Synthesis of biotinylated keratan sulfate repeating disaccharides

Article abstract of DOI:10.1080/09168451.2014.877834

We synthesized four types of keratan and keratan sulfate repeating disaccharides containing non-sulfate, Galβ1-4GlcNAcβ, and three types of sulfates, Gal6Sβ1-4GlcNAcβ, Galβ1-4GlcNAc6Sβ, and Gal6Sβ1-4GlcNAc6Sβ in an efficient and stereo-controlled manner.

Full text of DOI:10.1080/09168451.2014.877834

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Synthesis of biotinylated keratan sulfate repeating
disaccharides
Naoko Takedaab & Jun-ichi Tamuraac
a Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori
University, Tottori, Japan
b Japan Society for the Promotion of Science, Tokyo, Japan
c Faculty of Regional Sciences, Department of Regional Environment, Tottori University,
Tottori, Japan
Published online: 14 Apr 2014.
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To cite this article: Naoko Takeda & Jun-ichi Tamura (2014) Synthesis of biotinylated keratan sulfate repeating
disaccharides, Bioscience, Biotechnology, and Biochemistry, 78:1, 29-37, DOI: 10.1080/09168451.2014.877834
To link to this article: http://dx.doi.org/10.1080/09168451.2014.877834
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Bioscience, Biotechnology, and Biochemistry, 2014
Vol. 78, No. 1, 29–37
Synthesis of biotinylated keratan sulfate repeating disaccharides
Naoko Takeda1,2 and Jun-ichi Tamura1,3,
*
1Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan;
3
2Japan Society for the Promotion of Science, Tokyo, Japan; Faculty of Regional Sciences, Department of Regional
Environment, Tottori University, Tottori, Japan
Received September 25, 2013; accepted October 10, 2013
http://dx.doi.org/10.1080/09168451.2014.877834
We synthesized four types of keratan and keratan
sulfate repeating disaccharides containing non-sul-
fate, Galβ1-4GlcNAcβ, and three types of sulfates,
for biological use via a hydrophilic linker at the reduc-
ing terminal.
Gal6Sβ1-4GlcNAcβ,
Galβ1-4GlcNAc6Sβ,
and
Gal6Sβ1-4GlcNAc6Sβ in an efficient and stereo-con-
trolled manner. These disaccharides were conjugated
with biotin via a hydrophilic linker at the reducing
terminal.
Results and discussion
Based on the retrosynthetic analysis depicted in
Scheme 1, we attempted to synthesize the target com-
pounds (1–4) by adopting a common disaccharide unit
(18). Common disaccharide 18 was, respectively, pro-
tected with chemoselectively removable NAP and
TBDPS at the position 6 of the Gal and GlcNAc resi-
dues.
Key words: keratan sulfate; glycosaminoglycan;
glycosylation; biotin
Keratan sulfate (KS) is a member of the proteogly-
can family that has a linear glycan chain composed of
a repeating disaccharide unit, Galβ1-4GlcNAcβ. KS
repeating oligosaccharides are often sulfated at O-6 of
Gal and GlcNAc. A KS repeating disaccharide, the
minimum unit of the KS oligosaccharide, has four dif-
ferent types of sulfation patterns, including the non-sul-
fate. KS oligosaccharides have recently been shown to
have an inhibitory effect on axonal regrowth.1) How-
ever, the relationship between the factors regulating
neuronal actions and facile structures, including the sul-
fation patterns of KS oligosaccharides, in this interac-
tion has not yet been elucidated.
A few studies have investigated the synthesis of KS
oligosaccharides. In 1989, Ogawa’s group reported the
synthesis of the KS tetrasaccharide, GlcNAc6Sβ1-
3Gal6Sβ1-4GlcNAc6Sβ1-3Galβ,2,3) while Misra’s group
described the synthesis of the octyl glycosides of
N-acetyl lactosamine, Gal6Sβ1-4GlcNAcβ, and Galβ1-
4GlcNAc6Sβ, in 2004.4) However, these synthetic
approaches are not suitable for preparing KS conjugates
with four different types of sulfation patterns, including
the non-sulfate.
Scheme 2 shows that all the acetyl groups in known
thioglycoside 5 were removed by the Zemplén reaction.
The triol was regiospecifically benzylidenated at the 4
and 6 positions with benzaldehyde dimethylacetal
under acidic conditions to give 6 in a 78% yield in two
steps. The resulting OH-3 of 6 was benzylated with
NaH and BnBr to give 7 in an 86% yield. The benzyli-
dene acetal of 7 was removed and the primary alcohol
was selectively protected with TBDPS to afford 8 in a
69% yield in two steps. The glycosyl acceptor (8) was
coupled with 2,3,4,6-tetra-O-acetyl-α-D-galactopyrano-
syl trichloroacetimidate (9) in the presence of TMSOTf
at −20 °C. However, desired disaccharide 10 could
only be obtained in a 2% yield under these reaction
conditions. The dodecylthio group of 8 was transferred
to the anomeric position of 9 to give 12α and 12β in
respective 34 and 64% yields.
Similar intermolecular transfer of the thio group to
the anomeric position of the donor has been reported
for glycosylation with a thioglycoside.5–7) In 2000,
Yu’s group reported similar glycosylation with a tri-
chloroacetimidate donor and an acceptor having thio-
glycoside in the presence of TMSOTf (0.05 eq. vs. the
donor) at −10 °C to show intermolecular transfer of the
dodecylthio group.8) However, the coupling reaction at
−78 °C afforded the desired disaccharide in a 92%
yield without the formation of a side product.
We demonstrate in this study the efficient synthesis
of KS disaccharides with three possible types of sulfa-
tion patterns, as well as one without the sulfate. The
target KS disaccharides (1–4) were attached to biotin
*Corresponding author. Email: jtamura@rs.tottori-u.ac.jp
Abbreviations: KS, keratan sulfate; NAP, 2-naphthylmethyl; TBDPS, tert-butyldiphenylsilyl; TMSOTf, trimethylsilyl trifluoromethanesulfonate;
MBz, 4-methylbenzoyl; Z, benzyloxycarbonyl; Bn, benzyl; NIS, N-iodosuccinimide; TfOH, trifluoromethanesulfonic acid; DDQ, 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone; Phth, phthaloyl; DMAP; dimethylaminopyridine; THF, tetrahydrofurane; MS4A, molecular sieves 4A.
© 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

30
N. Takeda and J. Tamura
Scheme 1. Retrosynthetic analysis for disaccharides 1–4.
Scheme 2. Synthesis of disaccharide 11.
Note: Reagents and conditions: (a) NaOMe, MeOH; (b) PhCH(OMe)2, p-TsOH, 78% (2 steps); (c) BnBr, NaH, DMF, 0 °C-rt, 86%; (d) camphor-
sulfonic acid, CH2Cl2-MeOH; (e) TBDPSCl, imidazole, DMF, 69% (2 steps); (f) TMSOTf, CH2Cl2, −20 or −78 °C; (g) NaOMe, MeOH, 60% (2
steps).
We rationalized that intermolecular transfer of the
dodecylthio group was due to activation of the sulfur
atom on the dodecylthio group by the larger amount of
TMSOTf (0.5 eq.) and higher temperature (−20 °C).
We therefore adopted Yu’s conditions, lowered the
reaction temperature to −78 °C, and decreased the
amount of TMSOTf to 0.05 eq. We subsequently
obtained desired disaccharide 10 without intermolecular
transfer of the dodecylthio group; however, a portion
of the corresponding isomer having an ortho-ester

Synthesis of biotinylated keratan sulfate
31
linkage was generated due to the lower acidity used
Conclusion
(data not shown). We finally succeeded in suppressing
the ortho-ester formation by increasing the amount of
TMSOTf to 0.1 eq. Saponification then removed the
four acetyl groups of 10 to give desired tetraol 11 in a
60% yield in two steps.
