Synthesis of hyaluronic acid oligosaccharides with a GlcNAc-GlcA repeating pattern and their binding affinity with CD44

Article abstract of DOI:10.1039/d0ob01048k

Hyaluronic acid (HA) is a ubiquitous glycosaminoglycan in the extracellular matrix and a ligand of CD44, a transmembrane glycoprotein that is important in cell migration. Crystal and NMR studies found a hexasaccharide of the pattern (GlcA-GlcNAc)3 as the shortest HA that could bind to CD44, but molecular dynamics simulations indicated that a tetrasaccharide of the pattern (GlcNAc-GlcA)2 is the key structure interacting with CD44. Access to oligomers with such a repeat pattern is crucial in binding studies with CD44. Here we developed a synthetic procedure to afford the HA oligosaccharides with the GlcNAc-GlcA repeating unit and measured the binding interaction between these sugars and human CD44 by isothermal titration calorimetry (ITC). During the chemical synthesis, we successfully generated the β-glycosidic bond in the absence of neighbouring group participation and overcome the issues in the oxidation step. In addition, ammonia-free dissolving metal reduction for debenzylation and azido reduction has been applied in carbohydrate synthesis for the first time. ITC analysis revealed that the HA tetrasaccharide (GlcNAc-GlcA)2 could indeed interact and bind to the human CD44. This journal is

Full text of DOI:10.1039/d0ob01048k

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DOI: 10.1039/D0OB01048K
ARTICLE
Synthesis of hyaluronic acid oligosaccharides with GlcNAc–GlcA
repeating pattern and their binding affinity with CD44
Che-Jui Yeh,ab Medel Manuel L. Zulueta,c Yaw-Kuen Li b* and Shang-Cheng Hung a*
Received 00th January 20xx,
Accepted 00th January 20xx
Hyaluronic acid (HA) is a ubiquitious glycosaminoglycan in the extracellular matrix and a substrate of CD44, a transmembrane
glycoprotein that is important in cell migration. Crystal and NMR studies found the hexasaccharide of the pattern (GlcA–
GlcNAc)3 as the shortest HA that could bind to CD44, but molecular dynamics simulations indicated that a tetrasaccharide
of the pattern (GlcNAc–GlcA)2 is the key structure interacting with CD44. Access to oligomers with such a repeat pattern is
crucial in binding studies with CD44. Here we developed a synthetic procedure to afford the HA oligosaccharides with
GlcNAc–GlcA repeating unit and measured the binding interaction between these sugars and hCD44 by isothermal titration
calorimetry (ITC). During the chemical synthesis, we successfully generated the -glycosidic bond in the absence of
neighbouring group participation and overcome the issues in the oxidation step. In addition, ammonia-free dissolving metal
reduction for debenzylation and azido reduction was applied in carbohydrate synthesis for the first time. ITC analysis
revealed that the HA tetrasaccharide (GlcNAc–GlcA)2 could indeed interact and bind to the human CD44.
DOI: 10.1039/x0xx00000x
only in common cells, but also in tumour cells, especially, those
with highly expressed CD44 on the surface.15
Introduction
Although the interaction of CD44 and HA has been studied
Hyaluronic acid (HA) is an anionic, linear nonsulfated
glycosaminoglycan and a major component of the extracellular
matrix. It is composed of alternating (13) D-glucuronic acid
(GlcA) and (14) N-acetyl-D-glucosamine (GlcNAc) and can
weigh up to several million daltons. HA can take in water several
times heavier than itself to form a cushion and keep tissues
moist. It is elastic and lubricating enough to prevent the impacts
between bones in the articular cartilage and avoid frictions in
organs.1 Besides the physical properties, it has an essential
contribution to biological processes such as cell adhesion,2
proliferation,3 inflammation,4 and cell migration.5 In the process
of wound healing, for example, coagulation is enabled by the
HA–fibrin matrix formed.6 HA is hydrolysed later into
oligosaccharides for interaction with proteins, including CD44,
to induce inflammation and cell migration.
CD44 is a transmembrane glycoprotein with multiple
glycoforms differentiated by N- or O-glycosylation.7 Complex
splicing of the gene transcripts also results in different
isoforms.8 It plays an important role in neuronal axon guidance,9
lymphocyte activation,10 and cell migration.11 Interaction of HA
with CD44 induces a conformational change in the protein that
that increases its affinity to HA.12–14 This event results in signal
transduction, followed by cell migration. The process occurs not
for quite a while, the key structure is still unclear. Crystal and
NMR studies of CD44 and HA show that the latter must be at
least a hexasaccharide.16,17 On the other hand, theoretical
calculation indicated that the key HA structure of the
interaction is the tetrasaccharide (GlcNAc-GlcA)2.14 However,
this sequence pattern does not exist in human body because
hyaluronidases degrade HA mainly into the (GlcA-GlcNAc)n-type
oligosaccharides (Fig. 1). Only leech has so far been found able
to produce (GlcNAc-GlcA)n-type sugar in nature.18 We became
curious as to whether the (GlcNAc-GlcA)2 tetrasaccharide is
indeed the shortest length of HA for binding to CD44. After a
thorough search, this compound and its homologous variants
are neither commercially available nor are they accessible by
degradation even though there are new methods that can
control the degradation of high-molecular-weight HA into
homogeneous oligosaccharides.19 In the end, we decided to
develop a chemical strategy to obtain these sugars.
Several chemical methods of preparation of HA have been
reported previously.20 These methods may be classified on the
basis of the reducing end sugar: GlcA,21–26 GlcNAc,27–32 or
involving both.33–36 From these reports, a number of challenges
for the synthesis of HA oligosaccharides with (GlcNAc-GlcA)n
OH
OH
NaOOC
O
HO
NaOOC
O
HO
O
HO
O
O
HO
O
O
a. Genomics Research Center, Academia Sinica, 128, Section 2, Academia Road,
Taipei 115, Taiwan. E-mail: schung@gate.sinica.edu.tw.
O
AcHN
NHAc
OH
b. Department of Applied Chemistry, National Chiao Tung University, 1001, Ta-
Hsueh Road, Hsinchu 300, Taiwan. E-mail: ykl@faculty.nctu.edu.tw.
c. Institute of Chemistry, College of Science, University of the Philippines, Diliman,
Quezon City 1101, Philippines.
OH
-(GlcA-GlcNAc)-
Fig. 1 Sequence possibilities for the oligosaccharide repeating unit.
-(GlcNAc-GlcA)-
Electronic Supplementary Information (ESI) available: NMR spectra and ITC data.
See DOI: 10.1039/x0xx00000x
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ARTICLE
Journal Name
OH
O
pattern are highlighted. Use of glucuronic acid backbones at the
reducing end of glycosyl donors expected for such synthesis
encumbers the elongation procedures because of low
reactivity.20,27 However, using the more reactive glucose
backbone in donors may face difficult oxidation of multiple
primary alcohols at the late stage of the synthesis.26,34,35
Another challenge is building the -glycosidic bond of
glucosamine. Although previous studies reported that the
neighbouring group effect is effective in making the -glycosidic
bond,26–28,30 there are problems concerning the oxazoline
formation,25,28 purification, or NMR analysis.37 Using the azido
group can avoid those problems, but control of stereoselectivity
in glycosylation is not easy. Protecting groups also need to be
judiciously selected such that these moieties could be
selectively removed to form the precursor of glycosylation or
enable functional group transformation and remain inert in the
reaction environment when required by the process.
In this paper, we conceived a synthetic procedure for the
chemical preparation of HA disaccharide, tetrasaccharide, and
hexasaccharide with the (GlcNAc-GlcA)n repeat pattern. Along
the way, ammonia-free dissolving metal reduction for the
debenzylation and azido reduction in carbohydrates was
successfully implemented. The generated oligosaccharides
were examined for their binding interaction with human CD44
protein, in which observed the tetrasaccharide as the shortest
HA fragment that could bind to CD44
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DOI: 10.1039/D0OB01048K
NaOOC
O
HO
HO
O
O
O
NH2
5
AcHN
H
1: n = 1
2: n = 2
3: n = 3
HO
n
OAc
O
Ph
O
O
N3
O
O
BnO
O
O
NBn(Teoc)
5
BzO
4: n = 1
5: n = 2
6: n = 3
2-NAP
Ph
n
OAc
O
O
O
O
O
2-NAPO
HO
NBn(Teoc)
+
STol
BnO
N3
5
OBz
7
8
OAc
Ph
O
O
O
O
2-NAPO
HO
BnO
NH2
HO
+
+
OAc
N3
5
OBz
O
NH
CCl3
10
11
9
O
O
OH
OH
O
O
HO
HO
+
OH
HCl•H2N
O
O
12
13
Scheme 1 Retrosynthesis of the target compounds.
Results and discussion
We envisioned that the preparation of the desired HA
oligosaccharides 1, 2, and 3 can be approached from HA
oligosaccharide skeletons 4, 5, and 6 through selective alcohol
oxidation and final transformations (Scheme 1). The skeletons
would be elongated by -glycosylation of the linker derivative 8
and the disaccharide surrogate 7. The latter has a benzoyl (Bz)
group at O2 to give neighbouring group effect in forming the -
glycosidic bond. We also opted for the glucose backbone, which
is considerably more reactive than that with glucuronic acid
during glycosylation. Compound 7 is equipped with orthogonal
protecting groups to permit proper chain extension and
functionalization. The 2-naphthylmethyl (2-NAP) group could
be selectively cleaved by 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) to give a glycosyl acceptor without
affecting the benzyl (Bn) and benzylidene groups. Removal of
acetyl (Ac) groups at primary positions facilitated by Mg(OMe)2
should have higher reaction rate than the cleavage of Bz esters
and the 2-(trimethylsilyl)ethoxycarbonyloxy (Teoc) group. In
contrast to the phthalimido, trichloroacetyl, and
trichloroethylcarbamate protecting groups, the azido group can
survive strongly basic environments and can be reduced by
hydrogenolysis or Staudinger reaction.38 To build the -
glycosidic bond of compound 7, 4,6-O-benzylidene would be
installed on the 2-azido-2-deoxy-glucosamine 9 to reduce steric
hindrance of the -face during glycosylation. Linker 8, the
glucosamine-derived 9 and the glucose-derived 10 can be
prepared from the commercially available compounds 11–13.
Scheme 2 illustrates our preparation of the disaccharide
building block 7. The known 3-alcohol 1439 was obtained from
glucosamine hydrochloride (12) by using the typical diazo
transfer procedure,40 thioglycosylation,41 and benzylidene
formation.39 Further installation of a 2-NAP group gave the
thioglycoside 15 in 93% yield. The benzylglucofuranose 16,
obtained by typical benzylation of the diacetonide 13,42 was
hydrolysed and then rearranged by an acidic ion-exchange
resin, which does not require neutralization. Then,
regioselective diacetylation provided the 2,4-diol 17 in 53%
yield over two steps. To affect selective benzoylation, the
diacetylglucose 17 was dissolved in pyridine and slowly treated
with BzCl, giving the 2-benzoylglucose 10 in 61% yield.
Bearing the necessary functional groups, the thioglycoside
15 could already be used as glycosyl donor and form a link with
the acceptor 10. Nevertheless, the condensation of 10 and 15
using the promoter tandem N-iodosuccinimide (NIS) and
trifluoromethanesulfonic acid (TfOH) in the presence or
absence of the -inducing nitrile solvent43 could only provide
low to moderate yields and a nearly one-to-one proportion of
the  and  isomers. The alternate use of 1-benzenesulfinyl
piperidine (BSP), 2,4,6-tri-tert-butyl pyrimidine (TTBP), and
trifluoromethanesulfonic anhydride (Tf2O) promoter system,
which was used to enable -mannosylation,44 could adjust
neither the / ratio nor the yield for the disaccharide (24%,
/=1.5/1).
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/D0OB01048K
ARTICLE
2-NAPBr, NaH
Ph
O
N-Bromosuccinimide
Ph
O
Ph
O
O
O
O
O
O
O
2-NAPO
2-NAPO
HO
DMF, 0 °C
16 h, 93%
H2O/acetone = 1/10
rt, 3 h, 90%
STol
OH
N3
STol
N3
CCl3CN, DBU,
CH2Cl2, 0 °C, 1 h,
75%
N3
14
15
18
O
O
1. DOWEX 50X4 H+ resin,
H2 O, 70 °C, 1 d
Ph
O
OAc
O
O
OAc
O
OBn
O
O
BzCl
2-NAPO
HO
BnO
HO
BnO
OAc
N3
O
OAc
2. Ac2O, Et3N, DMF,
–10 to 0 °C, 8 h
53% (2 steps)
Pyridine, 0 °C,
6 h, 61%
O
NH
CCl3
OBz
OH
O
10
9
17
16
BF3•OEt2, 4 Å MS, CH2Cl2
–78 °C, 3 h, 81%
OAc
O
OAc
ZnI2, TMSSTol
Ph
O
Ph
O
O
O
O
O
O
O
BnO
O
BnO
2-NAPO
2-NAPO
STol
OAc
CH2Cl2, 30 min,
0 °C, 72%
N3
N3
OBz
OBz
7
19
Scheme 2 Preparation of the disaccharide donor 7. DMF, N,N-dimethylformamide.
With such a predicament, we opted to convert the
thioglycoside 15 into an -imidate that could proceed by an SN2-
like mechanism at glycosylation by using BF3·OEt2 as
promoter.45 The thioglycoside underwent hydrolysis and
reaction with trichloroacetonitrile in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) to form the imidate with
/ = 5/1 (1H NMR: H1 = 6.38 ppm, 3JH1,H2 = 3.7 Hz; H1 = 5.70
1. PhCHO, CSA, MeOH, 3 h;
NaBH4, 16 h
Bn
N
HO
NH2
HO
5
2. Teoc-OSu, Et3N, CH2Cl2,
0 °C, 16 h, 41% (2 steps)
5
Teoc
11
8
7, NIS, AgOTf, 3 Å MS, CH2Cl2,
–60 to –20 °C, 5 h, 88%
3
ppm, JH1,H2 = 6.6 Hz). The -isomer can be recycled, that is,
OAc
hydrolysed back into compound 18. The condensation of the -
imidate 9 and acceptor 10 fortunately led to the -disaccharide
Ph
O
O
N3
O
O
O
BnO
O
O
NBn(Teoc)
3
19 (1H NMR: H1’ = 4.37 ppm, JH1’,H2’ = 8.3 Hz) in 81% yield
5
BzO
R
n
without the -product. Using 1 equivalent ZnI2 in tandem with
trimethyl(4-methylphenylthio)silane (TMSSTol) to transform
the glycosyl acetate 19 into the thioglycoside 7 avoided the
hydrolysis of the benzylidene acetal.
4: R = 2-NAP, n = 1
20: R = H, n = 1
DDQ, H2 O/CH2 Cl2 = 1/10, rt, 2 h, 78%
7, NIS, TfOH, CH2Cl2, –78 to –40 °C, 3 h, 79%
DDQ, H2 O/CH2 Cl2 = 1/10, rt, 2 h, 69%
5: R = 2-NAP, n = 2
21: R = H, n = 2
The linker 8 was produced by installing Teoc group on the
benzylamine generated by reductive amination of 5-
aminopentanol (11) (Scheme 3). With NIS and silver
trifluoromethanesulfonate (AgOTf) as promoter, the
glycosylation of acceptor 8 and donor 7 offered the disaccharide
derivative 4 in 88% yield without orthoester formation (1H
NMR: H1 = 4.49 ppm, 3JH1,H2 = 7.8 Hz). Compound 4 was treated
with DDQ to form the disaccharide acceptor 20 in 78% yield.
Successive elongation of this acceptor with the thioglycoside
donor 7 punctuated by 2-NAP cleavage successfully delivered
the tetrasaccharide 5 and hexasaccharide 6. Selective hydrolysis
of the acetate groups at the primary position by Mg(OMe)2
furnished the compounds 22–24 in 89–99% yield.
7, NIS, TfOH, CH2Cl2, –78 to –40 °C, 3 h, 77%
6: R = 2-NAP, n = 3
Mg(OMe)2, CH2 Cl2, 0 °C, 2 to 4 h
OH
Ph
O
O
O
O
O
BnO
O
O
NBn(Teoc)
5
N3
BzO
2-NAP
n
22: n = 1, 99%
23: n = 2, 92%
24: n = 3, 89%
Scheme 3 Synthesis of HA-based oligomers and oxidation precursors. CSA, 10-
camphorsulfonic acid; Teoc-OSu, 1-[2-
(trimethylsilyl)ethoxycarbonyloxy]pyrrolidin-2,5-dione.
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ARTICLE
Table 1 Oxidation of HA skeletons
1. Reagent, Solvent media,
°C,
22
23
24
Ph
O
Teoc
N
h
t
MeOOC
O
BnO
T
O
25: n = 1
26: n = 2
27: n = 3
O
O
O
O
2. CH2N2, CH2Cl2 , ether,
30 min, 0 °C
Bn
N3
5
BzO
n
2-NAP
Entry
Compound
Reagent
Solvent media
T
0
0
t
2
2
2
2
2
48
5
2
2
Product
25
26
27
27
27
27
27
25
Yield (%)a
1
2
3
4
5
6
7
8
9
22
23
24
24
24
24
24
22
23
TEMPO, NaClO
TEMPO, NaClO
TEMPO, NaClO
NaBr, TBAB, NaHCO3, H2O, CH2Cl2
NaBr, TBAB, NaHCO3, H2O, CH2Cl2
NaBr, TBAB, NaHCO3, H2O, CH2Cl2
Toluene, phosphate buffer pH 6.8
CH2Cl2
44
53
0
0b
6
29
53
84
71
0
TEMPO, NaClO, NaClO2
PDC, Ac2O
rt
rt
rt
rt
rt
rt
TEMPO, BAIB
TEMPO, BAIB
TEMPO, BAIB
TEMPO, BAIB
CH2Cl2/H2O = 10/1
CH3CN/H2O = 10/1
CH3CN/H2O = 10/1
CH3CN/H2O = 10/1
26
a Isolated yield. b No reaction. TBAB, tetrabutylammonium bromide.
For the oxidation of the primary alcohol, the 2,2,6,6- gave better yield of compound 27 (29%, entry 6). Surprisingly,
tetramethylpiperidine-1-oxyl (TEMPO) free radical, NaClO, and the yield of compound 27 increased to 53% when the solvent in
NaClO2 combination was utilized as recommended in a previous the oxidation step was changed from CH2Cl2 to CH3CN (entry 7).
