Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device

Article abstract of DOI:10.1016/j.bmc.2008.11.004

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized a

Full text of DOI:10.1016/j.bmc.2008.11.004

Bioorganic & Medicinal Chemistry 17 (2009) 195–202
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of trimannoside-recognizing peptide sequences from a T7
phage display screen using a QCM device
Kazusa Nishiyama a, Yoichi Takakusagi b, Tomoe Kusayanagi b, Yuki Matsumoto b, Shiori Habu b,
Kouji Kuramochi b, Fumio Sugawara b, Kengo Sakaguchi b, Hideyo Takahashi c,
Hideaki Natsugari c, Susumu Kobayashi a,
*
a Department of Medicinal and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
b Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
c Laboratory of Synthetic Organic and Medicinal Chemistry, School of Pharmaceutical Sciences, Teikyo University, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage dis-
play screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a
self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode sur-
face of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying
PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44,
respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using
the respective single T7 phage and synthetic peptide also confirmed the specific binding of these
sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to
part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken
together, these results demonstrate the effectiveness of our T7 phage display environment for affinity
selection of binding peptides. We anticipate this screening result will also be extremely useful in the
development of inhibitors or drug delivery systems targeting polysaccharides as well as further investi-
gations into the function of carbohydrates in vivo.
Received 1 October 2008
Revised 4 November 2008
Accepted 4 November 2008
Available online 8 November 2008
Keywords:
T7 phage display
QCM
Self-assembled monolayer (SAM)
Trimannose
Polysaccharide
Peptide
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
sequent processing of the non-reducing end of the polysaccharide
chain by glycosidases and glycosyltransferases define the fate of
The biological role of sugar chains in higher organisms has been
intensively studied over a number of decades. Recent research has
revealed insights into the biological role of polysaccharides with
regard to the cell–cell interaction,1 pathogen invasion,2 serum sta-
bility of the proteins,3,4 and the mechanism of quality control of
glycoproteins in the endoplasmic reticulum (ER).5,6 Recent ad-
vances in glycoprotein research have also revealed that changes
in the glycosylation pattern of cellular glycoproteins are common
features of human cancer.1 Indeed, such changes can influence tu-
mor progression, suggesting that an inhibitor of glycosylation may
slow down the development of certain types of cancer.7
There have been significant advances in the development of oli-
gosaccharide libraries generated by chemical synthesis together
with efficient methods of isolating natural glycoproteins.8–11 These
developments have facilitated studies with regard to the biological
role of polysaccharides. Among them, the mechanism of quality
control of glycoproteins in ER has been gradually clarified over
the past decade. Glycosylation of the nascent polypeptide and sub-
the glycoprotein,12,13 which is cooperatively supported by the
function of polysaccharide-recognizing proteins called lectins. Var-
ious molecular chaperones, cargo receptors and ubiquitin ligases
contribute to maintaining the fidelity of glycoprotein synthesis in
the ER.5,10,14–18 In this mechanism, triglucosyl high-mannose type
oligosaccharide (Glc3Man9GlcNAc2, G3M9) is a common precursor
of the N-glycans (Fig. 1).5,10 This oligosaccharide comprises a trim-
annosyl core anchored by two N-acetylglucosamine (GlcNAc) units
for the asparagine (Asn) residue in the Asn-X-Ser/Thr motif of the
nascent polypeptide chain. At the opposite side of non-reducing
end are three branches, designated D1, D2, and D3 arms, composed
of glucose and mannose units (Fig. 1). The residues in these chains
are trimmed by glycosidases during the early stages of the biosyn-
thetic pathway and transmitted as a reporter for ER lectin-chaper-
ones, such as calreticulin (CRT) and calnexin (CNX).5,19
Identification of the (poly)saccharide-recognizing primary pep-
tide sequences would be an approach to investigate the precise
mode of binding of carbohydrate-related proteins, including lec-
tins.20,21 This information may also facilitate the discovery of
new species of proteins using a similarity search as well as gener-
ating peptides that can selectively target the polysaccharide chain
* Corresponding author. Tel.: +81 4 7124 1501x6543.
E-mail address: kobayash@rs.noda.tus.ac.jp (S. Kobayashi).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.004

196
K. Nishiyama et al. / Bioorg. Med. Chem. 17 (2009) 195–202
Figure 1. (A) Structure of triglucosyl high-mannose type oligosaccharide (Glc3Man9GlcNAc2, G3M9). Trimannosyl core is shown boxed. (B) Chemical structure of the
trimannosyl core.
for drug delivery or inhibit the corresponding function. Herein, we
attempted to identify peptide sequences that can specifically rec-
ognize trimannoside, the core structure in N-glycans as a scaffold
for branches D1–3 (Fig. 1B). A pea lectin, concanavalin A, is known
to specifically recognize this skeleton in many complex glycans. In-
deed, the mode of docking is well defined by X-ray crystallogra-
phy.22–24 In order to identify the binding-peptide sequence, we
used the T7 phage display technology.25,26 Recently, we have re-
ported the T7 phage display screen combined with the cuvette type
quartz-crystal microbalance (QCM) biosensor, which enables rapid
and efficient identification of binding proteins or peptides for small
organic molecules of interest.27,28 The target immobilization pro-
cess on the gold electrode surface of the sensor chip involves a
self-assembled monolayer (SAM). Importantly, SAM acts as a supe-
rior scaffold for the QCM biosensor by accumulating the substrate
of interest on the gold surface, thereby increasing the sensitivity of
the frequency changes.29 Thus, after synthesizing a trimannoside
derivative that can form SAM, we performed an affinity selection
using a HepG2-derived T7 phage pool. Herein, we describe the
experimental details, biological significance of the resulting pep-
tides and the effectiveness of our T7 phage display environment
for affinity selection of binding peptides.
trode surface. After washing the surface of the electrode, the sensor
chip was placed in the QCM apparatus using a buffer filled cuvette.
The QCM sensor was then fully stabilized. When an aliquot of pla-
que forming units (pfu) of a HepG2-derived T7 phage pool was in-
jected into the cuvette, a frequency change was detected (Fig. 2).
After monitoring the interaction for 10 min, the sensor chip was
detached from the device. The genomic DNA of trimannoside-rec-
ognizing phage on the gold electrode surface was then recovered
using the host Escherichia coli (BLT5615) solution and amplified.