In summary, we synthesized for the first time in an
efficient and stereo-controlled manner four types of kera-
tan and KS repeating disaccharides containing a non-sul-
fate, Galβ1-4GlcNAcβ, as well as three sulfates,
Gal6Sβ1-4GlcNAcβ, Galβ1-4GlcNAc6Sβ, and Gal6Sβ1-
4GlcNAc6Sβ, using a common disaccharide. These
disaccharides were linked to biotin via a hydrophilic lin-
ker to successfully give the target compounds (1-4).
The tetraol (11) was 2-naphthylildenated at the 4 and
6 positions of the Gal residue with 2-naphthaldehyde
under acidic conditions to give 13 in an 89% yield
(Scheme 3).9) The resulting diol was protected with
MBz to quantitatively give 14. Reductive ring opening
of the naphthylidene acetal using BH3·NMe3, AlCl3,
and a catalytic amount of H2O in THF10) afforded 6-O-
(2-naphthyl)methyl ether 15 in a 92% yield, without
any formation of the regioisomer. The resulting OH-4
of 15 was methylbenzoylated to give 16 in a 98%
yield. The disaccharide donor (16) was stereoselectively
coupled to HO(CH2)2NHZ in the presence of NIS and
TfOH to quantitatively give 17. We chemoselectively
removed TBDPS to give common disaccharide 18 in a
79% yield.
Scheme 4 shows that the liberated primary position
of 18 was protected with an acetyl group, and NAP
on the Gal residue was chemoselectively removed
with DDQ to give 19 in an 84% yield in two steps.
NAP on 18 was similarly removed to give 20 in a
79% yield. The free hydroxyl groups of 19, 18, and
20 were sulfated with SO3·Me3N in DMF to respec-
tively afford corresponding sulfates 21, 22, and 23 in
88%, quantitative and 90% yields. Phth, MBz, and Ac
of 18, 21, 22, and 23 were removed with 1,3-diami-
nopropane. The generated free amine was acetylated
with Ac2O and Et3N in MeOH. Bn, Z, and NAP were
removed by hydrogenolysis in the presence of Pd-
black. Finally, free amines on the aglycon were biotin-
ylated to quantitatively give the target compounds
(1–4). Selected 1H-NMR data for 1–4 are given in
Table 1. The chemical shifts of H-6′ (2), H-6 (3), and
of H-6 and 6′ (4), which were O-sulfated positions,
were shifted down-field.
Experimental
General methods. 1H-NMR assignments were con-
firmed by two-dimensional HH COSY experiments
using Bruker ADVANCE II 600 MHz spectrometers.
Silica gel chromatography and analytical TLC were
performed in a column of Silica Gel 60 (Merck) and
Silica Gel 60 N (spherical neutral; Kanto Kagaku). The
gel for size-exclusion chromatography (Sephadex
LH-20) was from GE Healthcare. Bond Elut was from
Agilent Technologies. Molecular sieves were from GL
Science and activated at 200 °C under reduced pressure
prior to their use. All reactions in organic solvents were
performed in a dry Ar-containing atmosphere. The
organic phase of the reaction mixture was successively
washed with aq. NaHCO3 and brine, and then dried
over anhyd. MgSO4 by the usual work-up.
Dodecyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-
thio-β-D-glucopyranoside (6).
To a solution of 5
(5.57 g, 8.99 mmol) in MeOH (212 mL) was added 0.5
M NaOMe (9 mL) while stirring at rt. After 2 h, the
reaction was quenched with Dowex 50W × 8 (H+
form), and the mixture was filtered. The volatiles were
removed under reduced pressure to give a triol (4.27 g)
which was dissolved in CH3CN (400 mL). To this solu-
tion, benzaldehyde dimethylacetal (2.6 mL, 17 mmol)
Scheme 3. Synthesis of common disaccharide 18.
Note: Reagents and conditions: (a) 2-naphthaldehyde, p-TsOH·H2O, CH3CN, 89%; (b) MBzCl, DMAP, pyridine-CH2Cl2, quant.; (c) BH3·Me3 N,
AlCl3, cat. H2O, THF, 92%; (d) MBzCl, DMAP, pyridine-CH2Cl2, 98%; (e) NIS, TfOH, MSAW300, Et2O-(CH2Cl)2, quant.; (f) 1 M tetra-n-butyl
ammonium fluoride, AcOH, THF, 79%.

32
N. Takeda and J. Tamura
Scheme 4. Synthesis of target compounds 1–4.
Note: Reagents and conditions: (a) Ac2O, pyridine, quant.; (b) DDQ, cat. H2O, CH2Cl2-MeOH, 84% for 19 (2 steps), 78% for 20; (c) SO3·
NMe3, DMF, 60 °C, 88% for 21, quant. for 22, 90% for 23; (d) 1,3-diaminopropane, EtOH, reflux; (e) Ac2O, Et3N, MeOH; (f) NaOH, MeOH (for
21, 22), 50% MeOH (for 23), reflux; (g) H2, Pd-black, aq. EtOH + dil. AcOH (for 18), aq. 2-PrOH + dil. AcOH (for 21, 22), dil. AcOH (for 23);
(h) NHS-PEG4-biotin, 1 M Na3PO4, 0.15 M NaCl.
Table 1. Selected chemical shifts (ppm) for 1–4 by 600 MHz 1H-
NMR.
Dodecyl
3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-
To
phthalimido-1-thio-β-D-glucopyranoside (7).
a
suspension of NaH (205.1 mg, 4.70 mmol, 55%) in
DMF (3.0 mL) was added 6 (1.37 g, 2.35 mmol) in dry
DMF (24 mL) at 0 °C, and the mixture was stirred for
1 h. BnBr was then added. After 1 h, unreacted NaH
was decomposed with MeOH, and the reaction mixture
was quenched with aq. NH4Cl and diluted with EtOAc.
The organic phase was washed with brine and dried
over anhyd. MgSO4. The volatiles were removed under
reduced pressure, and the residue was treated in a col-
umn of silica gel (7:1–4:1 n-hexane-EtOAc) to give 7
1
2
3
4
H-1
H-2
4.48
3.67
3.75–3.60
4.38
3.45
3.90, 3.75
1.96
4.33
4.51
2.90, 2.69
3.24
4.45
3.75
3.74, 3.59
4.37
3.57
4.13, 3.96
1.90
4.27
4.45
2.85, 2.63
3.36
4.51
3.7
4.35, 4.27
4.51
3.46
3.85–3.58
1.96
4.38
4.54
2.91, 2.71
3.28
4.49
3.69
4.35, 4.24
4.47
3.48
4.10, 3.93
1.94
4.35
4.54
2.93, 2.72
3.27
H-6ab
H-1′
H-2′
H-6′ab
NAc
H-b
H-c
H-d
H-e
1
(1.36 g) in an 86% yield. H-NMR δH (CDCl3): 7.86–
6.86 (m, 14H, Ar H), 5.63 (s, 1H, PhCH), 5.31 (d, 1H,
J1,2 = 10.68 Hz, H-1), 4.79, 4.50 (ABq, 2H, J = 12.36
Hz, PhCH2), 4.44 (br t, 1H, J = 9.45 Hz, H-3), 4.41
(dd, 1H, J5,6a = 10.62 Hz, Jgem = 4.92 Hz, H-6a), 4.27
(br t, 1H, J = 10.29 Hz, H-2), 3.82 (br t, 1H, J = 10.02
Hz, H-5), 3.80 (br t, 1H, J = 9.09 Hz, H-4), 3.70 (dd,
1H, J5,6b = 9.66 Hz, H-6b), 2.64 (m, 1H, 1/2SCH2),
2.55 (m, 1H, 1/2SCH2), 1.51–1.13 [m, 20H,
SCH2(CH2)10CH3], 0.87 (t, 3H, J = 14.16 Hz, CH3).
