Synthesis of an immunosuppressant SQAG9 and determination of the binding peptide by T7 phage display

Article abstract of DOI:10.1016/j.bmcl.2004.05.085

SQAG9, a new class of immunosuppressive sulfoquinovosylacylglycerol, and its biotinylated derivatives have been synthesized. A T7 Phage library, composed of random cDNA fragments from Drosophila melanogaster, displayed a possible binding peptide of 14 ami

Full text of DOI:10.1016/j.bmcl.2004.05.085

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4343–4346
Synthesis of an immunosuppressant SQAG9 and determination of
the binding peptide by T7 phage display
Takayuki Yamazaki, Satoko Aoki, Keisuke Ohta, Shinji Hyuma, Kengo Sakaguchi and
Fumio Sugawara*
Genome and Drug Research Center, Department of Applied Biological Science, Tokyo University of Science, Noda,
Chiba 278-8510, Japan
Received 24 March 2004; accepted 19 May 2004
Available online
Abstract—SQAG9, a new class of immunosuppressive sulfoquinovosylacylglycerol, and its biotinylated derivatives have been
synthesized. A T7 Phage library, composed of random cDNA fragments from Drosophila melanogaster, displayed a possible binding
peptide of 14 amino acids. The immobilized synthetic peptide on a sensor chip showed a dissociation constant of KD ¼ 1:5 ꢀ 10ꢁ6
against SQAG9 in a surface plasmon resonance experiment.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
graft rejection in vivo.9;10 In contrast, a-SQDG, a-
SQMG, and b-SQMG displayed no biological activity in
these tests. SQAG9 did not alter the expression of either
major histocompatibility antigen complex (MHC) class
I or class II molecules on the cell surface of the stimu-
lator cells and antigen-presenting cells (Fig. 1).
Sulfoquinovosylacylglycerol (SQAG) is a C-6 sulfonic
analogue of D-glucose bonded to a fatty acid mono- or
diester of glycerol. We recently carried out a screen for
inhibitors of DNA polymerases and identified several
natural derivatives of SQDG (sulfoquinovosyldiacyl-
glycerol) and SQMG (sulfoquinovosylmonoacylglyc-
erol).1–3 Since DNA polymerases are essential for DNA
replication and repair, inhibition of this activity will lead
to cell death. In 1997, Sahara et al. reported that SQMG
effectively suppressed the growth of solid tumors derived
from human lung cancer (adenocarcinoma cell line A-
549) in nude mice.4;5 Synthetic work on the a-anomers
of SQDG and SQMG and their biological activities were
reported elsewhere.6–8 However, anomeric b-isomers of
these compounds have not yet been found from natural
sources. We evaluated the immunosuppressive effect of a
synthetic b-isomer SQAG9 {3-O-(6-deoxy-6-sulfono-b-
Although SQAG9 inhibited the binding among allo-
geneic lymphocytes, the expression of known cell surface
accessory and adhesion molecules, such as CD4, CD28,
leukocyte function-associated antigen 1, intercellular
adhesion molecule 1, and CTLA-4, was not affected by
the treatment. Since neither binding proteins nor
SO3Na
6'
RO
4'
5'
O
3
1
D
-glucopyranosyl)-1,2-di-O-stearoylglycerol, 1}, which
2'
2
3'
1'
HO
O
OCOC17H35
was designated as b-SQDG by mixed lymphocyte reac-
tion (MLR) and rat allogeneic skin graft. SQAG9
inhibited human MLR in a dose-dependent manner
without overt cytotoxicity and prolonged rat skin allo-
OH
OOCOC17H35
O
1: R=H
2: R=
H
N
S
N
H
H
NH
H
HN
O
* Corresponding author. Tel.: +81-471-24-1501; fax: +81-471-23-9767;
e-mail: sugawara@rs.noda.tus.ac.jp
Both authors equally contributed on this paper.
O
Figure 1. Structure of SQAG9 (1) and the biotinylated derivative (2).
