THF-MeOH
(1:1)
CH2Cl2
49%
F
F F F F F F
F
F
F F F F F F F
5
quant.
4
O
1) DDQ, CH2Cl2-H2O
2) wash
5
1) , pyridine
F
F F F F F F F
2) wash
O
NH2
OPMB
N
H
3) Ac2O, pyridine
4) wash
F
F F F F F F F
6
Amino-PEGA resins
1) SO3·pyridine
pyridine
O
O
F
F F F F F F
F
F
F F F F F F
F
2) wash
O
O
OSO3Na
N
H
OH
O
N
H
F
F F F F F F
F
3) wash with sat. NaCl aq
4) wash
F
F F F F F F
F
8
7
(B)
1) Ac2O, pyridine
2) wash
NH2
N
H
Amino-PEGA resins
9
Scheme 1. Synthesis of PFOS analog-immobilized PEGA resin 8 (A) and acetylated control resin 9 (B).

Y. Miyano et al. / Bioorg. Med. Chem. 20 (2012) 3985–3990
3987
time (s)
100
20
-50
0
50
150
200
10
0
-10
-20
-30
-40
-50
-60
-70
2.5uM
5uM
7.5uM
10uM
12.5uM
15uM
20uM
25uM
30uM
Figure 2. Recovery rate of phage particles bound to 8 and 9 after each round of
biopanning. The phage random library was applied to 8 or 9. After removal of
unbound phage particles, bound phage particles were eluted and amplified by
infection of E. coli. The resulting phage particles were used in the next round of
selection. At each round, the recovery rates of the eluted phage particles to the
input phage particles from the resins were calculated.
Figure 3. SPR analysis of the binding between PFOS and a recombinant soluble
CD14 (sCD14 [1–335]). sCD14 [1–335] was immobilized on a CM5 sensor chip. PFOS
samples were injected over the immobilized sCD14 [1–335]. Response units (RU)
were calculated by subtracting the background response measured in the control
flow cell from the response in each sample flow cell. Binding of various
concentrations of PFOS to the sCD14 was analyzed. The concentrations of PFOS
were 2.5, 5.0, 7.5, 10.0, 12.5, 15, 20, 25 and 30 lM from top to bottom. Kinetic
studies were performed using the BIA evaluation software.
Table 1
Amino acid sequence of peptides displayed on the phage particle after the biopanning
using PEGA-supported PFOS analogue (8)
Phage clone
Sequencea
Frequencyb
1
PVSPFTCDLGARTSP
12/32
6/32
3/32
2/32
2/32
1/32
1/32
1/32
1/32
1/32
1/32
2
VRDVFSVCGGVSSCHESLRPHSSN
PAGISRELVDKLAAALE
LSRAGSLLGRSLRHA
SCTSYYAGSLNFSCL
LHSFDFFVTNVSFVV
SCTSYYVGSLNFSCL
PVLSPPPFDAGLVLKACGRTRVTS
SRFECNLGACPVRAC
FGCTLPIAVCRVADE
GCLSFTGYGPWWLPR
3c
4
5
6
7
8
9
10
11
a
b
c
Amino acid sequences were determined from DNA sequences of the insert DNA.
Frequency is the number of sequences among the total phage clones selected.
This phage clone does not contain the inserted DNA fragment.
Table 2
List of human proteins with high similarity to the selected peptidesa
Scoreb
Protein
777.78
678.85
647.31
627.60
622.22
612.54
595.34
574.55
561.29
Protocadherin Fat3
Figure 4. Inhibitory effects of PFOS on CD14-induced production of TNF-
mouse macrophage cell line RAW264.7. The indicated molecular ratios of PFOS and
CD14 protein were pre-incubated for 10 min at room temperature, and the mixture
a in the
Monocyte differentiation antigen CD14
Olfactory receptor 4K17
Adiponutrin
Integrin alpha-M precursor
Splice isoform of prostaglandin E2 receptor
Chondroitin sulfate synthase 1
Retinoblastoma-like protein 2
Cadherin 4
was incubated with the cells for 24 h. The TNF-
was measured by ELISA. Data represent means SD (n = 4).
a
concentration in the cell medium
⁄
, P <0.01 versus
untreated control (student’s t-test).
previous reports.35–37 The apparent dissociation constant value
for this binding was determined to be 5.83 ꢁ 10ꢂ5 M.
