Synthesis and interaction with midkine of biotinylated chondroitin sulfate tetrasaccharides

Article abstract of DOI:10.1016/j.bmcl.2011.12.054

Regiospecifically sulfated chondroitin sulfate repeating tetrasaccharides, CS-OO, GlcAβ-GalNAcβ-GlcAβ-GalNAcβ;CS-EE, GlcAβ-GalNAc(4S6S)β-GlcAβ-GalNAc(4S6S)β; and CS-AA, GlcAβ-GalNAc(4S)β-GlcAβ-GalNAc(4S)β, having biotin linked with a hydrophilic linker at

Full text of DOI:10.1016/j.bmcl.2011.12.054

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1371–1374
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and interaction with midkine of biotinylated chondroitin sulfate
tetrasaccharides
Jun-ichi Tamura a, , Nao Tsutsumishita-Nakai a, Yasuhiro Nakao a, Manami Kawano a, Saki Kato a,
⇑
Naoko Takeda a, Satomi Nadanaka b, Hiroshi Kitagawa b
a Department of Regional Environment, Tottori University, Tottori 680-8551, Japan
b Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Regiospecifically sulfated chondroitin sulfate repeating tetrasaccharides, CS-OO, GlcAb-GalNAcb-GlcAb-
GalNAcb;CS-EE, GlcAb-GalNAc(4S6S)b-GlcAb-GalNAc(4S6S)b; and CS-AA, GlcAb-GalNAc(4S)b-GlcAb-Gal-
NAc(4S)b, having biotin linked with a hydrophilic linker at the reducing terminal were synthesized
effectively by a coupling of the corresponding disaccharide units and regioselective sulfation. CS-EE
showed greater affinity for midkine than CS-AA and CS-OO.
Received 14 October 2011
Revised 12 December 2011
Accepted 12 December 2011
Available online 17 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Chondroitin sulfate
Glycosaminoglycan
Biotin
Midkine
Chondroitin sulfates (CSs) play pivotal roles in neuronal growth
control,1,2 lymphocyte homing,3 viral infections4 and cancer metas-
tasis.5 These biological processes are caused by interaction between
CSs and characteristic proteins. It is known that regiospecific sulfate
groups on glycans are involved in these interactions. It was also
revealed that midkine interacted with GlcAb-GalNAc(4S6S)b-type
CS, though only naturally occurring oligosaccharides were
employed in the experiments.6 It is difficult to achieve accurate re-
sults with natural substrates because of incomplete homogeneity
especially in relation to sulfation patterns.2 We have been synthesiz-
ing CS repeating oligosaccharides of different lengths.7–9 This Letter
describes the facile synthesis of CS repeating tetrasaccharides; CS-
OO, GlcAb-GalNAcb-GlcAb-GalNAcb (1); CS-EE, GlcAb-Gal-
NAc(4S6S)b-GlcAb-GalNAc(4S6S)b (2); and CS-AA, GlcAb-Gal-
NAc(4S)b-GlcAb-GalNAc(4S)b (3) (Fig. 1), having biotin attached
with a hydrophilic linker at the reducing terminal in order to clarify
the effect of the microenvironment on glycans in protein–carbohy-
drate interactions. Jacquinet and his co-workers have just reported
the synthesis of biotinylated CS oligosaccharides but with GalNAc-
GlcA type sequences having a hydrophobic linker.10,11 We investi-
gated the interaction of midkine with the synthesized substrates
(1–3) immobilized on plates for the first time.
O
a
HN
NH
R2O
m
c
R2O
b
R1O
H
H
H
R1O
g
r
HO
l
H
N
i
o
p
HO
N
j
e
O
HO
O
O
d
O
HO
O
O
O
O
S
O
O
O
q
n
h
k
f
O
HO
NHAc
COOH
O
NHAc
O
COOH
(1) R1=R2=H (CS-OO)
(2) R1=R2=SO3Na (CS-EE)
(3) R1=SO3Na, R2=H (CS-AA)
Figure 1. Target compounds (1–3).