After 30 min incubation at 37 °C, recovered phage particles were
arbitrarily extracted from the resulting T7 phage solution and the
enrichment was analyzed by sequencing the DNA encoding the fu-
sion protein of the phage capsid. Peptide sequences obtained after
six sets of individual one-cycle screens are summarized in Table 1.
Forty-four phage particles were arbitrarily extracted from the
resulting T7 phage solution and the enrichment was analyzed by
sequencing the DNA encoding the fusion protein of the phage cap-
sid. The T7 phage display screen using trimannoside-SAM and QCM
resulted in enrichment of T7 phages whose capsid fusion peptides
corresponded to PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-
mer), which were included at a rate of 17 and 9 out of 44 phage iso-
lates, respectively (Table 1).
2.3. Binding experiment using the respective single T7 phage
and synthetic peptide on a QCM apparatus
2. Results and discussion
2.1. Synthesis of trimannoside derivative
In order to characterize the interaction with trimannoside, T7
phage particles displaying PSVGLFTH (phage 1) or SVGLGLG
FSTVNCF (phage 2) was amplified by infection of host cells and
used for binding experiments. According to the same procedure
of screening, an aliquot of 1010 pfu/ml of each single phage pool
was individually injected into the cuvette and the frequency
change monitored for 10 min.27 Figure 3 shows the relative binding
of each phage to trimannoside as calculated from the frequency de-
crease 10 min after injection. The frequency change obtained with
control T7 phage (NSPAGISRELVDKLAAALE) is also shown as a ref-
erence. Phages 1 and 2, which were both significantly enriched in
the screen, showed an interaction with trimannoside, verifying
the result of the selection. Similarly, synthetic peptide that corre-
sponds to each sequence showed a binding to free trimannose,
confirming the specific interaction between the two molecules
(Fig. 4). The dissociation constant is predicted to be at the order
A trimannoside derivative (14) that forms SAM on the gold elec-
trode was synthesized as shown in Scheme 1. The trimannoside
was synthesized according to the previous report,30,31 and then
connected with a tetraethyleneglycol unit that can reduce non-
specific binding of T7 phages.29 This product was finally coupled
with compound 12, which facilitates chemisorption of the mole-
cule of interest onto the gold electrode surface via thiol–Au inter-
actions. Accumulation of the trimannoside on the gold electrode is
brought about by alignment of the C11 alkyl chains.29
2.2. Affinity selection of trimannoside-recognizing peptides
using a HepG2-derived T7 phage pool
The trimannoside derivative (14) was dissolved in 75% ethanol,
dropped onto the gold electrode and then incubated for 16 h under
a humid and shaded atmosphere at room temperature (rt), which
resulted in the formation of trimannoside-SAM on the gold elec-
of 10ꢀ4 M or more because 200
obtain the minimal response on QCM. However, because of low
lM of trimannose was needed to

K. Nishiyama et al. / Bioorg. Med. Chem. 17 (2009) 195–202
197
Scheme 1. Synthesis of trimannoside derivative (14). Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C, 72%; (b) Pd(PPh3)4, AcOH, 80 °C, 74%; (c) CCl3CN, DBU, CH2Cl2, rt,
84%; (d) TMSOTf, CH2Cl2, 0 °C, 72%; (e) Pd(OH)2/C, H2, EtOH–THF, rt; (f) WSCI, HOBt, DIPEA, CH2Cl2, rt, 86% (two steps); (g) Pd(OH)2/C, H2, EtOH–THF, rt, 88%; (h) WSCI, HOBt,
DIPEA, CH2Cl2, rt, 59% (i) NaOMe, MeOH, rt, 87%.
Table 1
Trimannoside-SAM-selected peptide sequence and enrichment ratio after six sets of
individual one-cycle screens
Phage number
Peptide sequence on phage capsid
Ratio
1
2
PSVGLFTH
SVGLGLGFSTVNCF
17/44
9/44
solubility of the synthetic peptides or requirement of a consider-
able amount of trimannose, accurate kinetic constant could not
be determined. Nevertheless, these experiments confirmed that
each peptide can selectively recognize trimannoside.
2.4. Biological significance of trimannoside-recognizing
peptide sequences
Figure 2. A representative QCM sensorgram obtained by an affinity selection of
peptide on the QCM apparatus (AffinixQ) using the trimannoside derivative and a
HepG2 cell-derived T7 phage pool (8 ll). The trimannoside-immobilized ceramic
sensor chip generated by SAM was first attached to the QCM apparatus. After
injecting the T7 phage pool at the indicated concentration, the frequency decrease
was monitored for 10 min.
Even though we used a T7 phage pool with mRNA derived from
HepG2 cells for the trimannoside-SAM screen, no phage that fully
matched lectins or enzymes were identified. We reasoned that
there must be many proteins that are not displayed on the T7

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K. Nishiyama et al. / Bioorg. Med. Chem. 17 (2009) 195–202
be useful in further investigations regarding the biochemistry of
carbohydrates in vivo, including analysis of the quality control pro-
cess of glycoproteins in the ER.
3. Conclusion
In the present study, we synthesized a trimannoside derivative
that forms SAM and performed a T7 phage display screen using a
QCM device. As a result of an affinity selection using a HepG2-de-
rived T7 phage pool we identified two peptides, PSVGLFTH (8-mer)
and SVGLGLGFSTVNCF (14-mer), as candidate trimannoside-recog-
nition sequences. A similarity search using these peptide se-
quences suggested that they correspond to part of the primary
sequence of carbohydrate-related proteins. We believe that this
preliminary examination will contribute to a better understanding
of the interaction between (poly)saccharides and these protein
mediators. Furthermore, these peptide sequences may be a useful
starting point for generating inhibitors that target molecules in-
volved in cancer progression. In conclusion, we have demonstrated
the effectiveness of the T7 phage display environment and ob-
tained crucial information regarding trimannoside-protein
interactions.
Figure 3. Relative binding of single phage clones to trimannoside-SAM. An aliquot
of 107 pfu/ml (final) of respective phage pool was injected into the cuvette and the
frequency changes were monitored for 10 min. The size of each frequency change is
compared with that using control phage (white bar) and shown as a black bar.
Phage 1: PSVGLFTH (8-mer), phage 2: SVGLGLGFSTVNCF (14-mer), phage (cont.):
NSPAGISRELVDKLAAALE (19-mer).
4. Experimental
4.1. Instruments
A 27-MHz QCM device, AffinixQ and ceramic sensor chip was
purchased from Initium Inc. (Tokyo, Japan). PCR was performed
using a PTC-200 (Peltier Thermal Cycler) (BIO-RAD, Hercules, CA).