a
The candidates for PFOS-binding proteins were obtained by analysis of selected
peptides using the FASTAskan program from the Receptor Ligand Contacts (RELIC).
b
Scores are generated by calculating the similarity between all peptides and each
Next, we investigated whether PFOS can inhibit the sCD14 func-
tion in mouse macrophage RAW264.7 cells. As shown in Figure 4,
segment of the protein sequence.
the sCD14 [1–335] significantly induced TNF-
a
production in the
on a CM5 sensor chip by the amine coupling reaction.33,34 Several
concentrations of PFOS were injected over the sCD14 [1–335]. As
shown in Figure 3, negative sensorgrams were observed. The neg-
ative response was enhanced at increasing concentrations of PFOS,
confirming a specific interaction between PFOS and sCD14 [1–335].
The negative sensorgrams might be explained by conformation
changes in the sCD14 [1–335] bound to PFOS, as described in
cultured cells (10 M of CD14-evoked TNF-
l
a level, 665.0 pg/mL).
This CD14 stimulation was inhibited by the pre-incubation of
CD14 protein and PFOS. When the molecular ratio of PFOS and
CD14 was more than 1, the TNF-
inhibited. In cultured macrophage RAW264.7 cells, PFOS was not
cytotoxic at 250 M (data not shown); therefore, the LD50 value
was >250 M. These results suggest that PFOS will directly bind
a production of the cells was
l
l

3988
Y. Miyano et al. / Bioorg. Med. Chem. 20 (2012) 3985–3990
sCD14 and prevent TNF-
a
production induced by CD14 in
4.2. Preparation of 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluoro-
macrophages.
1-(p-methoxybenzyloxy)decanol (2)
A solution of 1H,1H,10H,10H-perfluorodecane-1,10-diol (597
mg, 1.29 mmol), p-methoxybenzyl chloride (140 lL, 1.03 mmol)
3. Discussion
and potassium carbonate (536 mg, 3.88 mmol) in acetone (3 mL)
was refluxed for 72 h. The reaction was then quenched by the addi-
tion of water and diluted with EtOAc. The layers were separated, the
organic layer was washed with brine, dried (Na2SO4) and concen-
trated. The reside was purified by silica gel chromatography
(hexane/EtOAc = 3:1) to yield 2 (331 mg, 55%) as white solid.
Mp = 49.1–51.1 °C. 1H NMR (270 MHz, CDCl3) d 7.27 (2H, d,
J = 8.6 Hz), 6.90 (2H, d, J = 8.6 Hz), 4.60 (2H, s), 4.10 (2H, m), 3.90
(2H, t, J = 13.9 Hz), 3.82 (3H, s), 1.93 (1H, br m). IR (KBr) 3394,
2961, 2939, 2870, 2844, 1615, 1518, 1463, 1382, 1303, 1252, 1199,
1146, 1029, 973, 819 cmꢂ1. HRMS (ESI): calcd for C18H14O3F16Na
([M+Na]+) 605.0579, found 605.0582.
Here, we employed a combination of a T7 phage display tech-
nology and PFOS coupled to PEGA resin in order to identify ami-
no-acid sequences bound to the small ligand. Use of PEGA resin
facilitates preparation of a hydrophobic compound-immobilized
matrix as the resin can be used during both solid-phase synthesis
of the ligand in organic solution and in vitro selection of phage par-
ticles displaying oligopeptides in aqueous solution. The screen was
successfully achieved because the recovery rate of the eluted phage
particles from 8 against input phage particles increased with each
round of biopanning.