ESI-HRMS m/z [(M + Na)+]: calcd. for C40H49NO6SNa,
694.3173; found, 694.3168.
and a catalytic amount of p-TsOH (pH < 2) were added,
while stirring for 3 h. The reaction mixture was neutral-
ized with Et3N. The volatiles were removed under
reduced pressure, and the residue was treated in a col-
umn of silica gel (20:1–10:1 toluene-EtOAc) to give 6
(4.06 g) in a 78% yield in two steps. 1H-NMR δH
(CDCl3): 8.01–7.26 (m, 10H, Ar H), 5.57 (s, 1H,
PhCH), 5.39 (d, 1H, J1,2 = 10.62 Hz, H-1), 4.56 (br t,
1H, J = 9.57 Hz, H-3), 4.36 (dd, 1H, J5,6a = 5.04 Hz,
Jgem = 10.44 Hz, H-6a), 4.31 (br t, 1H, J = 10.29 Hz, H-
2), 3.80 (br t, 1H, J = 10.26 Hz, H-6b), 3.64 (m, 1H,
H-5), 3.61 (br t, 1H, J = 9.15 Hz, H-4), 2.68 (m, 1H, 1/
2SCH2), 2.59 (m, 1H, 1/2SCH2), 2.54 (s, 1H, 3-OH),
1.30–1.13 [m, 20H, SCH2(CH2)10CH3], 0.87 (t, 3H, J
= 14.10 Hz, CH3). ESI-HRMS m/z [(M + Na)+]: calcd.
for C33H43NO6SNa, 604.2703; found, 604.2690.
Dodecyl
3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-
deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (8).
To a solution of 7 (5.12 g, 7.62 mmol) in CH2Cl2 (48
mL) and MeOH (48 mL), a catalytic amount of cam-
phorsulfonic acid was added while stirring for 4 d. The
reaction was quenched with Et3N. The volatiles were

Synthesis of biotinylated keratan sulfate
33
removed under reduced pressure, and the residue was
treated in a column of silica gel (4:1–1:1 toluene-
EtOAc) to give a corresponding 4,6-diol (4.10 g) which
was dissolved in DMF (100 mL). Imidazole (1.06 g,
15.5 mmol) and TBDPSCl (2.0 mL, 7.7 mmol) were
added to the solution while stirring for 5 d. The reac-
tion mixture was diluted with EtOAc. The organic
phase was treated as described for general methods,
and the residue was then treated in a column of silica
gel (10:1–3:1 n-hexane-EtOAc) to give 8 (4.35 g) in a
69% yield (two steps) as a syrup. 1H-NMR δH
(CDCl3): 7.90–6.90 (m, 19H, Ar H), 5.23 (d, 1H,
J1,2 = 10.38 Hz, H-1), 4.78, 4.55 (ABq, 2H, J = 12.24
Hz, PhCH2), 4.29 (dd, 1H, J2,3 = 10.26 Hz, J3,4 = 8.46
Hz, H-3), 4.21 (br t, 1H, J = 10.35 Hz, H-2), 3.98 (dd,
1H, J5,6a = 4.56 Hz, Jgem = 10.62 Hz, H-6a), 3.92 (m,
11.04 Hz, H-6a), 4.03 (d, 1H, H-6b), 3.94 (d, 1H, J3′,4′
= 2.76 Hz, H-4′), 3.75 (m, 2H, H-6′ab), 3.69 (br t, 1H,
J= 8.61 Hz, H-2′), 3.57 (br d, 1H, J = 9.66 Hz, H-5),
3.41 (dd, 1H, J2′,3′ = 9.42 Hz, H-3′), 3.34 (br t, 1H,
J = 4.11 Hz, H-5′), 2.65 (m, 1H, 1/2SCH2), 2.58 (m,
1H, 1/2SCH2), 1.47 (m, 2H, SCH2CH2), 1.28–1.11 [m,
18H, SC2H4(CH2)9CH3], 1.08 [s, 9H, C(CH3)3], 0.87
(t, 3H, J = 14.16 Hz, CH3). ESI-HRMS m/z
[(M + Na)+]: calcd. for C55H73NO11SSiNa, 1006.4566;
found, 1006.4549.
Dodecyl 2,3,4,6-tetra-O-acetyl-1-thio-α-D-galactopyr-
anoside (12α).
1H-NMR δH (CDCl3): 5.71 (d, 1H,
J3,4 = 5.52 Hz, H-4), 4.45 (d, 1H, J1,2 = 3.18 Hz, H-1),
5.26 (dd, 1H, J2,3 = 10.86 Hz, H-3), 4.22 (dd, 1H, H-2),
4.59 (br t, 1H, J = 6.44 Hz, H-5), 4.11 (m, 2H, H-6ab),
2.56 (m, 1H, 1/2SCH2), 2.49 (m, 1H, 1/2SCH2), 2.15,
2.08, 2.05, 2.00 (4s, each 3H, 4Ac), 1.58 (m, 2H,
SCH2CH2), 1.38–1.23 [m, 18H, SC2H4(CH2)9CH3],
0.88 (t, 3H, J = 14.04 Hz, CH3). ESI-HRMS m/z [(M +
Na)+]: calcd. for C26H44O9SNa, 555.2598; found,
555.2585.
2H, H-4, 6b), 3.60 (m, 1H, H-5), 3.07 (d, 1H, J4,OH
=
2.28 Hz, OH-4), 2.62 (m, 1H, 1/2SCH2), 2.52 (m, 1H,
1/2SCH2), 1.43 (m, 2H, SCH2CH2), 1.30–1.13 [m,
18H, SC2H4(CH2)9CH3], 1.09 [s, 9H, C(CH3)3], 0.86
(t, 3H, J = 14.22 Hz, CH3). ESI-HRMS m/z [(M +
Na)+]: calcd. for C49H63NO6SSiNa, 844.4038; found,
844.4026.
Dodecyl β-D-galactopyranosyl-(1→4)-3-O-benzyl-6-
O-tert-butyldiphenylsilyl-2-deoxy-2-phthalimido-1-thio-
β-D-glucopyranoside (11). Method A: A solution of
8 (495.2 mg, 544.7 μmol) and 2,3,4,6-tetra-O-acetyl-α-
D-galactopyranosyl trichloroacetimidate (9, 668.9 mg,
1.36 mmol) in CH2Cl2 (55 mL) was stirred over
AW300 molecular sieves (1.44 g) for 30 min at rt. To
this solution, TMSOTf (173 μL, 679 μmol) was added
at −20 °C for 2.5 h. The reaction was quenched with
aq. NaHCO3 and diluted with CHCl3. The insoluble
materials were filtered through Celite. The organic
phase was treated as describe for general methods, and
the crude mixture was then treated in an LH-20 gel
permeation column (1:1 CHCl3-MeOH) and silica gel
column (6:1–5:1 n-hexane-EtOAc) to give 10 (9.7 mg,
2%), dodecyl thioglycosides (12α, 101.0 mg) and (12β,
184.3 mg) in respective 34 and 64% yields.
Method B: 4A molecular sieves (2.93 g) were added
to a solution of 8 (2.81 g, 3.42 mmol) and 9 (2.53 g,
5.13 mmol) in CH2Cl2 (233 mL), and the mixture was
stirred for 30 min at rt. To this mixture TMSOTf (46
μL, 0.26 mmol) was added while stirring at −78 °C for
2 h. The reaction was treated in the same way as that
described for Method A. The residue was purified
through a column of silica gel (15:1–5:1 toluene-
EtOAc) to give 10 (4.26 g). The disaccharide (10) was
diluted in MeOH (160 mL), and 0.5 M NaOMe (3.4
mL) was added to the solution while stirring at rt. After
4 h, the reaction was quenched with Dowex 50Wx8
(H+ form), and the mixture was filtered. The volatiles
of the filtrate were removed under reduced pressure,
and the residue was directly treated in a column of
silica gel to give 11 (2.02 g, 60% in two steps).
