Chemical synthesis of 4-azido-β-galactosamine derivatives for inhibitors of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase

Article abstract of DOI:10.1007/s10719-018-9839-2

Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-β-GalNAc derivatives and 4-azido-β-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-β-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC₅₀ value ranging from 0.800 to 0.828?mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.

Full text of DOI:10.1007/s10719-018-9839-2

Glycoconjugate Journal
https://doi.org/10.1007/s10719-018-9839-2
ORIGINAL ARTICLE
Chemical synthesis of 4-azido-β-galactosamine derivatives for inhibitors
of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase
Seanghai Hor1 & Takumi Kodama1 & Nobuo Sugiura2 & Hikaru Kondou1 & Mio Yanagida1 & Keiya Yanagisawa1
&
Aoki Shibasawa1 & Bunta Tsuzuki1 & Naoto Fukatsu1 & Kazuya Nagao1 & Kenji Yamana3 & Kazuya I. P. J. Hidari4
&
Hideto Watanabe2 & Osami Habuchi1,5 & Hirofumi Nakano1
Received: 8 June 2018 /Revised: 27 July 2018 /Accepted: 15 August 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its
regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main
structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding
physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-β-
GalNAc derivatives and 4-azido-β-GalNAc derivatives were synthesized for their potential application as inhibitors of
GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed
that 4-pivaloylamino- and 4-azido-β-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC50 value
ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.
.
.
.
Keywords Sulfotransferase N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase Inhibitor Chemical synthesis
Abbreviations
CS-A
PAP
PAPS
3′-phosphoadenosine 5′-phosphate
3′-phosphoadenosine 5′-phosphosulfate
chondroitin sulfate A
CS-E
chondroitin sulfate E
GalNAc4S
2-acetamido-2-deoxy-4-O-sulfonato-
D-galactopyranose
GalNAc4S-6ST N-acetylgalactosamine 4-sulfate
6-O-sulfotransferase
Introduction
GalNAc4S6S
2-acetamido-2-deoxy-4,6-di-O-sulfonato-
D-galactopyranose
Chondroitin sulfate E (CS-E), chondroitin sulfate isomer con-
taining GlcAβ1-3GalNAc4S6S repeating unit, is found in
various mammalian cells. CS-E is implicated in several phys-
iological functions in various mammalian systems such as
mast cell maturation [1], regulation of procoagulant activity
of monocytes, neuronal polarization [2], promotion of osteo-
blast differentiation by binding to both N-cadherin and
cadherin-11 [3], basic fibroblast growth factor [4], inhibition
of P-selectin binding to a human breast cancer cell line in vitro
[5], processes of neural development in brain [6], migration of
neuronal precursors during cortical development [7], pulmo-
nary metastasis [8], and enhancement of plasminogen activa-
tion [9]. In addition, CS-E has been reported to be strongly
expressed in human ovarian adenocarcinomas. CS-E is not
expressed in normal ovary [10] to function in metastasis of a
Lewis lung carcinoma cell line [11] and to prevent excitatory
amino acid-induced neuronal cell death [12]. Moreover, CS-E
GlcA
D-glucuronic acid
* Hirofumi Nakano
hnakano@auecc.aichi-edu.ac.jp
1
2
3
4
5
Department of Chemistry, Aichi University of Education, Igaya,
Kariya, Aichi 448-8542, Japan
Institute for Molecular Science of Medicine, Aichi Medical
University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan
Aichi Gakuin University, 12 Araike, Iwasaki-cho,
Nisshin, Aichi 470-0195, Japan
Junior College Division, University of Aizu, Ikki-machi,
Aizuwakamatsu, Fukushima 965-8570, Japan
Multidisciplinary Pain Center, Aichi Medical University, 1-1
Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Glycoconj J
expressed in murine osteosarcoma LM8G7 cells is involved in
focal formation of liver tumors [13].
indicate that β-GalNAc4S derivatives are possible specific
inhibitors of GalNAc4S-6ST [19]. After demonstrating that
phenyl GalNAc4S could serve as an acceptor for
GalNAc4S-6ST and thereby inhibit GalNAc4S-6ST competi-
tively, we compared the inhibitory effects of various glyco-
sides in which various hydrophobic aglycons were attached to
D-GalNAc4S via β-anomeric configuration. p-Nitrophenyl
and p-chlorophenyl D-GalNAc4S were stronger inhibitors
than phenyl D-GalNAc4S. Among the examined inhibitors
(Fig. 2) here, 3-estradiol β-D-GalNac4S was the strongest
inhibitor. The Ki of 3-estradiol β- D-GalNac4S for the com-
petitive inhibition was 0.008 mM, which was much lower
than that of phenyl D-GalNAc4S, 0.98 mM [20]. Inspired
by these results, we investigated and synthesized cost-
effective and simple compounds that can serve as effective
inhibitors for GalNAc4S-6ST.
In this report, we pursued multiple structural modification
strategies (Fig. 3) to discover a novel class of small molecular
inhibitor candidates for GalNAc4S-6ST. The sulfate group of
phenyl β-GalNAc4S (A, R1 = phenyl) was exchanged for
substituted amides at 4-position, resulting in 4-acylamino-β-
galactosamine derivatives B. In addition, 4-azido-β-
galactosamine derivatives C were generated via the modifica-
tion of GalNAc4S derivatives A by installing an azido group
and different aglycons (R1 = cyclohexyl, benzyl) into 4-
position and 1-position respectively. 4-Acylamino-β-
galactosamine derivatives B are sulfate analogue having de-
ceased negative charge and high cell membrane permeability.