HA synthesis.35 We later discovered that the oxidation could Furthermore, the yields of disaccharide 25 and tetrasaccharide
proceed toward the carboxylic acid even without NaClO2. 26 also increased considerably to 84% and 71%, respectively,
Because of the difficult purification and NMR analysis of the under such condition (entries 8 and 9).
carboxylate compound, subsequent methylation was carried
Hydrolysis of the esters in compounds 25–27 supplied the
out to afford the methyl esters 25–27 (Table 1). Although the carboxylates 28–30 in 65–93% yield (Scheme 4). Staudinger
stated oxidation condition provided the desired disaccharide 25 reaction, followed by N-acetylation produced the acetamides
(entry 1, 44%) and tetrasaccharide 26 (entry 2, 53%), 31–33 in 23–76% yield. Hydrogenolysis of 31–33 under
hexasaccharide 27 was not obtained even when the solvent was Pd(OH)2/C promotion produced compounds 34–36 in 18–50%
replaced by toluene and phosphate buffer (entries 3 and 4).46
yield. Finally, the Teoc group was eliminated by
Alternatively, we treated the hexasaccharide 24 with tetrabutylammoium fluoride (TBAF) to get the target molecules
TEMPO and [bis(acetoxy)iodo]benzene (BAIB) or pyridinium 1–3 in 65–99% yield.
dichromate (PDC) and Ac2O.32 The case with TEMPO and BAIB
1. PMe3•THF,
THF, MeOH,
25
26
27
Ph
O
Teoc
N
Ph
O
Teoc
N
NaOH(aq), MeOH
CHCl3, rt, 1–3 d
NaOH(a q), 3–5 h
NaOOC
O
BnO
NaOOC
O
BnO
O
O
O
O
O
O
O
O
O
O
2. Ac2O, Et3N,
N3
5
Bn
AcHN
5
Bn
2-NAP
HO
MeOH, rt, 30 min 2-NAP
HO
n
n
28: n = 1, 93%
29: n = 2, 81%
30: n = 3, 65%
31: n = 1, 76%
32: n = 2, 43%
33: n = 3, 23%
OH
O
OH
NaOOC
O
HO
Teoc
NH
NaOOC
O
HO
Pd(OH)2 /C, H2
1 M TBAF in THF
CH3CN, rt, 1–4 d
HO
O
O
HO
O
O
O
O
NH2
O
H2 O, MeOH, rt,
36–48 h
5
AcHN
5
AcHN
H
H
HO
HO
n
n
34: n = 1, 42%
35: n = 2, 50%
36: n = 3, 18%
1: n = 1, 65%
2: n = 2, 99%
3: n = 3, 90%
Scheme 4 Synthesis of the target molecules 1–3.
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ARTICLE
Table 3 Dissociation constants of the binding of HA-based sugars to hCD44 determined
through ITC.a
Table 2 Alternate procedure toward compounds 34–36.
28
34
35
36
KD (M)b
1. Reagent mixture, temp, time
Sugar
Disaccharide 1
Sugar-to-protein ratio
0
29
c
–
2. Ac2O, Et3N, MeOH, rt, 30 min
30
Tetrasaccharide 2
Hexasaccharide 3
LMW-HA (8–15 kDa)
1.05 (±0.05)
0.90 (±0.08)
0.17 (±0.02)
3.5 (±1.4)
29 (±6)
9.2 (±1.1)
Entry S.M.a
Reagent
temp
time
(h)
36
36
36
48
48
48
1
Product
Yield
(%)c
31
15
0
mixtureb
1
2
3
4
5
6
7
8
9
28
29
30
28
29
30
29
29
30
30
A
A
A
B
B
B
C
C
C
C
rt
rt
rt
rt
rt
34
35
36
34
35
36
35
35
36
36
a The values reported are averages of results from three experiments with standard
deviations indicated in parentheses. Measured as association constant, the
inverse of KD. c No binding detected.
b
58
d
–
the reaction at a temperature low enough for adequate stirring.
A reaction temperature controlled to –50 °C allowed the
solution to be stirred smoothly, and the tetrasaccharide 29
reacted completely in 1 h with 47% yield (entry 8). Applying the
same reaction condition to the hexasaccharide 30 supplied the
product 36 in 38% yield (entry 9). We noted here that the low
yield is caused by the incomplete debenzylation of the
hexasaccharide. Extending the reaction time from 1 h to 3 h
generated 36 in a far better 62% yield (entry 10). In the end, we
successfully improved the yield for the disaccharide,
tetrasaccharide, and hexasaccharide by using Pd black and
ammonia free dissolving metal reduction.
For the binding affinity measurements, isothermal titration
calorimetry (ITC) was used to detect the interaction between
the HA sugars and a commercially acquired hCD44. Binding for
disaccharide 1 is not evident, but exothermic binding to hCD44
are observable for tetrasaccharide 2 and hexasaccharide 3. In
Table 3, the binding ratios of tetrasaccharide and
hexasaccharide to hCD44 are both close to one. In contrast, that
of low-molecular-weight (LMW)-HA (8–15 kDa, about 40–75
sugar units per molecule) is 0.17, which suggests that one LMW-
HA molecule can interact with approximately six hCD44 units.
CD44 transitions between two stable conformations: an
d
rt
–
0 °C
–50 °C
–50 °C
–50 °C
37
47
38
62
1
1
3
10
a Starting material. b A: Pd(OH)2/C, H2, H2O, MeOH; B: Pd black, H2, H2O, MeOH; C:
Na(s), 15-crown-5, tetrahydrofuran. Isolated yields. No product because of
probable Pd black conjugation.
c
d
Although the syntheses of the HA-based compounds 1–3
were successful, the yields of Staudinger reaction and
hydrogenolysis dropped significantly as the sugar length
increased. Consequently, we modified the synthetic pathway at
these stages by first performing the simultaneous removal of
the benzyl-type and benzylidene groups and reduction of the
azido group and then, installation of the acetyl group on the
amino moiety. Thus, hydrogenolysis of the HA skeletons 28–30
by Pd(OH)2/C and then N-acetylation is expected to produce the
acetamides 34–36 (Table 2). The yield of disaccharide 34 is
similar to the yield of the original pathway (31% overall, entry
1), but the results for the tetrasaccharide 35 and, most
especially, the hexasaccharide 36 are not satisfactory (entries 2
and 3). These outcomes may be caused by the absorption of the
product by the activated carbon.47 When Pd black was used
instead of Pd(OH)2/C, the yield for the disaccharide 34 improved
to 58% (entry 4), which is higher than that of the initial route
(32%, 28→31→34). Unfortunately, no target products were
observed for the tetrasaccharide and hexasaccharide possibly
due to Pd black conjugation (entries 5 and 6).
The feasibility of ammonia-free dissolving metal reduction,
which uses Na(s) and 15-crown-5,48 was alternatively examined
for the transformation of compounds 29 and 30. It took 1 h to
remove all of the benzyl-type and benzylidene groups from
sugar at 0 °C. After quenching the reaction, the sodium salt was
easily separated by a C-18 column by virtue of the less polar
Teoc group. Then, the crude residue underwent N-acetylation
to provide the acetamide 35 in 37% yield (entry 7). Considering
that the reduction condition maybe too harsh 0 °C, we set up
ordered conformation with low affinity to HA and
a
conformation with partially disordered C-terminal exhibiting
high affinity to HA.12 Guvench et al.14 showed in their molecular
dynamics simulations that an HA tetrasaccharide segment of
the sequence (GlcNAc–GlcA)2 forms close associations with the
binding domain in CD44 with nearly similar minimum distances
for both the ordered and partially disordered states. Additional
sugar residues at the reducing and non-reducing ends are
relatively farther away from the protein. Our KD value for the
tetrasaccharide 2 (3.5 M) corroborates these observations.
The disaccharide 1 is indeed far too short to display any
meaningful association with CD44. Previous measurements of
binding affinity with polymeric HA showed a KD range of 13–27
M,17,49 which is quite consistent with our results (9.2 M). That
the tetrasaccharide 2 could fit closely to the binding groove may
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Page 6 of 19
ARTICLE
be the reason for the higher affinity. We also suspect that the Experimental procedures and characterization data
Journal Name
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DOI: 10.1039/D0OB01048K
notably lower affinity of our hexasaccharide 3 (29 M) and the
longer oligosaccharides of previous measurements17,50 have to
do with the extended sugar residues and their secondary
interaction with the protein that may influence the switching
between the ordered and partially disordered states. We hope
to investigate this behaviour in future studies.
4-Methylphenyl 2-azido-4,6-O-(benzylidene)-2-deoxy-3-O-(2-
naphthylmethyl)-1-thio-D-glucopyranoside (15). NaH (0.12 g, 3.0
mmol) was slowly added to the solution of compound 14 (1.0 g, 2.5
mmol) and BnBr (0.47 mL, 3.0 mmol) in anhydrous DMF (15 mL)
under nitrogen atmosphere at 0 °C. After 16 h, the mixture was
quenched with H2O (50 mL) and then extracted with ethyl acetate.
The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography (ethyl acetate/ hexane = 1/8, v/v) to yield
Conclusions
We successfully assembled HA-based disaccharide,
tetrasaccharide, and hexasaccharide with (GlcNAc-GlcA)n
repeat pattern. In this synthesis work, we were able to form the
-glycosidic bond between the glucosamine-derived donor 9
and the glucose-based acceptor 10 by SN2-like reaction without
neighbouring group effect. Our method for late-stage oxidation
worked well and the turnover at the debenzylation stage was
improved. After reviewing previous research papers, we believe
that this is the first application of ammonia-free dissolving
metal reduction (Na(s) and 15-crown-5) in the carbohydrate
field. Our ITC binding experiments also confirmed that the
tetrasaccharide 2 bearing the GlcNAc-GlcA repeat unit could
indeed effectively bind CD44 and, thus far, is the shortest HA
oligomer to exhibit this property.
the product 15 (1.3 g, 93%). For the -isomer: []2D4 +71.1 (c 0.79 in
CHCl3); IR (thin film): νmax/cm–1 3056, 2867, 2110, 1492, 1371, 1275,
1171, 1099; 1H NMR (400 MHz, CDCl3):  7.81-7.78 (3H, m, Ar-H),
7.76-7.73 (1H, m, Ar-H), 7.51-7.48 (3H, m, Ar-H), 7.45-7.43 (2H, m,
Ar-H), 7.39-7.36 (5H, m, Ar-H), 7.13-7.11 (2H, m, Ar-H), 5.59 (1H, s,
PhCH), 5.49 (1H, d, J 4.8, 1-H), 5.10-4.98 (2H, m, ArCH2), 4.44 (1H,
td, J 9.9, 4.9, 5-H), 4.22 (1H, dd, J 10.3, 5.0, 6-Ha), 4.03-3.95 (2H, m,
2-H, 3-H), 3.79-3.73 (2H, m, 4-H, 6-Hb), 2.32 (3H, s, CH3); 13C NMR
(100 MHz, CDCl3):  138.4 (C), 137.2 (C), 135.2 (C), 133.3 (C), 133.1
(CH), 130.0 (CH), 129.1 (CH), 128.3 (CH), 128.2 (CH), 128.0 (CH),
127.7 (CH), 127.0 (CH), 126.1 (CH), 126.0 (CH), 125.9 (CH), 101.6
(CH), 88.2 (CH), 82.8 (CH), 77.9 (CH), 77.2 (CH), 75.2 (CH2), 68.7
(CH2), 63.8 (CH), 21.1 (CH3); HRMS (ESI): m/z calcd for
C31H29N3O4NaS ([M+Na]+): 562.1776, found 562.1768. For the -
isomer: []2D4 –171.6 (c 4.86 in CHCl ); IR (thin film): νmax/cm–1 3480,
Experimental Section
3
3030, 2919, 2873, 2111, 1494, 1346, 1277, 1078; 1H NMR (400 MHz,
CDCl3):  7.81-7.74 (4H, m, Ar-H), 7.50-7.44 (7H, m, Ar-H), 7.40-7.38
(3H, m, Ar-H), 7.16-7.14 (2H, m, Ar-H), 5.57 (1H, s, PhCH), 5.05-4.95
(2H, m, ArCH2), 4.43 (1H, d, J 10.2, 1-H), 4.38 (1H, dd, J 10.5, 5.0, 6-
Ha), 3.78 (1H, t, J 10.3, 6-Hb), 3.69 (1H, t, J 9.1, 3-H), 3.63 (1H, t, J
9.2, 4-H), 3.43 (1H, dd, J 9.5, 5.0, 5-H), 3.36 (1H, t, J 9.4 Hz, 2-H),
2.35 (3H, s, Ar-CH3); 13C NMR (100 MHz, CDCl3):  139.1 (C), 137.1
(C), 135.0 (C), 134.5 (CH), 133.2 (C), 133.1 (C), 129.9 (CH), 129.1
(CH), 128.3 (CH), 128.2 (CH), 128.0 (CH), 127.7 (CH), 127.2 (CH),
126.6 (C), 126.1 (CH), 126.0 (CH), 125.96 (CH), 101.3 (CH), 86.7 (CH),
81.3 (CH), 80.9 (CH), 75.1 (CH2), 70.4 (CH), 68.5 (CH), 64.7 (CH), 21.2
(CH3).
ITC measurements
hCD44 (Gln21–Pro220) was brought from Acrobiosystem.
LMW-HA (molecular weight: 8–15 kDa) was brought from
Carbosynth. All ITC experiments were carried out at 25ꢀ°C with
sodium phosphate buffer [phosphate (50 mM), NaCl (62.3 mM),
pH 7.4]. The hCD44 protein solution (60-70 μM) was placed in
the calorimeter cell and titrated with the sugar solution (1–1.4
mM, 2 μL injections with 3 min spacing). The results of the
protein titration were subtracted by the data for the sugar
solution titrated into the buffer without protein and then fitted
by One site model.
General synthetic method
2-Azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)-D-
glucopyranose (18). NBS (8.3 g, 46 mmol) was added to a solution
of the thioglycoside 15 (5.0 g, 9.3 mmol) in the mixture of H2O and
THF (1/10, v/v, 495 mL) at room temperature. After 3 h, the
reaction was quenched with Na2S2O3(aq) and NaHCO3(aq). THF was
removed under reduced pressure. The mixture was extracted with
ethyl acetate. The organic layer was dried over anhydrous MgSO4,
CH2Cl2 was purified and dried from a safe purification system
filled with anhydrous Al2O3. All other reagents were obtained
from commercial sources and used without further purification.
Water was either distilled or Milli-Q-purified. Flash column
chromatography was carried out on Silica Gel 60 (230–400
mesh, E. Merck). TLC was performed on glass plates pre-coated
with Silica Gel 60 F254 (0.25 mm, E. Merck); detection was
executed by spraying with a solution of Ce(NH4)2(NO3)6, filtered, and concentrated under reduced pressure. The residue was
(NH4)6Mo7O24, and H2SO4 in water followed subsequent heating
on a hot plate. Specific rotations were taken at ambient
conditions and reported in 10−1·deg·cm2·g−1; the sample
concentrations are in g·dL−1. 1H and 13C NMR spectra were
recorded on 600 MHz spectrometers. Chemical shifts are in ppm
and J values are given in Hz. The hydrogen multiplicities of
carbon peaks were determined using DEPT-90 and DEPT-135
experiments, the spectra of which were herein provided
together with the power-gated-decoupled 13C NMR spectrum.
purified by silica gel chromatography (ethyl acetate/ hexane = 1/3,
v/v) to yield compound 18 (3.6 g, 90%, α/β = 1/1). []2D4 –78.9 (c 1.0
in CHCl3); IR (thin film): νmax/cm–1 3434, 3055, 1921, 1874, 2211,
2114, 1634, 1451, 1367, 1347, 1254, 1168, 1138, 1123, 1095, 1039,
1005, 962, 852, 815, 793, 770, 747, 695, 652, 473; 1H NMR (600
MHz, CDCl3):  7.82-7.79 (6H, m, Ar-H), 7.76-7.74 (2H, m, Ar-H),
7.51-7.43 (10H, m, Ar-H), 7.40-7.38 (6H, m, Ar-H), 5.60 (1H, s), 5.57
(1H, s), 5.26 (1H, m), 5.10 (1H, m), 5.06 (1H, m), 4.97 (1H, m), 4.96
6 | J. Name., 2012, 00, 1-3
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Page 7 of 19
Organic & Biomolecular Chemistry
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ARTICLE
(3H, s, OCOCH3), 2.03 (3H, s, OCOCH3); 13C NMR (150 MHz, CDCl3): 
DOI: 10.1039/D0OB01048K
171.7 (C), 169.5 (C), 138.0 (C), 128.4 (CH), 127.8 (CH), 93.7 (CH),
83.5 (CH), 74.7 (CH2),74.4 (CH),72.4 (CH), 69.2 (CH), 62.8 (CH2), 20.8
(CH3), 20.6 (CH3); HRMS (ESI): m/z calcd for C17H22O8Na ([M+Na]+):
377.1212, found 377.1209.
(1H, m), 4.58 (1H, m), 4.31-4.26 (2H, m), 4.15-4.09 (2H, m), 3.78-
3.71 (4H, m), 3.63-3.60 (2H, m), 3.49 (1H, m), 3.42-3.37 (2H, m),
3.07 (1H, m); 13C NMR (150 MHz, CDCl3):  137.1 (C), 137.0 (C),
135.2 (C), 135.1 (C), 133.2 (C), 133.2 (C), 133.1 (C), 129.13 (CH),
129.10 (CH), 128.3 (CH), 128.19 (CH), 128.16 (CH), 128.0 (CH),
127.66 (CH), 127.64 (CH), 127.1 (CH), 126.1 (CH), 126.04 (CH),
126.00 (CH), 125.95 (CH), 125.92 (CH), 101.5 (CH), 101.4 (CH), 96.5
(CH), 92.7 (CH), 82.7 (CH), 81.5 (CH), 78.8 (CH), 76.2 (CH), 75.1
(CH2), 74.9 (CH2), 68.9 (CH2), 68.4 (CH2), 67.3 (CH), 66.3 (CH), 63.6
(CH), 62.7 (CH); HRMS (ESI): m/z calcd for C13H18N3O4S ([M+H]+):
434.1710, found: 434.1712.
2-Azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl trichloroacetimidate (9). DBU (33.6 μL, 0.225
mmol) was added to a solution of the compound 18 (196 mg. 0.452
mmol) and trichloroacetonitrile (CCl3CN, 226 μL, 2.25 mmol) in
CH2Cl2 (2 mL) at 0 °C. After 1 h, the reaction was concentrated
under reduced pressure and then purified by silica gel
chromatography (ethyl acetate/hexane = 1/7, v/v) to give the -
trichloroacetimidate derivative 9 (196 mg, 75%); IR (thin film):
νmax/cm–1 3340, 3058, 2925, 2867, 2112, 1673, 1509, 1454, 1372,
1328, 1281, 1234, 1213, 1172, 1135, 1091, 1070, 1022, 990, 911,
856, 834, 819, 795, 754, 698, 646, 476; 1H NMR (600 MHz, CDCl3): 
8.73 (1H, s, N-H), 7.82-7.81 (3H, m, Ar-H), 7.77 (1H, m, Ar-H), 7.53-
7.50 (3H, m, Ar-H), 7.48-7.45 (2H, m, Ar-H), 7.40-7.38 (3H, m, Ar-H),
6.38 (1H, d, J 3.7, 1-H), 5.62 (1H, s, PhCH), 5.17 (1H, d, J 11.2,
ArCH2), 5.01 (1H, d, J 11.2, ArCH2), 4.34 (1H, dd, J 10.5, 4.8, 6-Ha),
4.07 (1H, td, J 10.0, 5.0, 5-H), 3.85 (1H, t, J 9.4, 4-H), 3.78 (1H, t, J
10.4, 6-Hb), 3.74 (1H, dd, J 9.9, 3.7, 2-H); 13C NMR (150 MHz, CDCl3):
 160.8 (C), 136.8 (C), 135.0 (C), 133.2 (C), 133.0 (C), 129.2 (CH),
128.3 (CH), 128.2 (CH), 128.0 (CH), 127.6 (CH), 127.0 (CH), 126.1
(CH), 126.0 (CH), 126.0 (CH), 125.9 (CH), 101.5 (CH), 94.8 (CH), 90.6
(C), 82.1 (CH), 76.4 (CH), 75.2 (CH2), 68.5 (CH2), 65.2 (CH), 62.5 (CH);
HRMS (ESI): m/z calcd for C26H24Cl3N4O5 ([M+H]+): 577.0807, found:
577.0795.