Analysis of selected peptides using the FASTAskan program
from RELIC provides candidates of PFOS-binding proteins. Among
the candidate binding proteins, PFOS interacts with sCD14 [1–
335] with an apparent dissociation constant of 5.83 ꢁ 10ꢂ5 M by
SPR analyses. CD14 is known to exist either in a glycosylphosphat-
idylinositol anchored form on the surface of cells (membrane
CD14, mCD14) or in a soluble form in blood (sCD14).38 Previous
studies showed that sCD14 binds lipopolysaccharide (LPS) in the
absence of lipopolysaccharide binding protein (LBP).39,40 The
LPS–sCD14 complexes are considered to be an important interme-
diate in the inflammatory responses of leukocytes to LPS.41
PFOS has been reported to alter inflammatory responses and pro-
4.3. Preparation of ethyl {2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-
hexadecafluoro-10-(p-methoxybenzyloxy)decanyloxy}acetate
(3)
A solution of 2 (331 mg, 0.57 mmol), ethyl bromoacetate (95 lL,
0.85 mmol) and potassium carbonate (393 mg, 2.84 mmol) in ace-
tone (5 mL) was stirred at room temperature for 18 h and then re-
fluxed for 6 h. Ethyl bromoacetate (60 lL, 0.54 mmol) was added to
the mixture, the resulting mixture was refluxed for 15 h. Since the
reaction was not completed, additional ethyl bromoacetate
(120 lL, 1.08 mmol) was added to the mixture. After the mixture
was refluxed for 5 h, the reaction was quenched by the addition
of water. The resulting mixture was diluted with EtOAc. The layers
were separated, the organic layer was washed with brine, dried
(Na2SO4) and concentrated. The reside was purified by silica gel
chromatography (hexane/EtOAc = 6:1) to yield 3 (293 mg, 77%) as
colorless oil. 1H NMR (270 MHz, CDCl3) d 7.26 (2H, d, J = 8.4 Hz),
6.90 (2H, d, J = 8.4 Hz), 4.61 (2H, s), 4.24 (2H, q, J = 7.1 Hz), 4.23
(2H, s), 4.11 (2H, t, J = 13.8 Hz), 3.89 (2H, t, J = 13.9 Hz), 3.82 (3H,
s), 1.29 (3H, t, J = 7.1 Hz). IR (KBr) 2943, 2850, 1754, 1614, 1588,
1514, 1465, 1377, 1213, 1146, 1035, 960, 823 cmꢂ1. HRMS (ESI):
calcd for C22H20O5F16Na ([M+Na]+) 691.0947, found 691.0925.
duction of cytokines in both PPAR-
a dependent and independent
manners.8,42,43 Recent studies showed that the immunotoxicity of
PFOS could be explained by its inhibitory effects on LPS-induced I-
j
B degradation.44,45 We found that pre-incubation of PFOS and
sCD14 [1–335] inhibited TNF-a production induced by the sCD14
in RAW264.7 cells. Since the critical micelle concentration of potas-
sium perfluorooctane sulfonate is reported to be 8 mM,46 PFOS
would not form micelles at the concentration used in this study
and act as a surfactant to sCD14 [1–335]. Taken together with our
SPR analyses, these results suggest that PFOS will directly bind
sCD14 and inhibit the function of sCD14, which might be another
mechanism for the immunotoxicity of PFOS.
In conclusion, we identified CD14 as a binding protein of PFOS
by a combination of solid-phase synthesis of a ligand with a T7
phage display screen. Although further study is required to eluci-
date the binding site of PFOS within CD14 as well as detailed mode
of action of PFOS, our results suggested that PFOS might cause the
immunotoxicity by impeding the function of CD14.
4.4. Preparation of {2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-
hexadecafluoro-10-(p-methoxybenzyloxy)decanyloxy}acetic
acid (4)
A solution of 3 (159 mg, 0.24 mmol) and 1 M aqueous LiOH
(1.0 mL, 1.00 mmol) in THF (1.5 mL) and MeOH (1.5 mL) was stir-
red at room temperature for 3 h. The mixture was then acidified
by the addition of 1 M HCl and diluted with EtOAc. The layers were
separated and the aqueous layer extracted twice with EtOAc. The
combined organic layer was washed with brine, dried (Na2SO4)
and concentrated to give 4 (242.3 mg, quant) as a colorless solid.
Mp = 77.5–78.5 °C. 1H NMR (270 MHz, CDCl3) d 7.26 (2H, d,
J = 8.6 Hz), 6.90 (2H, d, J = 8.6 Hz), 4.61 (2H, s), 4.32 (2H, s), 4.14
(2H, t, J = 13.6 Hz), 3.89 (2H, t, J = 13.7 Hz), 3.82 (3H, s). IR (KBr)
3480, 2994, 2961, 2937, 2838, 1730, 1614, 1588, 1518, 1459,
4. Experimental procedure
4.1. General methods for synthesis of a PEGA-supported PFOS-
analogue (8) and control resin (9)
All reagents were obtained from commercial sources and used
without further purification. Analytical thin-layer chromatography
was performed on Silica Gel 60 F254 plates (Merck, Darmstadt, Ger-
many). Flash chromatography was carried out on PSQ 100B (Fuji
Silysia Co., Japan). 1H NMR spectra were recorded on a Bruker
600 MHz spectrometer (Avance DRX-600) using a CDCl3 solution,
unless otherwise noted. Chemical shifts are expressed in d (ppm)
relative to Me4Si and coupling constants (J) are expressed in hertz.