⇑
Corresponding author.
E-mail address: jtamura@rstu.jp (J. Tamura).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.054

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J. Tamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1371–1374
Scheme 1. Synthesis of 1 and 2. Reagents and conditions: (a) TMSOTf, MSAW300, CH2Cl2, ꢀ20 °C to rt, 65% (for 6), 58% (for 8); (b) H2NNH2ꢁAcOH, toluene–EtOH, 77%; (c)
camphorsulfonic acid, CH2Cl2–MeOH, quant.; (d)SO3ꢁNMe3, DMF, 60 °C, 86%; (e)BH3ꢁNMe3, AlCl3, MS4A, CH2Cl2–Et2O, 13%; (f) aq LiOH, THF, then aq NaOH, CH2Cl2–MeOH,
quant. (for 14), 97% (for 15); (g) H2, Pd–C, cat. AcOH, aq EtOH; (h) NHS–PEO4–biotin, 0.1 M Na3PO4, 0.15 M NaCl, 93% (for 1), 69% (for 2).
Scheme 2. Sulfation at O-4 of GalN3 and GalNAc. Reagents and conditions: (a) camphorsulfonic acid, CH2Cl2–MeOH, 78%; (b) PivCl, pyridine, quant. (ꢀ40 °C, for 19), 73% (rt,
for 20); (c)SO3ꢁNMe3, DMF, 60 °C, 92% (for 21), 96% (for 23); (d)aq LiOH, THF, then aq NaOH, CH2Cl2–MeOH, 59%; (e) AcSH, 90%.

J. Tamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1371–1374
1373
Ph
Ph
O
OC(NH)CCl3
NHZ
O
HO
O
MBzO
MBzO
O
O
MBzO
O
NHZ
MBzO
O
O
O
O
O
a
RO
RO
COOMe
COOMe
N3
N3
(25) R=Lev
(26) R=Piv
(27)
R=Lev
b
(28)
R=H
(26)
OR2
R1O
O
MBzO
NHZ
MBzO
O
O
+
O
2
a
f
O
Piv
COOMe
N3
(28)
(29) R1,R2= >CHPh
c
d
e
1
2
(30)
(31)
R = R = H
1
2
R = H, R =Piv
(32) R1= SO3Na, R2= Piv
OH
g
NaO3SO
O
HO
NHZ
O
R
HO
(3)
O
O
O
h
H
COOH
2
(33)
(34)
R = N3
R = NHAc
Scheme 3. Synthesis of 3. Reagents and conditions: (a) TMSOTf, MSAW300, CH2Cl2, ꢀ20 °C to rt, 50% (for 27), 43% (for 29); (b)H2NNH2ꢁAcOH, toluene–EtOH, 84%; (c)
camphorsulfonic acid, CH2Cl2–MeOH, quant.; (d)PivCl, pyridine, quant.; (e)SO3ꢁNMe3, DMF, 60 °C, 92%; (f) aq LiOH, THF, then aq NaOH, CH2Cl2–MeOH, quant.; (g)Zn, aq AcOH,
62%; (h) H2, Pd–C, cat. AcOH, aq EtOH, then NHS–PEO4–biotin, 0.1 M Na3PO4, 0.15 M NaCl, 58% (for 2 steps).
THF12 to obtain 10 having di-O-benzyl ether at O-6 of both GalNAc
Table 1
residues in 13% yield. At the same time monobenzylated com-
pounds having intact benzylidene acetals were obtained in 36%
yield together with 30% of recovered 8. However, subsequent sul-
fation at two 4-OHs of 10 was unsuccessful. Although TLC showed
a complete change to a slower moving spot which suggested O-sul-
fation, 1H NMR showed no downfielded signals for H-4II and 4IV
(data not shown). The oxygen of the acetamide might be sulfated
as we have already reported,7 but no O-sulfation occurred even
with a longer reaction time.