View Article Online
6-O-Acetyl-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl acetate
(10). A solution of BzCl (0.30 mL, 2.9 mmol) in anhydrous CH2Cl2 (20
mL) was slowly added to a solution of the compound 17 (1.0 g, 2.8
mmol) in pyridine (10 mL) under nitrogen atmosphere at 0 °C. After
6 h, the reaction was quenched with MeOH (1.0 mL). The mixture
was concentrated under reduced pressure and purified by silica gel
chromatography (ethyl acetate/hexane = 1/10, v/v) to give the
product 10 (0.8 g, 62%). []2D4 +20.4 (c 3.5 in CHCl ); IR (thin film):
3
νmax/cm–1 3485, 1726, 1454, 1367, 1268, 1244; 1H NMR (600 MHz,
CDCl3):  7.98 (2H, m, Ar-H), 7.58 (1H, m, Ar-H), 7.46-7.42 (2H, m,
Ar-H), 7.19-7.14 (5H, m, Ar-H), 5.78 (1H, d, J 8.3, 1-H), 5.33 (1H, dd, J
9.1, 8.3, 2-H), 4.70 (2H, d, J 12, PhCH2), 4.53 (1H, dd, J 3.1, 12.3, 6-
Ha), 4.25 (1H, d, J 12.3, 6-Hb), 3.74-3.68 (1H, m, 3-H), 3.66-3.62 (2H,
m, 4-H, 5-H), 2.85-2.83 (1H, m, 4-OH), 2.12 (3H, s, OCOCH3), 1.99
(3H, s, OCOCH3); 13C NMR (150 MHz, CDCl3):  171.9 (C), 169.3 (C),
165.1 (C), 137.5 (C), 133.5 (CH), 129.8 (CH), 129.3 (C), 128.53 (CH),
128.46 (CH), 128.1 (CH), 128.0 (CH), 92.1 (CH), 81.5 (CH), 74.92
(CH), 74.89 (CH2), 72.2 (CH), 69.7 (CH), 62.5 (CH2), 20.8 (CH3); HRMS
(ESI): m/z calcd for C24H26O9Na ([M+Na]+): 481.1475, found
481.1476.
6-O-Acetyl-[2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2-
naphthylmethyl)--D-glucopyranosyl]-(1→4)-2-benzoyl-3-benzyl--
D-glucopyranosyl acetate (19). Method A: A mixture of
thioglycoside 15 (72.3 mg, 0.134 mmol) and acceptor 10 (50.2 mg,
0.109 mmol) in anhydrous CH2Cl2 (3 mL) was added to a reaction
flask containing freshly dried AW-300 (180 mg) at room
temperature under nitrogen atmosphere and stirred for 1 h. The
reaction flask was cooled to –40 °C and then NIS (36.2 mg, 0.161
mmol) and TfOH (3.8 uL, 0.0436 mmol) were sequentially added.
After 3 h, the donor 15 was consumed completely. The reaction was
quenched with Et3N and Na2S2O3(aq) and filtered through Celite. The
crude target product was extracted with CH2Cl2. The organic layer
was dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel
chromatography (ethyl acetate/ hexane = 1/3, v/v) to yield
compound 19 (39.2 mg, 41%, /=1.2/1). Method B: A solution of
thioglycoside 15 (73.0 mg, 0.135 mmol) in anhydrous CH2Cl2 (3.0
mL) was added to a reaction flask containing freshly dried 3 Å
molecular sieves (180 mg) at room temperature under nitrogen
atmosphere and stirred for 1 h. The reaction flask was cooled to -78
°C and then BSP (34.0 mg, 0.177 mmol), TTBP (49 mg, 0.197 mmol),
and Tf2O (26 uL, 0.158 mmol) were sequentially added. After 30
min, the acceptor 10 (50.3 mg, 0.109 mmol) was added to the
reaction vessel. After 3 h, the donor 15 was consumed completely.
The reaction was quenched with Et3N and Na2S2O3(aq) and filtered
through Celite. The mixture was extracted with CH2Cl2. The organic
layer was dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (ethyl acetate/ hexane = 1/3, v/v) to yield
6-O-Acetyl-3-O-benzyl-β-D-glucopyranosyl acetate (17). A solution
of glucofuranose 16 (1.5 g, 4.3 mmol) in H2O (15 mL) was mixed
with DOWEX 50WX4 H+ ion-exchange resin (1.5 g) and was heated
to 70 °C. After 1 day, the reaction was filtered through filter paper,
concentrated under reduced pressure, and dried in vacuo. The
crude residue and Et3N (2.1 mL, 15 mmol) was dissolved in
anhydrous DMF (10 mL) under nitrogen atmosphere at –10 °C. A
solution of Ac2O (0.70 mL, 7.4 mmol) in anhydrous CH2Cl2 (20 mL)
was slowly added to the solution of the crude residue at –10 °C. The
reaction was gradually warm to 0 °C and kept going for 8 h. The
reaction was quenched with MeOH (1.0 mL). The mixture was
concentrated under reduced pressure and purified by silica gel
chromatography (ethyl acetate/hexane = 3/1, v/v) to give the
product 17 (0.8 g, 53% in 2 steps); []2D4 –8.0 (c 5.2 in CHCl ); IR (thin
3
film): νmax/cm–1 3444, 1738, 1368, 1239, 1070; 1H NMR (600 MHz,
CDCl3):  7.36-7.30 (4H, m, Ar-H), 7.41-7.27 (1H, m, Ar-H), 5.48 (1H,
d, J 8.2, 1-H), 4.85 (1H, d, J 11.7, PhCH2), 4.83 (1H, d, J 11.7, PhCH2),
4.35 (1H, dd, J 12.3, 4.6, 6-Ha), 4.20 (1H, dd, J 2.0, 12.3, 6-Hb), 3.55
(1H, dd, J 7.7, 8.2, 2-H), 3.50 (1H, ddd, J 9.2, 4.6, 2.0, 5-H), 3.47-3.39
(2H, m, 3-H, 4-H), 3.14 (1H, d, J 3.5, 4-OH), 2.93 (1H, s, 2-OH), 2.08
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Journal Name
2.33 (3H, s, Ar-CH3), 2.12 (3H, s, OCOCH3); 13C NMR (150 MHz,
CDCl3):  170.4 (C), 165.0 (C), 138.3 (C), 137.D9O(CI:)1,01.13063.99/D(C0)O, 1B30510.048K
compound 19 (23.0 mg, 24%, /=1.5/1). Method C: A mixture of
imidate 9 (84.5 mg, 0.146 mmol) and acceptor 10 (53.6 mg, 0.117
mmol) in anhydrous CH2Cl2 (1.4 mL) was added to a reaction flask
containing freshly dried 3 Å molecular sieves (80 mg) at room
temperature under nitrogen atmosphere. After stirring for 1 h, the
reaction flask was cooled to –78 °C and BF3·Et2O (0.2 M in CH2Cl2,
292 L, 0.0730 mmol) was slowly added. After 3 h, the donor 9 was
consumed completely. The reaction was quenched with Et3N and
filtered through Celite. The mixture was concentrated under
reduced pressure. The residue was purified by silica gel
View Article Online
(C), 133.7 (CH), 133.1 (CH), 133.0 (CH), 129.8 (CH), 129.7 (CH), 129.4
(CH), 129.1 (CH), 128.4 (CH), 128.34 (CH), 128.25 (CH), 128.13 (CH),
128.06 (CH), 127.9 (CH), 127.6 (CH), 127.4 (CH), 127.4 (CH), 127.0
(CH), 126.03 (CH), 126.00 (CH), 125.94 (CH), 102.0 (CH), 101.3 (CH),
85.9 (CH), 82.0 (CH), 81.5 (CH), 79.1 (CH), 77.8 (CH), 77.0 (CH), 74.92
(CH2), 74.89 (CH2), 71.8 (CH), 68.2 (CH2), 66.7 (CH), 66.1 (CH), 62.3
(CH2), 21.1 (CH3), 20.9 (CH3), HRMS (ESI): m/z calcd for
C53H51N3O11SNa ([M+Na]+): 960.3137, found: 960.3137.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentanol (8).
CSA (45 mg, 0.097 mmol) was added to the solution of 5-amino-
pentanol 11 (2.0 g, 9.7 mmol) and benzaldehyde (2.4 mL, 12 mmol)
in MeOH (100 mL) at 0 °C. After 3 h, the reaction was treated with
NaBH4 (1.7 g, 23 mmol). The reaction was concentrated under
reduced pressure after 16 h and then dried in vacuo. Et3N (5.4 mL,
19 mmol) and Teoc-OSu (5.0 g, 9.7 mmol) were sequentially added
to the solution of the dried crude residue anhydrous CH2Cl2 (30 mL)
at 0 °C. After stirring 16 h, the reaction was quenched with H2O (50
mL) and the crude product was extracted with CH2Cl2. The organic
layer was dried over anhydrous MgSO4 and concentrated under
reduced pressure. The residue was purified by silica gel
chromatography (ethyl acetate/hexane = 1/2, v/v) to provide the
pentanol 8 (2.7g, 41% for 2 steps). IR (thin film): νmax/cm–1 3444,
3089, 3065, 3031, 2951, 2863, 1694, 1682, 1495, 1471, 1454, 1422,
1364, 1305, 1249, 1177, 1125, 1092, 937, 857, 837, 769, 751, 731,
699, 666, 616, 457; 1H NMR (600 MHz, CDCl3):  7.30-7.18 (5H, m,
Ar-H), 4.46 (2H, s, ArCH2), 4.19 (2H, s, CH2), 3.57 (2H, m, CH2), 3.18
(2H, d, CH2), 1.52 (4H, s, CH2), 1.30 (2H, s, CH2), 0.99 (2H, s, CH2),
0.01 (9H, s × 2, TMS);13C NMR (150 MHz, CDCl3):  156.8 (C), 138.1
(C), 127.7 (CH), 127.2 (CH), 63.6 (CH2), 62.6 (CH2), 49.9 (CH2), 46.5
(CH2), 32.3 (CH2), 27.3 (CH2), 22.9 (CH2), 22.8 (CH2), 17.8 (CH2), -1.5
(CH3); HRMS (ESI): m/z calcd for C18H31NO3SiNa ([M+Na]+):
360.1965, found: 360.1966.
chromatography (ethyl acetate/ hexane = 1/3, v/v) to yield
compound 19 (19.2 mg, 94%). []2D4 –34.0 (c 1.8 in CHCl ); IR (thin
3
film): νmax/cm–1 2877, 3223, 1738, 1602, 1453, 1368, 1267, 1231,
1175, 1094, 1064, 1004, 857, 820, 754, 699, 563, 476; 1H NMR (600
MHz, CDCl3):  7.93 (2H, m, Ar-H), 7.81-7.78 (2H, m, Ar-H), 7.76-7.73
(2H, m, Ar-H), 7.56 (1H, m, Ar-H), 7.48-7.40 (7H, m, Ar-H), 7.37-7.36
(3H, m, Ar-H), 7.12 (5H, m, Ar-H), 5.80 (1H, d, J 8.2, 1-H), 5.48 (1H, s,
PhCH), 5.34 (1H, t, J 8.8, 2-H), 5.04 (1H, d, J 11.3, ArCH2), 4.93 (1H,
d, J 11.3, ArCH2), 4.77 (1H, d, J 11.3, ArCH2), 4.62 (1H, d, J 11.3,
ArCH2), 4.57 (1H, dd, J 12.2, 1.7, 6-Ha), 4.37 (1H, d, J 8.3, 1’-H), 4.34
(1H, dd, J 8.0, 4.3 Hz, 6-Hb), 4.08 (1H, dd, J 10.6, 4.9, 1H; 6’-Ha), 4.01
(1H, t, J 9.1, 4-H), 3.85-3.81 (2H, m, 3-H, 5-H), 3.65-3.62 (2H, m, 3’-
H, 4’-H), 3.45-3.40 (2H, m, 2’-H, 6’-Hb), 3.23 (1H, m, 5’-H), 2.10 (3H,
s, OCOCH3); 1.99 (3H, s, OCOCH3); 13C NMR (150 MHz, CDCl3): 
170.5 (C), 169.3 (C), 165.0 (C), 137.8 (C), 137.0 (C), 135.0 (C), 133.4
(CH), 133.2 (C), 133.0 (C), 129.8 (CH), 129.2 (C), 129.2 (CH), 128.5
(CH), 128.3 (CH), 128.2 (CH), 128.0 (CH), 127.7 (CH), 127.6 (CH),
127.5 (CH), 127.12 (CH), 126.09 (CH), 126.06 (CH), 126.0 (CH), 102.1
(CH), 101.3 (CH), 91.9 (CH), 81.5 (CH), 80.3 (CH), 79.2 (CH), 77.5
(CH), 75.0 (CH2), 74.8 (CH2), 73.9 (CH), 71.8 (CH), 68.3 (CH2), 66.7
(CH), 66.2 (CH), 62.1 (CH2), 20.9 (CH3), 20.9 (CH3), HRMS (ESI): m/z
calcd for C48H48N3O13 ([M+H]+): 874.3182, found: 874.3159.
4-Methylphenyl [2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2-
naphthylmethyl)--D-glucopyranosyl]-(1→4)-6-acetyl-2-benzoyl-3-
benzyl-1-thio--D-glucopyranoside (7). ZnI2 (365 mg, 1.14 mmol)
was added to a solution of the compound 19 (1.00 g, 1.14 mmol)
and TMSSTol (674 mg, 3.43 mmol) in anhydrous CH2Cl2 (20 mL) at 0
°C. After stirring for 30 min, the reaction was filtered through Celite
and then quenched with Et3N. The mixture was concentrated under
reduced pressure and purified by silica gel chromatography (ethyl
acetate/hexane = 1/5, v/v) to give thioglycoside 7 (774 mg, 72%).
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-6-acetyl-2-benzoyl-3-benzyl--D-
glucopyranose (4). A mixture of donor 7 (504 mg, 0.538 mmol) and
acceptor 8 (363 mg, 1.08 mmol) in anhydrous CH2Cl2 (17 mL) was
added to a reaction flask containing freshly dried 3 Å molecular
sieves (500 mg) at room temperature under nitrogen atmosphere.
After stirring for 1 h, the reaction flask was cooled to –60 °C. The
reaction flask was sequentially added with NIS (145 mg, 0.645
mmol) and AgOTf (152 mg, 0.588 mmol) and was gradually warmed
to –20 °C. After 5 h, the donor 7 was consumed completely. The
reaction was filtered through Celite and quenched with NaHCO3(aq)
and Na2S2O3(aq). The crude target material was extracted with
CH2Cl2. The organic layer was dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The crude residue was
purified by silica gel chromatography (ethyl acetate/ hexane = 1/3,
[]2D4 –39.8 (c 2.0 in CHCl ); IR (thin film): νmax/cm–1 3062, 3028,
3
2920, 2872, 2112, 1732, 1601, 1584, 1493, 1452, 1367, 1315, 1265,
1173, 1093, 1030, 1003, 915, 856, 814, 754, 710, 698, 666, 562,
475; 1H NMR (600 MHz, CDCl3):  8.03 (2H, m, Ar-H), 7.83-7.75 (3H,
m, Ar-H), 7.58 (1H, m, Ar-H), 7.51-7.43 (7H, m, Ar-H), 7.39-7.37 (5H,
m, Ar-H), 7.15-7.14 (5H, m, Ar-H), 7.08 (2H, m, Ar-H), 5.50 (1H, s,
PhCH), 5.21 (1H, t, J 9.4 Hz, 2-H), 5.07 (1H, d, J 11.3, ArCH2), 4.95
(1H, d, J 11.3, ArCH2), 4.78 (1H, d, J 11.0, ArCH2), 4.73 (2H, m, 1-H, 6-
Ha), 4.60 (1H, d, J 11.0, ArCH2), 4.39 (1H, d, J 8.1, 1’-H), 4.32 (1H, dd,
J 11.9, 4.7, 6-Hb), 4.08 (1H, dd, J 10.4, 4.7, 6’-Ha), 3.94 (1H, t, J 9.2, 4-
H), 3.80 (1H, t, J 8.8, 3-H), 3.71 (1H, m, 5-H), 3.66 (2H, m, 3’-H, 4’-H),
3.46 (1H, t, J 5.7, 2’-H), 3.41 (1H, t, J 10.3, 6’-Hb), 3.25 (1H, m, 5’-H),
v/v) to yield compound 4 (545 mg, 88%). []2D4 –43.7 (c 3.4 in CHCl );
3
IR (thin film): νmax/cm–1 3456, 3063, 3030, 2951, 2870, 2112, 1732,
1693, 1603, 1585, 1496, 1469, 1453, 1421, 1367, 1314, 1267, 1249,
1173, 1094, 1094, 1031, 1002, 857, 838, 754, 710, 698, 666, 602,
8 | J. Name., 2012, 00, 1-3
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Page 9 of 19
Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
560, 475; 1H NMR (600 MHz, CDCl3):  7.92 (2H, m, Ar-H), 7.81-7.73
(4H, m, Ar-H), 7.48-7.43 (6H, m, Ar-H), 7.36-7.34 (5H, m, Ar-H), 7.30-
7.27 (2H, m, Ar-H), 7.23-7.21 (1H, m, Ar-H), 7.16-7.13 (7H, m, Ar-H),
5.48 (1H, s, PhCH), 5.19 (1H, t, J 7.9, 2-H), 5.04 (1H, d, J 11.3, ArCH2),
62.40 (CH2), 50.1 (CH2), 46.5 (CH2), 28.9 (CH), 27.6 (CHV2)ie,w2A2r.t9icl(eCOHn2li)n,e
DOI: 10.1039/D0OB01048K
20.8 (CH3), 17.7 (CH2), -1.6 (CH3); HRMS (ESI): m/z calcd for
C13H18N3O4SNa ([M+H]+): 1033.4237, found: 1033.4240.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
4.93 (1H, d, J 11.3, ArCH2), 4.75 (1H, d, J 11.3, ArCH2), 4.61-4.60 (2H, azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
m, 6-Ha, ArCH2), 4.49 (1H, d, J 7.8, 1-H), 4.39 (1H, d, J = 8.1, 1’-H),
4.34-4.30 (3H, m, 6-Hb, ArCH2), 4.16-4.13 (2H, m, ArCH2), 4.07 (1H,
dd, J 10.6, 4.8, 6’-Ha), 3.96 (1H, t, J 9.1, 4-H), 3.81-3.75 (2H, m, 3-H,
CH2), 3.63-3.61 (3H, m, 3’-H, 5-H, 4’-H), 3.45-3.36 (3H, m, 2’-H, 6’-
Hb, CH2), 3.24-3.22 (1H, m, 5’-H), 2.98-2.87 (2H, m, CH2), 2.08 (3H, s,
OCOCH3); 1.44 (2H, m, CH2), 1.32 (2H, m, CH2), 1.10 (2H, m, CH2),
0.95 (2H, m, CH2), 0.01-0.01 (9H, m, TMS); 13C NMR (150 MHz,
CDCl3):  170.5 (C), 165.0 (C), 157.0 (C), 138.1 (C), 138.0 (C), 137.0
(C), 135.0 (C), 133.2 (C), 133.0 (CH), 129.8 (C), 129.6 (CH), 129.1
(CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 127.9 (CH),
127.6 (CH), 127.5 (CH), 127.4 (CH), 127.1 (CH), 126.1 (CH), 126.0
(CH), 126.0 (CH), 125.9 (CH), 102.0 (CH), 101.3 (CH), 101.0 (CH),
81.5 (CH), 80.5 (CH), 79.2 (CH), 77.9 (CH), 77.2 (CH), 76.8 (CH), 75.0
(CH2), 74.5 (CH2), 73.1 (CH), 73.0 (CH), 69.8 (CH2), 68.3 (CH2), 66.7
(CH), 66.2 (CH), 63.5 (CH2), 62.4 (CH2), 50.2 (CH), 46.6 (CH2), 29.0
(CH2), 27.2 (CH2), 23.0 (CH2), 20.9 (CH3), 17.8 (CH2), -1.5 (CH3);
HRMS (ESI): m/z calcd for C64H74N4O14SiNa ([M+Na]+): 1173.4863,
found: 1173.4877.
glucopyranosyl]-(1→4)-(6-acetyl-2-benzoyl-3-benzyl--D-
glucopyranosyl)-(1→3)-[2-azido-4,6-O-benzylidene-2-deoxy--D-
glucopyranosyl]-(1→4)-6-acetyl-2-benzoyl-3-benzyl--D-
glucopyranose (5). A mixture of donor 7 (2.77 g, 2.95 mmol) and
acceptor 20 (2.44 g, 2.46 mmol) in anhydrous CH2Cl2 (60 mL) was
added to a reaction flask containing freshly dried 3 Å molecular
sieves (1.50 g) at room temperature under nitrogen atmosphere.