One of our purposes was to synthesize selective cell mem-
brane permeable inhibitors for GalNAc4S-6ST.
N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase
(GalNAc4S-6ST: EC 2.8.2.33) is the sulfotransferase respon-
sible for biosynthesis of highly sulfated CS-E that is expressed
in the anterior visceral endoderm and accordingly is possible
to play an active role during early mouse development [14].
Sulfated carbohydrate chains in glycoproteins and glycolipids
play important roles in infection by microorganisms and dis-
eases. Inhibitors of sulfotransferases, which are responsible
for biosynthesis of these carbohydrate chains, are medical
agents against such infections and diseases [15]. Previously,
4-[6-(morpholin-4-yl)-5-nitropyridin-2-yl] morpholine and
5-(4-fluorophenyl)-3-(4-methylphenyl)-1-(4-nitrophenyl)-
4,5-dihydro-1H-pyrazole, have been reported, which serve as
inhibitors for GalNAc4S-6ST [16, 17]. Recently, (2E)-3-(3-
bromo-4-hydroxy-5-methoxyphenyl)-2-cyano-N-(2,4-
dichlorophenyl)prop-2-enamide has been found to be the first
cell-permeable, small molecule to selectively inhibit the activ-
ity of GAG sulfotransferases as well as to reduce CS-E and
overall sulfation levels on cell-surface [18]. We previously
cloned GalNAc4S-6ST, that transfers sulfate from 3′-
phosphoadenosine 5′-phosphosulfate (PAPS) to the C-6 hy-
droxy group of the GalNAc4S residue of chondroitin sulfate A
(CS-A). Thus CS-E containing GlcA-GalNAc4S6S repeating
units is formed (Fig. 1).
The development of specific inhibitors of GalNAc4S-6ST
is important to investigate the function of CS-E. Because
GalNAc4S-6ST requires a group attached to the C-4 hydroxy
group of the GalNAc residue as the acceptor, the sulfated
GalNAc residue is expected to interact with GalNAc4S-6ST
and affect its activity. We furthermore synthesized phenyl α-
or β-2-acetamido-2-deoxy-D-galactopyranosides containing
a sulfate group at the C-3, C-4, or C-6 hydroxy group and
examined their inhibitory activities against recombinant
GalNAc4S-6ST. We found that phenyl β-GalNAc4S inhibited
GalNAc4S-6ST competitively and also served as an acceptor.
The sulfated product derived from phenyl β-GalNAc4S was
identical to phenyl β-GalNAc4S6S. These observations
Results and discussion
Synthesis of amide derivatives B
As described in scheme 1, 2,2,2-trichloroethoxycarbonyl
(Troc) protecting group having temporary amino-masking
functionality was used as because it performed neighboring
Fig. 1 Function of GalNAc4S-
6ST

Glycoconj J
mixture of 95% EtOH-H2O to give amine. Subsequently, the
amino group was selectively N-acylated with acetic anhy-
dride, benzoyl chloride, and pivaloyl chloride to afford com-
pound 8, 9, and 10, respectively.
Fig. 2 Previous inhibitors A
Synthesis of 4-azido-β-galactosamine derivatives C
group participation and accordingly allowed the introduction
of β-glycosides. Phenyl glycoside 2 that has been reported
previously [21] can be prepared by β-selective glycosylation
of phenol with imidate donor 1 at −20 °C in CH2Cl2 in the
presence of BF3•OEt2. We also conducted a TfOH-promoted
glycosylation of phenol with thioglycoside donor at −20 °C to
0 °C in the presence of NIS in CH2Cl2. A complex mixture,
however, was provided. Conversion the phenyl glycoside 2 to
acetylated glucosamine 3 that has been documented [22] was
accomplished by a two-step procedure. First, the Troc
protecting group was deprotected with Zn and AcOH in
MeOH and CH2Cl2. The acetylation of the amino group with
Ac2O was then performed in the presence of pyridine. Then,
the transesterification of the acetyl esters of 3 afforded known
compound 4 using sodium methoxide in MeOH [22–28].
Compound 4 has acetamido group at 2-position and three
hydroxy groups. Subsequently, attention was focused on the
regioselective installation of azido into the compound at 4-
position. Numerous of conditions were examined to improve
the selectivity of 3,6-di-O-pivaloylation of compound 4. We
found that the treatment of compound 4 using pivaloyl chlo-
ride in pyridine at 0 °C formed 3,4,6-tri-O-pivaloylated com-
pound. A high selectivity was obtained when compound 4
was reacted with pivaloyl chloride in pyridine at −20 °C to
give compound 5. Compound 6 was afforded via the reaction
between compound 5 and Tf2O at −10 °C in the presence of
pyridine and CH2Cl2 followed by SN2 reaction with sodium
azide. Compound 7 was easily prepared in 91% yield by
depivaloylation of 6 using sodium methoxide in MeOH. The
azido moiety of 7 was reduced with hydrogen over Pd-C in the
As shown in scheme 2, NIS-promoted glycosylation of select-
ed alcohols (in this study: cyclohexanol, benzyl alcohol) with
glycosyl donor 11 in CH2Cl2 at −10 °C gave compound 12a
[29–34] in 90% yield, and 12b [35–37]. Next, the Ac
protecting groups of 12a were removed by sodium methoxide
in MeOH to give GalNAc derivative 13a in a near-quantitative
yield. Selective 3,6-di-O-pivaloylation of 13a with pivaloyl
chloride and pyridine in CH2Cl2 at 0 °C afforded glucosamine
14a in good yield. In this approach, to avoid pivaloylation of
hydroxy group at 4-position due to the steric hindrance which
might be caused by Troc group, the selective 3,6-di-O-
pivaloylation had been conducted before removing the Troc
group. Then, removal of Troc group of 14a using Zn and
AcOH in MeOH and CH2Cl2 gave a free amine derivative
that was converted into acetamido derivative 15a by reaction
with Ac2O in the presence of pyridine in CH2Cl2. Compound
16a was obtained by the treatment of 15a with Tf2O at −10 °C
in the presence of pyridine and CH2Cl2 followed by azidation
with NaN3 at rt. in DMF. Thence, compound 17a was obtain-
ed by depivaloylation of 16a.The same reaction sequence in
high yield was applied to precursor 12b to afford 13b [37],
14b, 15b [38, 39], 16b, and 17b.