Compound 11. 1H-NMR δH (CDCl3): 7.83–6.88 (m,
19H, Ar H), 5.20 (d, 1H, J1,2 = 10.50 Hz, H-1), 4.90,
4.50 (ABq, 2H, J = 12.18 Hz, PhCH2), 4.76 (d, 1H,
J1′,2′ = 7.80 Hz, H-1′), 4.42 (br t, 1H, J= 9.48 Hz, H-3),
4.28 (br t, 1H, J = 8.94 Hz, H-4), 4.27 (br t, 1H, J =
Dodecyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyr-
anoside (12β).
J3,4 = 3.30 Hz, H-4), 5.24 (br t, 1H, J= 9.96 Hz H-2),
1H-NMR δH (CDCl3): 5.43 (d, 1H,
5.05 (dd, 1H, J2,3 = 9.66 Hz, H-3), 4.48 (d, 1H, J1,2
10.02 Hz, H-1), 4.16 (dd, 1H, J5,6b = 6.60 Hz, Jgem
=
=
11.34 Hz, H-6b), 4.11 (dd, 1H, J5,6a = 6.60 Hz, H-6a),
3.93 (t, 1H, H-5), 2.64–2.73 (m, 2H, SCH2), 2.16,
2.07, 2.05, 2.00 (4s, each 3H, 4Ac), 1.38–1.25 [m,
20H, SCH2(CH2)10CH3], 0.88 (t, 3H, J = 14.04 Hz,
CH3). ESI-HRMS m/z [(M + Na)+]: calcd. for
C26H44O9SNa, 555.2598; found, 555.2598.
Dodecyl 4,6-O-(2-naphthylidene)-β-D-galactopyrano-
syl-(1→4)-3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-deo
xy-2-phthalimido-1-thio-β-D-glucopyranoside (13). To
a solution of 11 (1.18 g, 1.20 mmol) in CH3CN, 2-
naphthaldehyde (374.8 mg, 2.40 mmol) and p-TsOH
(22.8 mg, 0.12 mmol) were added while stirring for 2 h.
The reaction mixture was neutralized with Et3N. The
volatiles were removed under reduced pressure, and the
residue was treated in a column of silica gel (4:1–2:1
toluene-EtOAc) to give 13 (1.20 g) in an 89% yield
1
which was used without further purification. H-NMR
δH (CDCl3): 7.84–6.80 (m, 26H, Ar H), 5.67 (s, 1H,
NpCH), 5.21 (d, 1H, J1,2 = 10.56 Hz, H-1), 5.10, 4.64
(ABq, 2H, J = 12.24 Hz, PhCH2), 4.85 (d, 1H, J1′,2′
7.74 Hz, H-1′), 4.45 (dd, 1H, J2,3 = 10.02 Hz, J3,4
=
=
8.82 Hz, H-3), 4.40 (d, 1H, Jgem = 12.36 Hz, H-6′a),
4.34 (t, 1H, H-4), 4.29 (br d, 2H, J = 10.20 Hz, H-
2,6a), 4.20 (d, 1H, J3′,4′ = 3.66 Hz, H-4′), 4.04 (d, 1H,
Jgem = 12.36 Hz, H-6b), 4.01 (d, 1H, H-6′b), 3.79 (t,
1H, H-2′), 3.54 (m, 2H, H-5, 3′), 3.36 (s, 1H, H-5′),
2.66 (m, 1H, 1/2SCH2), 2.58 (m, 2H, 1/2SCH2, OH-
2′), 2.51 (d, 1H, J3′,OH = 9.36 Hz, OH-3′), 1.52–1.30
[m, 20H, SCH2(CH2)10CH3], 1.10 [s, 9H, C(CH3)3],
0.87 (t, 3H, J = 14.16 Hz, CH3). ESI-HRMS m/z [(M +
Na)+]: calcd. for C66H79NO11SSiNa, 1144.5035; found,
1144.5013.
10.43 Hz, H-2), 4.22 (dd, 1H, J5,6a = 2.04 Hz, Jgem
=

34
N. Takeda and J. Tamura
Dodecyl 2,3-di-O-(4-methyl)benzoyl-4,6-O-(2-naphth
Dodecyl
2,3,4-tri-O-(4-methyl)benzoyl-6-O-(2-
ylidene)-β-D-galactopyranosyl-(1→4)-3-O-benzyl-6-O-te
rt-butyldiphenylsilyl-2-deoxy-2-phthalimido-1-thio-β-D-g
naphthylmethyl)-β-D-galactopyranosyl-(1→4)-3-O-ben-
zyl-6-O-tert-butyldiphenylsilyl-2-deoxy-2-phthalimido-1-
lucopyranoside (14).
Compound 13 (1.10 g, 0.98
thio-β-D-glucopyranoside (16).
To a solution of 15
mmol) was dissolved in CH2Cl2 (5.0 mL) and pyridine
(30 mL). p-Toluoylchloride (312 μL, 2.35 mmol) and a
catalytic amount of DMAP were added to the solution
while stirring for 5 d. The reaction mixture was diluted
with CHCl3. The organic phase was successively
washed with 1 M HCl, aq. NaHCO3 and brine, and
dried over anhyd. MgSO4. The volatiles were removed
under reduced pressure, and the residue was treated in
a column of silica gel (10:1 toluene-EtOAc) to quanti-
(152.2 mg, 111.8 μmol) in CH2Cl2 (1.5 mL) and pyri-
dine (1.0 mL) p-toluoylchloride (18 μL, 0.13 mmol)
and a catalytic amount of DMAP were added while
stirring for 6 d. The reaction mixture was treated in the
same way for the synthesis of 14. The crude mixture
was treated in an LH-20 gel permeation column (1:1
CHCl3-MeOH) to give 16 (161.2 mg) in a 98% yield.
1H-NMR δH (CDCl3): 7.90–6.90 (m, 38H, Ar H), 5.95
(d, 1H, J3′,4′ = 3.54 Hz, H-4′), 5.76 (dd, 1H, J1′,2′ = 8.04
Hz, J2′,3′ = 10.38 Hz, H-2′), 5.47 (dd, 1H, H-3′), 5.25
(d, 1H, H-1′), 5.12 (d, 1H, J1,2 = 10.14 Hz, H-1), 4.98,
4.69 (ABq, 2H, J = 11.88 Hz, ArCH2), 4.67, 4.48
(ABq, 2H, J = 11.58 Hz, ArCH2), 4.46 (br t, 1H, J =
1
tatively give 14 (1.35 g). H-NMR δH (CDCl3): 7.85–
6.75 (m, 34H, Ar H), 5.91 (dd, 1H, J1′,2′ = 8.04 Hz,
J2′,3′ = 10.44 Hz, H-2′), 5.65 (s, 1H, NpCH), 5.29 (dd,
1H, J3′,4′ = 3.66 Hz, H-3′), 5.25 (d, 1H, H-1′), 5.14,
4.72 (ABq, 2H, J = 12.30 Hz, PhCH2), 5.12 (d, 1H,
J1,2 = 10.26 Hz, H-1), 4.60 (d, 1H, H-4′), 4.53 (d, 1H,
Jgem = 12.36 Hz, H-6′a), 4.47 (dd, 1H, J3,4 = 8.40 Hz,
J4,5 = 9.72 Hz, H-4), 4.32 (dd, 1H, J2,3 = 10.26 Hz, H-
3), 4.26 (t, 1H, H-2), 4.10 (d, 1H, H-6b′), 3.92 (d, 1H,
Jgem = 10.56 Hz, H-6a), 3.83 (d, 1H, H-6b), 3.57 (s,
1H, H-5′), 3.23 (d, 1H, H-5), 2.62 (m, 1H, 1/2SCH2),
2.53 (m, 1H, 1/2SCH2), 2.32, 2.29 (2 s, each 3H,
2MePh), 1.48–1.18 [m, 20H, SCH2(CH2)10CH3], 1.14
[s, 9H, C(CH3)3], 0.87 (t, 3H, J = 14.22 Hz, CH3). ESI-
HRMS m/z [(M + Na)+]: calcd. for C82H91NO13SSiNa,
1380.5873; found, 1380.5852.