KD values for the interaction of the synthesized tetrasa-
charides and midkine in the BIAcore system
CS-type
KD (nM)
OO
AA
AE
EE
2.03 ꢂ 103
17.8
11.6
2.62
We re-examined the O-sulfation with a disaccharide having lib-
erated 4-OH at the GalNAc residue (20) which was synthesized by
regioselective pivaloylation at the primary position of 187 with Piv-
Cl in pyridine at ꢀ40 °C in 73% yield (Scheme 2). We observed a
similar phenomenon on TLC during the sulfation of 20 and
obtained a polar product in 92% yield (as a monosulfate) which
was subjected to a saponification reaction. The final product
proved to be non-sulfated 227 (59% yield) as expected. This result
indicates the polar product obtained by the sulfation to have an
imine-like structure (21).
To avoid sulfation at the acetamide we changed the synthetic
route as pictured in Scheme 2, employing azide instead of acetam-
ide on the galactosamine residue. The benzylidene acetal of the
known disaccharide (16)7 was removed under acidic conditions
to give 17 in 78% yield. We obtained 19 by the regioselective piva-
loylation of 17 at 6-OH. The sulfation of 19 at 4-OH afforded sulfate
(23) in 96% yield. The azide of 23 was successfully converted to
acetamide with AcSH13 to give 24 in 90% yield, thus proving that
this strategy is reasonable for obtaining the 4-O-sulfated CS.
We prepared a tetrasaccharide (8) common to the three target
compounds. The diaosyl trichloroacetimidate8 was coupled with
HO(CH2)2NHZ in the presence of TMSOTf to give 6 in 65% yield
(Scheme 1). The levulinoyl group was chemoselectively removed
with H2NNH2ꢁAcOH to afford 7 in 77% yield. Another imidate hav-
ing a pivaloyl ester at the non-reducing end (5)9 was coupled with
7 in the same manner as for the synthesis of 6 to give the tetrasac-
charide 8 in 58% yield. We removed both benzylidene acetals un-
der acidic conditions to obtain
9 in a quantitative yield.
Unprotected 12 was also quantitatively obtained by saponification
of 9. Subsequent hydrogenolysis afforded free amine (14) which
was coupled with a biotin linker to obtain the target compound
(1, 93%, 2 steps). Conversely, the tetraol (9) was sulfated with
SO3ꢁNMe3 in DMF at 60 °C to give 11 in 86% yield. The same sapon-
ification procedure afforded 13 in 97% yield. Subsequent hydrogen-
olysis and biotinylation gave the target compound (2, 69%, 2 steps).
We tried to obtain another target compound, CS-AA (3). The
benzylidene acetal of 8 was reductively and regioselectively
opened with selected amounts of BH3ꢁNMe3, AlCl3 and H2O in

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J. Tamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1371–1374
3. Kawashima, H. Biol. Pharm. Bull. 2006, 29, 2343.
As shown in Scheme 3, the imidate (25)8 having azide was ste-
4. Uyama, T.; Ishida, M.; Izumikawa, T.; Trybala, E.; Tufaro, F.; Bergström, T.;
Sugahara, K.; Kitagawa, H. J. Biol. Chem. 2006, 281, 38668.
5. Sugahara, K. N.; Hirata, T.; Tanaka, T.; Ogino, S.; Takeda, M.; Terasawa, H.;
Shimada, I.; Tamura, J.; ten Dam, G. B.; van Kuppevelt, T. H.; Miyasaka, M.
Cancer Res. 2008, 68, 7191.
6. Yamamoto, H.; Muramatsu, H.; Nakanishi, T.; Natori, Y.; Sakuma, S.; Ishiguro,
N.; Muramatsu, T. Biolchem. Biophys. Res. Commn. 2006, 351, 915.