After stirring for 1 h, the reaction flask was cooled to –78 °C and NIS
(776 mg, 3.45 mmol) and TfOH (44.0 L, 0.492 mmol) were
sequentially added. The reaction was gradually warmed to –40 °C
and kept going for 3 h. The reaction was filtered through Celite and
quenched with NaHCO3(aq) and Na2S2O3(aq). The crude product was
extracted with CH2Cl2. The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by silica gel chromatography (ethyl acetate/
hexane = 1/2, v/v) to yield compound 5 (3.52 g, 79%). []2D4 –26.5 (c
2.4 in CHCl3); IR (thin film): νmax/cm–1 3063, 3031, 2950, 2874, 2112,
1738, 1732, 1693, 1602, 1585, 1496, 1454, 1418, 1367, 1314, 1267,
1249, 1174, 1094, 1030, 1003, 839, 753, 710, 699, 666, 604, 552,
476; 1H NMR (600 MHz, CDCl3):  8.07(2H, m, Ar-H), 7.96 (2H, m, Ar-
H), 7.83-7.76 (4H, m, Ar-H), 7.58 (1H, m, Ar-H), 7.53-7.43 (10H, m,
Ar-H), 7.40-7.35 (8H, m, Ar-H), 7.33-7.30 (2H, m, Ar-H), 7.26 (1H, m,
Ar-H), 7.20-7.16 (12H, m, Ar-H), 5.52 (1H, s, PhCH), 5.50 (1H, s,
PhCH), 5.32 (1H, t, J 8.8, 2’’-H), 5.22 (1H, t, J 8.4, 2-H), 5.07 (1H, d, J
11.4, ArCH2), 4.96 (1H, d, J 11.4, ArCH2), 4.85 (1H, d, J 7.9, 1’’-H),
4.80 (1H, d, J 11.3, ArCH2), 4.74 (1H, d, J 11.2, ArCH2), 4.65 (1H, d, J
11.3, ArCH2), 4.60 (1H, d, J 11.2, ArCH2), 4.53 (1H, d, J 11.0, 6-Ha),
4.49 (1H, d, J 7.9, 1-H), 4.40-4.35 (5H, m, 1’-H, 1’’’-H, 6-Hb, 6’’-Ha,
CH2), 4.22-4.09 (6H, m, 6’-Ha, 6’’-Hb, 6’’’-Ha, CH2), 4.01 (1H, t, J 9.2,
4’’-H), 3.97 (1H, t, J 8.9, 4-H), 3.81 (1H, m, CH2), 3.81 (2H, m, 3’’-H,
CH2), 3.75 (1H, t, J 8.8, 3-H), 3.68-3.61 (5H, m, 3’-H, 3’’’-H, 4’’’-H, 5-
H, 5’’-H), 3.48-3.43 (4H, m, 2’’’-H, 4’-H, 6’-Hb, 6’’’Hb), 3.38 (1H, s,
CH2), 3.34 (1H, t, J 8.7, 2’-H), 3.25-3.21 (2H, m, 5’-H, 5’’’-H), 3.03-
2.94 (2H, m, CH2), 2.07 (3H, s, OCOCH3), 2.02 (3H, s, OCOCH3), 1.46
(2H, m, CH2), 1.36 (2H, m, CH2), 1.13 (2H, m, CH2), 1.01 (2H, m, CH2),
0.05 (9H, m, TMS); 13C NMR (150 MHz, CDCl3):  170.2 (C), 170.1 (C),
164.9 (C), 164.8 (C), 156.8 (C), 137.9 (C), 137.82 (C), 137.75 (C),
136.8 (C), 136.7 (C), 134.9 (C), 133.0 (C), 132.89 (C), 132.85 (CH),
129.6 (C), 129.6 (C), 129.4 (CH), 129.4 (CH), 129.1 (CH), 128.9 (CH),
128.22 (CH), 128.19 (CH), 128.11 (CH), 128.07 (CH), 128.0 (CH),
127.9 (CH), 127.7 (CH), 127.5 (CH), 127.4 (CH), 127.31 (CH), 127.27
(CH), 127.24 (CH), 127.20 (CH), 126.9 (CH), 126.8 (CH), 125.9 (CH),
125.80 (CH), 125.76 (CH), 101.9 (CH), 101.7 (CH), 101.3 (CH), 101.1
(CH), 100.8 (CH), 100.7 (CH), 81.3 (CH), 80.3 (CH), 80.0 (CH), 79.2
(CH), 79.1 (CH), 78.9 (CH), 77.4 (CH), 77.3 (CH), 74.7 (CH2), 74.4
(CH2), 73.2 (CH), 72.9 (CH), 72.8 (CH), 69.5 (CH2), 68.1 (CH2), 66.5
(CH), 66.2 (CH), 66.1 (CH), 66.0 (CH), 63.3 (CH2), 62.0 (CH2), 61.8
(CH2), 50.0 (CH2), 46.4 (CH2), 28.8 (CH), 27.4 (CH2), 22.8 (CH2), 20.7
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy--D-glucopyranosyl]-(1→4)-6-
acetyl-2-benzoyl-3-benzyl--D-glucopyranoside (20). A solution of
compound 4 (518 mg, 0.450 mmol) in a mixed solvent CH2Cl2 and
water (10/1, v/v, 5.5 mL) was added with DDQ (153 mg, 2.01 mmol)
in three equal portions and in half-hour intervals at room
temperature. After 2 h, the reaction was quenched with NaHCO3(aq)
and Na2S2O3(aq). The crude product was extracted with CH2Cl2. The
organic layer was dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (ethyl acetate/hexanes = 1/2, v/v)
to give 3'-alcohol 20 (349 mg, 78%). []2D4 –4.4 (c 2.1 in CHCl ); IR
3
(thin film): νmax/cm–1 3444, 3064, 3031, 2951, 2113, 1732, 1688,
1603, 1496, 1453, 1422, 1367, 1314, 1267, 1250, 1171, 1093, 1056,
1029, 996, 838, 753, 711, 699, 665, 603, 555; 1H NMR (600 MHz,
CDCl3):  7.94 (2H, m, Ar-H), 7.48-7.42 (3H, m, Ar-H), 7.36-7.22 (8H,
m, Ar-H), 7.17-7.12 (7H, m, Ar-H), 5.41 (1H, s, PhCH), 5.22 (1H, dd, J
9.0, 8.9, 2-H), 4.77 (1H, d, J 11.3, ArCH2), 4.63 (1H, d, J 11.3, ArCH2),
4.60 (1H, d, J 11.9, 6-Ha), 4.52 (1H, d, J 7.9, 1-H), 4.41 (1H, d, J 8.1,
1’-H), 4.35-4.32 (2H, m, ArCH2), 4.33 (1H, dd, J 12.1, 4.6, 6-Hb), 4.17
(2H, m, CH2), 4.08 (1H, dd, J 10.6, 4.9, 6’-Ha), 3.98 (1H, t, J 9.1, 4-H),
3.81 (1H, m, CH2), 3.79 (1H, t, J 8.8, 3-H), 3.68-3.64 (2H, m, 5-H, 3’-
H), 3.44-3.34 (4H, m, 2’-H, 4’-H, 6’Hb, CH2), 3.22-3.18 (2H, m, 5’-H,
CH2), 3.00-2.89 (2H, m, CH2), 2.09 (3H, s, OCOCH3); 1.45 (2H, m,
CH2), 1.33 (2H, m, CH2), 1.10 (2H, m, CH2), 0.97 (2H, m, CH2), 0.02
(9H, m, TMS); 13C NMR (150 MHz, CDCl3):  170.5 (C), 165.0 (C),
157.0 (C), 138.03 (C), 137.99 (C), 136.7 (C), 133.0 (CH), 129.7 (C),
129.6 (CH), 129.3 (CH), 128.3 (CH), 128.2 (CH), 128.0 (CH), 127.6
(CH), 127.4 (CH), 127.0 (CH), 126.1 (CH), 101.9 (CH), 101.8 (CH),
100.9 (CH), 80.5 (CH), 80.3 (CH), 77.8 (CH), 74.4 (CH2), 73.0 (CH),
72.3 (CH), 69.7 (CH2), 68.2 (CH2), 67.0 (CH), 66.1 (CH), 63.4 (CH2),
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Organic & Biomolecular Chemistry
Page 10 of 19
ARTICLE
Journal Name
(CH3), 20.6 (CH2), 17.6 (CH3), -1.7 (CH3); HRMS (ESI): m/z calcd for
C99H109N7O25SiNa ([M+Na]+): 1847.7165, found: 1847.7163.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy--D-glucopyranosyl]-(1→4)-6-
(acetyl-2-benzoyl-3-benzyl--D-glucopyranosyl)-(1→3)-[2-azido-
4,6-O-benzylidene-2-deoxy--D-glucopyranosyl]-(1→4)-6-acetyl-2-
benzoyl-3-benzyl--D-glucopyranose (21). DDQ (150 mg, 0.661
mmol) was added to a solution of compound 5 (265 mg, 0.145
mmol) in a mixed solvent CH2Cl2 and water (10/1, v/v, 4.4 mL) in
three equal portions and in half-hour intervals at room
glucopyranosyl]-(1→4)-6-acetyl-2-benzoyl-3-benzyl--D-
glucopyranose (6). A mixture of donor 7 (85D.7OmI: 1g0,.91013.49/Dm0OoBl)0a1n04d8K
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acceptor 21 (126 mg, 74.8 mol) in anhydrous CH2Cl2 (4.6 mL) was
added to a reaction flask containing freshly dried 3 Å molecular
sieves (115 mg) at room temperature under nitrogen atmosphere.
After stirring for 1 h, the reaction flask was cooled to –78 °C and NIS
(24.0 mg, 107 mol) and TfOH (1.3 L, 18 mol) were sequentially
added. The reaction was gradually warmed up to –40 °C and kept
going for 3 h. After the donor 7 was consumed completely, the
reaction was filtered through Celite and quenched with NaHCO3(aq)
and Na2S2O3(aq). The crude product was extracted with CH2Cl2. The
organic layer was dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography (ethyl acetate/ hexane = 1/2, v/v) to yield
temperature. After 2 h, the reaction was quenched with the
addition of NaHCO3(aq) and Na2S2O3(aq). The crude product was
extracted with CH2Cl2. The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (ethyl
acetate/hexanes = 2/3, v/v) to give the 3’’’-alcohol 21 (168 mg,
compound 6 (145 mg, 77%). []2D4 –16.0 (c 1.7 in CHCl ); IR (thin
3
film): νmax/cm–1 3064, 3031, 2925, 2873, 2113, 1738, 1732, 1693,
1602, 1585, 1496, 1454, 1417, 1367, 1315, 1267, 1250, 1174, 1094,
1031, 1003, 918, 839, 753, 710, 699, 666, 603, 552, 476; 1H NMR
(600 MHz, CDCl3):  8.02(2H, m, Ar-H), 8.00 (2H, m, Ar-H), 7.92 (2H,
69%). []2D4 –1.9 (c 2.1 in CHCl ); IR (thin film): νmax/cm–1 3444, 2952,
3
2113, 1731, 1682, 1645, 1497, 1453, 1422, 1367, 1315, 1267, 1173,
1093, 1030, 1002, 838, 753, 699, 666; 1H NMR (600 MHz, CDCl3): 
8.02 (2H, m, Ar-H), 7.92 (2H, d, J 7.3, Ar-H), 7.56 (1H, m, Ar-H), 7.47- m, Ar-H), 7.82-7.79 (4H, m, Ar-H), 7.60-7.55 (2H, m, Ar-H), 7.49-7.41
7.41 (7H, m, Ar-H), 7.35-7.32 (8H, m, Ar-H), 7.30-7.27 (2H, m, Ar-H),
7.23 (1H, m, Ar-H), 7.16-7.10 (12H, m, Ar-H), 5.47 (1H, s, PhCH),
5.41 (1H, s, PhCH), 5.27 (1H, dd, J 8.9, 8.2, 2’’-H), 5.15 (1H, t, J 9.1,
2-H), 4.80 (1H, d, J 7.9 Hz, 1’’-H), 4.75 (1H, d, J 11.3, ArCH2), 4.69
(1H, d, J 11.2, ArCH2), 4.60 (1H, d, J 11.2, ArCH2), 4.55 (1H, d, J 11.3,
ArCH2), 4.48 (1H, d, J 11.3, 6-Ha), 4.45 (1H, d, J 7.9, 1-H), 4.34-4.31
(14H, m, Ar-H), 7.37-7.33 (8H, m, Ar-H), 7.30-7.28 (4H, m, Ar-H),
7.23 (1H, m, Ar-H), 7.18-7.09 (18H, m, Ar-H), 5.48 (1H, s, PhCH),
5.47 (1H, s, PhCH), 5.45 (1H, s, PhCH), 5.26 (1H, t, J 8.5, 2’’’’-H), 5.21
(1H, t, J 8.7, 2’’-H), 5.15 (1H, t, J 8.7, 2-H), 5.04 (1H, d, J 11.4, ArCH2),
4.93 (1H, d, J 11.4, ArCH2), 4.79-4.73 (3H, m, 1’’-H, 1’’’’-H, ArCH2),
4.70-4.67 (2H, m, ArCH2), 4.61 (1H, d, J 11.2, ArCH2), 4.55 (1H, d, J
(5H, m, 1’-H, 1’’’-H, 6-Hb, 6’’-Ha, CH2), 4.17-4.04 (6H, m, 6’-Ha, 6’’-Hb, 11.8, ArCH2), 4.53 (1H, d, J 11.6, ArCH2), 4.47 (1H, d, J 11.6, 6-Ha),
6’’’-Ha, CH2), 3.96 (1H, t, J 9.1, 4’’-H), 3.92 (1H, t, J 9.0, 4-H), 3.77
(1H, m, CH2), 3.76 (1H, t, J 8.9, 3’’-H), 3.71 (1H, t, J 8.8, 3-H), 3.65-
3.54 (4H, m, 3’-H, 3’’’-H, 5-H, 5’’-H), 3.44-3.37 (4H, m, 2’’’-H, 4’-H,
6’-Hb, 6’’’Hb), 3.34-3.27 (3H, m, 2’-H, 4’’’-H, CH2), 3.20-3.15 (2H, m,
5’-H, 5’’’-H), 3.00 (1H, s, CH2), 2.89 (1H, m, CH2), 2.04 (3H, s,
OCOCH3), 1.99 (3H, s, OCOCH3), 1.42 (2H, m, CH2), 1.31 (2H, m,
4.45 (1H, d, J 7.9, 1-H), 4.35-4.30 (5H, m, 1’’’-H, 1’’’’’-H, 6-Hb, 6’’-Ha,
CH2), 4.23-4.04 (9H, m, 1’-H, 6’-Ha, 6’’-Hb, 6’’’-Ha, 6’’’’-Ha, 6’’’’’-Ha,
CH2), 3.96 (1H, t, J 9.1, 4’’-H), 3.93-3.89 (2H, m, 4-H, 4’’’’-H), 3.86
(1H, m, 6’’’’-H), 3.78-3.74 (2H, m, 3’’’’-H, CH2), 3.70 (1H, t, J 8.9, 3-
H), 3.68 (1H, t, J 8.9, 3’’-H), 3.65-3.53 (7H, m, 3’-H, 3’’’-H, 3’’’’’-H, 4’-
H, 4’’’-H, 4’’’’’-H 5-H), 3.44-3.38 (5H, m, 2’’’’’-H, 5’’-H, 6’-Hb, 6’’’-Hb,
6’’’’’-Hb), 3.34 (1H, s, CH2), 3.30-3.13 (6H, m, 2’-H, 2’’’-H, 5’-H, 5’’’-H,
5’’’’-H, 5’’’’’-H), 2.99-2.89 (2H, m, CH2), 2.04 (3H, s, OCOCH3), 1.98
(3H, s, OCOCH3), 1.94 (3H, s, OCOCH3), 1.42 (2H, m, CH2), 1.30 (2H,
CH2), 1.10 (2H, m, CH2), 0.96 (2H, m, CH2), 0.01 (9H, m, TMS); 13
C
NMR (150 MHz, CDCl3):  170.4 (C), 170.3 (C), 165.1 (C), 164.9 (C),
156.5 (C), 138.1 (C), 137.9 (C), 137.9 (C), 136.8 (C), 136.6 (C), 133.03
(CH) 133.00 (CH) 129.8 (C), 129.7 (C), 129.59 (CH) 129.55 (CH)
129.30 (CH) 129.25 (CH) 128.36 (CH) 128.33 (CH) 128.28 (CH)
128.25 (CH) 128.1 (CH) 127.6 (CH) 127.5 (CH) 127.4 (CH) 127.1 (CH)
126.1 (CH) 126.0 (CH) 102.0 (CH) 101.84 (CH) 101.79 (CH) 101.5
(CH) 101.0 (CH) 100.9 (CH) 80.5 (CH) 80.4 (CH) 80.2 (CH) 79.4 (CH)
79.3 (CH) 77.5 (CH) 77.3 (CH) 74.54 (CH2), 74.51 (CH2), 73.3 (CH)
73.1 (CH) 72.9 (CH) 72.2 (CH) 69.7 (CH2), 68.2 (CH2), 67.0 (CH) 66.4
(CH) 66.3 (CH) 66.2 (CH) 63.5 (CH2), 62.2 (CH2), 62.0 (CH2), 60.3
(CH2), 50.2 (CH2), 45.9 (CH2), 28.9 (CH2), 27.6 (CH2), 22.9 (CH2), 20.8
(CH3), 20.8 (CH3), 17.7 (CH2), 14.1 (CH3), -1.6 (CH3); HRMS (ESI): m/z
m, CH2), 1.08 (2H, m, CH2), 0.97 (2H, m, CH2), 0.01 (9H, m, TMS); 13
C
NMR (150 MHz, CDCl3):  170.4 (C), 170.3 (C), 170.2 (C), 165.08 (C),
165.06 (C), 164.9 (C), 156.5 (C), 138.1 (C), 138.0 (C), 137.9 (C), 137.8
(C), 136.9 (C), 136.8 (C), 135.0 (C), 133.2 (C), 133.0 (CH), 129.8 (C),
129.8 (C), 129.63 (CH), 129.59 (CH), 129.3 (CH), 129.2 (CH), 129.1
(CH), 128.4 (CH), 128.34 (CH), 128.27 (CH), 128.26 (CH), 128.13
(CH), 128.09 (CH), 128.07 (CH), 127.9 (CH), 127.63 (CH), 127.58
(CH), 127.57 (CH), 127.5 (CH), 127.4 (CH), 127.1 (CH), 127.0 (CH),
126.02 (CH), 125.98 (CH), 125.95 (CH), 102.0 (CH), 101.93 (CH),
101.89 (CH), 101.49 (CH), 101.47 (CH), 101.3 (CH), 101.1 (CH), 101.0
calcd for C88H101N7O25SiNa ([M+Na]+): 1707.6539, found: 1707.6544. (CH), 100.9 (CH), 81.5 (CH), 80.5 (CH), 80.3 (CH), 80.2 (CH), 79.4
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-(6-acetyl-2-benzoyl-3-benzyl--D-
glucopyranosyl)-(1→3)-[2-azido-4,6-O-(benzylidene)-2-deoxy--D-
glucopyranosyl]-(1→4)-(6-acetyl-2-benzoyl-3-benzyl--D-
glucopyranosyl)-(1→3)-[2-azido-4,6-O-(benzylidene)-2-deoxy--D-
(CH), 79.34 (CH), 79.29 (CH), 79.1 (CH), 77.5 (CH), 77.4 (CH), 77.1
(CH), 74.9 (CH2), 74.7 (CH2), 74.57 (CH2), 74.56 (CH2), 73.4 (CH), 73.2
(CH), 73.1 (CH), 73.03 (CH), 72.96 (CH), 69.7 (CH2), 68.29 (CH2),
68.25 (CH2), 66.7 (CH), 66.4 (CH), 66.3 (CH), 66.2 (CH), 63.5 (CH2),
62.2 (CH2), 62.0 (CH2), 61.7 (CH2), 50.2 (CH2), 46.6 (CH2), 29.6 (CH2),
29.0 (CH2), 27.6 (CH2), 23.0 (CH2), 20.83 (CH3), 20.80 (CH3), 20.77
10 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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ARTICLE
(CH3), 17.8 (CH2), -1.5 (CH3); HRMS (ESI): m/z calcd for
film): νmax/cm–1 3440, 2953, 2111, 1726, 1645, 1634, 1V4ie9w6,Ar1ti4cl5e3O,nline
C134H144N10O36SiNa ([M+Na]+): 2520.9417, found: 2520.9437.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-2-benzoyl-3-benzyl--D-glucopyranoside
(22). Magnesium methoxide (7–8%) in MeOH (2.00 mL, 1.63 mmol)
was added to a solution of compound 4 (188 mg, 0.163 mmol) in
CH2Cl2 (4 mL) under nitrogen atmosphere at 0 °C. After 2 h, the
reaction was quenched with HCl(aq) until the solution was neutral.