Sequencing analysis was carried out using an ABI PRISM 3100 Ge-
netic Analyzer (Applied Biosystems, Foster City, CA). Centrifugation
was performed using a MX-201 centrifuge (TOMY Industry, Tokyo,
Japan).
4.2. General (chemistry)
All non-aqueous reactions were carried out using freshly dis-
tilled solvents under an atmosphere of argon. All reactions were
monitored by TLC, which was carried out on Silica Gel 60 F254
plates (E. Merck, Darmstadt, Germany). Flash chromatography sep-
arations were performed on BW820 (Fuji Silysia Co., Ltd, Kasugai,
Japan).
Figure 4. Binding analysis between trimannose and immobilized synthetic peptide.
Trimannose (final concentration 200 lM) was added to the cuvette. Interaction
with synthetic peptide immobilized on a gold electrode of a ceramic sensor chip
was then monitored until the frequency change reaches equilibrium. N = 3; data
represented as means SE.
The NMR spectra (1H, 13C) were determined on a Bruker
600 MHz or 400 MHz spectrometer (Avance DRX-600, Avance
DRX-400) or a JEOL 400 MHz spectrometer (JNM-LD400), using
CDCl3 (with TMS for 1H NMR and chloroform-d for 13C NMR as
the internal reference) solution, unless otherwise noted. Chemical
shifts were expressed in parts per million (ppm) and coupling con-
stants are in Hertz. Optical rotations were recorded using CHCl3 as
a solvent of a JASCO P-1030 digital polarimeter at rt, using the so-
dium D line. Infrared spectra (IR) were recorded on a Jasco FT/IR-
410 spectrometer using NaCl (neat) or KBr pellets (solid), and re-
ported as wavenumbers (cmꢀ1). Mass spectra (MS) were obtained
on LCMS-IT-TOF (Shimazu, Kyoto, Japan). Sodium trifluoroacetate
(TFA–Na) was used as internal standard for high resolution MS.
phage capsid or selected in the screen because of problems associ-
ated with misfolding, low solubility or abundance. Nonetheless,
the binding experiment clearly demonstrated the interaction be-
tween trimannoside and the obtained phages displaying short pep-
tides (Fig. 3). Thus, based on the available chemical and biological
information concerning the polysaccharides, we dissected the sig-
nificance of the obtained peptide sequences.
A similarity search of human proteins using PSVGLFTH or
SVGLGLGFSTVNCF as a query sequence against the SwissProt data-
base identified approximately 500 potential hits. Tables 2 and 3
summarizes the carbohydrate-related proteins ranked by PSI-
BLAST. In addition to mannose-related enzymes (bold in Tables 2
and 3), other hexose-related enzymes or lectins are included in this
list (plain), suggesting that the primary sequence corresponding to
the affinity-selected peptides may be important for recognition of
the hexose skeleton (Suppl. Tables 1 and 2). Furthermore, a series
of E3 ubiquitin ligases in the ubiquitin–proteasome pathway,
which are involved in the degradation of misfolded N-glycans in
the ER,16,17 were also identified as candidate hits in this similarity
search. Thus, the partial sequence identified in this screen may be a
common feature of proteins that recognize the N-glycan compo-
nent of glycoproteins. The information obtained in this study will
4.3. Synthesis of trimannoside-SAM compound
4.3.1. Allyl 2,3,4,6-tetra-O-acetyl-
[2,3,4,6-tetra-O-acetyl- -mannopyranosyl-(1 ? 6)]-2,4-di-O-
benzoyl- -mannopyranoside (3)
TMSOTf (0.03 mmol) was added to a mixture of 130,31 (248 mg,
0.58 mmol), 2,3,4,6-tetra-O-acetyl- -mannopyranosyl-2,2,2-tri-
a-D-mannopyranosyl-(1 ? 3)-
a-D
a-D
a
-D
chloroacetimidate 2 (857 mg, 1.74 mmol) and 4 Å molecular sieves
in anhydrous CH2Cl2 (8 ml) at 0 °C under an Ar atmosphere. The

K. Nishiyama et al. / Bioorg. Med. Chem. 17 (2009) 195–202
199
Table 2
Summary of human carbohydrate-related protein that contain a sequence resembling PSVGLFTH
No.
Score
Protein name [Homo sapiens]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
19.3
17.6
16.8
16.8
16.8
16.8
15.5
15.5
15.5
15.5
15.5
15.5
15.5
15.5
15.5
15.5
15.5
15.5
15.1
14.6
14.6
14.6
14.6
14.6
14.6
Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase
Inositol-trisphosphate 3-kinase B
GPI mannosyltransferase 4
E3 ubiquitin–protein ligase HUWE1
Sialidase-3 (Membrane sialidase)
Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 3
Sugar phosphate exchanger 2
UDP-glucuronosyltransferase 3A2 precursor
Thioesterase superfamily member 5
E3 ubiquitin–protein ligase HECTD1
N-Acetyl-beta-glucosaminyl-glycoprotein 4-beta-N-acetylgalactosaminyltransferase 2 (NGalNAc-T2)
UDP-glucuronosyltransferase 3A1 precursor
Lipopolysaccharide-binding protein precursor (LBP)
Phosphatidylinositol N-acetylglucosaminyltransferase subunit H
Protein O-mannosyl transferase 2
Dolichol phosphate-mannose biosynthesis regulatory protein
Sugar phosphate exchanger 3
Platelet glycoprotein 4
6-phosphofructokinase, muscle type
Ubiquitin-conjugating enzyme E2 O (Ubiquitin–protein ligase O)
Glycoprotein endo-alpha-1,2-mannosidase-like protein
Glycoprotein endo-alpha-1,2-mannosidase (Mandaselin)
Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (P-Rex1)
Phosphatidylinositol-glycan-specific phospholipase D precursor (PI-G PLD)
Probable E3 ubiquitin–protein ligase HERC2
Table 3
Summary of human carbohydrate-related protein that contains a sequence resembling SVGLGLGFSTVNCF
No.