7. Tamura, J.; Neumann, K. W.; Kurono, S.; Ogawa, T. Carbohydr. Res. 1998, 305, 43.
8. Tamura, J.; Tokuyoshi, M. Biosci. Biotech. Biochem. 2004, 68, 2436.
9. Tamura, J.; Nakada, Y.; Taniguchi, K.; Yamane, M. Carbohydr. Res. 2008, 343,
39.
10. Vibert, A.; Lopin-Bon, C.; Jacquinet, J.-C. Chem. Eur. J. 2009, 15, 9561.
11. Vibert, A.; Lopin-Bon, C.; Jacquinet, J.-C. Eur. J. Org. Chem. 2011, 4183.
12. Sherman, A. A.; Mironov, Y. V.; Yudina, O. N.; Nifantiev, N. E. Carbohydr. Res.
2003, 338, 697.
reoselectively coupled with HO(CH2)2NHZ in the same manner as
for the synthesis of 6 to give 27 in 50% yield. The levulinoyl group
of 27 was removed in 84% yield and the other imidate (26) was
coupled in the same manner as above to afford the tetrasaccharide
(29) in 43% yield. Two benzyliden acetals of 29 were removed to
obtain 30 and the primary alcohols of which were pivaloylated
to give 31 in a quantitative yield (2 steps). The two liberated hy-
droxyl groups were sulfated with SO3ꢁNMe3 in DMF to give 32 in
92% yield. All the acyl groups were quantitatively removed by
saponification to obtain 33.
We then examined the chemoselective reduction of the azide of
33 in the presence of Z. Z is thought to usually resist hydrogenoly-
sis in the presence of a Lindlar catalyst,14 but was completely re-
moved in our case. It seems that this reaction deeply depends on
the lot of the reagent or degree of Pd by PbSO4. An alternative Pd
catalyst, Pd–C/Ph2S,15 afforded the same result. We also tried the
reduction of azide with AcSH, but obtained a very low yield. Naka-
hara’s group achieved the reduction of azide with Zn and AcOH in
ethyl acetate.16 We employed Zn in AcOH but this afforded a
complicated reaction mixture. However, aq AcOH (AcOH:
H2O = 5:1) instead of glacial AcOH completely improved the reac-
tion to give 34 in 62% yield after N-acetylation. The final removal
of Z by hydrogenolysis and subsequent biotinylation at the non-
reducing end as above afforded CS-AA (3) in 58% yield (2 steps).
We confirmed the structures of 1–3 by 1H NMR spectroscopy and
TOF-MS.17
The tetrasaccharides having a biotin linker were immobilized
on plates and their affinity for midkine was investigated.18 We also
prepared a CS-AE type substrate, GlcAb-GalNAc(4S)b-GlcAb-Gal-
NAc(4S6S)b, by digestion with 6-O-sulfatase. Midkine exhibited
interaction with these tetrasaccharides. The KD values are summa-
rized in Table 1. Midkine had the highest affinity for CS-EE among
the tetraosyl biotins with other types of sulfation patterns. The KD
value of CS-AE is larger than that of CS-EE. It seems that the 6-O-
sulfate at the non-reducing terminal is important for binding to
midkine. The sulfate at the 4-position at the non-reducing end is
also involved in the interaction with midkine.
In summary, we synthesized chondroitin sulfate tetrasaccha-
rides having different sulfation patterns via the 2+2 coupling of
disaccharide units. These oligosaccharides are linked to biotin via
a hydrophilic linker. We clarified that midkine preferred the highly
sulfated EE-type sequence to the AE, AA and OO-type chondroitin
tetrasaccharides.
13. Rosen, T.; Lico, I. M.; Chu, D. T. W. J. Org. Chem. 1988, 53, 1580.
14. Wuts, P. G. M.; Green, T. W. Green’s Protective Groups in Organic Synthesis; John
Wiely & Sons: Weinheim, 2007.