9.06 Hz, H-4), 4.34 (dd, 1H, J2,3 = 10.14 Hz, J3,4
=
8.28 Hz, H-3), 4.30 (t, 1H, H-2), 4.08 (br t, 1H, J =
6.39 Hz, H-6′a), 3.85 (br d, 2H, H-6ab), 3.66 (dd, 1H,
J5′,6′b = 9.66 Hz, Jgem = 5.70 Hz, H-6′b), 3.57 (dd, 1H,
J5′,6′a = 7.68 Hz, H-5′), 3.24 (br d, 1H, J = 9.78 Hz, H-
5), 2.63 (m, 1H, 1/2SCH2), 2.55 (m, 1H, 1/2SCH2),
2.30, 2.30, 2.29 (3 s, each 3H, 3MePh), 1.52–1.12 [m,
20H, SCH2(CH2)10CH3], 1.08 [s, 9H, C(CH3)3], 0.87
(t, 3H, J = 14.28 Hz, CH3). ESI-HRMS m/z [(M +
Na)+]: calcd. for C90H99NO14SSiNa, 1500.6448; found,
1500.6411.
2-(N-Benzyloxycarbonyl)aminoethyl 2,3,4-tri-O-(4-
methyl)benzoyl-6-O-(2-naphthylmethyl)-β-D-galactopyr-
anosyl-(1→4)-3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-
Dodecyl 2,3-di-O-(4-methyl)benzoyl-6-O-(2-naphthyl-
methyl)-β-D-galactopyranosyl-(1→4)-3-O-benzyl-6-O-te
rt-butyldiphenylsilyl-2-deoxy-2-phthalimido-1-thio-β-D-g
deoxy-2-phthalimido-β-D-glucopyranoside (17).
AW
300 molecular sieves (993.2 mg) were added to a solu-
tion of 16 (819.1 mg, 553.9 μmol) and benzyl N-(2-
hydroxyethyl)carbamate (162.2 mg, 830.9 μmol) in
(CH2Cl)2 (8.5 mL) and Et2O (29 mL), and the mixture
was stirred for 30 min at rt. To this mixture, NIS
(249.8 mg, 1.11 mmol) and TfOH (15 μL, 0.16 mmol)
were added while stirring for 6 h at rt. The reaction
mixture was treated in the same way as that for the
synthesis of 11. The crude mixture was treated in a col-
umn of silica gel (20:1–15:1 toluene-EtOAc) to quanti-
tatively give 17 (867.6 mg). 1H-NMR δH (CDCl3):
7.77–6.74 (m, 43H, Ar H), 5.95 (d, 1H, J3′,4′ = 3.36 Hz,
H-4′), 5.76 (dd, 1H, J1′,2′ = 8.04 Hz, J2′,3′ = 10.38 Hz,
H-2′), 5.47 (dd, 1H, H-3′), 5.27 (d, 1H, H-1′), 5.05 (d,
1H, J1,2 = 8.58 Hz, H-1), 4.98, 4.67 (ABq, 2H, J =
11.97 Hz, ArCH2), 4.89 (m, 3H, NH, ArCH2), 4.68,
4.48 (ABq, 2H, J = 12.24 Hz, ArCH2), 4.46 (br t, 1H,
lucopyranoside (15).
To a solution of 14 (1.35 g,
0.98 mmol) in THF (37 mL) was added BH3·NMe3
(427.3 mg, 5.88 mmol) while stirring for 30 min. To
this mixture, AlCl3 (786.6 mg, 5.88 mmol) and H2O (1
drop) were added while stirring overnight. The reaction
was quenched with aq. NH4Cl and diluted with EtOAc.
The organic phase was successively washed with aq.
NaHCO3 and brine, and dried over anhyd. MgSO4.
The volatiles were removed under reduced pressure,
and the residue was treated in an LH-20 gel permeation
column (1:1 CHCl3-MeOH) to give 15 (1.23 g) in a
1
92% yield. H-NMR δH (CDCl3): 7.83–6.79 (m, 34H,
Ar H), 5.80 (dd, 1H, J1′,2′ = 8.04 Hz, J2′,3′ = 10.32 Hz,
H-2′), 5.21 (dd, 1H, J3′,4′ = 3.18 Hz, H-3′), 5.25 (d, 1H,
H-1′), 5.11 (d, 1H, J1,2 = 10.02 Hz, H-1), 4.94, 4.60
(ABq, 2H, J = 12.30 Hz, ArCH2), 4.71, 4.67 (ABq, 2H,
J = 12.18 Hz, ArCH2), 4.44 (dd, 1H, J3,4 = 8.40 Hz,
J4,5 = 9.72 Hz, H-4), 4.42 (dd, 1H, J4′,OH = 3.84 Hz, H-
4′), 4.32 (br t, 1H, J = 10.20 Hz, H-3), 4.26 (br t, 1H, J
= 10.20 Hz, H-2), 3.76 (m, 5H, H-6ab,5′,6′ab), 3.22 (d,
1H, H-5), 2.73 (d, 1H, OH-4′), 2.60 (m, 1H, 1/2SCH2),
2.52 (m, 1H, 1/2SCH2), 2.35, 2.29 (2 s, each 3H,
2MePh), 1.47–1.06 [m, 20H, SCH2(CH2)10CH3], 1.06
[s, 9H, C(CH3)3], 0.86 (t, 3H, J = 14.28 Hz, CH3). ESI-
HRMS m/z [(M + Na)+]: calcd. for C82H93NO13SSiNa,
1382.6029; found, 1382.6003.
J = 9.18 Hz, H-4), 4.28 (dd, 1H, J2,3 = 10.80 Hz, J3,4
=
8.82 Hz, H-3), 4.17 (dd, 1H, H-2), 4.07 (br t, 1H, J =
6.84 Hz, H-5′), 3.89 (br d, 1H, J = 10.38 Hz, H-6a),
3.81 (br d, 1H, J = 11.40 Hz, H-6b), 3.70 (m, 1H, 1/
2OCH2), 3.67 (dd, 1H, J5′,6′a = 5.52 Hz, Jgem = 9.54 Hz,
H-6′a), 3.58 (dd, 1H, J5′,6′b = 7.68 Hz, H-6′b), 3.45 (m,
1H, 1/2OCH2), 3.24 (m, 3H, H-5, NHCH2), 2.28 (s,
9H, 3MePh), 1.09 [s, 9H, C(CH3)3]. ESI-HRMS m/z
[(M + Na)+]: calcd. for C88H86N2O17SiNa, 1493.5588;
found, 1493.5549.

Synthesis of biotinylated keratan sulfate
35
2-(N-Benzyloxycarbonyl)aminoethyl 2,3,4-tri-O-(4-
methyl)benzoyl-6-O-(2-naphthylmethyl)-β-D-galacto-
pyranosyl-(1→4)-3-O-benzyl-2-deoxy-2-phthalimido-
β-D-glucopyranoside (18). Compound 17 (867.6 mg,
553.9 μmol) was dissolved in THF (4.0 mL) and AcOH
(320 μL, 5.55 mmol). To this mixture, 1 M TBAF (2.8
mL, 2.8 mmol) was added while stirring for 13 d. The
reaction mixture was diluted with CHCl3. The organic
phase was washed with brine and dried over MgSO4.