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.085

4344
T. Yamazaki et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4343–4346
receptors of SQDG and SQMG were identified, a phage
display cloning method was used to identify a peptide
fragment(s) involved in the binding process. Here we
report synthetic work on the biotinylated derivatives of
SQAG9 (2) and the results of an affinity guided phage
display experiment.11;12
sphere of hydrogen, to yield 1 (SQAG9) and 11,
respectively.
1.2. Biotinylated derivatives of SQAG9
The stereochemistry at C-4 of the glucose moiety did not
affect the DNA polymerase inhibitory activity in our
previous structure–activity relationships (SAR) study.
For the binding protein assay, we prepared a SQAG9
derivative, which was biotinylated at C40 on the carbo-
hydrate ring (Scheme 2). 1-O-Allyl-2,3-di-O-benzyl-6-O-
1.1. Synthesis of SQAG9
The synthetic route for the b-anomeric isomers of
SQDG and SQMG are essentially the same as those of
the a-anomers6;8 (Scheme 1). Briefly, the treatment of 1-
tosyl-b-
able 1-O-allyl-2,3-di-O-benzyl-b-
D
-glucose (12) was prepared from readily avail-
D
O-allyl-2,3,4-tri-O-benzyl-b-
D
-glucose (3)13 with TsCl in
-glucose by treatment
the presence of DMAP in pyridine gave the 6-O-tosylate
(4). Reaction with AcSK in EtOH gave 5. Oxidation of
the olefin with OsO4 in the presence of NMO, in a
solution of water and tert-BuOH, afforded a diastereo-
meric diol derivative 6 in a ratio of 1:1 determined by 1H
NMR.14 The diol (6) gave the monoester (7) and diester
(9) derivatives, in a ratio of 31:69, respectively, by a
condensation reaction with stearic acid (2.0 equiv) in the
presence of EDCI (1.2 equiv) and DMAP (catalytic
amount) in CH2Cl2. After separating 8 and 10 by silica
gel column chromatography, oxidation of the acetylthio
derivative with oxone gave the sulfonic analogue of
glucose. The protective group was subsequently
removed, by reductive cleavage with Pd/C in an atmo-
with TsCl in the presence of DMAP in pyridine. The
condensation reaction of 12 and Cbz–alanine in CH2Cl2
gave 13, which was converted to the acetylthio deriva-
tive with AcSK in EtOH to give 14. Oxidation of the
olefin by OsO4 in the presence of NMO, in a solution of
water and tert-BuOH, afforded a diastereomeric diol
derivative 15. Treatment of 15 in pyridine with an excess
amount of stearoyl chloride in CH2Cl2 gave the diester
(16). Oxidation of the acetylthio derivative with oxone
gave the sulfonic analogue of glucose (17). The protec-
tive group was then removed, by reductive cleavage with
Pd/C in an atmosphere of hydrogen, to give 18. Treat-
ment of 18 with biotin 4-nitrophenyl ester, in the pres-
ence of Et3N in DMF, gave the biotinylated derivative
of SQAG9 (2).
RO
BnO
BnO
O
O
b
R2
O
BnO
R1O
BnO
O
BnO
4 R=Ts
3 R=H
5 R=AcS
OH
a
12 R1=H R2=OTs
13 R1=COCH2CH2NHCbz R2=OTs
14 R1=COCH2CH2NHCbz R2=SAc
c
a
b
AcS
O
BnO
BnO
O
OH
c
BnO
d
6
O
OR1
R2
O
BnO
OR3
R1
Cbz-HN
O
O
OR1
O
BnO
R2O
R2O
O
OR4
15 R1=H R2=SAc
d
e
OR2
16 R1=COC17H35 R2=SAc
17 R1=COC17 35 R2=SO3Na
H
7 R1=AcS R2=Bn R3=R4=COC17H35
8 R1=SO3Na R2=Bn R3=R4=COC17H35
e
f
f
O
SO3Na
OCOC17H35
1
R1=SO3Na R2=H R3=R4=COC17H35
O
H2N
O
O
OCOC17H35
HO
9 R1=AcS 2'=Bn R3=H R4=COC17H35
10 R1=SO3Na R2=Bn R3=H R4=COC17H35
11 R1=SO3Na R2=H R3=H R4=COC17H35
OH
g
k
18
g
2
Scheme 1. Syntheses of SQAG9 (1) and its monoester derivative (11).