Biological activity
The compounds (7, 8, 9, 10, 17a, and 17b) were tested as
inhibitors of GalNAc4S-6ST. The result of the GalNAc4S-
6ST test was expressed as the inhibition that indicates the
inhibitor concentration at which 50% of inhibitory activity
occurs (IC50) (Figs. 4 and 5). The inhibition is then compared
Fig. 3 Structural modification

Glycoconj J
Scheme 1 Synthesis of amide
derivatives 8, 9, and 10. i) Phenol,
BF3·OEt2, MS4A, CH2Cl2,
−20 °C, 2.5 h; ii) 1) Zn, AcOH,
MeOH, CH2Cl2, rt., 2.5 h, 2)
Ac2O, pyridine, rt., 2.5 h; iii)
NaOMe, MeOH, rt., 1.5 h; iv)
PivCl, pyridine, −20 °C, 14 h; v)
1) Tf2O, pyridine, CH2Cl2,
−10 °C, 1 h, 2) NaN3, DMF, rt.,
28 h; vi) NaOMe, MeOH, rt.,
27.5 h; vii) 1) H2, Pd-C,
95%EtOH-H2O, rt., 14.5 h, 2) (a)
Ac2O, 0 °C, 2 h, (b) BzCl, 0 °C,
2 h, (c) PivCl, 0 °C, 40 min
with that of a reference inhibitor such as benzyl D-GalNAc4S,
which is a small molecular inhibitor against GalNAc4S-6ST,
to give relative inhibition. The results of the tested compounds
are summarized in Table 1 below.
Compounds 7, 10, 17a, and 17b showed approximately
1.36-fold improvement in activity relative to benzyl D-
GalNAc4S, which were more effective than compounds
8, 9. This result implied that 4-azido-β-GalNAc deriva-
tives are more preferable for the activity because of the
linear structure of azido group to enhance its proximity to
the GalNAc4S-6ST binding site and the assistance from
the binding site-orientated 6-hydroxy group’s lone pairs.
The result also suggested that functional groups at 4-
position were more influential in inhibitory activity than
aglycons. Moreover, modification of the linear azido group
to 1,1,1-trimethylacetamido group (10) that has bulky and
restrictive conformation led to no change in potency. The
modification to acetamido (8) and benzamido (9) groups
decreased the potency by 42 and 26%, respectively. These
results suggest that the bulky group at 4-position restricted
its conformation and the rotation of 6-hydroxy group re-
sulted in strong hydrogen bonds. Thus, specific structure
and electronic properties of the amide would be important
for the interaction with GalNAc4S-6ST.
Scheme 2 Synthesis of 4-azido-β-galactosamine derivatives 17a and
17b. i) R1OH, NIS, TfOH, MS4A, CH2Cl2, −10 °C, 1 to 23 h; ii)
NaOMe, MeOH, −5 to 0 °C, 1.5 to 2.5 h; iii) PivCl, pyridine, CH2Cl2,
−10 to 0 °C, 3 to 20 h; iv) 1) Zn, AcOH, MeOH, CH2Cl2, rt., 2 to 4 h, 2)
Ac2O, pyridine, CH2Cl2, rt., 16 to 18 h; v) 1) Tf2O, pyridine, CH2Cl2,
−10 °C, 1.5 to 2.5 h, 2) NaN3, DMF, rt., 17 to 17.5 h; vi) NaOMe, MeOH,
0 °C, 17.5 to 22.5 h

Glycoconj J
Fig. 4 Inhibitory effects on
GalNAc4S-6ST by azido and
amido derivatives 7, 8, 9, and 10.
Bars show standard deviation of
triplicate measurements in each
experiment
Conclusion
Experimental
In summary, we described a new approach for the chemical
synthesis of 4-azido-β-GalNAc derivatives. In addition, we
discovered that 4-azido-β-GalNAc derivatives greatly
inhibited the activity of GalNAc4S-6ST. A specific functional
group rather than aglycon was important for the inhibitory
activity. Moreover, specific structure and electronic properties
of the amide would be important for the interaction with
GalNAc4S-6ST. The applications of click chemistry and
Staudinger ligation reaction on these molecules to afford li-
braries of potential inhibitors for GalNAc4S-6ST and
monosaccharide-linked biomolecules are currently underway.
General methods
All solvents were of reagent grade quality and purchased
commercially. Structures of synthetic compounds were
1
confirmed by H NMR, 13C NMR, and two-dimensional
NMR (COSY, HSQC, HMBC, and NOESY) spectroscopy.