15. Mori, A.; Mizusaki, T.; Kawase, M.; Maegawa, T.; Monguchi, Y.; Takao, S.;
Takagi, Y.; Sajiki, H. Adv. Synth. Catal. 2008, 350, 406.
16. Ueki, A.; Nakahara, Yu.; Hojo, H.; Nakahara, Yo. Tetrahedron 2007, 63, 2170.
17. 1H NMR assignments were confirmed by two-dimensional HH COSY
experiments using BRUKER ADVANCE II 600 MHz spectrometers with tert-
BuOH as an internal standard (1.23 ppm) in D2O. As an example of signal
assignments, 1III stands for a proton at C-1 of sugar residue III. Positions of the
protons in the aglycon are depicted in Scheme 1. Compound 1: 1H NMR: d 4.60
(dd, 1H, Jb,c = 7.86 Hz, Jc,d = 5.04 Hz, H-c), 4.51 (br d, 2H, J = 7.98 Hz, H-1I
,
or III
1IV), 4.50 (br d, 1H, J = 6.67 Hz, H-1III or I), 4.45 (d, 1H, J1,2 = 8.40 Hz, H-1II), 4.41
(dd, 1H, Jb,e = 4.56 Hz, H-b), 4.09 (d, 1H, J3,4 = 3.00 Hz, H-4IV), 3.98 (dd, 1H,
J1,2 = 8.52 Hz, J2,3 = 10.74 Hz, H-2IV), 3.93 (m, 1H, H-r), 3,91 (d, 1H, J3,4 = 3.12 Hz,
H-4II), 3.88 (d, 1H, J4,5 = 9.79 Hz, H-5I), 3.86 (dd, 1H, J2,3 = 10.80 Hz, H-2II), 3.83–
3.65 (m, 7H, H-4I, III, 5 III, 6II, IVx4), 3.80 (m, 1H, H-3IV), 3.76 (m, 5H, H-n, r), 3.68
(m, 2H, H-3II, 5IV), 3.68 (br s, 12H, H-m), 3.64–3.57 (m, 4H, H-3I, III, l ꢂ 2), 3.43
(m, 4H, H-k, q), 3.36 (m, 2H, H-2I, III), 3.32 (m, 1H, H-e), 2.98 (dd, 1H,
Jgem = 13.02 Hz, H-d), 2.77 (d, 1H, H-d0), 2.52 (br t, 2H, J = 6.09 Hz, H-o), 2.26 (br
t, 2H, J = 7.30 Hz, H-i), 2.02, 1.99 (2s, 3H ꢂ 2, 2NAc), 1.70 (m, 1H, H-f), 1.64 (m,
2H, H-h), 1.58 (m, 1H, H-f0), 1.40 (m, 2H, H-g).MALDI-TOFMS: m/z calcd for
C
51H84N6O30SNa, 1315.49; found, 1315.49 [M+Na+].Compound 2: 1H NMR: d
4.74 (s, 1H, H-4II), 4.67 (d, 1H, J3,4 = 2.28 Hz, H-4IV), 4.59 (m, 2H, H-1II, c), 4.54
(d, 1H, J1,2 = 9.78 Hz, H-1IV), 4.52 (d, 1H, J1,2 = 9.72 Hz, H-1I III), 4.52 (d, 1H,
or
J1,2 = 7.74 Hz, H-1III or I), 4.42 (dd, 1H, Jb,c = 7.87 Hz, Jb,e = 4.50 Hz, H-b), 4.02 (d,
1H, J = 9.78 Hz, H-5I), 4.01 (brd, 1H, J = 3.90 Hz, H-2II), 3.95 (d, 1H, J4,5 = 9.86 Hz,
H-5 III), 3.92 (m, 1H, H-r), 3.88 (dd, 1H, J2,3 = 12.43 Hz, H-2IV), 3.86 (m, 1H, H-
3IV), 3.84–3.65 (m, 6H, H-5II, IV, 6II, IV ꢂ 2), 3.82 (m, 2H, H-4I, III), 3.81 (m, 1H, H-
3II), 3.77 (m, 3H, H-n, r), 3.67 (m, 14H, H-3I, III, m), 3.62 (m, 2H, H-l), 3.42 (m,