The volatiles were removed under reduced pressure,
and the residue was treated in an LH-20 gel permeation
column (1:1 CHCl3-MeOH) to give 18 (538.4 mg) in a
that for the synthesis of 19, and the residue was treated
in an LH-20 gel permeation column (1:1 CHCl3-
1
MeOH) to give 20 (52.9 mg) in a 78% yield. H-NMR
δH (CDCl3): 7.95–6.78 (m, 26H, Ar H), 5.85 (dd, 1H,
J1′,2′ = 7.95 Hz, J2′,3′ = 10.32 Hz, H-2′), 5.77 (d, 1H,
J3′,4′ = 3.06 Hz, H-4′), 5.59 (dd, 1H, H-3′), 5.24 (br t, J
= 5.46 Hz, NH), 5.16 (d, 1H, J1,2 = 7.62 Hz, H-1), 5.15
(d, 1H, H-1′), 4.92, 4.65 (ABq, 2H, J = 12.12 Hz,
PhCH2), 4.82, 4.66 (ABq, 2H, J = 12.36 Hz, PhCH2),
4.27 (m, 1H, H-3), 4.21 (m, 1H, H-2), 4.11 (m, 1H, H-
6′a), 3.85 (br d, 1H, J = 12.18 Hz, H-6a), 3.75 (br s,
1H, H-6b), 3.51 (m, 3H, H-5′,6′b, 1/2OCH2), 3.33 (br
d, 1H, J = 6.12 Hz, H-4), 3.20 (m, 1H, 1/2NCH2), 3.10
(m, 1H, 1/2NCH2), 2.89 (br s, 1H, H-5), 2.38, 2.33,
2.28 (3 s, each 3H, 3MePh). ESI-HRMS m/z [(M +
Na)+]: calcd. for C61H60N2O17Na, 1115.3784; found,
1115.3760.
1
79% yield. H-NMR δH (CDCl3): 7.83–6.75 (m, 33H,
Ar H), 5.91 (d, 1H, J3′,4′ = 3.24 Hz, H-4′), 5.91 (dd, 1H,
J1′,2′ = 7.98 Hz, J2′,3′ = 10.38 Hz, H-2′), 5.56 (dd, 1H,
H-3′), 5.06 (d, 1H, J1,2 = 8.34 Hz, H-1), 5.06 (s, 1H,
NH), 5.04 (d, 1H, H-1′), 4.97, 4.67 (ABq, 2H, J =
12.45 Hz, ArCH2), 4.89 (m, 2H, ArCH2), 4.59, 4.43
(ABq, 2H, J = 12.36, Hz, ArCH2), 4.30 (br t, 1H, J =
9.04 Hz, H-3), 4.15 (br t, 1H, H-5′), 4.12 (m, 1H, H-2),
4.04 (br t, 1H, J= 9.27 Hz, H-4), 3.70 (br s, 2H, H-
6ab), 3.34 (m, 1H, 1/2OCH2), 3.55 (m, 3H, H-6′ab, 1/
2OCH2), 3.28 (m, 2H, H-5, 1/2NCH2), 3.16 (m, 1H, 1/
2NCH2), 2.37, 2.32, 2.29 (3 s, each 3H, 3MePh), 1.99
(br s, 1H, OH-6). ESI-HRMS m/z [(M + Na)+]: calcd.
for C77H68N2O17Na, 1255.4410; found, 1255.4380.
O-Sulfation of 19, 18 and 20. The starting material
was dissolved in DMF (2.0 mL per 50 mg of the start-
ing material). To this solution, SO3·Me3N (20 equiv.
per hydroxyl group) was added while stirring at 60 °C
for 1–3 h. The reaction mixture was treated in an LH-
20 gel permeation column (1:1 CHCl3-MeOH) to give
the corresponding sulfate (21, 22, and 23) in respective
88%, quantitative and 90% yields.
2-(N-Benzyloxycarbonyl)aminoethyl 2,3,4-tri-O-(4-me
thyl)benzoyl-β-D-galactopyranosyl-(1→4)-6-O-acetyl-3-
O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside
(19). To a solution of 18 (130.6 mg, 105.9 μmol) in
CH2Cl2 (2.0 mL), pyridine (90 μL) and Ac2O (90 μL)
were added while stirring overnight. The volatiles were
removed under reduced pressure. To the residue in
CH2Cl2 (2.1 mL) and MeOH (0.6 mL), DDQ (24.0 mg,
106 μmol) and H2O (1 drop) were added while stirring
for 3 d. The reaction mixture was diluted in CHCl3.
The organic phase was treated as described for the gen-
eral methods. The residue was treated in an LH-20 gel
permeation column (1:1 CHCl3-MeOH) to give 19
2-(N-Benzyloxycarbonyl)aminoethyl
methyl)benzoyl-6-O-sulfo-β-D-galactopyranosyl-(1→4)-6-
O-acetyl-3-O-benzyl-2-deoxy-2-phthalimido-β-D-gluco-
2,3,4-tri-O-(4-
pyranoside, trimethylamine salt (21).
1H-NMR δH
(CDCl3): 7.91–6.85 (m, 26H, Ar H), 5.93 (br s, 1H, H-
4′), 5.78 (dd, 1H, J1′,2′ = 7.92 Hz, J2′,3′ = 10.32 Hz, H-
2′), 5.46 (dd, 1H, J3′,4′ = 2.94 Hz, H-3′), 5.06 (d, 2H,
J1,2 = 12.06 Hz, H-1, NH), 5.00, 4.59 (ABq, 2H, J =
12.06 Hz, PhCH2), 4.94, 4.90 (ABq, 1H, J = 12.30 Hz,
PhCH2), 4.92 (d, 1H, H-1′), 4.39 (m, 1H, H-5′), 4.33
(d, 1H, Jgem = 11.28 Hz, H-6b), 4.27 (m, 2H, H-3,6′a),
4.17 (dd, 1H, J5,6a = 4.32 Hz, H-6a), 4.15 (m, 1H, H-6′
b), 3.96 (m, 2H, H-2,4), 3.62 (m, 1H, 1/2CH2), 3.53
(m, 1H, 1/2CH2), 3.48 (m, 2H, H-5,5′), 3.21 (br s, 2H,
CH2), 2.77 (t, 9 H, NMe3), 2.37, 2.32, 2.29 (3 s, each
3H, 3MePh), 2.05 (s, 3H, Ac). ESI-HRMS m/z [(M +
Na)+]: calcd. for C63H61N2O21SNa1,2259.3277; found,
1259.3248.
1
(84.1 mg) with an 84% yield in two steps. H-NMR δH
(CDCl3): 7.93–6.80 (m, 26H, Ar H), 5.87 (dd, 1H,
J1′,2′ = 7.92 Hz, J2′,3′ = 10.38 Hz, H-2′), 5.72 (d, 1H,
J3′,4′ = 3.30 Hz, H-4′), 5.50 (dd, 1H, H-3′), 5.07 (d, 1H,
J1,2 = 8.58 Hz, H-1), 5.07 (br s, 1H, NH), 4.95, 4.90
(ABq, 2H, J = 12.24 Hz, PhCH2), 4.89, 4.52 (ABq, 2H,
J = 12.06 Hz, PhCH2), 4.86 (d, 1H, H-1′), 4.35 (m, 1H,
H-6a), 4.31 (m, 1H, H-3), 4.16 (m, 1H, H-6b), 4.11
(m, 1H, H-2), 3.92 (m, 2H, H-4,5′), 3.64 (m, 1H, 1/
2OCH2), 3.53 (m, 2H, H-5, 1/2OCH2), 3.46 (m, 1H,
H-6′a), 3.36 (m, 1H, H-6′b), 3.20 (m, 2H, NCH2), 2.54
(br s, 1H, 6′-OH), 2.38, 2.37, 2.28 (3 s, each 3H,
3MePh), 2.01 (s, 3H, Ac). ESI-HRMS m/z [(M + Na)+]:
calcd. for C63H62N2O18Na, 1157.3890; found,
1157.3862.