1H and 13C NMR spectra were recorded using a Bruker
AVANCE III instrument operating at 400.13 and
100.62 MHz, respectively. Chemical shifts were referenced
to TMS in CDCl3 and δ values (ppm) of water in D2O (1H:
δ = 4.70) as internal standard. Electrospray ionization
Fig. 5 Inhibitory effects on
GalNAc4S-6ST by benzyl
GalNAc4S and azido compounds
7, 17a, and 17b. Values are
averages of three determinations.
Benzyl GalNAc4S
7 R1 = Ph
120%
Bars show standard deviation of
triplicate measurements in each
100%
experiment
17a R1 = c-Hex
17b R1 = Bn
80%
60%
40%
20%
0%
0
0.5
1
1.5
2
2.5
Inhibitor concentration (mM)

Glycoconj J
Table 1 Inhibition of GalNAc4S-6ST
General procedure C (azidation)
Test compound (R1, R4)
IC50 (mM) Relative inhibition
Trifluoromethanesulfonic anhydride was added to a cooled
solution of respective monosaccharide in pyridine and
CH2Cl2. The mixture was stirred at temperature and reaction
time as recorded individually. After quenching with 5% aq
HCl, the mixture was extracted with CHCl3. The organic layer
was washed with 5% aq HCl, saturated aq NaHCO3, and
saturated aq NaCl, before it was dried over Na2SO4 and con-
centrated under reduced pressure. The residue was dissolved
in DMF, and sodium azide was added to the resulting solution.
The mixture was stirred at temperature and reaction time as
described individually. Then, the reaction mixture was extract-
ed using ethyl acetate. The organic layer was washed with
saturated aq NaHCO3, saturated aq NaCl, dried over
Na2SO4, and concentrated under reduced pressure to afford
the respective crude product that was purified as reported
individually.
−
Benzyl D-GalNAc4S (Bn, OSO3
7 (Ph, N3)
8 (Ph, NHAc)
)
1.09
1
0.800
2.59
1.47
0.802
0.804
0.828
1.36
0.421
0.741
1.36
1.36
1.32
9 (Ph, NHBz)
10 (Ph, NHPiv)
17a (c-Hex, N3)
17b (Bn, N3)
(ESI) and atmospheric pressure chemical ionization (APCI)
mass spectra were obtained on a Bruker Daltonics micrOTOF-
QII. Thin layer chromatography (TLC) was performed on pre-
coated Silica gel 60 F254 plates. Column chromatography was
performed on silica gel 60 N (spherical neutral) that was pur-
chased from Kanto Chemical Company, Japan.
General procedure a (Troc removal and N-acetylation)
General procedure D (deacylation)
Activated zinc and AcOH were add to a solution of re-
spective monosaccharide in CH2Cl2 and MeOH. The mix-
ture was stirred at temperature and reaction time as de-
scribed individually. After quenching with saturated aq
NaHCO3, the mixture was passed through a column of
the Celite washing with chloroform. The filtrate was
washed using saturated aq NaHCO3, saturated aq NaCl,
dried over Na2SO4, and concentrated under reduced pres-
sure. The residue was dissolved in CH2Cl2, and the solu-
tion was cooled to 0 °C. After adding pyridine and Ac2O,
the solution was stirred at temperature and reaction time
as described individually. The reaction mixture was
quenched using MeOH before concentrated under reduced
pressure. The residue was dissolved in CHCl3. The
resulting solution was washed with 5% aq HCl, saturated
aq NaHCO3, and saturated aq NaCl, before it was dried
over Na2SO4, and concentrated under reduced pressure to
provide the respective crude product that was purified as
described individually.
A solution of the respective starting material in dry MeOH
was treated with 1.0 M sodium methoxide in MeOH. The
mixture was then stirred at temperature and reaction time as
reported individually. Next, the mixture was neutralized with
Amberlite IRC-50 H+ resin and concentrated under dimin-
ished pressure to give the respective crude product that was
purified as described individually.
General procedure E (reduction and N-acylation)
Pd-C (5%) was added to a solution of the respective mono-
saccharide in 95% v/v EtOH-H2O. The mixture was stirred
under hydrogen at temperature and reaction time as described
individually. The reaction mixture was filtered through Celite
and concentrated under reduced pressure. The residue was
dissolved in EtOH, and the solution was cooled to 0 °C.
Ac2O was added to the solution. The mixture was stirred at
temperature and reaction time as documented individually.
The mixture was concentrated under reduced pressure to give
the respective crude product that was purified as described
individually.
General procedure B (pivaloylation)
Pivaloyl chloride was added to a cooled solution of respec-
tive monosaccharide in dry pyridine. The mixture was
stirred at temperature and reaction time as mentioned indi-
vidually. After the addition of MeOH, the mixture was
concentrated under diminished pressure. The residue was
diluted with CHCl3 and washed with 2.5% aq HCl, satu-
rated aq NaHCO3, and saturated aq NaCl. The organic
layer was dried over Na2SO4 and concentrated under re-
duced pressure to give the respective crude product that
was purified as described individually.
General procedure F (glycosylation)
A mixture of thioglycoside, respective alcohol, N-
iodosuccinimide, and MS4A in dry CH2Cl2 was stirred at
temperature and reaction time as described individually. A
solution of 1.0 M trifluoromethanesulfonic acid in CH2Cl2
was added to the solution and stirred. After quenching with
saturated aq NaHCO3, the reaction mixture was passed
through a column of the Celite washing CHCl3. The filtrate
was washed with 5% aq Na2S2O3, saturated aq NaHCO3,

Glycoconj J
saturated aq NaCl, before it was dried over Na2SO4 and thence
concentrated under reduced pressure. The respective crude
product was afforded and purified as described individually.