The organic solvent was removed under reduced pressure. The
crude product was extracted with CH2Cl2. The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel
1420, 1367, 1315, 1269, 1175, 1095, 1071, 1002, 913, 837, 819,
DOI: 10.1039/D0OB01048K
752, 699, 666; 1H NMR (600 MHz, CDCl3):  8.04 (2H, m, Ar-H), 7.91
(2H, d, J 7.4, Ar-H), 7.82-7.74 (4H, m, Ar-H), 7.57 (1H, m, Ar-H), 7.49-
7.28 (10H, m, Ar-H), 7.37-7.33 (8H, m, Ar-H), 7.30-7.28 (2H, m, Ar-
H), 7.23 (1H, m, Ar-H), 7.12-7.09 (12H, m, Ar-H), 5.48 (1H, s, PhCH),
5.42 (1H, s, PhCH), 5.23 (1H, t, J 8.6 Hz, 2’’-H), 5.12 (1H, t, J 8.9, 2-H),
5.03 (1H, d, J 11.5, ArCH2), 4.92 (1H, d, J 11.5, ArCH2), 4.79-4.75 (2H,
m, 1’’-H, ArCH2), 4.71 (1H, d, J 11.3, ArCH2), 4.60 (1H, d, J 11.3,
ArCH2), 4.53 (2H, m, 1’’’-H, ArCH2), 4.47-4.44 (2H, m, 1-H, 1’-H), 4.35
(2H, m, CH2), 4.42-4.11 (4H, m, 6’-Ha, 6’’’-Ha, CH2), 4.06 (1H, t, J 9.2,
4-H), 4.01 (1H, t, J 9.2, 4’’-H), 3.90 (1H, d, J 11.94, 6-Ha), 3.74-3.73
(3H, m, 3-H, 6-Hb, CH2), 3.69-3.65 (3H, m, 3’’-H, 6’’-Ha, 6’’-Hb), 3.63
(1H, t, J 10.2, 3’-H), 3.60 (1H, t, J 9.2, 4’-H), 3.55 (1H, t, J 9.2, 3’’’-H),
3.51 (1H, t, J 9.1, 4’’’-H), 3.44-3.37 (5H, m, 2’-H, 5-H, 6’-Hb, 6’’’-Hb,
CH2), 3.32-3.27 (3H, m, 5’-H, 5’’-H, 5’’’-H), 3.25 (1H, t, J = 8.7 Hz, 2’’’-
H), 3.01-2.89 (2H, m, CH2), 1.43 (2H, m, CH2), 1.31 (2H, m, CH2), 1.11
(2H, m, CH2), 0.97 (2H, m, CH2), 0.01 (9H, m, TMS); 13C NMR (150
MHz, CDCl3):  165.2 (C), 165.0 (C), 156.6 (C), 138.2 (C), 138.1 (C),
137.08 (C), 137.05 (C), 135.1 (C), 133.2 (C), 133.1 (C), 133.0 (CH),
129.9 (C), 129.8 (C), 129.7 (CH), 129.6 (CH), 129.4 (CH), 129.1 (CH),
128.42 (CH), 128.39 (CH), 128.34 (CH), 128.28 (CH), 128.26 (CH),
128.14 (CH), 128.04 (CH), 128.01 (CH), 127.95 (CH), 127.7 (CH),
127.64 (CH), 127.60 (CH), 127.4 (CH), 127.12 (CH), 127.07 (CH),
126.1 (CH), 126.03 (CH), 125.99 (CH), 125.9 (CH), 102.0 (CH), 101.71
(CH), 101.66 (CH), 101.5 (CH), 101.3 (CH), 101.2 (CH), 81.7 (CH),
80.3 (CH), 80.0 (CH), 79.4 (CH), 79.1 (CH), 79.0 (CH), 76.4 (CH), 75.2
(CH), 75.1 (CH), 74.9 (CH2), 74.5 (CH2), 74.4 (CH2), 73.4 (CH), 73.0
(CH), 69.6 (CH2), 68.4 (CH2), 68.3 (CH2), 66.8 (CH), 66.6 (CH), 66.4
(CH), 66.0 (CH), 63.5 (CH2), 60.7 (CH2), 49.9 (CH2), 46.6 (CH2), 29.0
(CH2), 27.0 (CH2), 22.9 (CH2), -1.5 (CH3); HRMS (ESI): m/z calcd for
C95H105N7O23SiNa ([M+Na]+): 1763.6954, found: 1763.6958.
chromatography (ethyl acetate/ hexane = 1/2, v/v) to give alcohol
22 (179 mg, 99%). []2D4 –49.5 (c 3.1 in CHCl ); IR (thin film): νmax/cm–
3
1 3444, 2953, 2111, 1729, 1660, 1645, 1634, 1453, 1421, 1367,
1314, 1268, 1172, 1093, 1030, 1001, 837, 752, 698, 666; 1H NMR
(600 MHz, CDCl3):  7.95 (2H, m, Ar-H), 7.83-7.75 (4H, m, Ar-H),
7.52-7.45 (6H, m, Ar-H), 7.38-7.35 (5H, m, Ar-H), 7.32-7.30 (2H, m,
Ar-H), 7.26-7.25 (1H, m, Ar-H), 7.16-7.14 (7H, m, Ar-H), 5.50 (1H, s,
PhCH), 5.20 (1H, t, J 8.5 Hz, 2-H), 5.07 (1H, d, J 11.4, ArCH2), 4.95
(1H, d, J 11.4, ArCH2), 4.81 (1H, d, J 11.4, ArCH2), 4.65 (1H, d, J 11.4,
ArCH2), 4.59 (1H, d, J 8.0, 1’-H), 4.54 (1H, d, J 7.9, 1-H), 4.37-4.34
(2H, m, ArCH2), 4.21-4.12 (3H, m, 6’-Ha, CH2), 4.12 (1H, t, J 9.2, 4-H),
4.03-3.98 (2H, m, 6-Ha, 6-Hb), 3.82 (1H, m, CH2), 3.79 (1H, t, J 9.2, 3-
H), 3.69-3.65 (2H, m, 3’-H, 4’-H), 3.51-3.35 (5H, m, 2’-H, 5-H, 5’-H
6’Hb, CH2), 3.03-2.92 (2H, m, CH2), 1.47 (2H, m, CH2), 1.35 (2H, m,
CH2), 1.14 (2H, m, CH2), 0.99 (2H, m, CH2), 0.03 (9H, m, TMS); 13
C
NMR (150 MHz, CDCl3):  164.9 (C), 156.5 (C), 138.2 (C), 138.1 (C),
137.1 (C), 135.1 (C), 133.2 (C), 133.0 (C), 132.94 (CH), 129.85 (C),
129.6 (CH), 129.0 (CH), 128.4 (CH), 128.2 (CH), 128.1 (CH), 128.0
(CH), 127.9 (CH), 127.6 (CH), 127.3 (CH), 127.1 (CH), 127.0 (CH),
126.1 (CH), 126.0 (CH), 125.9 (CH), 101.7 (CH), 101.3 (CH), 101.1
(CH), 81.7 (CH), 80.2 (CH), 79.1 (CH), 75.3 (CH), 74.9 (CH2), 74.3
(CH2), 73.1 (CH), 69.6 (CH2), 68.4 (CH), 66.8 (CH), 65.9 (CH), 63.5
(CH2), 60.8 (CH2), 49.9 (CH2), 46.6 (CH2) 29.0 (CH2), 27.0 (CH2), 22.9
(CH2), 17.8 (CH2), -1.5 (CH3); HRMS (ESI): m/z calcd for
C62H72N4O13SiNa ([M+Na]+): 1159.4707, found: 1131.4775.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-(2-O-benzoyl-3-O-benzyl--D-
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-(2-O-benzoyl-3-O-benzyl--D-
glucopyranosyl)-(1→3)-[2-azido-4,6-O-benzylidene-2-deoxy-3-O-
(2-nathphylmethyl)--D-glucopyranosyl]-(1→4)-(2-O-benzoyl-3-O-
benzyl--D-glucopyranosyl)-(1→3)-[2-azido-4,6-O-benzylidene-2-
deoxy--D-glucopyranosyl]-(1→4)-2-O-benzoyl-3-O-benzyl--D-
glucopyranoside (24). Magnesium methoxide (7-8%) in MeOH (15.2
mL, 12.4 mmol) was added to a solution of compound 6 (1.02 g,
0.413 mmol) in CH2Cl2 (15.2 mL) under nitrogen atmosphere at 0 °C.
After 4 h, the reaction was quenched with HCl(aq) until the solution
was neutral. The organic solvent was removed under reduced
pressure. The crude product was extracted with CH2Cl2. The organic
layer was dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (ethyl acetate/ hexane=3/4, v/v) to give compound
glucopyranosyl)-(1→3)-[2-azido-4,6-O-benzylidene-2-deoxy--D-
glucopyranosyl]-(1→4)-2-O-benzoyl-3-O-benzyl--D-
glucopyranoside (23). Magnesium methoxide (7-8%) in MeOH (630
μL, 576 mol) was added to a solution of compound 5 (52.6 mg,
28.8 mol) in CH2Cl2 (2 mL) under nitrogen atmosphere at 0 °C.
After 2 h, the reaction was quenched with HCl(aq) until the solution
was neutral. The organic solvent was removed under reduced
pressure. The crude product was extracted with CH2Cl2. The organic
layer was dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (ethyl acetate/ hexane = 2/3, v/v) to give
24 (877 mg, 89%). []2D4 –38.4 (c 4.2 in CHCl ); IR (thin film): νmax/cm–
3
1 3418, 3064, 3031, 2929, 2881, 2222, 2112, 1728, 1694, 1603,
1585, 1496, 1453, 1418, 1367, 1315, 1269, 1176, 1095, 1030, 1003,
839, 753, 699, 667, 620, 553, 477; 1H NMR (600 MHz, CDCl3):  8.06-
8.03 (4H, m, Ar-H), 7.92 (2H, m, Ar-H), 7.83-7.75 (4H, m, Ar-H), 7.59-
7.55 (2H, m, Ar-H), 7.50-7.42 (14H, m, Ar-H), 7.37-7.32 (11H, m, Ar-
compound 23 (46.8 mg, 92%). []2D4 –14.8 (c 2.0 in CHCl ); IR (thin
3
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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Organic & Biomolecular Chemistry
Page 12 of 19
ARTICLE
Journal Name
H), 7.31-7.29 (2H, m, Ar-H), 7.25 (1H, m, Ar-H), 7.17-7.09 (17H, m,
5.47 (1H, s, PhCH), 5.21 (1H, t, J 7.8, 2-H), 5.03 (1H, d, VJi1ew1.A5rt,icAlerCOHnli2n)e,
DOI: 10.1039/D0OB01048K
Ar-H), 5.49 (1H, s, PhCH), 5.42 (1H, s, PhCH), 5.41 (1H, s, PhCH), 5.24 4.91 (1H, d, J 11.5, ArCH2), 4.78 (1H, d, J 11.4, ArCH2), 4.62 (1H, d, J
(1H, t, J 8.5, 2’’’’-H), 5.20 (1H, t, J 8.8, 2’’-H), 5.14 (1H, t, J = 8.8 Hz,
2-H), 5.05 (1H, d, J 11.5, ArCH2), 4.93 (1H, d, J 11.5, ArCH2), 4.80 (1H, (2H, m, ArCH2), 4.30 (1H, t, J 8.9, 4-H), 4.18-4.15 (2H, m, CH2), 4.11
d, J 11.3, ArCH2), 4.75-4.70 (4H, m, 1’’-H, 1’’’’-H, ArCH2), 4.62 (1H, d, (1H, dd, J 10.4, 4.7, 6’-Ha), 4.07 (1H, d, J 9.2, 5-H), 3.83-3.78 (5H, m,
J 11.3, ArCH2), 4.55-4.52 (3H, m, 1’-H, ArCH2), 4.47-4.45 (2H, m, 1-H, 3-H, CH2, OCH3), 3.61 (1H, t, J 9.2, 4’-H), 3.56 (1H, t, J 9.2, 3’-H),
11.4, ArCH2), 4.59 (1H, d, J 7.5, 1-H), 4.50 (1H, d, J 8.0, 1’-H), 4.36
1’’’’’-H), 4.42 (1H, d, J 8.0, 1’’’-H), 4.36 (2H, m, CH2), 4.19-4.13 (5H,
m, 6’-Ha, 6’’’-Ha, 6’’’’’-Ha, CH2), 4.06 (1H, t, J 9.2, 4-H), 4.01 (1H, t, J
9.2, 4’’’’-H), 3.98 (1H, t, J 9.2, 4’’-H), 3.90 (1H, d, J 11.7, 6-Ha), 3.78-
3.72 (3H, m, 3’’’’-H, 6-Hb, CH2), 3.70-3.48 (7H, m, 3-H, 3’’-H, 3’’’’’-H,
4’’’’’-H, 6’’-Ha, 6’’’’-Ha, 6‘’-Hb), 3.56-3.48 (4H, m, 3’-H, 3’’’-H, 4’-H,
4’’’-H), 3.47-3.36 (7H, m, 2’’’’’-H, 5-H, 6’-Hb, 6’’’-Hb, 6’’’’-H, 6’’’’’-Hb,
CH2), 3.33-3.20 (7H, m, 2’-H, 2’’’-H, 5’-H, 5’’-H, 5’’’-H, 5’’’’-H, 5’’’’’-
H), 3.01-2.90 (2H, m, CH2), 1.43-1.30 (4H, m, CH2), 1.13-1.10 (2H, m,
CH2), 0.99-0.96 (2H, m, CH2), 0.02 (9H, m, TMS); 13C NMR (150 MHz,
3.43-3.67 (3H, m, 2’-H, 6’-Hb, CH2), 3.30 (1H, td, J 7.4, 4.9, 5’-H),
3.01-2.91 (2H, m, CH2), 1.44 (2H, m, CH2), 1.34 (2H, m, CH2), 1.11
(2H, m, CH2), 0.96 (2H, m, CH2), 0.01 (9H, m, TMS); 13C NMR (150
MHz, CDCl3):  168.7 (C), 164.9 (C), 157.0 (C), 138.1 (C), 138.0 (C),
137.1 (C), 135.2 (C), 133.2 (C), 133.1 (C), 133.0 (C), 129.73 (C),
129.66 (CH), 129.1 (CH), 128.4 (CH), 128.31 (CH), 128.26 (CH), 128.1
(CH), 127.9 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.1 (CH),
126.9 (CH), 126.0 (CH), 125.9 (CH), 102.2 (CH), 101.4 (CH), 101.3
(CH), 81.5 (CH), 79.5 (CH), 79.0 (CH), 75.0 (CH2), 74.5 (CH), 74.4
CDCl3):  165.14 (C), 165.10 (C), 164.9 (C), 156.5 (C), 138.2 (C), 138.1 (CH2), 72.9 (CH), 69.9 (CH), 68.3 (CH), 66.7 (CH), 66.1 (CH), 63.5
(C), 138.0 (C), 137.1 (C), 137.0 (C), 135.1 (C), 133.2 (C), 133.03 (C),
132.99 (CH), 129.81 (C), 129.77 (C), 129.74 (C), 129.69 (CH), 129.6
(CH), 129.4 (CH), 129.3 (CH), 129.0 (CH), 128.40 (CH), 128.37 (CH),
128.29 (CH), 128.26 (CH), 128.2 (CH), 128.1 (CH), 128.02 (CH),
127.97 (CH), 127.9 (CH), 127.7 (CH), 127.62 (CH), 127.55 (CH), 127.4
(CH), 127.1 (CH), 127.0 (CH), 126.1 (CH), 126.00 (CH), 125.95 (CH),
125.9 (CH), 102.00 (CH), 101.7 (CH), 101.6 (CH), 101.43 (CH), 101.41
(CH), 101.3 (CH), 101.1 (CH), 101.0 (CH), 100.9 (CH), 81.6 (CH), 80.3
(CH), 80.0 (CH), 79.3 (CH), 79.3 (CH), 79.0 (CH), 79.0 (CH), 76.6 (CH),
76.5 (CH), 75.2 (CH), 75.1 (CH), 75.0 (CH), 74.9 (CH2), 74.5 (CH2),
74.4 (CH2), 73.4 (CH), 73.3 (CH), 73.00 (CH), 69.6 (CH2), 68.4 (CH2),
68.3 (CH2), 66.8 (CH), 66.54 (CH), 66.48 (CH), 66.32 (CH), 66.29 (CH),
65.9 (CH), 63.5 (CH2), 60.7 (CH2), 60.6 (CH2), 50.2 (CH2), 46.6 (CH2),
29.0 (CH2), 27.0 (CH2), 22.9 (CH2), 17.8 (CH2), -1.5 (CH3); HRMS (ESI):
m/z calcd for C128H139N10O33Si ([M+H]+): 2372.9301, found:
2372.9293.