Reagents and conditions: (a) TsCl, pyridine, DMAP, rt (89.8%); (b)
AcSK, EtOH, reflux (86.1%); (c) OsO4, NMO, tert-BuOH, water, rt
(74.7%); (d) C17H35COOH, EDCI, DMAP, CH2Cl2, rt, 8 (91.0%,
diester 30.9%; monoester 69.1%); (e) oxone, AcOK, AcOH (83.4%); (f)
H2, Pd/C, EtOH, rt (52.6%); (g) oxone, AcOK, AcOH (85.9%); (h) H2,
Pd/C, EtOH, rt (80.9%).
Scheme 2. Synthesis of the biotinylated derivative of SQAG9 (2).
Reagents and conditions: (a) Cbz-b-Ala, EDCI, DMAP, pyridine,
CH2Cl2, rt (89%); (b) AcSK, DMF, 80 °C (81%); (c) OsO4, NMO, tert-
BuOH, water, rt (72%); (d) C17H35COCl, pyridine, CH2Cl2, rt (quant);
(e) oxone, AcOK, AcOH, rt (45%); (f) H2, Pd/C, EtOH, CHCl3, rt
(16%); (g) biotin 4-nitrophenyl ester, Et3N, DMF, rt (98%).

T. Yamazaki et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4343–4346
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1.3. Selection and sequence analysis of high affinity phage
A biotinylated derivative of SQAG9 (2) was immobi-
lized on a streptavidine-coated well, and then incubated
with a T7 phage library composed of cDNA fragment
inserts from Drosophila melanogaster. False positives
were removed by a series of washes using different buf-
fers. A mixture of phage particles, apparently exhibiting
affinity to biotinylated SQAG9, was then subjected to a
further four rounds of screening. The phage titer of the
washed and eluted solutions was checked at each round.
The cDNA inserts from recovered phage were amplified
by the polymerase chain reaction (PCR) and analyzed
by agarose gel electrophoresis. After the fifth round of
screening, the selected phage DNA encoded the 14
amino acid sequence, NSRMRVRNATTYNS. No
other specific peptides were detected in this experiment.
RU
700
600
500
400
300
200
100
0
29.4
23.5
17.6
7.4 µM
- 100
- 200
- 300
- 25
25
75
125
175
225
s
Time
1.4. Surface plasmon resonance (SPR) analysis
A surface plasmon resonance (SPR) biosensor chip was
employed to analyze the interaction between SQAG9 (1)
and a synthetic peptide, NSRMRVRNATTYNS. Four
different concentrations of 1 (29.4, 23.5, 17.6, and
7.4 lM) were used for the binding analysis to a conju-
gated peptide on a CM5 sensor chip. SQAG9 bound to
the peptide with a high rate of association and a low rate
of dissociation. The kinetic constant for the interaction
was determined from the SPR association and dissoci-
ation curves obtained by varying the concentration of 1.
The determined KD value of 1.5 ꢀ 10ꢁ6 indicated that
SQAG9 tightly bound the peptide. Comparison of the
peptide sequence against the SwissProt protein database
indicates homology to a small inducible cytokine. A full
analysis of the binding between SQAG9 and the
chemokine and its immunosuppressant activity will be
published elsewhere (Fig. 2).
Figure 2. SPR analysis of the binding of SQAG9 to the immobilized
peptide on a CM5 sensor chip.
2.32 (m, 2H, COCH2), 1.62–1.58 (m, 2H, COCH2CH2),
1.28 (br, 28H, –CH2–), 0.91–0.87 (m, 3H, Me); HRMS
calcd for C27H51O11S (MꢁNa)ꢁ 583.3157, found
583.3186.