(11.252 g, 0.172 mol, 17 equiv) and acetic acid (10 mL) at
room temperature for 2.5 h followed by acetylation with acetic
anhydride (2.80 mL, 3.02 g, 29.6 mmol, 2.9 equiv) in the
presence of pyridine (0.780 mL, 0.764 g, 9.66 mmol, 0.96
equiv) at room temperature for 2.5 h to give, after separated
by silica gel column chromatography eluting with a gradient
from 33% v/v ethyl acetate-hexane to 100% ethyl acetate,
compound 3 (3.312 g, 7.82 mmol, 78%) as colorless crystals.
General procedure G (zinc activation)
Zinc powder was added to a 5% aq HCl and then the mixture
was sonicated for 30 min. Next, the mixture was filtered with
suction. The residue (activated zinc) was first washed using
H2O, MeOH, and finally Et2O.
1
Rf = 0.40 (5% v/v methanol-chloroform); H NMR (CDCl3,
400.13 MHz): δ 1.96 (s, 3 H, 2-NHCOCH3), 2.05 (s, 3 H, 4-
OCOCH3), 2.07 (s, 3 H, 3-OCOCH3), 2.08 (s, 3 H, 6-
OCOCH3), 3.87 (ddd, 1 H, J4,5 = 9.9 Hz, J5,6b = 5.4 Hz,
Phenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxy)carbonylamino-β-D-glucopyranoside (2).
Boron trifluoride diethyl etherate (0.325 mL, 367 mg,
2.59 mmol, 2.0 equiv) was added to a cooled (−20 °C) mixture
of 1 (824 mg, 1.32 mmol, 1.0 equiv), phenol (0.500 mL, 536 mg,
5.69 mmol, 4.3 equiv), and MS4A in dichloromethane (10 mL).
The mixture was stirred for 2.5 h. After quenching with saturated
aq NaHCO3 (50 mL), the reaction mixture was passed through a
column of the Celite washing with chloroform (50 mL). The
organic layer was washed using saturated aq NaHCO3, saturated
aq NaCl, before it was dried over Na2SO4 and thence concentrat-
ed under reduced pressure to give, after separated by silica gel
column chromatography eluting with 50% v/v ethyl acetate-hex-
ane, product 2 (643 mg, 1.15 mmol, 88%) as colorless crystals.
J
5,6a = 2.5 Hz, H-5), 4.13 (dt, 1 H, J2,3 = 10.5 Hz, J2,NH
8.7 Hz, J1,2 = 8.2 Hz, H-2), 4.16 (dd, 1 H, J6a,6b = 12.2 Hz,
5,6a = 2.5 Hz, H-6a), 4.30 (dd, 1 H, J6a,6b = 12.2 Hz, J5,6b
=
J
=
5.4 Hz, H-6b), 5.15 (t, 1 H, J4,5 = 9.9 Hz, J3,4 = 9.3 Hz, H-4),
5.27 (d, 1 H, J1,2 = 8.2 Hz, H-1), 5.41 (dd, 1 H, J2,3 = 10.5 Hz,
J
3,4 = 9.3 Hz, H-3), 5.60 (d, 1 H, J2,NH = 8.7 Hz, 2-
NHCOCH3), 6.99 (d, 2 H, Jo,m = 8.5 Hz, o-arom. H), 7.06
(t, 1 H, Jm,p = 7.4 Hz, p-arom. H), 7.28 (dd, 2 H, Jo,m
=
8.5 Hz, Jm,p = 7.4 Hz, m-arom. H); 13C NMR (CDCl3,
100.62 MHz): δ 20.65 (q, 6-OCOCH3), 20.71 (q, 3,4-
OCOCH3), 23.37 (q, 2-NHCOCH3), 54.86 (d, C-2), 62.15
(t, C-6), 68.51 (d, C-4), 71.98 (d, C-3), 72.03 (d, C-5), 98.94
(d, C-1), 116.89 (d, o-arom. CH), 123.18 (d, p-arom. CH),
129.55 (d, m-arom. CH), 157.00 (s, arom. C), 169.42 (s, 4-
OCOCH3), 170.32 (s, 2-NHCOCH3), 170.64 (s, 6-OCOCH3),
170.87 (s, 3-OCOCH3); HRMS (APCI) m/z [M + H]+ calcd
for C20H26NO6+ 424.1602, found 424.1613.
1
Rf = 0.48 (50% v/v ethyl acetate-hexane); H NMR (CDCl3,
400.13 MHz): δ 2.06 (s, 3 H, 4-OCOCH3), 2.07 (s, 3 H, 3-
OCOCH3), 2.09 (s, 3 H, 6-OCOCH3), 3.87 (ddd, 1 H, J4,5
10.1 Hz, J5,6b = 5.3 Hz, J5,6a = 2.2 Hz, H-5), 3.90 (dt, 1 H,
2,3 = 10.5 Hz, J1,2 = 8.2 Hz, J2,NH = 8.0 Hz, H-2), 4.17 (dd, 1
H, J6a,6b = 12.2 Hz, J5,6a = 2.2 Hz, H-6a), 4.32 (dd, 1 H, J6a,6b
=
J
=
12.2 Hz, J5,6b = 5.3 Hz, H-6b), 4.71 (d, 1 H, Jgem = 12.0 Hz, Ha
of Troc), 4.76 (d, 1 H, Jgem = 12.0 Hz, Hb of Troc), 5.15 (t, 1 H,
Phenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (4)
J4,5 = 10.1 Hz, J3,4 = 9.7 Hz, H-4), 5.23 (d, 1 H, J2,NH = 8.0 Hz,
2-NHCOOCH2CCl3), 5.25 (d, 1 H, J1,2 = 8.2 Hz, H-1), 5.43 (t, 1
H, J2,3 = 10.5 Hz, J3,4 = 9.7 Hz, H-3), 6.98–7.01 (m, 2 H, o-arom.