(CH2), 52.8 (CH3), 49.9 (CH2), 45.9 (CH2), 29.0 (CH2), 27.3 (CH2), 23.0
(CH2), 17.8 (CH2), -1.5 (CH3); HRMS (ESI): m/z calcd for
C64H74N4O14SiNa ([M+Na]+): 1173.4863, found: 1173.4877.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-(methyl 2-benzoyl-3-O-benzyl--D-
glucopyranosyluronate)-(1→3)-[2-azido-4,6-O-benzylidene-2-
deoxy--D-glucopyranosyl]-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-
-D-glucopyranosyluronate (26). TEMPO (7.7 mg, 49.3 mol) and
BAIB (159 mg, 492 mol) were added to a solution of
tetrasaccharide 23 (172 mg, 98.5 mol) in mixed solvent of CH3CN
and H2O (10/1, v/v, 3.7 mL) at 0 °C. After 2 h, the reaction was
quenched with Na2S2O3(aq) and NaHCO3(aq). The crude product was
extracted with ethyl acetate. The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated under reduced
pressure. A solution of the crude product in CH2Cl2 (5 mL) was
added to a solution of diazomethane in ether (8 mL, 0.591 mmol) at
0 °C. After 20 min, the reaction solution was quenched with acetic
acid. The resulting mixture was concentrated under reduced
pressure and purified by silica gel column chromatography (ethyl
acetate/hexanes = 1/3, v/v) to yield the methyl ester 26 (126 mg,
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-methyl 2-benzoyl-3-benzyl--D-
glucopyranosyluronate (25). TEMPO (13.9 mg, 88.8 mol) and BAIB
(286 mg, 888 mol) were added to a solution of disaccharide 22
(395 mg, 0.356 mmol) in mixed solvent of CH3CN and H2O (10/1,
v/v, 10.5 mL) at 0 °C. After 2 h, the reaction was quenched with
Na2S2O3(aq) and NaHCO3(aq). The crude product was extracted with
ethyl acetate. The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. A solution of
the crude product in CH2Cl2 (10 mL) was added to a solution of
diazomethane in ether (12 mL, 2.14 mmol) at 0 °C. After 20 min, the
reaction was quenched with acetic acid. The resulting mixture was
concentrated under reduced pressure and purified by silica gel
column chromatography (ethyl acetate/hexanes = 1/3, v/v) to yield
the methyl ester 25 (341 mg, 84% in 2 steps). []2D4 –43.7 (c 1.5 in
CHCl3); IR (thin film): νmax/cm–1 3456, 3063, 3030, 2951, 2870, 2112,
1732, 1693, 1603, 1585, 1496, 1469, 1453, 1421, 1367, 1314, 1267,
1249, 1173, 1094, 1094, 1031, 1002, 857, 838, 754, 710, 698, 666,
602, 560, 475; 1H NMR (600 MHz, CDCl3):  7.92 (2H, m, Ar-H), 7.81-
7.73 (4H, m, Ar-H), 7.49-7.43 (6H, m, Ar-H), 7.37-7.35 (5H, m, Ar-H),
7.30-7.28 (2H, m, Ar-H), 7.23 (1H, m, Ar-H), 7.22-7.12 (7H, m, Ar-H),
71% in 2 steps). []2D4 –32.8 (c 1.2 in CHCl ); IR (thin film): νmax/cm–1
3
3063, 3030, 2952, 2925, 2856, 2222, 2114, 1955, 1732, 1693, 1602,
1496, 1454, 1439, 1421, 1373, 1315, 1267, 1223, 1174, 1094, 1029,
1003, 915, 857, 839, 755, 710 ,699, 666, 551, 476; 1H NMR (600
MHz, CDCl3):  8.01 (2H, m, Ar-H), 7.90 (2H, m, Ar-H), 7.82-7.74 (4H,
m, Ar-H), 7.55 (1H, m, Ar-H), 7.48-7.43 (10H, m, Ar-H), 7.36-7.33
(8H, m, Ar-H), 7.29-7.28 (2H, m, Ar-H), 7.23 (1H, m, Ar-H), 7.17-7.10
(12H, m, Ar-H), 5.47 (1H, s, PhCH), 5.42 (1H, s, PhCH), 5.30 (1H, t, J
8.7, 2’’-H), 5.17 (1H, t, J 8.8, 2-H), 5.03 (1H, d, J 11.5, ArCH2), 4.91
(1H, d, J 11.5, ArCH2), 4.85 (d, J 7.9, 1’’-H), 4.75 (1H, d, J 11.4,
ArCH2), 4.71 (1H, d, J 11.4, ArCH2), 4.59 (1H, d, J 11.4, ArCH2), 4.54
(1H, d, J 11.3, ArCH2), 4.51 (1H, d, J 7.5, 1-H), 4.42 (1H, d, J 8.0, 1’’’-
H), 4.39 (1H, d, J 8.0, 1’-H), 4.35 (2H, m, CH2), 4.27 (1H, t, J 8.8, 4’’-
H), 4.21-4.15 (3H, m, 4-H, CH2), 4.11-4.07 (2H, m, 6’-Ha, 6’’’-Ha),
3.94-3.92 (2H, m, 5-H, 5’’-H), 3.78-3.75 (2H, m, 3’’-H, CH2), 3.70 (1H,
t, J 8.9, 3-H), 3.66 (3H, s, OCH3), 3.62-3.51 (6H, m, 3’-H, 3’’’-H, 4’’’-H,
OCH3), 3.41-3.33 (4H, m, 2’’’-H, 6’-H, 6’’’-H, CH2), 3.30-3.20 (3H, m,
12 | J. Name., 2012, 00, 1-3
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Page 13 of 19
Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
2’-H, 5’-H, 5’’’-H), 3.00-2.90 (3H, m, CH2), 1.41 (2H, m, CH2), 1.30
4’’’’’-H, OCH3), 3.39 (1H, t, J 10.3, 6’-Hb), 3.36-3.25 (8HV,imew,A2rt’i-cHle,O5n’l-ine
DOI: 10.1039/D0OB01048K
(2H, m, CH2), 1.08 (2H, m, CH2), 0.96 (2H, m, CH2, 0.01 (9H, m, TMS); H, 6’’’-Hb, 6’’’’’-Hb, CH2, OCH3), 3.20-3.15 (4H, m, 2’’’-H, 2’’’’’-H, 5’’’-
13C NMR (150 MHz, CDCl3):  168.6 (C), 168.4 (C), 164.90 (C), 164.86
(C), 156.5 (C), 138.1 (C), 137.94 (C), 137.89 (C), 137.04 (C), 136.98
(C), 135.22 (C), 133.19 (C), 133.1 (CH), 133.04 (C), 132.96 (CH),
129.9 (C), 129.7 (CH), 129.6 (CH), 129.1 (CH), 128.9 (CH), 128.41
(CH), 128.37 (CH), 128.30 (CH), 128.25 (CH), 128.2 (CH), 128.12
(CH), 128.08 (CH), 128.05 (CH), 127.9 (CH), 127.7 (CH), 127.6 (CH),
127.4 (CH), 127.1 (CH), 127.0 (CH), 126.9 (CH), 126.1 (CH), 126.0
(CH), 125.9 (CH), 102.3 (CH), 102.1 (CH), 101.5 (CH), 101.4 (CH),
101.3 (CH), 101.0 (CH), 81.5 (CH), 79.5 (CH), 79.5 (CH), 79.3 (CH),
79.0 (CH), 78.9 (CH), 78.8 (CH), 75.0 (CH2), 74.5 (CH2), 74.4 (CH),
74.3 (CH2), 74.2 (CH), 73.1 (CH), 72.8 (CH), 69.9 (CH2), 68.3 (CH2),
68.1 (CH2), 66.7 (CH), 66.1 (CH), 66.0 (CH), 63.5 (CH2), 52.6 (CH3),
52.4 (CH3), 50.2 (CH2), 45.9 (CH2), 28.9 (CH2), 27.6 (CH2), 23.0 (CH2),
17.8 (CH2), -1.5 (CH3); HRMS (ESI): m/z calcd for C97H105N7O25SiNa
([M+Na]+): 1819.6852, found: 1819.6849.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-(methyl 2-benzoyl-3-O-benzyl--D-
glucopyranosyluronate)-(1→3)-[2-azido-4,6-O-benzylidene-2-
deoxy--D-glucopyranosyl]-(1→4)-(methyl 2-benzoyl-3-O-benzyl--
D-glucopyranosyluronate)-(1→3)-[2-azido-4,6-O-benzylidene-2-
deoxy--D-glucopyranosyl]-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-
-D-glucopyranosyluronate (27). TEMPO (19.2 mg, 0.123 mmol)
and BAIB (384 mg, 1.19 mmol) were added to a solution of
hexasaccharide 26 (362 mg, 0.153 mmol) in mixed solvent of CH3CN
and H2O (10/1, v/v, 33 mL) at 0 °C. After 5 h, the reaction was
quenched with Na2S2O3(aq) and NaHCO3(aq). The crude product was
extracted with ethyl acetate. The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated under reduced
pressure. A solution of crude product in CH2Cl2 (30 mL) was added
to a solution of diazomethane in ether (10 mL, 0.765 mmol). After
20 min, the reaction was quenched with acetic acid. The mixture
was concentrated under reduced pressure and purified by silica gel
column chromatography (ethyl acetate/hexanes = 1/2, v/v) to yield
the methyl ester 27 (199 mg, 53% in 2 steps). []2D4 –32.5 (c 5.3 in
CHCl3); IR (thin film): νmax/cm–1 3032, 2951, 2115, 1735, 1691, 1602,
1497, 1453, 1368, 1315, 1267, 1223, 1174, 1094, 1072, 1029, 1004,
840, 753, 710, 699, 551, 476; 1H NMR (600 MHz, CDCl3):  8.01 (2H,
m, Ar-H), 7.97 (2H, m, Ar-H), 7.88 (2H, m, Ar-H), 7.81-7.73 (4H, m,
Ar-H), 7.58-7.52 (2H, m, Ar-H), 7.47-7.39 (12H, m, Ar-H), 7.38-7.31
(13H, m, Ar-H), 7.30-7.26 (2H, m, Ar-H), 7.21-7.21 (1H, m, Ar-H),
7.11-7.05 (17H, m, Ar-H), 5.46 (1H, s, PhCH), 5.40 (1H, s, PhCH), 5.38
(1H, s, PhCH), 5.28 (1H, t, J 8.6, 2’’’’-H), 5.23 (1H, t, J 8.8, 2’’-H), 5.15
(1H, t, J 8.4, 2-H), 5.02 (1H, d, J 11.5, ArCH2), 4.90 (1H, d, J 11.5,
ArCH2), 4.83 (1H, d, J 7.9, 1’’’’-H), 4.77-4.74 (2H, m, 1’’-H, ArCH2),
4.68 (1H, d, J 11.2, ArCH2), 4.67 (1H, d, J 11.2 Hz, ArCH2), 4.49 (1H,
d, J 11.4, ArCH2), 4.53-4.48 (3H, m, 1-H, ArCH2), 4.41 (1H, d, J 8.1, 1’-
H), 4.36-4.28 (4H, m, 1’’’-H, 1’’’’’-H, CH2), 4.25 (1H, t, J 8.9, 4’’’’-H),
4.18-4.13 (4H, m, 4-H, 4’’-H, CH2), 4.09 (1H, dd, J 9.8, 4.9, 6’’’’’-Ha),
4.07-4.04 (2H, m, 6’-Ha, 6’’’-Ha), 3.92-3.90 (2H, m, 5-H, 5’’-H), 3.79-
3.73 (3H, m, 3’’’’-H, 5’’-H, CH2), 3.69-3.64 (5H, m, 3-H, 3’’-H, OCH3),
3.60 (1H, t, J 9.2, 4’-H), 3.55-3.43 (8H, m, 3’-H, 3’’’-H, 3’’’’’-H, 4’’’-H,
H, 5’’’’’-H), 3.98-2.87 (2H, m, CH2), 1.42-1.38 (2H, m, CH2), 1.28 (2H,
m, CH2), 1.08-1.03 (2H, m, CH2), 0.95-0.91 (2H, m, CH2), 0.01 (m, 9H;
TMS); 13C NMR (150 MHz, CDCl3):  168.6 (C), 168.4 (C), 168.3 (C),
164.90 (C), 164.87 (C), 157.0 (C), 138.1 (C), 137.93 (C), 137.86 (C),
137.04 (C), 136.97 (C), 135.2 (C), 133.2 (C), 133.10 (CH), 133.05 (C),
133.0 (C), 129.9 (C), 129.8 (C), 129.70 (C), 129.66 (CH), 129.6 (CH),
129.1 (CH), 128.94 (CH), 128.85 (CH), 128.42 (CH), 128.37 (CH),
128.31 (CH), 128.28 (CH), 128.25 (CH), 128.2 (CH), 128.10 (CH),
128.07 (CH), 128.05 (CH), 128.0 (CH), 127.9 (CH), 127.7 (CH), 127.63
(CH), 127.60 (CH), 127.5 (CH), 127.4 (CH), 127.1 (CH), 126.9 (CH),
126.04 (CH), 126.03 (CH), 125.94 (CH), 125.91 (CH), 125.86 (CH),
102.3 (CH), 102.2 (CH), 102.1 (CH), 101.6 (CH), 101.5 (CH), 101.4
(CH), 101.3 (CH), 101.0 (CH), 100.9 (CH), 81.5 (CH), 79.54 (CH),
79.51 (CH), 79.4 (CH), 79.3 (CH), 79.1 (CH), 78.90 (CH), 78.85 (CH),
75.0 (CH2), 74.6 (CH2), 74.5 (CH2), 74.40 (CH), 74.35 (CH2), 74.2 (CH),
74.1 (CH), 73.0 (CH), 72.9 (CH), 72.7 (CH), 70.0 (CH2), 68.3 (CH2),
68.1 (CH2), 66.7 (CH), 66.0 (CH), 63.5 (CH2), 52.7 (CH3), 52.4 (CH3),
52.3 (CH3), 50.2 (CH2), 45.9 (CH2), 29.7 (CH2), 28.9 (CH2), 27.2 (CH2),
23.0 (CH2), 17.8 (CH2), -1.5 (CH3); HRMS (ESI): m/z calcd for
C131H140N10O36Si ([M+2H+]): 1228.9611, found: 1228.9610.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-3-benzyl--D-glucopyranosyluronate
sodium salt (28). A mixture of 5 N NaOH(aq), H2O, and MeOH
(1/2/15, v/v/v, 50 mL) was added to a solution of compound 25
(341 mg, 299 mol) in CHCl3 (10 mL) at room temperature. After 1
day, H2O (50 mL) was added to the reaction. The organic solvent
was removed under reduced pressure. The crude product was
extracted with CH2Cl2. The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by Sephadex LH-20 column (MeOH) and then
passed through a column of Biorad 50X80 Na+ resin (MeOH/H2O =
20/1) to give the carboxylate 28 (291 mg, 93%). 1H NMR (600 MHz,
CD3OD):  7.69-7.57 (4H, m, Ar-H), 7.36-7.11 (18H, m, Ar-H), 5.44
(1H, m, PhCH), 4.90-4.86 (3H, m, ArCH2), 4.72-4.70 (1H, m, ArCH2),
4.63 (1H, d, J 7.6, 1’-H), 4.36-4.35 (2H, m, ArCH2), 4.22 (1H, d, J 7.7,
1-H), 4.10 (2H, s, CH2), 4.00-3.97 (2H, m, 4-H), 3.76-3.74 (1H, m, 5-
H), 3.69 (1H, m, CH2), 3.57-3.53 (2H, m, 3’-H, 5’-H), 3.45-3.37 (4H,
m, 3-H, 4’-H, 6’-Hb, CH2), 3.30-3.25 (2H, m, 2-H, 2’-H), 3.08 (2H, m,
CH2), 1.46-1.39 (2H, m, CH2), 1.18 (2H, m, CH2), 0.92-0.87 (2H, m,
CH2), -0.07 (9H, m, TMS); 13C NMR (150 MHz, CD3OD):  175.5 (C),
158.5 (C), 140.6 (C), 139.5 (C), 139.2 (C), 137.2 (C), 134.8 (C), 134.6
(C), 130.1 (CH), 129.8 (C), 129.33 (CH), 129.26 (CH), 129.1 (CH),
128.8 (CH), 128.5 (CH), 128.0 (CH), 127.5 (CH), 127.3 (CH), 127.2
(CH), 127.1 (CH), 104.8 (CH), 102.8 (CH), 102.7 (CH), 84.2 (CH), 83.0
(CH), 80.3 (CH), 79.6 (CH), 77.8 (CH), 76.0 (CH2), 75.7 (CH2), 75.0
(CH), 71.6 (CH2), 71.0 (CH2), 69.7 (CH2), 68.4 (CH), 67.5 (CH2), 65.1
(CH2), 51.4 (CH2), 47.5 (CH2), 30.9 (CH2), 29.0 (CH2) 24.4 (CH2), 18.8
(CH2), -1.3 (CH3); HRMS (ESI): m/z calcd for C55H66N4O13SiNa
([M+H]+): 1041.4288, found: 1041.4286.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 13
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Organic & Biomolecular Chemistry
Page 14 of 19
ARTICLE
Journal Name
4.17 (1H, d, J = 7.8 Hz, 1-H), 4.10-4.02 (8H, m, 4-H, 4’’-H, 4’’’’-H, 6’-
DOI: 10.1039/D0OB01048K
Ha, 6’’’-Ha, 6’’’’’-Ha, CH2), 3.69-3.62 (6H, m, 3’-H, 3’’’-H, 5-H, 5’’-H,
glucopyranosyl]-(1→4)-(3-O-benzyl--D-glucopyranosyluronate)-
(1→3)-[2-azido-4,6-O-benzylidene-2-deoxy--D-glucopyranosyl]-
(1→4)-3-O-benzyl--D-glucopyranosyluronate disodium salt (29). A
mixture of 5 N NaOH(aq), H2O, and MeOH (1/2/15, v/v/v, 4mL) was
added to a solution of compound 26 (126 mg, 69.9 mol) in CHCl3
(1.6 mL) at room temperature. After 2 days, H2O (10 mL) was added
to the reaction. The organic solvent was removed under reduced
pressure. The crude product was extracted with CH2Cl2. The organic
layer was dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by Sephadex LH-
20 column (MeOH) and then passed through a column of Biorad
50X80 Na+ resin (MeOH/H2O = 20/1) to give the carboxylate 29
(90.4 mg, 81%). 1H NMR (600 MHz, CD3OD):  7.77-7.65 (4H, m, Ar-
H), 7.41-7.19 (28H, m, Ar-H), 5.51 (1H, s, PhCH), 5.45 (1H, s, PhCH),
4.95-4.78 (4H, m, ArCH2), 4.62-4.54 (5H, m, 1’-H, 1’’-H, 1’’’-H,
ArCH2), 4.44 (2H, s, ArCH2), 4.27 (1H, m, 1-H), 4.17 (1H, s, CH2), 4.08-
4.05 (4H, m, 4-H, 4’’-H, 6’-Ha, 6’’’-Ha), 3.81-3.73 (3H, m, 5-H, 5’’-H,
CH2), 3.66-3.44 (12H, m, 2’-H, 3-H, 3’-H, 3’’-H, 3’’’-H, 4’-H, 4’’’-H, 5’-
H, 5’’’-H, 6’-Hb, 6’’’-Hb, CH2), 3.36-3.27 (3H, m, 2-H, 2’-H, 2’’’-H), 3.17
(2H, m, CH2), 1.53-1.47 (4H, m, CH2), 1.24 (2H, m, CH2), 1.00-0.95
(2H, m, CH2), 0.02 (9H, m, TMS); 13C NMR (150 MHz, CD3OD): 
176.2 (C), 175.5 (C), 159.0 (C), 140.7 (C), 140.6 (C), 139.5 (C), 139.2
(C), 138.9 (C), 137.1 (C), 134.8 (C), 134.7 (C), 130.1 (CH), 130.0 (CH),
129.8 (CH), 129.31 (CH), 129.29 (CH), 129.23 (CH), 129.19 (CH),
129.12 (CH), 129.11 (CH), 128.90 (CH), 128.8 (CH), 128.6 (CH), 128.5
(CH), 128.1 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH), 127.2 (CH),
127.1 (CH), 105.0 (CH), 103.0 (CH), 102.8 (CH), 102.5 (CH), 84.3
(CH), 84.1 (CH), 83.0 (CH), 80.2 (CH), 80.1 (CH), 79.3 (CH), 78.9 (CH),
78.8 (CH), 76.1 (CH2), 75.7 (CH2), 75.6 (CH2), 75.0 (CH), 74.8 (CH),
70.9 (CH2), 69.7 (CH2), 68.5 (CH), 68.4 (CH), 68.3 (CH), 68.1 (CH),
65.1 (CH2), 51.3 (CH2), 48.2 (CH2), 30.5 (CH2), 28.6 (CH2), 24.4 (CH2),
18.8 (CH2), -1.3 (CH3); HRMS (ESI): m/z calcd for C81H92N7O23Si
([M+H]+): 1604.5804, found: 1604.5808.