Score
Protein name [Homo sapiens]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
19.7
19.3
19.3
18.0
18.0
18.0
18.0
17.6
17.6
17.6
17.6
17.6
17.2
17.2
17.2
16.8
16.8
16.8
16.8
16.8
16.8
16.8
16.8
16.3
Glycosyltransferase 54 domain-containing protein precursor
Glucose-1,6-bisphosphate synthase
Ubiquitin–protein ligase E3C
Ubiquitin carboxyl-terminal hydrolase 24 (ubiquitin thioesterase 24)
Lysosomal alpha-mannosidase precursor
Alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase B
Globoside alpha-1,3-N-acetylgalactosaminyltransferase 1
Beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase 7
Probable O-sialoglycoprotein endopeptidase 2
E3 ubiquitin–protein ligase RNF19A
Fructose-1,6-bisphosphatase 1 (FBPase 1)
Hexokinase-3
Probable E3 ubiquitin–protein ligase HERC2
Probable E3 ubiquitin–protein ligase MYCBP2
Glucose-6-phosphate translocase (glucose-5-phosphate transporter)
Beta-galactosidase-1-like protein 3
Chondroitin sulfate synthase 1
Phosphatidylinositol 4-kinase type 2-beta
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-4
Mannose-P-dolichol utilization defect 1 protein (Suppressor of Lec15 and Lec35 glycosylation mutation homolog) (SL15)
Chondroitin sulfate synthase 2
Trehalase precursor
Sialic acid-binding Ig-like lectin 11 precursor
Fructosamine-3-kinase
mixture was stirred for 1 h, and then neutralized with saturated
aqueous NaHCO3. The mixture was extracted with CH2Cl2, and
the organic phase was dried, filtered, and concentrated in vacuo.
The residue was purified by silica gel chromatography (hexane/
(3H, s), 1.86 (3H, s), 1.83 (3H, s); 13C NMR (150 MHz, CDCl3) d
171.1, 170.6, 170.5, 169.9, 169.7, 169.6, 169.5, 169.1, 169.0,
165.9, 165.3, 133.6, 133.5, 132.9, 129.9, 129.8, 129.1, 128.7,
128.5, 118.7, 99.4, 97.0, 96.2, 75.4, 71.8, 69.5, 69.3, 69.2, 68.9,
68.7, 68.6, 68.5, 68.2, 66.6, 65.9, 65.8, 62.2, 62.1, 60.3, 20.8, 20.6,
20.6, 20.6, 20.5, 20.5, 20.4, 20.4; IR (neat) 3024, 2955, 1755,
1603, 1452, 1372, 1232, 1047, 978, 937, 756, 715 cmꢀ1; HRMS
(ESI) calcd for C51H60O26 [M+Na]+: 1111.3271, found 1111.3242.
EtOAc = 1:1) to yield 3 (452 mg, 72%). ½a D27
ꢁ
+22.56 (c = 0.25 in
CHCl3); 1H NMR (600 MHz, CDCl3) d 8.14 (2H, m), 8.04 (2H, m),
7.54 (6H, m), 5.97 (1H, m), 5.62 (1H, t, J = 10.2 Hz), 5.52 (1H, dd,
J = 3.6 Hz, 1.8 Hz), 5.41 (1H, m), 5.36 (1H, m), 5.31 (1H, m), 5.25
(1H, m), 5.23 (1H, dd, J = 4.8 Hz, 1.8 Hz), 5.62 (3H, m), 4.99 (1H,
d, 1.8 Hz), 4.88 (1H, dd, J = 4.8 Hz, 1.8 Hz), 4.80 (1H, d, J = 1.2 Hz),
4.51 (1H, dd, J = 10.2 Hz, 3.6 Hz), 4.16 (5H, m) 4.07 (1H, m), 4.03
(1H, dd, J = 13.8 Hz, 1.8 Hz), 3.99 (1H, dd, J = 14.4 Hz, 2.4 Hz), 3.91
(1H, dd, J = 10.8 Hz, 7.2 Hz), 3.60 (1H, dd, J = 10.8 Hz, 2.4 Hz), 2.12
(3H, s), 2.11 (3H, s), 2.05 (3H, s), 1.98 (3H, s), 1.94 (3H, s), 1.93
4.3.2. 2,3,4,6-tetra-O-acetyl-
[2,3,4,6-tetra-O-acetyl- -mannopyranosyl-(1 ? 6)]-2,4-di-O-
benzoyl- -mannopyranose (4)
a-D-mannopyranosyl-(1 ? 3)-
a-D
a-D
To a solution of 3 (286 mg, 0.26 mmol) in anhydrous AcOH
(2.6 ml) was added Pd(PPh3)4 (91 mg, 0.08 mmol) at rt. The mix-

200
K. Nishiyama et al. / Bioorg. Med. Chem. 17 (2009) 195–202
ture was stirred for 1 h at 80 °C. Toluene was added and then the
solvents were evaporated in vacuo to give a residue, which was
purified by silica gel column chromatography (hexane/
(1H, dd, J = 10.2 Hz, 2.4 Hz), 3.91 (2H, m), 3.63 (1H, m), 3.59 (1H,
dd, J = 10.8 Hz, 1.8 Hz), 3.49 (2H, m), 2.12 (3H, s), 2.11 (3H, s),
2.05 (3H, s), 1.98 (3H, s), 1.94 (3H, s), 1.91 (3H, s), 1.88 (3H, s),
1.83 (3H, s); 13C NMR (150 MHz, CDCl3) d 170.6, 170.5, 170.2,
169.9, 169.7, 169.6, 169.2, 168.9, 165.9, 165.4, 133.7, 133.6,
129.9, 129.9, 129.0, 128.8, 128.7, 128.5, 99.6, 97.4, 97.0, 75.9,
71.8, 69.7, 69.4, 69.3, 69.2, 68.9, 68.6, 68.3, 66.4, 65.9, 65.8, 65.2,
62.3, 62.1, 60.6, 48.3, 28.8, 20.8, 20.8, 20.7, 20.6, 20.6, 20.5, 20.5;
IR (neat) 2939, 2100, 1749, 1603, 1452, 1371, 1226, 1047, 756,
EtOAc = 1:2) to give 4 (205 mg, 74%). ½a D24
ꢀ7.61 (c = 1.0 in CHCl3);
ꢁ
1H NMR (600 MHz, CDCl3) d 8.14 (2H, m), 8.04 (2H, m), 7.58 (4H,
m), 7.45 (2H, m), 5.58 (1H, t, J = 10.2 Hz), 5.54 (1H, dd, J = 3.6 Hz,
1.8 Hz), 5.45 (1H, m), 5.36 (1H, dd, J = 10.2 Hz, 3.6 Hz), 5.23 (1H,
t, J = 10.2 Hz), 5.20 (1H, dd, J = 3.6 Hz, 1.8 Hz), 5.10 (2H, m), 4.99
(1H, d, J = 1.8 Hz), 4.89 (1H, m), 4.81 (1H, d, J = 1.8 Hz), 4.57 (1H,
dd, J = 10.2 Hz, 3.6 Hz), 4.38 (1H, m), 4.21 (1H, dd, J = 12.0 Hz,
5.4 Hz), 4.10 (4H, m), 3.99 (1H, dd, J = 12.0 Hz, 1.8 Hz), 3.89 (1H,
dd, J = 10.8 Hz, 7.2 Hz), 3.66 (1H, dd, J = 10.8 Hz, 1.8 Hz), 2.13 (3H,
s), 2.12 (3H, s), 2.05 (3H, s), 1.99 (3H, s), 1.98 (3H, s), 1.94 (3H,
s), 1.86 (3H, s), 1.83 (3H, s); 13C NMR (150 MHz, CDCl3) d 171.2,
170.1, 170.1, 170.0, 169.9, 169.8, 169.1, 169.1, 165.9, 165.4,
133.6, 133.6, 130.0, 130.0, 128.7, 128.6, 128.6, 128.5, 99.4, 97.4,
92.2, 74.7, 72.1, 69.6, 69.4, 69.3, 69.2, 69.1, 68.5, 68.4, 66.3, 66.1,
62.4, 62.3, 60.4, 60.4, 21.1, 20.9, 20.8, 20.7, 20.7, 20.6, 20.5, 20.4;
IR (neat) 3470, 3026, 2941, 1745, 1603, 1452, 1371, 1226, 1047,
715 cmꢀ1
;
HRMS (ESI) calcd for C51H61O26N3 [M+Na]+:
1154.3441, found 1154.3429.