2-(N-Benzyloxycarbonyl)aminoethyl
2,3,4-tri-O-(4-
methyl)benzoyl-6-O-(2-naphthylmethyl)-β-D-galactopyra-
nosyl-(1→4)-3-O-benzyl-2-deoxy-2-phthalimido-6-O-
sulfo-β-D-glucopyranoside, trimethylamine salt (22).
1H-NMR δH (CD3OD): 7.96–6.56 (m, 33H, Ar H),
5.84 (d, 1H, J3′,4′ = 3.36 Hz, H-4′), 5.64 (dd, 1H, J1′,2′
= 7.80 Hz, J2′,3′ = 10.26 Hz, H-2′), 5.56 (dd, 1H, H-3′),
5.37 (d, 1H, H-1′), 5.01 (d, 1H, J1,2 = 8.52 Hz, H-1),
4.89, 4.47 (ABq, 1H, J = 11.64 Hz, ArCH2), 4.69 (s,
2H, ArCH2), 4.61, 4.36 (ABq, 2H, J = 12.18 Hz,
ArCH2), 4.35 (d, 1H, Jgem = 10.50 Hz, H-6a), 4.33 (br
t, J = 6.60 Hz, H-5′), 4.17 (dd, 1H, J2,3 = 10.50 Hz, J3,4
= 9.18 Hz, H-3), 4.09 (br t, 1H, J = 9.42 Hz, H-4), 4.03
(d, 1H, H-6b), 3.92 (dd, 1H, H-2), 3.60 (m, 1H, 1/
2CH2), 3.55 (dd, 1H, J5′,6′a = 5.82 Hz, Jgem = 9.54 Hz,
H-6′a), 3.49 (dd, 1H, J5′,6′b = 7.50 Hz, H-6′b), 3.37 (m,
2-(N-Benzyloxycarbonyl)aminoethyl
2,3,4-tri-O-(4-
methyl)benzoyl-β-D-galactopyranosyl-(1→4)-3-O-benzyl-
2-deoxy-2-phthalimido-β-D-glucopyranoside (20). To a
solution of 18 (77.0 mg, 62.4 μmol) in CH2Cl2 (1.3
mL) and MeOH (0.4 mL), DDQ (16.8 mg, 74.9 μmol)
and H2O (1 drop) were added while stirring overnight.
The reaction mixture was treated in the same way as

36
N. Takeda and J. Tamura
2H, 1/2CH2, H-5), 3.30 (m, 2H, CH2), 2.77 (s, 9 H,
NMe3), 2.27, 2.18, 2.15 (3 s, each 3H, 3MePh). ESI-
HRMS m/z [(M + Na)+]: calcd. for C72H68N2O20SNa,
1357.3798; found, 1357.3782.
added 1,3-diaminopropane (290 μL, 3.9 mmol). The
reaction mixture was heated overnight under reflux,
and then the volatiles were removed under reduced
pressure. To the residue in MeOH (2.3 mL), Ac2O (0.3
mL) and Et3N (0.3 mL) were added while stirring over-
night. The volatiles were removed again, and the resi-
due was subjected to reverse-phase chromatography in
a Bond Elut C8 column (0–90% MeOH). To a solution
of the product in MeOH (0.6 mL) was added 0.5 M
NaOH (250 μL). The reaction mixture was heated
under reflux for 9 h and then quenched with 1% AcOH.
The volatiles were removed under reduced pressure,
and the residue was purified through a Bond Elut C8
column (0–90% MeOH) as already described. The
product was dissolved in 50% aq. 2-PrOH (1.0 mL)
and then hydrogenated in the presence of a catalytic
amount of Pd-black in an H2 atomosphere while stir-
ring for 1 d. H2O (1.0 mL) and AcOH (1 drop) were
added to the reaction mixture, and the reaction was
continued for 2 d. The insoluble materials were
removed on Celite, and the volatiles were removed
under reduced pressure. The product was dissolved in
1 M Na3PO4 and 0.15 M NaCl (0.8 mL). To this solu-
tion, NHS-PEG4-biotin (27.7 mg, 47.1 μmol) was added
while stirring overnight. The reaction mixture was trea-
ted in an LH-20 gel permeation column (H2O) to quan-
2-(N-Benzyloxycarbonyl)aminoethyl
2,3,4-tri-O-(4-
methyl)benzoyl-6-O-sulfo-β-D-galactopyranosyl-(1→4)-3-
O-benzyl-2-deoxy-2-phthalimido-6-O-sulfo-β-D-glucopyr-
anoside, bistrimethylamine salt (23).
1H-NMR δH
(CD3OD): 7.95–6.75 (m, 26H, Ar H), 5.83 (d, 1H,
J3′,4′ = 3.18 Hz, H-4′), 5.67 (dd, 1H, J1′,2′ = 7.92 Hz,
J2′,3′ = 10.26 Hz, H-2′), 5.56 (dd, 1H, H-3′), 5.40 (d,
1H, H-1′), 5.01 (d, 1H, J1,2 = 8.58 Hz, H-1), 4.92, 4.57
(ABq, 1H, J = 12.00 Hz, PhCH2), 4.69 (s, 2H, PhCH2),
4.39 (br t, 1H, J = 6.63 Hz, H-5′), 4.35 (br d, 1H, J =
9.30 Hz, H-6a), 4.14 (m, 2H, H-3,6′a), 4.01 (m, 3H, H-
4,6b,6′b), 3.86 (dd, 1H, J2,3 = 10.44 Hz, H-2), 3.57 (m,
1H, 1/2OCH2), 3.36 (m, 1H, 1/2OCH2), 3.35 (d, 1H,
J5,6a = 9.96 Hz, H-5), 2.99 (t, 2H, NCH2), 2.82 (s, 18
H, 2NMe3), 2.25, 2.18, 2.17 (3 s, each 3H, 3MePh).
ESI-HRMS
m/z
[(M + Na)+]:
calcd.
for
C61H58N2O23S2Na1, 3319.2559; found, 1319.2536.
Biotinylated KS (0S) disaccharide (1). To a solu-
tion of 18 (57.2 mg, 46.4 μmol) in EtOH (2.4 mL) was
added 1,3-diaminopropane (232 μL, 2.8 mmol). The
reaction mixture was heated overnight under reflux,
and then the volatiles were removed under reduced
pressure. To the residue in MeOH (5.0 mL), Ac2O (0.2
mL) and Et3N (0.2 mL) were added while stirring over-
night. The volatiles were removed again, and the resi-
due was subjected to reverse-phase chromatography in
a Bond Elut C8 column (0–90% MeOH). The product
was dissolved in 50% aq. EtOH (2.0 mL) and then
hydrogenated in the presence of a catalytic amount of
Pd-black in an H2 atomosphere, while stirring for 2 d.
H2O (1.0 mL) and AcOH (1 drop) were added to the
reaction mixture, and the reaction was continued for
4 d. The insoluble materials were removed on Celite,
and the volatiles were removed under reduced pressure.
The product having free amine at the end of the linker
was dissolved in 1 M Na3PO4 and 0.15 M NaCl (1.5
mL). To this solution, NHS-PEG4-biotin (60.4 mg,
99.7 μmol) was added while stirring overnight. The
reaction mixture was treated in an LH-20 gel perme-
ation column (H2O) to quantitatively give 1 from 18.