2.3. Compound 11
[3-O-(6-Deoxy-6-sulfono-b-D-glucopyranosyl)-1,2-di-O-
20
stearoyl-glycerol sodium salt]: ½aꢂ ꢁ0.44 (c 0.98,
D
CHCl3–MeOH–H2O, 70:30:4); 1H NMR (400 MHz,
CDCl3þCD3ODþD2O) d 5.31–5.25 (m, 1H, H2), 4.45–
4.38 (m, 1H, H3a), 4.34–4.32 (m, 1H, H10), 4.26–4.16
(m, 1H, H3b), 4.05–4.00 (m, 1H, H1a), 3.78–3.72 (m,
2H, H1b and H50), 3.46–3.40 (m, 1H, H30), 3.35–3.31
(m, 1H, H60a), 3.29–3.24 (m, 2H, H20 and H40), 3.11–
3.06 (m, 1H, H60), 2.37–2.27 (m, 4H, COCH2), 1.64–1.58
(m, 4H, COCH2CH2), 1.28 (br, 56H, –CH2–), 0.91–0.87
(m, 6H, Me); HRMS calcd for C45H85O12S (MꢁNa)ꢁ
849.5767, found 849.5844.
2. Experimental
2.1. NMR and MS data
Synthetic compounds were characterized by NMR and
ESIMS. Mass spectral data was collected on an ABI
Q-Star, operated in negative ion mode, using a glycerol
matrix. NMR data was obtained on a BRUKER DRX-
1
400 (400 MHz for H) in either CD3OD or a mixture of
2.4. Compound 2 (Biotinylated derivative of SQAG9)
deuterium solvents. Chemical shifts were expressed as d
ppm from TMS as internal standard. Optical rotation
values were determined on a JASCO polarimeter P1010.
20
D
1
½aꢂ ꢁ0.38 (c 0.34, CHCl3–MeOH–H2O, 70:30:4); H
NMR (400 MHz, CDCl3þCD3OD) d 5.34–5.26 (m, 1H,
H2), 4.77–4.44 (overlapping solvent peak, 1H, H40),
4.55–4.52 (m, 1H, H8000), 4.48–4.42 (m, 1H, H3a), 4.38–
4.35 (m, 2H, H10 and H7000), 4.21–4.16 (m, 1H, H3b),
4.07–4.03 (m, 1H, H1a), 3.97–3.92 (m, 1H, H50), 3.82–
3.75 (m, 1H, H1b), 3.62–3.57 (m, 1H, H30), 3.52–3.45
(m, 2H, H300a and H300b), 3.38–3.31 (overlapping solvent
peak, 1H, H20), 3.23–3.18 (m, 1H, H6000), 3.09–3.01 (m,
2H, H60a and H60b), 2.97–2.93 (m, 1H, H90a), 2.66–2.72
(m, 1H, H9000b), 2.66–2.63 (m, 2H, H200a and H200b),
2.37–2.31 (m, 4H, COCH2), 2.26–2.23 (m, 2H, H2000),
2.2. Compound 1
[3-O-(6-Deoxy-6-sulfono-b-D-glucopyranosyl)-1-O-stea-
20
royl-glycerol sodium salt]: ½aꢂ þ0.04 (c 1.03, MeOH);
D
1H NMR (400 MHz, CD3OD) d 4.30–4.28 (m, 1H, H10),
4.10–3.56 (m, 6H, H50, H1a, H1b, H2, H3a, and H3b),
3.37–3.31 (m, 2H, H3 and H6a), 3.21–3.16 (m, 1H, H20),
3.11–3.05 (m, 1H, H40), 2.94–2.88 (m, 1H, H60), 2.30–

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T. Yamazaki et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4343–4346
1.78–1.60 (m, 6H, COCH2CH2, H3000a, H3000b, H4000a,
and H4000b), 1.48–1.40 (m, 2H, H3000a and H3000b), 1.28
(br, 56H, –CH2–), 0.91–0.88 (m, 6H, Me).