4.3.5. Azide derivative 10
The mixture of azide 7 (266 mg, 0.24 mmol) and Pd on C
(130 mg) in THF/EtOH (1:1) was stirred under hydrogen atmo-
sphere at rt for 50 min. The catalyst was filtered off, and the filtrate
was condensed in vacuo to give a crude preparation of 8 as syrup.
The crude 8 and 14-azido-3,6,9,12-tetraoxatetradecanoic acid 929
(0.71 mmol, 195 mg) was dissolved in dry CH2Cl2 (3 ml) at rt. Then,
756, 716 cmꢀ1
;
HRMS (ESI) calcd for C48H56O26 [M+Na]+:
WSCI–HCl (90 mg, 0.47 mmol), DIPEA (130 ll, 0.71 mmol), and
1071.2958, found 1071.2938.
HOBt (0.47 mmol, 64 mg) was added to the mixture. After 12 h,
the mixture was diluted with CH2Cl2, washed with H2O, and the or-
ganic layer was dried, filtered, and concentrated in vacuo. The
crude residue was purified by silica gel chromatography (CH2Cl2/
MeOH = 9:1) to obtain 10 (294 mg, 90%, two steps yield from 7).
4.3.3. 2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl-(1 ? 3)-
[2,3,4,6-tetra-O-acetyl-
benzoyl- -mannopyranosyl trichloroacetimidate (5)
To a stirred solution of 4 (325 mg, 0.31 mmol) in anhydrous
CH2Cl2 (3 ml) was added trichloroacetonitrile (12.5 l, 1.24 mmol)
and DBU (8.8 l, 0.06 mmol) at 0 °C. The mixture was stirred for 1 h
a-D-mannopyranosyl-(1 ? 6)]-2,4-di-O-
a-D
½
a 2D1 = +6.36 (c = 1.0 in CHCl3); 1H NMR (600 MHz, CDCl3) d 8.15
ꢁ
l
(2H, m), 8.07 (2H, m), 7.63 (1H, m), 7.59 (1H, m), 7.55 (2H, m),
7.46 (2H, m), 7.12 (1H, br m), 5.66 (1H, t, J = 9.6 Hz), 5.49 (1H,
dd, J = 3.0 Hz, 1.8 Hz), 5.34 (1H, dd, J = 10.2 Hz, 3.6 Hz), 5.24 (2H,
m), 5.10 (2H, m), 5.03 (1H, d, J = 1.8 Hz), 5.02 (1H, d, J = 1.8 Hz),
4.87 (1H, dd, J = 3.0 Hz, 1.8 Hz), 4.80 (1H, d, J = 1.8 Hz), 4.46 (1H,
dd, J = 9.6 Hz, 3.0 Hz), 4.09 (3H, m), 4.02 (5H, m), 3.96 (1H, m),
3.90 (1H, m), 3.85 (1H, m), 3.69 (15H, m), 3.58 (2H, m), 3.48 (1H,
m), 3.38 (4H, m), 2.11 (3H, s), 2.10 (3H, s), 2.06 (3H, s), 1.98 (3H,
s), 1.92 (3H, s), 1.90 (3H, s), 1.88 (3H, s), 1.82 (3H, s); 13C NMR
(150 MHz, CDCl3) d 170.6, 170.5, 169.9, 169.9, 169.8, 169.7,
169.6, 169.2, 169.0, 165.9, 165.3, 133.6, 133.5, 129.9, 129.1,
128.8, 128.7, 128.5, 99.5, 97.4, 97.1, 76.1, 71.8, 70.9, 70.6, 70.5,
70.2, 70.0, 70.0, 69.4, 69.4, 69.3, 69.2, 68.9, 68.5, 68.4, 68.3, 66.4,
66.0, 65.9, 65.8, 62.3, 62.1, 53.4, 50.6, 35.9, 29.5, 20.8, 20.7, 20.7,
20.6, 20.6, 20.6, 20.5, 20.4; IR (neat) 3385, 2934, 2878, 2100,
1739, 1672, 1602, 1539, 1452, 1371, 1226, 1043, 979, 756,
l
at rt. The solvents were removed in vacuo. The residue was purified
by silica gel column chromatography (hexane/EtOAc = 1:1 to 1:2)
to give 5 (308 mg, 83%). 1H NMR (600 MHz, CDCl3) d 8.95 (1H, s),
8.17 (2H, m), 8.05 (2H, m), 7.61 (4H, m), 7.47 (2H, m), 6.47 (1H,
d, J = 1.8 Hz), 5.45 (1H, t, J = 9.6 Hz), 5.67 (1H, dd, J = 3.6 Hz,
1.8 Hz), 5.29 (1H, dd, J = 10.2 Hz, 3.6 Hz), 5.25 (1H, t, J = 10.2 Hz),
5.20 (1H, dd, J = 3.6 Hz, 1.8 Hz), 5.15 (1H, t, J = 9.6 Hz), 5.11 (1H,
dd, J = 9.6 Hz, 3.6 Hz), 5.07 (1H, d, J = 1.8 Hz), 4.91 (1H, dd,
J = 3.0 Hz, 2.4 Hz), 4.78 (1H, d, 1.8 Hz), 4.53 (1H, dd, J = 9.6 Hz,
3.6 Hz), 4.32 (1H, m), 4.18 (2H, m), 4.07 (2H, m), 3.96 (1H, dd,
J = 12.0 Hz, 1.8 Hz), 3.94 (1H, dd, J = 12.0 Hz, 2.4 Hz), 3.90 (1H, dd,
J = 11.4 Hz, 6.6 Hz), 3.67 (1H, dd, J = 11.4 Hz, 1.8 Hz), 2.11 (3H, s),
2.08 (3H, s), 2.06 (3H, s), 1.98 (3H, s), 1.95 (3H, s), 1.92 (3H, s),
1.89 (3H, s), 1.84 (3H, s); 13C NMR (150 MHz, CDCl3) d 170.6,
170.5, 169.9, 169.7, 169.6, 169.5, 169.2, 169.1, 165.7, 165.2,
159.6, 133.9, 133.7, 130.2, 129.9, 129.0, 128.9, 128.7, 128.6,
128.5, 128.5, 128.2, 99.7, 97.2, 94.3, 75.6, 72.0, 70.3, 69.4, 69.2,
68.9, 68.4, 68.2, 68.0, 67.9, 66.3, 65.9, 65.7, 62.2, 61.9, 53.4, 20.8,
20.7, 20.6, 20.6, 20.6, 20.5; HRMS (ESI) calcd for C50H56O26NCl3
[M+Na]+: 1214.2054, found 1214.2053.