4H, H-k, q), 3.38 (m, 2H, H-2I, III), 3.33 (m, 1H, H-e), 2.99 (dd, 1H,
Jgem = 13.08 Hz, Jc,d = 4.98 Hz, H-d), 2.77 (d, 1H, H-d0), 2.52 (t, 2H, J = 6.15 Hz,
H-o), 2.27 (t, 2H, J = 7.32 Hz, H-i), 2.02, 2.00 (2s, 3H ꢂ 2, 2NAc), 1.72 (m, 1H, H-
f), 1.62 (m, 2H, H-h), 1.59 (m, 1H, H-f0), 1.41 (m, 2H, H-g). MALDI-TOFMS: m/z
calcd for C51H80N6O42S5Na5, 1723.19; found, 1723.24 [M+Na+]. Compound 3:
1H NMR: d 4.76 (m, 1H, H-4II), 4.71 (d, 1H, J3,4 = 2.10 Hz, H-4IV), 4.62 (m, 1H, H-
1II), 4.60 (m, 1H, H-c), 4.60 (d, 1H, J1,2 = 7.80 Hz, H-1IV), 4.55 (d, 1H,
or
or
J1,2 = 7.98 Hz, H-1I
III), 4.54 (d, 1H, J1,2 = 7.92 Hz, H-1III
I), 4.42 (dd, 1H,
Jb,c = 7.92 Hz, Jb,e = 4.50 Hz, H-b), 4.29–4.20 (m, 4H, H-6II, ꢂ 2), 4.11 (m, 2H,
IV
H-5II, IV), 4.03 (m, 2H, H-2II,3II), 4.02 (d, 1H, J = 9.66 Hz, H-5I), 3.97 (d, 1H,
J4,5 = 9.78 Hz, H-5 III), 3.92 (m, 1H, H-r), 3.88 (m, 2H, H-2IV, 3IV), 3.83 (m, 2H, H-
4I, III), 3.77 (m, 3H, H-n, r), 3.71–3.64 (m, 14H, H-3I,
, m), 3.62 (t, 2H,
III
J = 5.34 Hz, H-l), 3.42 (m, 4H, H-k, q), 3.39 (m, 2H, H-2I, III), 3.33 (m, 1H, H-e),
2.99 (dd, 1H, Jgem = 13.08 Hz, Jc,d = 4.98 Hz, H-d), 2.77 (d, 1H, H-d0), 2.52 (t, 2H,
J = 6.09 Hz, H-o), 2.27 (t, 2H, J = 7.30 Hz, H-i), 2.02, 2.00 (2s, 3H ꢂ 2, 2NAc), 1.72
(m, 1H, H-f), 1.64 (m, 2H, H-h), 1.57 (m, 1H, H-f0), 1.41 (m, 2H, H-g).
Acknowledgment
This study was supported in part by a Grant-in-aid for Scientific
Research on Innovative Areas 3301, MEXT, Japan.
18. Streptavidin-coated sensor chip (Sensor Chip SA)(GE Healthcare) was washed
with 1 M NaCl at a flow rate of 30 ll/min according to the manufacture’s
instructions. Biotinylated tetrasaccharides diluted in HBS-EP buffer (10
mMHEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4) were
perfused and allowed to interact with the surface of the sensor at a flow rate of
References and notes
30
midkine concentrations (90, 180, and 360 nM)at 30
applied to the sensor chip (30 l/min) for 2 min and left to dissociate for 3 min.
Surfaces were regenerated using 1 M NaCl. Data were analyzed by BIA
evaluation 3.0 software using a 1:1 Langmuir binding model.
l
l/min. Kinetic analyses were performed using a range of recombinant
ll/min. Midkine was
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