View Article Online
5’’’’-H, CH2), 3.55-3.33 (15H, m, 2’’-H, 2’’’-H, 2’’’’-H, 2’’’’’-H, 3-H, 3’’-
H, 3’’’’-H, 3’’’’’-H, 4’-H, 4’’’-H, 4’’’’’-H, 6’-Hb, 6’’’-Hb, 6’’’’’-Hb, CH2),
3.28-3.25 (5H, m, 2-H, 2’-H, 5’-H, 5’’’-H, 5’’’’’-H), 3.14-3.09 (2H, m,
CH2), 1.46-1.40 (4H, m, CH2), 1.19-1.18 (2H, m, CH2), 0.93 (2H, m,
CH2), -0.05--0.01 (9H, m, TMS); 13C NMR (150 MHz, CD3OD):  175.9
(C), 175.4 (C), 175.3 (C), 159.0 (C), 140.9 (C), 140.79 (C), 140.75 (C),
139.6 (C), 139.3 (C), 139.0 (C), 137.3 (C), 134.9 (C), 134.7 (C), 130.1
(CH), 129.8 (CH), 129.34 (CH), 129.26 (CH), 129.19 (CH), 129.16
(CH), 129.13 (CH), 129.06 (CH), 128.9 (CH), 128.8 (CH), 128.5 (CH),
128.4 (CH), 128.0 (CH), 127.7 (CH), 127.5 (CH), 127.4 (CH), 127.1
(CH), 127.0 (CH), 104.8 (CH), 104.3 (CH), 104.1 (CH), 102.8 (CH),
102.5 (CH), 102.5 (CH), 102.2 (CH), 84.4 (CH), 84.2 (CH), 83.1 (CH),
80.8 (CH), 80.7 (CH), 80.5 (CH), 79.6 (CH), 79.4 (CH), 79.3 (CH), 79.3
(CH), 79.20 (CH), 79.17 (CH), 75.8 (CH2), 75.6 (CH2), 75.5 (CH2), 75.1
(CH), 74.9 (CH), 70.7 (CH2), 69.8 (CH2), 69.7 (CH2), 68.7 (CH), 68.4
(CH), 67.7 (CH), 67.5 (CH), 65.1 (CH2), 51.4 (CH2), 48.2 (CH2), 47.6
(CH2), 30.6 (CH2), 29.2 (CH2), 24.5 (CH2), 18.8 (CH2), -1.3 (CH3);
HRMS (ESI): m/z calcd for C107H121N10O33Si ([M+H+]): 2100.7746,
found: 2100.7766.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
acetamido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--
D-glucopyranosyl]-(1→4)-3-benzyl--D-glucopyranosyluronate
sodium salt (31). PMe3 (1.0 M in THF, 0.656 mL, 0.656 mmol),
MeOH (82 L), and 0.1 N NaOH(aq) (82 L) were sequentially added
to a solution of carboxylate 28 (17.2 mg, 16.5 mol) in THF (656 L).
After 3 h, the reaction was concentrated under reduced pressure
and dried in vacuo. To a solution of the dried crude residue and
Et3N (69.0 L, 496 mol) in MeOH (1.0 mL) was added Ac2O (23.4
L, 248 mol) in three equal portions and in half-hour intervals at
room temperature. After 1.5 h, the reaction was concentrated
under reduced pressure and purified by silica gel column
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--D-
glucopyranosyl]-(1→4)-(3-O-benzyl--D-glucopyranosyluronate)-
(1→3)-[2-azido-4,6-O-benzylidene-2-deoxy--D-glucopyranosyl]-
(1→4)-(3-O-benzyl--D-glucopyranosyluronate)-(1→3)-[2-azido-
4,6-O-benzylidene-2-deoxy--D-glucopyranosyl]-(1→4)-3-O-benzyl-
-D-glucopyranosyluronate trisodium salt (30). A mixture of 5 N
NaOH(aq), H2O, and MeOH (1/2/15, v/v/v, 1mL) was added to a
solution of compound 27 (10 mg, 4.07 mol) in CHCl3 (0.5 mL) at
room temperature. After 3 days, H2O (10 mL) was added to the
chromatography (MeOH/CHCl3 = 1/15, v/v) and then passed
through a column of Biorad 50X80 Na+ resin (MeOH/H2O = 20/1) to
yield the acetamide 31 (13.2 mg, 76%). HRMS (ESI): m/z calcd for
C57H70N2NaO14Si ([M+H+]): 1057.4489, found: 1057.4481.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
acetamido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--
D-glucopyranosyl]-(1→4)-(3-O-benzyl--D-glucopyranosyluronate)-
(1→3)-[2-acetamido-4,6-O-benzylidene-2-deoxy--D-
glucopyranosyl]-(1→4)-3-O-benzyl--D-glucopyranosyluronate
disodium salt (32). PMe3 (1.0 M in THF, 40 L, 40 mol), MeOH (40
reaction. The organic solvent was removed under reduced pressure. L), and 0.1 N NaOH(aq) (40 L) were sequentially added to a
The crude product was extracted with CH2Cl2. The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by Sephadex LH-20
column (MeOH) and then passed through a column of Biorad 50X80
Na+ resin (MeOH/H2O = 20/1) to give the carboxylate acid 30 (5.7
mg, 65%). 1H NMR (600 MHz, CD3OD):  7.68 (1H, m, Ar-H), 7.64-
7.63 (2H, m, Ar-H), 7.58 (1H, m, Ar-H), 7.38-7.30 (15H, m, Ar-H),
7.25-7.12 (23H, m, Ar-H), 5.42 (1H, s, PhCH), 5.39 (1H, s, PhCH), 5.37
(1H, s, PhCH), 4.87-4.80 (4H, m, ArCH2), 4.71 (3H, m, 1’-H, 1’’’-H,
1’’’’’-H), 4.67-4.60 (6H, m, 1’’-H, 1’’’’-H, ArCH2), 4.36 (2H, s, ArCH2),
solution of carboxylate 29 (6.4 mg, 4.0 mol) in THF (0.32 mL). After
3 h, the reaction was concentrated under reduced pressure and
dried in vacuo. To a solution of dried crude residue and Et3N (37 L,
240 mol) in MeOH (1.0 mL) was added Ac2O (11 L, 120 mol) in
three equal portions and in half-hour intervals at room
temperature. After 1.5 h, the reaction was concentrated under
reduced pressure and purified by silica gel column chromatography
(MeOH/CHCl3 = 1/12, v/v) and then passed through a column of
Biorad 50X80 Na+ resin (MeOH/H2O = 20/1) to yield the acetamide
14 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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ARTICLE
32 (2.8 mg, 43%). HRMS (ESI): m/z calcd for C85H102N3O25Si ([M+H+]): passed through a column of Biorad 50X80 Na+ resinV(ieHw2AOrt)ictleoOgnilivnee
1
DOI: 10.1039/D0OB01048K
1592.6566, found: 1592.6575.
disaccharide 34 (4.0 mg, 58%). H NMR (600 MHz, D2O):  4.53 (1H, d,
J 8.5 Hz, 1’-H), 4.43 (1H, d, J 8.0 Hz, 1-H), 4.17 (2H, m, CH2), 3.92-3.86
(2H, m, 6’-Ha, CH2), 3.77-3.62 (5H, m, 2’-H, 4-H, 5-H, 6’-Hb, CH2), 3.57
(1H, t, J 9.2, 3-H), 3.51 (1H, t, J 8.7, 3’-H), 3.46-3.45 (2H, m, 4’-H, 5’-
H), 3.31 (1H, t, J 8.2, 2-H), 3.01 (2H, t, J 6.7, CH2), 2.04 (3H, s, OCOCH3),
1.61 (2H, m, CH2), 1.49 (2H, m, CH2), 1.36 (2H, m, CH2), 0.98 (2H, t, J
7.9, CH2), 0.02 (9H, s, TMS); 13C NMR (150 MHz, D2O):  175.2 (C),
174.7 (C), 159.3 (C), 102.7 (CH), 101.1 (CH), 80.3 (CH), 77.0 (CH), 76.2
(CH), 74.2 (CH), 74.1 (CH), 73.1 (CH), 70.9 (CH2), 70.0 (CH), 64.3 (CH2),
60.8 (CH2), 55.7 (CH), 40.5 (CH2), 28.9 (CH2), 28.7 (CH2), 22.7 (CH3),
22.5 (CH2), 12.3 (CH2), -2.1 (CH3); HRMS (ESI): m/z calcd for
C32H52N2O14SiNa ([M+H]+): 717.3270, found: 717.3261.
N-Benzyl-N-2-(trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-
acetamido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthylmethyl)--
D-glucopyranosyl]-(1→4)-(3-O-benzyl--D-glucopyranosyluronate)-
(1→3)-[2-acetamido-4,6-O-benzylidene-2-deoxy--D-
glucopyranosyl]-(1→4)-(3-O-benzyl--D-glucopyranosyluronate)-
(1→3)-[2-acetamido-4,6-O-benzylidene-2-deoxy--D-
glucopyranosyl]-(1→4)-3-O-benzyl--D-glucopyranosyluronate
trisodium salt (33). PMe3 (1.0 M solution in THF, 98.8 L, 98.8
mol), MeOH (98.8 L), and 0.1 N NaOH(aq) (98.8 L) were
sequentially added to a solution of carboxylate 30 (14.3 mg, 6.59
mol) in THF (800 L). After 5 h, the reaction was concentrated
under reduced pressure and dried in vacuo. To a solution of dried
crude residue and Et3N (92.6 L, 594 mol) in MeOH (1.0 mL) was
added with Ac2O (27.9 L, 297 mol) in three equal portions and in
half-hour intervals at room temperature. After 1.5 h, the reaction
was concentrated under reduced pressure and purified by silica gel
column chromatography (MeOH/CHCl3 = 1/8, v/v) and then passed
through a column of Biorad 50X80 Na+ resin (MeOH/H2O = 20/1) to
yield the acetamide 33 (3.4 mg, 23%). HRMS (ESI): m/z calcd for
C113H134N4O36Si ([M+2H+]): 1075.4268, found: 1075.4259.
N-2-(Trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-acetamido-2-
deoxy--D-glucopyranosyl]-(1→4)--D-glucopyranosyluronate
sodium salt (34). Method A: A mixture of the acetamide 31 (13.2 mg,
12.5 mol) and 20% Pd(OH)2 on carbon (39.6 mg) in a MeOH (1 mL)
was equipped with a hydrogen balloon at room temperature. After
16 h, H2O (5 mL) was added to the reaction and kept going for 1 day
under hydrogen. The mixture was filtered through Celite and the
filtrate was concentrated under reduced pressure. The residue was
purified by Sephadex G-10 (H2O) and then passed through a column
of Biorad 50X80 Na+ resin (H2O) to give disaccharide 34 (3.9 mg, 42%).
Method B: A mixture of compound 28 (19.1 mg, 18.3 mol) and 20%
Pd(OH)2 on carbon (57.3 mg) in MeOH (1 mL) was equipped with a
hydrogen balloon at room temperature. After 16 h, H2O (5 mL) was
added to the reaction and kept going for 1 day under hydrogen. The
mixture was filtered through Celite, and the filtrate was
concentrated under reduced pressure. Ac2O (25.8 μL, 275 mol) and
Et3N (85.6 μL, 0.549 mmol) were added to the solution of the crude
residue in MeOH (1 mL) in three equal portions and in 10-min
intervals. After 30 min, the mixture was concentrated under reduced
pressure. The residue was purified by Sephadex G-10 (H2O) and then
passed through a column of Biorad 50X80 Na+ resin (H2O) to give
disaccharide 36 (4.2 mg, 31%). Method C: A mixture of compound 28
(9.7 mg, 9.3 mol) and Pd black (50 mg) in a MeOH (1.5 mL) was
equipped with a hydrogen balloon at room temperature. After 16 h,
H2O (7.5 mL) was added to the reaction and kept going for 1 day
under hydrogen. The mixture was filtered through Celite, and the
filtrate was concentrated under reduced pressure. Ac2O (13.1 μL, 140
mol) and Et3N (43.5 μL, 280 mol) were added to the solution of
crude residue in MeOH (1 mL) in three equal portions and in 10-min
intervals. After 30 min, the mixture was concentrated under reduced
pressure. The residue was purified by Sephadex G-10 (H2O) and then
N-2-(Trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-acetamido-
2-deoxy--D-glucopyranosyl]-(1→4)-(-D-glucopyranosyluronate)-
(1→3)-[2-acetamido-2-deoxy--D-glucopyranosyl]-(1→4)--D-
glucopyranosyluronate disodium salt (35). Method A: A mixture of
the acetamide 32 (2.8 mg, 1.7 mol) and 20% Pd(OH)2 on carbon
(8.4 mg) in a MeOH (1 mL) was equipped with a hydrogen balloon
at room temperature. After 16 h, H2O (5 mL) was added to the
reaction and kept going for 1 day under hydrogen. The mixture was
filtered through Celite and the filtrate was concentrated under
reduced pressure. The residue was purified by Sephadex G-10 (H2O)
and then passed through a column of Biorad 50X80 Na+ resin (H2O)
to give disaccharide 35 (0.9 mg, 50%). Method B: A mixture of
compound 29 (22.0 mg, 0.0137 mmol) and 20% Pd(OH)2 on carbon
(75.0 mg) in MeOH (1 mL) was equipped with a hydrogen balloon at
room temperature. After 16 h, H2O (5 mL) was added to the
reaction and kept going for 1 day under hydrogen. The mixture was
filtered through Celite, and the filtrate was concentrated under
reduced pressure. Ac2O (38.9 μL, 0.411 mmol) and Et3N (129 μL,
0.822 mmol) were added to the solution of the crude residue in
MeOH (1 mL) in three equal portions and in 10-min intervals. After
30 min, the whole mixture was concentrated under reduced
pressure. The residue was purified by Sephadex G-10 (H2O) and
then passed through a column of Biorad 50X80 Na+ resin (H2O) to
give the tetrasaccharide 35 (2.3 mg, 15%). Method C: A solution of
Na(s) dispersion in oil (0.34 mL, 5.7 mmol) and 15-crown-5 (1.0 mL,
5.1 mmol) in THF (1 mL) was added to a solution of compound 29
(9.8 mg, 6.1 mol) in THF (0.5 mL) at –50 °C. After 1 h, the reaction
was quenched by sequential addition of isopropyl alcohol, MeOH,
and 2 N HCl(aq). The organic solvent was removed under reduced
pressure. The mixture was washed by CHCl3. The water layer was
concentrated under reduced pressure and purified by C-18 column
(H2O/MeOH = 1/0 and 0/1) to give a crude residue. To a solution of
the crude residue in a mixed solvent of 5 N NaOH(aq), H2O and
MeOH (1/2/15, v/v/v, 2 mL) was added Ac2O (17.3 μL, 183 mol) in
three equal portions and in 10-min intervals at room temperature.
After 30 min, the reaction mixture was concentrated under reduced
pressure and sequentially purified by C-18 column (H2O/MeOH=1/0
and 9/1), Biorad 50X80 Na+ resin column (H2O), and Sephadex G-10
column (H2O) to give product 35 (3 mg, 47%). 1H NMR (600 MHz,
D2O):  4.57-4.52 (2H, m, 1’-H, 1’’’-H), 4.49-4.46 (2H, m, 1-H, 1’’-H),
4.18 (2H, m, CH2), 3.92-3.82 (4H, m, 2’’’-H, 6’-Ha, 6’’’-Ha, CH2), 3.78-
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
3.66 (9H, m, 2’-H, 3’-H, 3’’’-H, 4-H, 4’’-H, 5-H, 5’’-H, 6’-Hb, 6’’’-Hb,
CH2), 3.59 (2H, m, 3-H, 3’’-H), 3.10 (2H, m, CH2), 2.08 (3H, s,
OCOCH3), 2.04 (3H, s, OCOCH3), 2.01 (3H, s, OCOCH3), 1.61 (2H, m,
CH2 ), 1.49 (2H, m, CH2), 1.36 (2H, m, CH2), 0.99 (2H, m, CH2), 0.03
(9H, m, TMS); 13C NMR (150 MHz, D2O):  174.8 (C), 173.6 (C), 103.0
(CH), 102.3 (CH), 100.80 (CH), 100.75 (CH), 82.3 (CH), 80.1 (CH),
79.8 (CH), 75.8 (CH), 75.6 (CH), 75.3 (CH), 75.2 (CH), 73.8 (CH), 73.7
(CH), 73.5 (CH), 72.6 (CH), 72.2 (CH), 70.6 (CH2), 69.6 (CH), 68.3
(CH), 63.9 (CH2), 60.4 (CH2), 55.3 (CH), 54.3 (CH), 40.1 (CH2), 28.5
(CH2), 28.3 (CH2), 22.4 (CH3), 22.3 (CH3), 22.1 (CH), 16.9 (CH2), -2.5
(CH3); HRMS (ESI): m/z calcd for C39H67N3Na2O25Si ([M+H]+):
1050.3545, found: 1050.3539.
N-2-(Trimethylsilyl)ethoxycarbonyl-5-aminopentyl [2-acetamido-
2-deoxy--D-glucopyranosyl]-(1→4)-(-D-glucopyranosyluronate)-
(1→3)-[2-acetamido-2-deoxy--D-glucopyranosyl]-(1→4)-(-D-
glucopyranosyluronate)-(1→3)-[2-acetamido-2-deoxy--D-
glucopyranosyl]-(1→4)--D-glucopyranosyluronate trisodium salt
(36). Method A: A mixture of the acetamide 33 (3.4 mg, 1.5 mol)
and 20% Pd(OH)2 on carbon (10 mg) in MeOH (1 mL) was equipped
with a hydrogen balloon at room temperature. After 16 h, H2O (5
mL) was added to the reaction and kept going for 1.5 day under
hydrogen. The mixture was filtered through Celite and the filtrate
was concentrated under reduced pressure. The residue was purified
by Sephadex G-10 (H2O) and then passed through a column of
Biorad 50X80 Na+ resin (H2O) to give disaccharide 36 (0.4 mg, 18%).
Method B: A solution of Na(s) dispersion in oil (0.340 mL, 5.74 mmol)
and 15-crown-5 (1.00 mL, 5.05 mmol) in THF (1 mL) was added to a
solution of compound 29 (14.5 mg, 6.69 mol) in THF (0.5 mL) at –
50 °C. After 3 h, the reaction was quenched by sequentially adding
IPA, MeOH and 2 N HCl(aq). The organic solvent was removed under
reduced pressure. The mixture was washed by CHCl3. The water
layer was concentrated under reduced pressure and purified by C-
18 column (H2O/MeOH = 1/0 and 0/1) to give a crude residue. To a
(CH2), 28.7 (CH2), 28.5 (CH2), 22.6 (CH3), 22.4 (CH3), 17V.1iew(CAHrt2ic)l,e-O2n.2line
(CH3); HRMS (ESI): m/z calcd for C53H86N4O36Si ([M+H ]): 1451.4479,
found: 1451.4486.