715 cmꢀ1
;
HRMS (ESI) calcd for C61H80O31N4 [M+Na]+:
1387.4704, found 1387.4701.
4.3.6. Protected trimannoside derivative (13)
The mixture of 10 (200 mg, 0.15 mmol) and Pd on C (100 mg) in
THF/EtOH (1:1) was stirred was stirred under hydrogen atmo-
sphere at rt for 60 min. The catalyst was filtered off, and the filtrate
was condensed in vacuo to give 11 as syrup. A mixture of 11
(172 mg) and 1229 (0.04 mmol, 30 mg) in dry THF (4 ml) was stir-
red at rt for 12 h. Then the mixture was concentrated, and the res-
idue was purified by silica gel chromatography to yield 13
4.3.4. 3-Azidopropyl-2,3,4,6-tetra-O-acetyl-
(1 ? 3)-[2,3,4,6-tetra-O-acetyl- -mannopyranosyl-(1 ? 6)]-
2,4-di-O-benzoyl-
a-D-mannopyranosyl-
a-D
a-D-mannopyranoside (7)
A solution of 3-azido-1-propanol 632,33 (173 mg, 1.71 mmol)
and 5 (227 mg, 0.19 mmol) in dry CH2Cl2 (2 ml), containing 4 Å
molecular sieves, was stirred under Ar at 0 °C. Then TMSOTf
(78.2 mg, 52%, two steps yield). ½a D21
ꢁ
+6.61 (c = 1.25 in CHCl3); 1H
NMR (600 MHz, CDCl3) d 8.14 (2H ꢂ 2, m), 8.07 (2H ꢂ 2, m), 7.60
(2H ꢂ 2, m), 7.55 (2H ꢂ 2, m), 7.46 (2H ꢂ 2, m), 7.11 (1H ꢂ 2, br
m), 6.19 (1H ꢂ 2, br m), 5.65 (1H ꢂ 2, t, J = 9.6 Hz), 5.49 (1H ꢂ 2,
dd, J = 3.0 Hz, 1.8 Hz), 5.34 (1H ꢂ 2, dd, J = 10.2 Hz, 3.6 Hz), 5.24
(2H ꢂ 2, m), 5.10 (2H ꢂ 2, m), 5.03 (1H ꢂ 2, d, J = 1.8 Hz), 5.02
(1H ꢂ 2, d, J = 1.8 Hz), 4.87 (1H ꢂ 2, dd, J = 3.0 Hz, 1.8 Hz), 4.80
(1H ꢂ 2, d, J = 1.8 Hz), 4.46 (1H ꢂ 2, dd, J = 9.6 Hz, 3.0 Hz), 4.14
(3H ꢂ 2, m), 4.02 (5H ꢂ 2, m), 3.96 (1H ꢂ 2, dd, J = 12.0 Hz,
2.4 Hz), 3.90 (1H ꢂ 2, dd, J = 10.8 Hz, 6.6 Hz), 3.85 (1H ꢂ 2, m),
3.68 (8H ꢂ 2, m), 3.63 (5H ꢂ 2, m), 3.58 (2H ꢂ 2, m), 3.55
(2H ꢂ 2, m), 3.49 (1H ꢂ 2, m), 3.42 (2H ꢂ 2, m), 2.67 (2H ꢂ 2, t,
J = 7.2 Hz), 2.12 (2H ꢂ 2, t, J = 7.2 Hz), 2.11 (3H ꢂ 2, s), 2.10
(2.8 ll, 0.015 mmol) was added, and the reaction mixture was stir-
red for 1 h. The mixture was neutralized with aqueous NaHCO3.