Melting point (mp) data were determined with a Yanaco MP-3S
instrument and are uncorrected. Infrared (IR) spectra were re-
corded on a Jasco FT/IR-410 spectrometer, using NaCl (neat) or
KBr pellet (solid). Mass spectra (MS) were obtained on an Applied
Biosystems mass spectrometer (API QSTAR pulsar i) under high-
resolution conditions.
1367, 1315, 1251, 1214, 1139, 1032, 995, 975, 951, 828 cmꢂ1
.
HRMS (ESI): calcd for C20H16O5F16Na ([M+Na]+) 663.0634, found
663.0614.
4.5. Preparation of p-nitrophenyl {2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-
hexadecafluoro-10-(p-methoxybenzyloxy)decanyloxy}acetate
(5)
To a solution of 4 (154.5 mg, 0.24 mmol) and p-nitrophenol
(67.8 mg, 0.48 mmol) in CH2Cl2 (4 mL) was added N,N0-

Y. Miyano et al. / Bioorg. Med. Chem. 20 (2012) 3985–3990
3989
dicyclohexylcarbodiimide (102 mg, 0.49 mmol) followed by 4-
dimethylaminopyridine (31.2 mg, 2.6 mmol). The mixture was
stirred at room temperature for 5 h. The reaction was quenched
by the addition of water. The resulting mixture was diluted with
EtOAc. The layers were separated, the organic layer was washed
with brine, dried (Na2SO4) and concentrated. The reside was puri-
fied by silica gel chromatography (hexane/EtOAc = 3.5:1) to yield 5
(78 mg, 49%) as yellow solid. Mp = 41.5–42.5 °C. 1H NMR
(270 MHz, CDCl3) d 8.30 (2H, d, J = 9.1 Hz), 7.34 (2H, d, J = 9.1 Hz),
7.26 (2H, d, J = 8.6 Hz), 6.90 (2H, d, J = 8.6 Hz), 4.61 (2H, s), 4.55
(2H, s), 4.21 (2H, t, J = 13.5 Hz), 3.90 (2H, t, J = 13.9 Hz), 3.82 (3H,
s). IR (KBr) 3006, 2938, 2842, 1775, 1618, 1592, 1537, 1518,
1486, 1459, 1351, 1250, 1207, 1145, 1031, 966, 920, 859, 822 cm
ꢂ1. HRMS (ESI): calcd for C26H19NO7F16Na ([M+Na]+) 784.0798,
found 784.0772.
buffer, heated to 95 °C for 5 min and annealed by cooling the mix-
ture to 37 °C. The single-stranded regions were converted to du-
plex DNA by continuing the incubation at 37 °C for 2 h in the
presence of dNTPs (2.5 mM) and Klenow enzyme (0.5 mU/lL).
After the reaction, double-stranded DNA was recovered by EtOH
precipitation. The recovered DNA was then digested separately
with EcoRI and HindIII restriction enzymes and inserted into the
T7select10-3b vector, according to the manufacturer’s instructions.
The primary titer of this T7 phage pool was 1.6 ꢁ 107 pfu/mL. For
the screening procedure, the phage pool was amplified up to
1.7 ꢁ 1010 pfu/mL using Escherichia coli (BLT5615) as the host
strain.
4.11. Procedure for the T7 random phage display screening
using the PEGA-supported PFOS analogue
4.6. Preparation of resin 6
PEGA-supported PFOS analogue (8) and control resin (9) (5 mg
each) were pre-equilibrated with buffer A (50 mM Tris–HCl (pH
8.0), 150 mM NaCl; 1 mL) at 4 °C. For the removal of resin-binding
Amino-PEGA resin (0.42 g, 0.021 mmol) was washed with CHCl3
(ꢁ3) and pyridine (ꢁ3) and swollen in pyridine (2 mL). To the sus-
pension of the resin was added 5 (32 mg, 0.042 mmol), and the
suspension stirred at room temperature for three days. Then the
suspension was filtered and washed with CHCl3 three times. The
resulting resin 6 was dried and used for the next reaction. The fil-
trate was concentrated, and the residue was purified by silica gel
chromatography (hexane/EtOAc = 1:1) to give p-nitrophenol
(4.5 mg, 77% from 5) and the recovered 5 (6.4 mg).
phage clones, the phage library (500
the control resin (60 L each) for 2 h at room temperature. The re-
sin was precipitated and the supernatant was used for screening.