1H-NMR δH (D2O, selected): 4.51 (dd, 1H, Jb,c = 7.92
Hz, Jc,d = 4.86 Hz, H-c), 4.48 (d, 1H, J1,2 = 8.37 Hz, H-
1), 4.38 (d, 1H, J1′,2′ = 7.86 Hz, H-1′), 4.33 (dd, 1H,
Jb,e = 4.50 Hz, H-b), 4.00 (m, 1H, H-r), 3.90 (dd, 1H,
J5′,6′a = 2.16 Hz, Jgem = 12.18 Hz, H-6′a), 3.84 (d, 1H,
J3′,4′ = 3.36 Hz, H-4′), 3.79 (m, 1H, H-r), 3.75 (dd, 1H,
J5′,6′b = 5.10 Hz, H-6′b), 3.75–3.60 (m, 2H, H-6ab),
3.67 (m, 2H, H-2,3), 3.58 (dd, 1H, J2′,3′ = 9.90 Hz, H-
3′), 3.52 (m, 3H, H-4,5,5′), 3.45 (dd, 1H, H-2′), 3.24
1
titatively give 2 from 21. H-NMR δH (D2O, selected):
4.45 (m, 2H, H-c, 1), 4.37 (d, 1H, J1′,2′ = 8.37 Hz, H-
1′), 4.27 (m, 1H, H-b), 4.13 (d, 1H, Jgem = 4.74 Hz, H-
6′a), 3.96 (d, 1H, H-6′b), 3.75 (m, 1H, H-2), 3.74–3.59
(m, 2H, H-6ab), 3.57 (m, 1H, H-2′), 3.36 (m, 1H, H-e),
2.85 (dd, 1H, Jc,d = 5.10 Hz, Jgem = 13.02 Hz, H-d),
2.63 (d, 1H, H-d′), 1.90 (s, 3H, NAc). ESI-HRMS m/z
[(M + Na)+]: calcd. for C37H64N5O21S2Na2, 1024.3325;
found, 1024.3331.
Biotinylated KS (6S) disaccharide (3). To a solu-
tion of 22 (64.1 mg, 46.7 μmol) in EtOH (2.7 mL) was
added 1,3-diaminopropane (234 μL, 2.8 mmol). The
reaction mixture was heated overnight under reflux,
and then the volatiles were removed under reduced
pressure. To the residue in MeOH (1.5 mL) were added
Ac2O (0.2 mL) and Et3N (0.2 mL) while stirring over-
night. The volatiles were removed again, and the resi-
due was subjected to reverse-phase chromatography in
a Bond Elut C8 column (0–90% MeOH). To a solution
of the product in MeOH (0.6 mL) was added 0.5 M
NaOH (250 μL). The reaction mixture was heated
under reflux for 6 h and then quenched with 1% AcOH.
The volatiles were removed under reduced pressure,
and the residue was purified through Bond Elut C8 (0–
90% MeOH) as already described. The product was
dissolved in 50% 2-PrOH (1.0 mL) and then hydroge-
nated in the presence of a catalytic amount of Pd-black
in an H2 atmosphere while stirring for 2 d. H2O (1.0
mL) and AcOH (1 drop) were added to the reaction
mixture, and the reaction was continued for 4 d. The
insoluble materials were removed on Celite, and the
volatiles were removed under reduced pressure. The
product having free amine at the end of the linker was
dissolved in 1 M Na3PO4 and 0.15 M NaCl (0.8 mL).
To this solution, NHS-PEG4-biotin (9.9 mg, 18.5 μmol)
was added while stirring overnight. The reaction
(m, 1H, H-e), 3.17 (m, 2H, H-q), 2.90 (dd, 1H, Jgem
=
13.02 Hz, H-d), 2.69 (d, 1H, H-d′), 1.96 (s, 3H, NAc).
ESI-HRMS m/z [(M + Na)+]: calcd. for C37H65
N5O18SNa, 922.3938; found, 922.3925.
Biotinylated KS (6′S) disaccharide (2). To a solu-
tion of 21 (80.9 mg, 65.4 μmol) in EtOH (3.5 mL) was

Synthesis of biotinylated keratan sulfate
37
mixture was treated in an LH-20 gel permeation
column (H2O) to quantitatively give 3 from 22.
1H-NMR δH (D2O, selected): 4.54 (m, 1H, H-c), 4.51
(m, 2H, H-1,1′), 4.38 (m, 1H, H-b), 4.35 (m, 1H, H-
6a), 4.27 (br s, 1H, H-6b), 3.86 (br d, 1H, J3′,4′ = 3.48
Hz, H-4′), 3.85–3.58 (m, 2H, H-6′ab),3.70 (m, 1H, H-
2), 3.68 (m, 1H, H-3), 3.62 (m, 1H, H-3′), 3.63 [dd,
1H, J1′,2′, J2′,3′ = 9.87, 7.98 Hz (reversible), H-2′], 3.28
1H, H-d′), 1.94 (s, 3H, NAc). ESI-HRMS m/z [(M +
Na)+]: calcd. for C37H63N5O24S3Na1, 3126.2713; found,
1126.2700.
Acknowledgments
This study was supported by grant aid for scientific
research in innovative areas (23110003) from MEXT,
Japan. N.T. is grateful for the JSPS Research Fellow-
ship for Young Scientists. We thank Mrs. Mayumi Ike-
nari (Research Center for Bioscience and Technology,
Division of Instrumental Analysis, Tottori University)
for performing the ESI-HRMS measurements.
(m, 1H, H-e), 2.91 (dd, 1H, Jc,d = 4.95 Hz, Jgem
=
13.02 Hz, H-d), 2.71 (d, 1H, H-d′), 1.96 (s, 3H, NAc).
ESI-HRMS
m/z
[(M + Na)+]:
calcd.
for
C37H64N5O21S2Na2, 1024.3325; found, 1024.3325.
Biotinylated KS (6,6′-diS) disaccharide (4).
To a
solution of 23 (67.1 mg, 49.0 μmol) in EtOH (2.8 mL)
was added 1,3-diaminopropane (245 μL, 2.9 mmol).
The reaction mixture was heated overnight under
reflux, and then the volatiles were removed under
reduced pressure. To the residue in MeOH (1.5 mL),
Ac2O (0.2 mL) and Et3N (0.2 mL) were added while
stirring overnight. The volatiles were removed again,
and the residue was subjected to Bond Elut C8 reverse-
phase chromatography (0–90% MeOH). To a solution
of the product in 50% aq. MeOH (1.5 mL) was added
0.5 M NaOH (200 μL). The reaction mixture was
heated under reflux for 8 h and then quenched with 1%
AcOH. The volatiles were removed under reduced
pressure, and the residue was purified through Bond
Elut C8 (0–90% MeOH) as already described. The
product was dissolved in H2O (1.5 mL) and AcOH (1
drop) and then hydrogenated in the presence of a cata-
lytic amount of Pd-black in an H2 atmosphere while
stirring for 4 d. The insoluble materials were removed
on Celite, and the volatiles were removed under
reduced pressure. The product having free amine at the
end of the linker was dissolved in 1 M Na3PO4 and
0.15 M NaCl (0.3 mL). To this was added NHS-PEG4-
biotin (2.6 mg, 4.6 μmol) while stirring overnight. The
reaction mixture was treated in an LH-20 gel perme-
ation column (H2O) to quantitatively give 4 from 23.
1H-NMR δH (D2O, selected): 4.54 (dd, 1H, Jb,c = 7.92
Hz, Jc,d = 4.86 Hz, H-c), 4.49 (d, 1H, J1,2 = 7.92 Hz, H-
1), 4.47 (d, 1H, J1′,2′ = 7.86 Hz, H-1′), 4.35 (m, 2H, H-
b,6a), 4.24 (m, 1H, H-6b), 4.10 (m, 1H, H-6′a), 3.94
(d, 1H, J3′,4′ = 3.06 Hz, H-4′), 3.93 (dd, 1H, H-6′b),
3.84 (m, 1H, H-r), 3.70 (m, 1H, H-3), 3.69 (br t, 1H, J
= 5.94 Hz, H-2), 3.63 (m, 1H, H-3′), 3.48 (dd, 1H,
J2′,3′ = 10.02 Hz, H-2′), 3.33 (m, 2H, H-q), 3.27 (m,
1H, H-e), 2.93 (dd, 1H, Jgem = 13.02 Hz, H-d), 2.72 (d,
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