The organic layer was dried, filtered, and concentrated. The residue
was purified by column chromatography (hexane/EtOAc = 1:1) to
obtain 7 (155 mg, 72%). ½a D26
ꢁ
+12.48 (c = 0.5 in CHCl3); 1H NMR
(600 MHz, CDCl3) d 8.14 (2H, m), 8.04 (2H, m), 7.61 (2H, m), 7.54
(2H, m), 7.47 (2H, m), 5.59 (1H, t, J = 10.2 Hz), 5.50 (1H, dd,
J = 3.6 Hz, 1.8 Hz), 5.34 (1H, dd, J = 10.2 Hz, 3.6 Hz), 5.24 (1H, t,
J = 10.2 Hz), 5.22 (1H, dd, J = 3.6 Hz, 1.8 Hz), 5.11 (2H, m), 5.04
(1H, d, J = 1.8 Hz), 5.02 (1H, d, J = 1.8 Hz), 4.88 (1H, m), 4.80 (1H,
d, J = 1.8 Hz), 4.44 (1H, dd, J = 10.2 Hz, 3.6 Hz), 4.08 (8H, m), 3.95

K. Nishiyama et al. / Bioorg. Med. Chem. 17 (2009) 195–202
201
(3H ꢂ 2, s), 2.06 (3H ꢂ 2, s), 1.98 (3H ꢂ 2, s), 1.95 (2H ꢂ 2, m), 1.92
(3H ꢂ 2, s), 1.89 (3H ꢂ 2, s), 1.88 (3H ꢂ 2, s), 1.82 (3H ꢂ 2, s), 1.66
(2H ꢂ 2, m), 1.62 (2H ꢂ 2, m), 1.37 (2H ꢂ 2, m), 1.27 (10H ꢂ 2, br
s); 13C NMR (150 MHz, CDCl3) d 173.2 (ꢂ2), 170.5 (ꢂ2), 170.5
(ꢂ2), 169.9 (ꢂ2), 169.8 (ꢂ2), 169.7 (ꢂ2), 169.6 (ꢂ2), 169.2 (ꢂ2),
169.0 (ꢂ2), 165.9 (ꢂ2), 165.3 (ꢂ2), 133.6 (ꢂ2), 133.5 (ꢂ2), 130.0
(ꢂ2), 129.9 (ꢂ 2), 129.1 (ꢂ2), 128.8 (ꢂ2), 128.5 (ꢂ2), 99.5 (ꢂ2)
97.4 (ꢂ2), 97.0 (ꢂ2), 76.1 (ꢂ2), 71.8 (ꢂ2), 70.8 (ꢂ2), 70.6 (ꢂ 2),
70.5 (ꢂ2), 70.4 (ꢂ2), 70.4 (ꢂ2), 70.3 (ꢂ2), 70.1 (ꢂ2), 69.9 (ꢂ2),
69.4 (ꢂ2), 69.3 (ꢂ2), 69.3 (ꢂ2), 69.2 (ꢂ2), 69.0 (ꢂ2), 68.6 (ꢂ2),
68.4 (ꢂ2), 68.3 (ꢂ2), 66.4 (ꢂ2), 66.0 (ꢂ2), 65.9 (ꢂ2), 65.8 (ꢂ2),
62.3 (ꢂ2), 62.1 (ꢂ2), 39.1 (ꢂ2), 36.6 (ꢂ2), 35.9 (ꢂ2), 29.4 (ꢂ2),
29.4 (ꢂ2), 29.4 (ꢂ2), 29.3 (ꢂ2), 29.3 (ꢂ2), 29.2 (ꢂ2), 29.2 (ꢂ2),
29.2 (ꢂ2), 28.5 (ꢂ2), 25.7 (ꢂ2), 20.8 (ꢂ2), 20.7 (ꢂ2), 20.6 (ꢂ2),
20.6 (ꢂ2), 20.5 (ꢂ2), 20.5 (ꢂ2), 20.4 (ꢂ2), 20.4 (ꢂ2); IR (neat)
2928, 2363, 1751, 1655, 1543, 1256, 1371, 1228, 1091, 1051,
756, 713 cmꢀ1; HRMS (ESI) calcd for C144H202O64N4S2 [M+Na]+:
3098.2023, found 3098.2014.
the recovery of bound phages, 10
l
l of host E. coli (BLT5615) solu-
tion (cultured for 30 min at 37 °C in the presence of 1 mM of IPTG
beforehand) was dropped onto the gold electrode and then incu-
bated at 37 °C for 30 min. To the resulting solution was then added
another 200 ll of LB medium. An aliquot of phage was then ex-
tracted from this solution and subjected to PCR analysis followed
by agarose gel electrophoresis and DNA sequencing.
4.6. Agarose gel electrophoresis and DNA sequencing
Agarose gel electrophoresis and DNA sequencing were per-
formed as described previously.29
4.7. Preparation of peptide
The proposed trimannside-recognizing peptide PSVGLFTH (8-
mer), SVGLGLGFSTVNCF (14-mer) or the control peptide
NSPAGISRELVDKLAAALE (19-mer) was synthesized by the Fmoc
method using a peptide synthesizer PS-3 (Aloka, Tokyo, Japan).28
By using Fmoc-Gly-Wang-resin (0.61 mmol/g, Carbiochem, San
Diego, CA) and adding 0.4 mmol of each amino acid (Carbiochem)
sequentially, the peptide chain (PSVGLFTH, SVGLGLGFSTVNCF or
NSPAGISRELVDKLAAALE) was extended from the C terminal to
the N terminal end by repeating the process of Fmoc deprotection
using 20% piperidine in DMF (6 ml), activation by HBTU (0.4 mmol)
and 0.4 M 4-methylmorpholine in DMF (3 ml), and amino acid cou-
pling with the resin. The resultant material was treated with 4 ml
of cleavage cocktail (0.75 g phenol, 0.25 ml 1,2-ethanedithiol,
0.5 ml thioanisole in 10 ml of 95% TFA) for 3 h to cleave the peptide
from the resin and deprotect the side chains. After cold ether pre-
cipitation, the precipitant was washed with ether three times and
recovered for HPLC purification.