H), 7.04–7.09 (m, 1 H, p-arom. H), 7.26–7.31 (m, 2 H, m-arom.
H); 13C NMR (CDCl3, 100.62 MHz):δ 20.64 (q, 4-OCOCH3),
20.66 (q, 3-OCOCH3), 20.71 (q, 6-OCOCH3), 56.33 (d, C-2),
62.06 (t, C-6), 68.54 (d, C-4), 71.50 (d, C-3), 72.01 (d, C-5),
74.45 (t, 2-NHCOOCH2CCl3), 95.35 (s, 2-NHCOOCH2CCl3),
99.00 (d, C-1), 116.96 (d, o-arom. CH), 123.36 (d, p-arom. CH),
129.56 (d, m-arom. CH), 153.99 (s, 2-NHCOOCH2CCl3),
156.88 (s, arom. C), 169.47 (s, 4-OCOCH3), 170.58 (s, 3-
OCOCH3), 170.61 (s, 6-OCOCH3); HRMS (APCI) m/z [M +
H]+ calcd for C21H25Cl3NO10+ 556.0539, found 556.0538.
Following general procedure D, compound 3 (3.312 g,
7.822 mmol, 1.0 equiv) was treated with 1.0 M NaOMe-
MeOH (1.50 mL, 1.50 mmol, 0.19 equiv) at room temperature
for 1.5 h to provide 4 (2.126 g, 7.151 mmol, 91%) as a color-
less solid. Rf = 0.40 (20% v/v methanol-chloroform); 1H NMR
(D2O, 400.13 MHz): δ 1.91 (s, 3 H, 2-NHCOCH3), 3.43 (t, 1
H, J4,5 = 9.5 Hz, J3,4 = 9.0 Hz, H-4), 3.51 (ddd, 1 H, J4,5
=
9.5 Hz, J5,6a = 5.3 Hz, J5,6b = 2.1 Hz, H-5), 3.53 (dd, 1 H,
J
2,3 = 10.1 Hz, J3,4 = 9.0 Hz, H-3), 3.67 (dd, 1 H, J6a,6b
=
=
12.3 Hz, J5,6a = 5.3 Hz, H-6a), 3.83 (dd, 1 H, J6a,6b
12.3 Hz, J5,6b = 2.1 Hz, H-6b), 3.86 (dd, 1 H, J2,3 = 10.1 Hz,
1,2 = 8.4 Hz, H-2), 5.04 (d, 1 H, J1,2 = 8.4 Hz, H-1), 6.96 (d, 2
J
H, Jo,m = 7.6 Hz, o-arom. H), 7.03 (t, 1 H, Jp,m = 7.5 Hz, p-
arom. H), 7.24–7.29 (m, 2 H, m-arom. H); 13C NMR (D2O,
100.62 MHz): δ 22.04 (q, 2-NHCOCH3), 55.48 (d, C-2),
60.49 (t, C-6), 69.65 (d, C-4), 73.51 (d, C-3), 76.06 (d, C-5),
99.57 (d, C-1), 116.62 (d, o-arom. CH), 123.41 (d, p-arom.
CH), 129.92 (d, m-arom. CH), 156.66 (s, arom. C), 174.87 (s,
2-NHCOCH3); HRMS (APCI) m/z [M + H]+ calcd for
C14H20NO6+ 298.1285, found 298.1296.
Phenyl
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyr
anoside (3)
Following general procedure A, a solution of compound 2
(5.606 g, 10.07 mmol, 1.0 equiv) in dichloromethane
(10 mL) and methanol (20 mL) was treated with activated zinc

Glycoconj J
Phenyl
8.3 Hz, H-2), 4.25 (dd, 1H, J6a,6b = 11.2 Hz, J5,6a
5.5 Hz, H-6a), 4.32 (dd,1 H, J6a,6b = 11.2, Hz, J5,6b
=
=
2-acetamido-2-deoxy-3,6-di-O-pivaloyl-β-D-glucopyr
anoside (5)