SQAG9 was performed in running buffer (10 mM HE-
PES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Sur-
factant P 20, 8% DMSO) at a flow rate of 20 lL/min at
25 °C. BIAEVALUATION 3.1 software was used to
determine the kinetic parameters.
2.5. Construction of T7 phage library from D. melano-
gaster
Poly(A)þRNA, random primers, 50-methylated dCTP,
T4 DNA polymerase, EcoRI//HindIII linkers, EcoRI,
HindIII, T7Select10-3b vector, and T7 packaging ex-
tracts were used.15 Aliquots (80 lg) of total RNA, ex-
tracted from D. melanogaster Kc cells, were used to
construct the cDNA library. Oligotex-dt30 <super> was
used for a second round of isolation, with minimal loss
of material, to produce poly(A)þRNA suitable for
random primed cDNA synthesis. cDNA synthesis was
primed with 4 lg of poly(A)þRNA using random
primers. 50-Methylated dCTP was then incorporated
into both strands, without extraction or precipitation
between the first and second strand synthesis. The cDNA
was then treated with T4 DNA polymerase to generate
flush ends and ligated with directional EcoRI/HindIII
linkers. Following linker ligation, the cDNA was di-
gested sequentially with HindIII and EcoRI, then in-
serted into EcoRI/HindIII digested T7Select10-3b vector
arms. The cDNA was cloned into the EcoRI/HindIII
sites of the T7 phage 10-3B vector and packaged into
phage.16 The titer of this library was 1.6 ꢀ 1010 pfu/mL.
Acknowledgements
Grant of this work was partially supported by Uehara
Memorial Life Science Foundation, Toyo Suisan Co.
Ltd, and Grant-in-Aid of JSPS.
References and notes
1. Mizushina, Y.; Watanabe, I.; Ohta, K.; Takemura, M.;
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2.6. Biopanning and DNA sequence analysis
Following five rounds of selection, 47 plaques were
randomly picked from LB plates and each dissolved in
phage extraction buffer (washing: 100 mM Tris–HCl,
pH 8.0, eluting: 1.5 M NaI). The candidate clones were
amplified and used to check their affinity to the bioti-
nylated derivative of SQAG9 (2). In order to disrupt the
phages, the extract was heated at 65 °C for 10 min. After
amplifying the phage DNA, the fragments were purified
with ExoSAP-IT and EtOH precipitated. The PCR
fragments were sequenced on an ABI Prism3100 Genetic
Analyzer. Based on the sequence results, the amino acid
sequence displayed on the T7 phage capsid was deter-
mined.
10. Matsumoto, T.; Sahara, H.; Fujita, T.; Shimozawa, K.;
Hanashima, S.; Yamazaki, T.; Takahashi, S.; Sugawara,
F.; Mizushina, Y.; Ohta, K.; Takahashi, N.; Gasa, S.;
Jimbow, K.; Sakaguchi, K.; Sato, N. Transplantation
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2.7. SPR Analysis
Binding analysis between SQAG9 and the peptide was
performed using a Biosensor Biacore 3000. The syn-
thetic peptide (332 lg/mL, 170 lL) in coupling buffer
(10 mM Na2CO3–NaHCO3, pH 8.5) was injected over a
CM5 sensor chip at 10 lL/min to capture the protein on
the carboxymethyl dextran matrix of the chip by using
amine coupling. The surface was activated by injecting a
solution containing 0.2 M EDC and 50 mM NHS for
14 min. The peptide was injected and the surface was
then blocked by injecting 1 M ethanolamine at pH 8.5
for 14 min. This reaction immobilized about 1500 re-
sponse units (RU) of the peptide. Binding analysis of
15. Novagen, T7 Select System Manual, TB178, 2000.
16. Danner, S.; Belasco, J. G. Proc. Natl. Acad. Soc. U.S.A.
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