+
DOI: 10.1039/D0OB01048K
5-Aminopentyl [2-acetamido-2-deoxy--D-glucopyranosyl]-(1→4)-
-D-glucopyranosyluronate sodium salt (1). TBAF (1.0 M in THF, 65
L, 65 mol) was added to a solution of compound 34 (1.9 mg, 2.6
mol) in CH3CN (2 mL) at room temperature. After 1 day, the
mixture was concentrated under reduced pressure, purified by
Sephadex G-10 column (H2O), and then passed through a column of
Biorad 50X80 Na+ resin (H2O) to provide product 1 (0.80 mg, 65%).
1H NMR (600 MHz, D2O):  4.57 (1H, d, J 8.4, 1’-H), 4.48 (1H, d, J 7.9,
1-H), 3.96-3.91 (2H, m, 6’-Ha, CH2), 3.80-3.71 (5H, m, 2’-H, 4-H, 5-H,
6’-Hb, CH2), 3.61 (1H, t, J 9.1, 3-H), 3.55 (1H, t, J 9.5 Hz, 3’-H), 3.49-
3.48 (2H, m, 4’-H, 5’-H), 3.35 (1H, t, J 8.5, 2-H), 3.01 (2H, m, CH2),
2.07 (3H, s, OCOCH3), 1.69 (4H, m, CH2), 1.47 (2H, m, CH2); 13C NMR
(150 MHz, D2O):  175.3 (C), 174.7 (C), 102.7 (CH), 101.2 (CH), 80.4
(CH), 77.1 (CH), 76.2 (CH), 74.3 (CH), 74.2 (CH), 73.1 (CH), 70.6
(CH2), 70.1 (CH), 60.9 (CH2), 55.7 (CH), 39.7 (CH2), 28.5 (CH2), 26.7
(CH2), 22.8 (CH3), 22.3 (CH2); HRMS (ESI): m/z calcd for C19H35N2O12
([M+H]+): 483.2195, found: 483.2191.
5-Aminopentyl [2-acetamido-2-deoxy--D-glucopyranosyl]-(1→4)-
(-D-glucopyranosyluronate)-(1→3)-[2-acetamido-2-deoxy--D-
glucopyranosyl]-(1→4)--D-glucopyranosyluronate disodium salt
(2). TBAF (1.0 M in THF, 230 L, 230 mol) was added to a solution
of compound 35 (9.6 mg, 9.1 mol) in CH3CN (5 mL) under room
temperature. After 1 day, the mixture was concentrated under
reduced pressure, purified by Sephadex G-10 column (H2O), and
then passed through a column of Biorad 50X80 Na+ resin (H2O) to
provide product 2 (8.0 mg, 99%). 1H NMR (600 MHz, D2O):  4.60-
4.55 (2H, m, 1’-H, 1’’’-H), 4.50-4.47 (2H, m, 1-H, 1’’-H), 3.96-3.85
(4H, m, 2’’’-H, 6’-Ha, 6’’’-Ha, CH2), 3.81-3.70 (9H, m, 2’-H, 3’’’-H, 4-H,
4’’-H, 5-H, 5’’-H, 6’-Hb, 6’’’-Hb, CH2), 3.62-3.59 (2H, m, 3-H, 3’’-H),
3.57-3.48 (5H, m, 3’-H, 4’-H, 4’’’-H, 5’-H, 5’’’-H), 3.39-3.34 (2H, m, 2-
H, 2’-H), 3.02 (2H, m, CH2), 2.08 (3H, s, OCOCH3), 2.05 (3H, s,
OCOCH3), 1.69 (4H, m, CH2), 1.48 (2H, m, CH2); 13C NMR (150 MHz,
D2O):  175.3 (C), 174.6 (C), 103.5 (CH), 102.8 (CH), 101.1 (CH),
101.0 (CH), 82.8 (CH), 80.5 (CH), 80.2 (CH), 77.1 (CH), 76.7 (CH), 76.3
(CH), 75.7 (CH), 74.3 (CH), 74.2 (CH), 73.9 (CH), 73.1 (CH), 72.9 (CH),
70.6 (CH2), 70.1 (CH), 68.8 (CH), 60.9 (CH2), 55.8 (CH), 54.7 (CH),
39.7 (CH2), 28.5 (CH2), 26.6 (CH2), 22.8 (CH3), 22.8 (CH3), 22.3 (CH2);
HRMS (ESI): m/z calcd for C13H18N3O4SNa ([M+H]+): 884.3145,
found: 884.3119.
solution of the crude residue in a mixed solvent of 5 N NaOH(aq)
,
H2O and MeOH (1/2/15, v/v/v, 2 mL) was added Ac2O (28.5 μL, 301
mol) in three equal portions and in 10-min intervals at room
temperature. After 30 min, the reaction mixture was concentrated
under reduced pressure and sequentially purified by C-18 column
(H2O/MeOH = 1/0 and 0/1), Biorad 50X80 Na+ resin column (H2O),
and Sephadex G-10 column (H2O) to give the product 36 (5.3 mg,
62%) 1H NMR (600 MHz, D2O):  4.56-4.52 (3H, m, 1’-H, 1’’’-H, 1’’’’’-
H), 4.47-4.43 (3H, m, 1-H, 1’’-H, 1’’’’-H), 4.19-4.18 (2H, m, CH2),
3.92-3.88 (4H, m, 6’-Ha, 6’’’-Ha, 6’’’’’-Ha, CH2), 3.85-3.82 (2H, m, 2’’’-
H, 2’’’’’-H), 3.77-3.64 (14H, m, 2’-H, 3’-H, 3’’’-H, 3’’’’’-H, 4-H, 4’’-H,
4’’’’-H, 5-H, 5’’-H, 5’’’’-H, 6’-Hb, 6’’’-Hb, 6’’’’’-Hb, CH2), 3.59-3.56 (3H,
m, 3-H, 3’’-H, 3’’’’-H), 3.54-3.45 (6H, m, 4’-H, 4’’’-H, 4’’’’’-H, 5’-H,
5’’’-H, 5’’’’’-H), 3.36-3.30 (3H, m, 2-H, 2’’-H, 2’’’’-H), 3.11-3.09 (2H,
m, CH2), 2.05-2.02 (9H, m, OCOCH3), 1.63-1.60 (2H, m, CH2), 1.51-
1.48 (2H, m, CH2), 1.39-1.35 (2H, m, CH2), 1.01-0.99 (2H, m, CH2),
0.04-0.03 (9H, m, TMS); 13C NMR (150 MHz, D2O):  175.1 (C), 174.4
(C), 171.2 (C), 161.1 (C), 103.4 (CH), 102.5 (CH), 100.8 (CH), 82.8
(CH), 80.3 (CH), 80.1 (CH), 79.9 (CH), 76.9 (CH), 76.5 (CH), 76.1 (CH),
75.5 (CH), 74.1 (CH), 73.7 (CH), 73.0 (CH), 72.7 (CH), 70.7 (CH2), 69.9
(CH), 68.6 (CH), 64.2 (CH2), 60.7 (CH2), 55.6 (CH), 54.5 (CH), 40.3
Aminopentyl [2-acetamido-2-deoxy--D-glucopyranosyl]-(1→4)-(-
D-glucopyranosyluronate)-(1→3)-[2-acetamido-2-deoxy--D-
glucopyranosyl]-(1→4)-(-D-glucopyranosyluronate)-(1→3)-[2-
acetamido-2-deoxy--D-glucopyranosyl]-(1→4)--D-
glucopyranosyluronate trisodium salt (3). TBAF (1.0 M in THF, 38
L, 38 mol) was added to a solution of compound 36 (2.1 mg, 1.5
mol) in CH3CN (0.7 mL) at room temperature. After 1 day, the
mixture was concentrated under reduced pressure, purified by
Sephadex G-10 column (H2O), and then passed through a column of
Biorad 50X80 Na+ resin (H2O) to provide product 3 (1.7 mg, 90%). 1H
NMR (600 MHz, D2O):  4.57-4.52 (3H, m, 1’-H, 1’’’-H, 1’’’’’-H), 4.47-
16 | J. Name., 2012, 00, 1-3
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15 L. T. Senbanjo and M. A. Chellaiah, Front. Cell Dev. Biol., 2017,
4.44 (3H, m, 1-H, 1’’-H, 1’’’’-H), 3.92-3.88 (4H, m, 6’-Ha, 6’’’-Ha, 6’’’’’-
Ha, CH2), 3.85-3.82 (2H, m, 2’’’-H, 2’’’’’-H), 3.77-3.67 (13H, m, 2’-H,
3’’’-H, 3’’’’’-H, 4-H, 4’’-H, 4’’’’-H, 5-H, 5’’-H, 5’’’’-H, 6’-Hb, 6’’’-Hb,
6’’’’’-Hb, CH2), 3.59-3.56 (3H, m, 3-H, 3’’-H, 3’’’’-H), 3.54-3.45 (7H, m,
3’-H, 4’-H, 4’’’-H, 4’’’’’-H, 5’-H, 5’’’-H, 5’’’’’-H), 3.36-3.30 (3H, m, 2-H,
2’’-H, 2’’’’-H), 2.99-2.97 (2H, m, CH2), 2.05-2.02 (9H, m, OCOCH3),
1.70-1.63 (4H, m, CH2), 1.46-1.43 (2H, m, CH2); 13C NMR (150 MHz,
D2O):  174.90 (C), 174.84 (C), 174.2 (C), 103.1 (CH), 102.3 (CH),
100.6 (CH), 100.5 (CH), 82.5 (CH), 82.3 (CH), 80.1 (CH), 79.9 (CH),
79.7 (CH), 76.7 (CH), 76.3 (CH), 73.9 (CH), 73.8 (CH), 73.5 (CH), 72.7
(CH), 72.4 (CH), 70.2 (CH2), 69.6 (CH), 68.4 (CH), 60.5 (CH2), 55.3
(CH), 54.2 (CH), 39.3 (CH2), 28.1 (CH2), 26.2 (CH2), 22.4 (CH3), 22.3
(CH3), 21.9 (CH2); HRMS (ESI): m/z calcd for C47H74N4O34Si ([M+H+]):
1307.3872, found: 1307.3831.
View Article Online
5, 18.
DOI: 10.1039/D0OB01048K
16 L. K. Liu and B. Finzel, Acta. Crystallogr. F Struct. Biol.
Commun., 2014, 70, 1155–1161.
17 S. Banerji, A. J. Wright, M. Noble, D. J. Mahoney, I. D.
Campbell, A. J. Day and D. G. Jackson, Nat. Struct. Mol. Biol.,
2007, 14, 234–239.
18 R. Stern, G. Kogan, M. J. Jedrzejas and L. Soltés, Biotechnol.
Adv., 2007, 25, 537–557.
19 J. Li, M. Qiao, Y. Ji, L. Lin, X. Zhang and R. J. Linhardt, Int. J. Biol.
Macromol., 2020, 152, 199–206.
20 M. Mende, C. Bednarek, M. Wawryszyn, P. Sauter, M. B.
Biskup, U. Schepers and S. Bräse, Chem. Rev., 2016, 116,
8193–8255.
21 T. M. Slaghek, T. K. Hyppoenen, T. Ogawa, J. P. Kamerling and
F. G. Vliegenthart, Tetrahedron Lett., 1993, 34, 7939–7942.
22 T.M. Slagheka, T. K. Hyppönen, T. Ogawa, J. P. Kamerling and
J. F.G.Vliegenthart, Tetrahedron: Asymmetry, 1994, 5, 2291–
2301.
23 G. Blatter and J. C. Jacquinet, Carbohydr. Res., 1996, 288, 109–
125.
24 K. M. Halkes, T. M. Slaghek, T. K. Hypponen, P. H. Kruiskamp,
T. Ogawa, J. P. Kamerling and J. F. G. Vliegenthart, Carbohydr.
Res., 1998, 309, 161–174.
Conflicts of interest
There are no conflicts to declare.
25 J. Dinkelaar, J. D. C. Codee, L. J. van den Bos, H. S. Overkleeft
and G. A. van der Marel, J. Org. Chem., 2007, 72, 5737–5742.
26 G. Macchione, J. L. de Paz and P. M. Nieto, Carbohydr. Res.,
2014, 394, 17–25.
27 J. Dinkelaar, H. Gold, H. S. Overkleeft, J. D. C. Codee and G. A.
van der Marel, J. Org. Chem., 2009, 74, 4208–4216.
28 X. Lu, M. N. Kamat, L. Huang and X. Huang, J. Org. Chem.,
2009, 74, 7608–7617.
Acknowledgements
This work was supported by the Ministry of Science and
Technology of Taiwan, Academia Sinica and National Chiao Tung
University.
29 H. Gold, S. Munneke, J. Dinkelaar, H. S. Overkleeft, J. M. F. G.
Aerts, J. D. C. Codee and G. A. van der Marel, Carbohydr. Res.,
2011, 346, 1467–1478.
30 M. T. C. Walvoort, A. G. Volbeda, N. R. M. Reintjens, H. van
der Elst, O. J. Plante, H. S. Overkleeft, G. A. van der Marel and
J. D. C. Codee, Org. Lett., 2012, 14, 3776–3779.
31 A. R. de Jong, A. G. Volbeda, B. Hagen, H. van den Elst, H. S.
Overkleeft, G. A. van der Marel and J. D. C. Codée, Eur. J. Org.
Chem., 2013, 29, 6644–6655.
32 G. Gu, P. J. Adabala, M. G. Szczepina, S. Borrelli and B. M.
Pinto, J. Org. Chem., 2013, 78, 8004–8019.
33 B. K. Yeung, D. C. Hill, M. Janicka and P. A. Petillo, Org. Lett.,
2000, 2, 1279–1282.
34 E. R. Palmacci and P. H. Seeberger, Tetrahedron, 2004, 60,
7755–7766.
35 L. Huang and X. Huang, Chem. Eur. J., 2007, 13, 529–540.
36 A. F. Lopez, J. C. Jacquinet and C. Lopin-Bon, Eur. J. Org. Chem.
2013, 2013, 6934-6947.
37 P. T. Nyffeler, C.-H. Liang, K. M. Koeller and C. H. Wong, J. Am.
Chem. Soc., 2002, 124, 10773–10778.
38 T.-Y. Huang, D. Irene, M. M. L. Zulueta, T.-J. Tai, S.-H. Lain, C.-
P. Cheng, P.-X. Tsai, S.-Y. Lin, Z.-G. Chen, C.-C. Ku, C.-D. Hsiao,
C.-L. Chyan and S.-C. Hung, Angew. Chem. Int. Ed., 2017, 56,
4192–4196.
Notes and references
1
2
3
4
J. R. E. Fraser, T. C. Laurent and U. B. G. Laurent, J. Intern.
Med., 1997, 242, 27–33.
H. Ponta, L. Sherman and P. A. Herrlich, Nat. Rev. Mol. Cell
Biol., 2003, 4, 33–45.
J.-X. Tan, X.-Y. Wang, X.-L. Su, H.-Y. Li, Y. Shi, L. Wang and G.-
S. Ren, PLoS One, 2011, 6, e22836.
C. G. Johnson, V. P. Stober, J. M. Cyphert-Daly, C. S. Trempus,
G. P. Flake, V. Cali, I. Ahmad, R. J. Midura, M. A. Aronica, S.
Matalon and S. Garantziotis, Am. J. Physiol. Lung Cell Mol.
Physiol., 2018, 315, L787–L798.
5
6
7
8
9
P. Takabe, G. Bart, A. Ropponen, K. Rilla, M. Tammi, R. Tammi
and S. Pasonen-Seppänen, Exp. Cell Res., 2015, 337, 1–15.
M. Dobaczewski, M. Bujak, P. Zymek, G. Ren, M. L. Entman
and N. G. Frangogiannis, Cell Tissue Res., 2006, 324, 475–488.
B. W. Mallard and J. Tiralongo, Glycoconjugate J., 2017, 34,
441–452.
L. Wang, X. Zuo, K. Xie and D. Wei, Methods Mol. Biol., 2018,
1692, 31–42.
J. Dzwonek and G. M. Wilczynski, Front. Cell. Neurosci., 2015,
9, 175.
10 C. M. McKee, M. B. Penno, M. Cowman, M. D. Burdick, R. M.
Strieter, C. Bao and P. W. Noble, J. Clin. Invest., 1996, 98,
2403–2413.
11 L. O. Orestes and S. A. Leopoldo, Front. Immunol., 2017, 8,
1731.
12 S. Ogino, N. Nishida, R. Umemoto, M. Suzuki, M. Takeda, H.
Terasawa, J. Kitayama, M. Matsumoto, H. Hayasaka, M.
Miyasaka and I. Shimada, Structure, 2010, 18, 649–656.
13 M. Takeda, S. Ogino, R. Umemoto, M. Sakakura, M. Kajiwara,
K. N. Sugahara, H. Hayasaka, M. Miyasaka, H. Terasawa and I.
Shimada, J. Biol. Chem., 2006, 281, 40089–40095.
14 F. W. JamisonII, T. J. Foster, J. A. Barker, R. D. Hills Jr. and O.
Guvench, J. Mol. Biol., 2011, 406, 631–647.
39 C.-S. Chao, Y.-F. Yen, W.-C. Hung and K.-K. T. Mong, Adv.
Synth. Catal., 2011, 353, 879−884.
40 P. B Alper, S.-C. Hung and C.-H. Wong, Tetrahedron Lett.,
1996, 37, 6029–6032.
41 C.-A. Tai, S. S. Kulkarni and S.-C. Hung, J. Org. Chem. 2003, 68,
8719–8722.
42 S.-C. Hung, X.-A. Lu, J.-C. Lee, M. D.-T. Chang, S.-l. Fang, T.-c.
Fan, M. M. L. Zulueta and Y.-Q. Zhong, Org. Biomol. Chem.,
2012, 10, 760–772.
43 C.-S. Chao, C.-Y. Lin, S. Mulani, W.-C. Hung and K.-K. Mong,
Chem. Eur. J., 2011, 17, 12193–12202.
This journal is © The Royal Society of Chemistry 20xx
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Page 18 of 19
ARTICLE
Journal Name
44 D. Crich and M. Smith, J. Am. Chem. Soc., 2002, 124, 8867–
8869.
View Article Online
DOI: 10.1039/D0OB01048K
45 L. Cirillo, E. Bedini, A. Molinaro and M. Parrilli, Tetrahedron
Lett., 2010, 51, 1117–1120.
46 K. Furukawa, M. Shibuya and Y. Yamamoto, Org. Lett., 2015,
17, 2282–2285.
47 J. A. Cifonelli and M. Mayeda, Biochim. Biophys. Acta., 1957,
24, 397–400.
48 P. Lei, Y. Ding, X. Zhang, A. Adijiang, H. Li, Y. Ling and J. An,
Org. Lett. 2018, 20, 3439–3442.
49 M. Takeda, H. Terasawa, M. Sakakura, Y. Yamaguchi, M.
Kajiwara, H. Kawashima, M. Miyasaka and I. Shimada, J.
Biomol. NMR, 2004, 29, 97–98.
50 L.-K. Liu and B. C. Finzel, J. Med. Chem., 2014, 57, 2714–2725.
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DOI: 10.1039/D0OB01048K
Table of Contents
A practical route for the synthesis of hyaluronic acid oligosaccharides was developed and a
tetrasaccharide (GlcNAcβ1→4GlcAβ1→3GlcNAcβ1→4GlcAβ1→) was identified to be the
minimum sugar unit to bind with human CD44 (KD = 3.5 μM) through the measurement of
Isothermal Titration Calorimeter.