The supernatant (500
L ꢁ 2) was incubated with each resin
(60 L each) for 4 h at room temperature. A hole was made in
l
L ꢁ 2) was incubated with
l
l
l
the bottom of a tube, and the resin was then centrifuged at
3300g for 1 min to remove the phage solution. The resin was
washed with buffer B (50 mM Tris–HCl (pH 8.0), 150 mM NaCl,
0.1% Tween20; 600
lL) 12 times. The bound phage particles were
4.7. Preparation of resin 7
eluted by 20 min incubation with 1% SDS (100
lL). The eluted
phage particles were amplified by infection into E. coli strain
BLT5615 as host cells. The amplified phage was applied by subse-
quent rounds of biopanning. Four rounds of biopanning were
performed.
Resin 6 was swollen in CH2Cl2–H2O (10:1; 4.4 mL). To the sus-
pension of the resin was added 2,3-dichloro-5,6-dicyano-p-benzo-
quinone (17.1 mg, 0.021 mmol), and the mixture was stirred at
room temperature for 24 h. Then the suspension was filtered and
washed with CHCl3 three times, MeOH–H2O three times, and CHCl3
twice. The resulting resin 7 was dried and used for the next reac-
tion. The organic layer was separated from the filtrate. The organic
layer was washed with saturated NaHCO3, brine, dried (Na2SO4)
and concentrated. The residue was purified by preparative TLC
(hexane/EtOAc = 1:1) to give anisaldehyde (1.8 mg, 80% calculated
from amino-PEGA resin).
4.12. Characterization of binding phage
After the fourth round of biopanning, the eluted phage particles
were applied to Luria-Bertani plates containing 50 lg/mL carbeni-
cillin. Thirty-two clones were randomly selected from the plate
and each clone was dissolved in phage extraction solution
(20 mM Tris–HCl pH 8.0, 100 mM NaCl, 6 mM MgSO4). Phage
DNA carrying insert DNAs was amplified by PCR using forward pri-
mer (50-TGCTAACTTCCAAGCGGACC-30) and reverse primer (50-
TTGCCCAGAACTCCCCAA-30). The PCR product was purified with
ExoSAP-IT (GE Healthcare) and used for cycle sequence PCR with
a BigDye Terminator 3.1 Cycle Sequence Kit (Applied BioSystems).
The resulting fragments were precipitated with ethanol. The puri-
fied PCR products were sequenced on an ABI PRISM 3100 Genetic
Analyzer (Applied BioSystems). From the results, the deduced ami-
no acid sequence present on the T7 phage capsid was determined.
4.8. Preparation of resin 8
Resin 7 was swollen in pyridine (4 mL). To the suspension of the
resin was added sulfur trioxide pyridine complex (12.0 mg,
0.075 mmol), and the mixture was stirred at room temperature
for 24 h. Then the suspension was filtered and washed with CHCl3
three times, MeOH three times, MeOH–H2O three times, H2O twice,
saturated aqueous solution of NaCl and H2O twice. The resulting
resin 7 was dried and used for affinity selection.
4.13. Analysis of selected peptides using the Receptor Ligand
Contacts (RELIC) bioinformatics
4.9. Preparation of resin 9
Amino-PEGA resin (1.0 g) was washed with CHCl3 twice and
pyridine twice and swollen in pyridine (5.0 mL). To the suspension
of the resin was added Ac2O (5.0 mL), and the suspension was stir-
red at room temperature for 3 h. Then the suspension was filtered
and washed with CHCl3 three times, MeOH three times, and CHCl3
twice. The resulting resin was dried in vacuo.
Peptide sequences obtained by phage DNA sequencing were ana-
lyzed using the RELIC program according to the supplier’s manuals.