6.9 Hz, H-6b), 5.26 (d, 1 H, J1,2 = 8.3 Hz, H-1), 5.55
(dd, 1 H, J2,3 = 11.1 Hz, J3,4 = 3.7 Hz, H-3), 5.59 (d, 1
Following general procedure B, compound 4 (2.126 g,
7.151 mmol, 1.0 equiv) was reacted with pivaloyl chloride
(3.60 mL, 3.528 g, 29.26 mmol, 4.1 equiv) in dry pyridine
(30 mL, 0.24 M) at −20 °C for 14 h to give, after silica gel
chromatography eluting with a gradient from 30% v/v ethyl
acetate-hexane to 100% ethyl acetate, compound 5 (2.798 g,
6.010 mmol, 84%) as colorless crystals. Rf = 0.33 (50% v/v
H, J2,NH = 8.5 Hz, 2-NHCOCH3), 6.99 (d, 2 H, Jo,m =
7.7 Hz, o-arom. H), 7.03 (t, 1 H, Jm,p = 7.3 Hz, p-arom.
H), 7.24 (t, 2 H, Jo,m = 7.7 Hz, Jm,p = 7.3 Hz, m-arom. H);
13C NMR (CDCl3, 100.62 MHz): δ 23.32 (q, 2-
NHCOCH3), 27.04 (q, 3-OCOC (CH3)3), 27.12 (q, 6-
OCOC (CH3)3), 38.75 (s, 6-O-COC (CH3)3), 39.24 (s,
3-O-COC (CH3)3), 52.08 (d, C-2), 60.49 (d, C-4), 63.00
(t, C-6), 71.04 (d, C-5), 71.21 (d, C-3), 98.89 (d, C-1),
116.90 (d, o-arom. CH), 123.02 (d, p-arom. CH), 129.42
(d, m-arom. CH), 157.03 (s, arom. C), 170.17 (s, 2-
NHCOCH3), 178.00 (s, 6-O-COC (CH3)3), 178.09 (s, 3-
O-COC (CH3)3); HRMS (FAB) m/z [M + H]+ calcd for
1
ethyl acetate-hexane); H NMR (CDCl3, 400.13 MHz): δ
1.205 (s, 9 H, 6-OCOC (CH3)3), 1.208, (s, 9 H, 3-OCOC
(CH3)3), 1.89 (s, 3 H, 2-NHCOCH3), 3.26 (d, 1 H, J4,OH
6.2 Hz, 4-OH), 3.58 (td, 1 H, J4,5 = 9.6 Hz, J3,4 = 9.2 Hz,
4,OH = 6.2 Hz, H-4), 3.79 (ddd, 1 H, J4,5 = 9.6 Hz, J5,6a
6.6 Hz, J5,6b = 2.0 Hz, H-5), 4.20 (dt, 1 H, J2,3 = 10.4 Hz,
=
J
=
+
C24H35N4O7 491.2500, found 491.2486.
J
2,NH = 9.0 Hz, J1,2 = 8.3 Hz, H-2), 4.31 (dd, 1 H, J6a,6b
=
=
12.0 Hz, J5,6a = 6.6 Hz, H-6a), 4.47 (dd, 1 H, J6a,6b
Phenyl
12.0 Hz, J5,6b = 2.0 Hz, H-6b), 5.14 (d, 1 H, J1,2 = 8.3 Hz,
H-1), 5.28 (dd, 1 H, J2,3 = 10.4 Hz, J3,4 = 9.2 Hz, H-3), 6.05
2-acetamido-4-azido-2,4-dideoxy-β-D-galactopyrano
side (7)
(d, 1 H, J2,NH = 9.0 Hz, 2-NHCOCH3), 7.00 (d, 2 H, Jo,m
=
7.7 Hz, o-arom. H), 7.02 (t, 1 H, Jp,m = 7.4 Hz, p-arom. H),
7.24 (t, 2 H, Jo,m = 7.7 Hz, Jp,m = 7.4 Hz, m-arom. H); 13C
NMR (CDCl3, 100.62 MHz): δ 23.17 (q, 2-NHCOCH3),
27.01 (q, 3-OCOC (CH3)3), 27.14 (q, 6-OCOC (CH3)3),
38.85 (s, 6-OCOC (CH3)3), 39.00 (s, 3-OCOC (CH3)3),
54.07 (d, C-2), 63.56 (t, C-6), 69.54 (d, C-4), 74.36 (d, C-5),
74.73 (d, C-3), 99.25 (d, C-1), 116.80 (d, o-arom. CH), 122.86
(d, p-arom. CH), 129.40 (d, m-arom. CH), 157.29 (s, arom.
C), 170.13 (s, 2-NHCOCH3), 179.01 (s, 6-OCOC (CH3)3),
179.86 (s, 3-OCOC (CH3)3); HRMS (FAB) m/z [M + H]+
calcd for C24H36NO8+ 466.2435, found 466.2455.
Following general procedure D, compound 6 (646 mg,
1.32 mmol, 1.0 equiv) in dry methanol (13 mL, 0.10 M) was
treated with 1.0 M NaOMe-MeOH (5.27 mL, 5.27 mmol, 4.0
equiv) at room temperature for 27.5 h to provide, after silica
gel chromatography eluting with a gradient from 10% v/v to
50% v/v methanol-chloroform, compound 7 (388 mg,
1.20 mmol, 91%) as a colorless solid. Rf = 0.50 (20% v/v
methanol-chloroform); H NMR (CD3OD, 400.13 MHz):
δ 2.00 (s, 3 H, 2-NHCOCH3), 3.72–3.79 (m, 3 H, H-5, H-
6a, H-6b), 4.00 (d, 1 H, J3,4 = 3.5 Hz, H-4), 4.07 (dd, 1 H,
1
J
2,3 = 10.6 Hz, J3,4 = 3.5 Hz, H-3), 4.13 (dd, 1 H, J2,3 =
10.6 Hz, J1,2 = 8.0 Hz, H-2), 5.02 (d, 1 H, J1,2 = 8.0 Hz, H-
1), 7.00–7.03 (m, 3 H, o-arom. H, p-arom. H), 7.28 (t, 2 H,
Phenyl
2-acetamido-4-azido-2,4-dideoxy-3,6-di-O-pivaloyl-β-
J
m,p = 7.8 Hz, Jo,m = 8.2 Hz m-arom. H); 13C NMR (CD3OD,
D-galactopyranoside (6)
100.62 MHz): δ 21.57 (q, 2-NHCOCH3), 53.23 (d, C-2),
60.74 (t, C-6), 62.39 (d, C-4), 71.51 (d, C-3), 73.76 (d, C-5),
99.75 (d, C-1), 116.34 (d, o-arom. CH), 122.21 (d, p-arom.