4.3.7. Trimannoside derivative (14)
A mixture of 13 (45 mg, 0.015 mmol) and NaOMe (4 mg,
0.07 mmol) in MeOH (1.5 ml) was stirred at rt for 2 days. Then
the mixture was concentrated, and the residue was purified by re-
verse phase silica gel chromatography (H2O/MeOH = 1:4 to 1:1) to
yield trimannoside derivative 14 (26 mg, 87%). ½a D27
ꢁ
+38.1 (c = 0.1
in CHCl3); 1H NMR (600 MHz, CDCl3) d 5.08 (1H ꢂ 2, d, J = 1.2 Hz),
4.83 (1H ꢂ 2, d, J = 1.2 Hz), 4.69 (1H ꢂ 2, d, J = 1.2 Hz), 4.03
(2H ꢂ 2, s), 3.98 (1H ꢂ 2, dd, J = 3.6 Hz, 1.8 Hz), 3.90 (1H ꢂ 2, dd,
J = 10.8 Hz, 6.0 Hz), 3.86 (1H ꢂ 2, dd, J = 3.6 Hz, 1.8 Hz), 3.80
(5H ꢂ 2, m), 3.74 (4H ꢂ 2, m), 3.66 (15H ꢂ 2, m), 3.56 (2H ꢂ 2, t,
J = 5.4 Hz), 3.46 (1H ꢂ 2, m), 3.41 (2H ꢂ 2, m), 3.34 (2H ꢂ 2, m),
2.67 (2H ꢂ 2, t, J = 7.2 Hz), 2.19 (2H ꢂ 2, t, J = 7.8 Hz), 1.82
(2H ꢂ 2, m), 1.67 (2H ꢂ 2, m), 1.59 (2H ꢂ 2, br m), 1.39 (2H ꢂ 2,
br m), 1.31 (10H ꢂ 2, br s); 13C NMR (150 MHz, CDCl3) d 176.4
(ꢂ2), 172.7 (ꢂ2), 103.9 (ꢂ2), 101.9 (ꢂ2), 101.4 (ꢂ2), 80.8 (ꢂ2),
74.9 (ꢂ2), 74.4 (ꢂ2), 74.1 (ꢂ2), 73.5 (ꢂ2), 72.6 (ꢂ2), 72.5 (ꢂ2),
72.1 (ꢂ2), 72.1 (ꢂ2), 72.0 (ꢂ2), 71.6 (ꢂ2), 71.5 (ꢂ2), 71.4 (ꢂ2),
71.4 (ꢂ2), 71.3 (ꢂ2), 71.2 (ꢂ2), 70.6 (ꢂ2), 68.8 (ꢂ2), 68.6 (ꢂ2),
67.5 (ꢂ2), 67.3 (ꢂ2), 66.5 (ꢂ2), 62.9 (ꢂ2), 40.3 (ꢂ2), 39.8 (ꢂ2),
37.4 (ꢂ2), 37.1 (ꢂ2), 30.8 (ꢂ2), 30.6 (ꢂ2), 30.6 (ꢂ2), 30.4 (ꢂ2),
30.4 (ꢂ2), 30.3 (ꢂ2), 30.3 (ꢂ2), 30.2 (ꢂ2), 29.4 (ꢂ2), 27.0 (ꢂ2);
IR (neat) 3358, 2924, 2855, 1653, 1458, 1101 cmꢀ1; HRMS (ESI)
calcd for C84H154O44N4S2 [M+Na]+: 1985.9299, found 1985.9318.
4.8. Peptide purification
The peptide was purified using a reverse phase preparative
HPLC instrument (SSC-3461, Senshu Scientific, Tokyo, Japan)
equipped with a CAPCELL PAK C-18 column (/20 ꢂ 250 mm,
UG 120 Å, Shiseido, Tokyo, Japan) that was kept at 40 °C.28 A bin-
ary gradient with a flow rate of 4 ml/min was employed; A
phase: 0.05% TFA aq, B phase: 0.05% TFA in MeOH. The gradient
condition was as follows; PSVGLFTH: 5%
B
(0 min)ꢀ100% B
(20 min)ꢀ100% B (30 min), SVGLGLGFSTVNCF: 80% B (0 min)
ꢀ80% B (30 min), NSPAGISRELVDKLAAALE: 65% B (0 min)ꢀ85%
B (30 min). The UV absorption at 210 or 280 nm was monitored
using a UV detector (SSC-5200, Senshu Scientific). The peaks de-
tected at 22 min (PSVGLFTH), 16 min (SVGLGLGFSTVNCF) and
28 min (NSPAGISRELVDKLAAALE) under each gradient condition
were fractionated and dried up to obtain the purified peptides.
The identity of the peptide was verified by ESI-IT-MS [Esquire
3000 Plus, Bruker Daltonics, Billerica, MA].
4.4. Construction of a T7 phage pool
A T7 phage pool was constructed using HepG2 cell-derived
cDNA according to the previous reports.29 The primary titer of this
T7 phage pool was 5.0 ꢂ 106 pfu/ml. For the screening procedure,
the phage pool was amplified up to 1.28 ꢂ 108 pfu/ml using
E. coli (BLT5615) as the host strain.
4.5. Procedure for the T7 phage display screen using a cuvette
type QCM apparatus
4.9. Kinetic analysis by QCM
The synthetic peptide was immobilized on gold electrode sur-
A 20
l
l aliquot of trimannoside derivative (14) (1 mM in 75%
face of
a
sensor chip [NSPSVGLFTH: 193 Hz (5.2 pmol),
EtOH) was dropped onto the gold electrode of the ceramic sensor
chip and left for 16 h under a humid and shaded atmosphere at
rt. The surface of the electrode was washed for 10 min in buffer
(10 mM Tris–HCl, pH 8.0, 200 mM NaCl), which was stirred at
1000 rpm. The sensor chip was setup for the QCM apparatus with
the cuvette containing 8 ml of buffer. The QCM sensor was then al-
SVGLGLGFSTVNCF: 243 Hz (5.0 pmol), NSPAGISRELVDKLAAALE:
542 Hz (8.1 pmol)]. After immersing the sensor chip in 8 ml of sal-
ine solution (200 mM NaCl in 10 mM Tris–HCl, pH 8.0), 8 ll of tri-
mannose (200 mM) were added in buffer at 25 °C and monitored
until the frequency change reaches equilibrium.
lowed to fully stabilize. An aliquot of 8
l
l of T7 phage pool
4.10. Bioinformatics tool
(1.28 ꢂ 108 pfu/ml) was then injected into the cuvette. Frequency
changes, caused by binding to the trimannoside immobilized on
the gold electrode surface were then monitored for 10 min. For
We searched for human proteins containing identical or similar
sequences to the peptides identified from the phage screening pro-

202
K. Nishiyama et al. / Bioorg. Med. Chem. 17 (2009) 195–202
11. Ito, Y.; Hagihara, S.; Arai, M. A.; Matsuo, I.; Takatani, M. Glycoconjug. J. 2004, 21,
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12. Totani, K.; Ihara, Y.; Matsuo, I.; Koshino, H.; Ito, Y. Angew. Chem. Int. Ed. Engl.
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13. Totani, K.; Ihara, Y.; Matsuo, I.; Ito, Y. J. Biol. Chem. 2006, 281, 31502.
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cedure, NSPSVGLFTH (8-mer) or SVGLGLGFSTVNCF (14-mer), using
the Position-Specific Iterated Basic Local Alignment Search Tool
(PSI-BLAST, NCBI) (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi)
and the SwissProt database.
Acknowledgments
16. Yoshida, Y.; Chiba, T.; Tokunaga, F.; Kawasaki, H.; Iwai, K.; Suzuki, T.;
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This work was partially supported by a Grant-in-Aid for Scientific
Research (The Ministry of Education, Culture, Sports, Science and
Technology of Japan, Japan Society for the Promotion of Science).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.11.004.
21. Ishikawa, D.; Kikkawa, H.; Ogino, K.; Hirabayashi, Y.; Oku, N.; Taki, T. FEBS Lett.
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