The peptide sequences (PVSPFTCDLGARTSP, VRDVFSVCGGVSSCH,
LSRAGSLLGRSLRHA, SCTSYYAGSLNFSCL, LHSFDFFVTNVSFVV, SCTS-
YYVGSLNFSCL, PVLSPPPFDAGLVLK, SRFECNLGACPVRAC, FGCTLPI-
AVCRVADE, GCLSFTGYGPWWLPR) were used as a query for a FAS-
TAskan search.32
4.10. Construction of a random DNA phage library
4.14. Surface plasmon resonance
For the preparation of a duplex DNA library, oligonucleotide
GGGGATCCGAATTCT(NNK)15TGAAAGCTTCTCGAGGG (0.056 pM)
and CCCTCGAGAAGCTTTCA (0.56 pM) were mixed with Klenow
A soluble form of recombinant CD14 that lacked the C-terminal
21 amino acids (sCD14 [1–335]) was purchased from ITSI-

3990
Y. Miyano et al. / Bioorg. Med. Chem. 20 (2012) 3985–3990
8. DeWitt, J. C.; Shnyra, A.; Badr, M. Z.; Loveless, S. E.; Hoban, D.; Frame, S. R.;
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Luster, M. I. Crit. Rev. Toxicol. 2009, 39, 76.
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Biosciences. Surface plasmon resonance (SPR) analysis was per-
formed on a BIAcore 3000 (GE Healthcare). The surface of a CM5
chip was activated by injecting a solution containing 0.2 M N-
ethyl-N0-(3-dimethylaminopropyl) carbodiimide hydrochloride
and 50 mM N-hydroxysuccinimide for 10 min. Protein diluted with
10 mM sodium acetate buffer at pH 4.0–6.0 was injected and the
surface was then blocked by injecting 1 M ethanolamine at pH
8.5 for 10 min. sCD14 protein was immobilized on the chip at a le-
vel of approximately 15,000 response units (RU). Perfluorooctane-
sulfonic acid (Wako Pure Chemical Industries, Ltd) at various
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concentrations (2.5, 5.0, 7.5, 10.0, 12.5, 15, 20, 25 and 30
lM) in
PBS (pH 7.4) were flowed at 20 L/min. To regenerate the sensor
l
chip we used 25 mM NaOH and 0.5 mM NaCl and replicates of each
experiment were performed more than two times. After the nega-
tive sensorgrams were mirrored, dissociation constant (KD) re-
sponse curves were generated by subtraction of the background
signal generated simultaneously on the control flow cell. We used
BIA evaluation 4.1 software (GE Healthcare) to determine the ki-
netic parameters.
4.15. Measurement of TNF-a level in the cell culture medium of
mouse macrophages
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A mouse macrophage cell line, RAW264.7, was obtained from
the American Type Culture Collection (ATCC) (Manassas). The cells
were cultured in Eagle’s Minimum Essential Medium (MEM) sup-
plemented with 4.5 g glucose per liter plus 10% fetal calf serum,
5 lM L-glutamine, 50 units/mL penicillin and 50 units/mL strepto-
mycin. The cells were cultured at 37 °C in standard medium in a
humidified atmosphere of 5% CO2–95% air. RAW264.7 cells were
placed in a 12-well plate at 1 ꢁ 105 cells/well and incubated for
24 h. The various molecular ratios of CD14 protein and perfluo-
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32. Because the clone 3 does not have the inserted DNA fragment, the clone 3
peptide was excluded from the FASTAskan analysis. The clone 1, 4, 5, 6, 7, 9, 10,
and 11 peptides as well as amino acid sequences of the first 15 residues of
clone 2 and clone 8 peptides were used as a query for the FASTAskan analysis
(see also Section 4.13.)
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After stimulation with CD14 (final concentration of 10
24 h, the cell culture medium was collected to measure the amount
of TNF- secreted. The concentration of TNF- in the culture med-
lM) for
a
a
ium was quantified by using a commercially available enzyme-
linked immunosorbent assay (ELISA) development system (Bay
Bioscience Co., Ltd) in accordance with the manufacturer’s
protocol.
Acknowledgments
This work was partially supported by Grant-in-Aid for Young
Scientists (B) (22710219) (K.K.). We are grateful to Professor Akito
Ishida (Kyoto Prefectural University) for valuable discussions on
the SPR experiments.
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