CH), 129.07 (d, m-arom. CH), 157.68 (s, arom. C), 172.81 (s,
2 - N H C O C H 3 ) ; H R M S ( FA B ) m / z [ M + N a ] +
C14H18N4O5Na+ 345.1169, found 345.1185.
Following general procedure C, compound 5 (0.532 g,
1.14 mmol, 1.0 equiv) in pyridine (2 mL) and dichloro-
methane (2 mL) was reacted with trifluorometha
nesulfonic anhydride (0.288 mL, 0.495 g, 1.76 mmol,
1.5 equiv) at −10 °C for 1 h followed by treatment with
sodium azide (167.6 mg, 2.578 mmol, 2.3 equiv) in DMF
(5 mL) at room temperature for 28 h to afford, after silica
gel column chromatography eluting with 35% v/v ethyl
acetate-hexane, compound 6 (0.476 g, 0.970 mmol,
85%) as colorless crystals. Rf = 0.48 (50% v/v ethyl ace-
tate-hexane); 1H NMR (CDCl3, 400.13 MHz): δ 1.22 (s, 9
H, 6-OCOC (CH3)3), 1.26 (s, 9 H, 3-OCOC (CH3)3), 1.93
(s, 3 H, 2-NHCOCH3), 4.01 (dd, 1 H, J5,6b = 6.9 Hz,
Phenyl
2,4-diacetamido-2,4-dideoxy-β-D-galactopyranoside
(8)
The general procedure E with compound 7 (32 mg,
99.3 μmol, 1.0 equiv), 5% Pd-C (34 mg) and acetic an-
hydride (0.10 mL, 108 mg, 1.06 mmol, 10.7 equiv),
followed by silica gel column chromatography eluting
with 5% v/v methanol-chloroform gave 8 (20 mg,
59.1 μmol, 60%) as colorless crystals. Rf = 0.41 (20% v/
J
5,6a = 5.5 Hz, H-5), 4.02 (d, 1 H, J3,4 = 3.7 Hz, H-4),
4.24 (dt, 1 H, J2,3 = 11.1 Hz, J2,NH = 8.5 Hz, J1,2
=

Glycoconj J
v methanol-chloroform); 1 H N MR (CD 3 O D,
Phenyl
400.13 MHz): δ 2.01 (s, 3 H, 2-NHCOCH3), 2.09 (s, 3
2-acetamido-2,4-dideoxy-4-pivaloylamino-β-D-galac
H, 4-NHCOCH3), 3.58 (dd, 1 H, J6a,6b = 11.7 Hz, J5,6a
5.6 Hz, H-6a), 3.64 (dd, 1 H, J6a,6b = 11.7 Hz, J5,6b
6.9 Hz, H-6b), 3.80 (ddd, 1 H, J5,6b = 6.9 Hz, J5,6a
5.6 Hz, J4,5 = 1.4 Hz, H-5), 3.92 (dd, 1 H, J2,3
10.8 Hz, J3,4 = 4.4 Hz, H-3), 4.16 (dd, 1 H, J2,3
10.8 Hz, J1,2 = 8.5 Hz, H-2), 4.47 (dd, 1 H, J3,4
=
=
=
=
=
=
topyranoside (10)
The general procedure E with compound 7 (31 mg,
96.2 μmol, 1.0 equiv), 5% Pd-C (34 mg) and pivaloyl chloride
(0.24 mL, 235 mg, 1.95 mmol, 20 equiv), followed by silica
gel column chromatography eluting with a gradient from 5%
v/v to 10% v/v methanol-chloroform gave 10 (8 mg,
21.0 μmol, 22%) as colorless crystals. Rf = 0.60 (20% v/v
4.4 Hz, J4,5 = 1.4 Hz, H-4,), 5.00 (d, 1 H, J1,2 = 8.5 Hz,
H-1), 7.02 (t, 1 H, Jm,p = 7.3 Hz, p-arom. H), 7.04 (d, 2 H,
1
J
o,m = 8.7 Hz, o-arom. H), 7.29 (dd, Jo,m = 8.7 Hz, Jm,p
=
methanol-chloroform); H NMR (CD3OD, 400.13 MHz):
7.3 Hz, m-arom. H); 13C NMR (CD3OD, 100.62 MHz), δ
21.08 (q, 4-NHCOCH3), 21.55 (q, 2-NHCOCH3), 50.13
(d, C-4), 53.29 (d, C-2), 60.83 (t, C-6), 70.21 (d, C-3),
74.94 (d, C-5), 100.24 (d, C-1), 116.29 (d, o-arom. CH),
122.18 (d, p-arom. CH), 129.10 (d, m-arom. CH), 157.78
(s, arom. C), 172.78 (s, 2-NHCOCH3), 173.70 (s, 4-
NHCOCH3); HRMS (FAB) m/z [M + H]+ calcd for
δ 1.17 (s, 9 H, 4-NHCOC (CH3)3), 1.89 (s, 3 H, 2-
NHCOCH3), 3.46 (dd, 1 H, J6a,6b = 11.8 Hz, J5,6a = 5.8 Hz,
H-6a), 3.50 (dd, 1 H, J6a,6b = 11.8 Hz, J5,6b = 6.5 Hz, H-6b),
3.72 (td, 1 H, J5,6b = 6.5 Hz, J5,6a = 5.8 Hz, J4,5 = 1.3 Hz, H-5),
3.84 (dd, 1 H, J2,3 = 10.9 Hz, J3,4 = 4.5 Hz, H-3), 3.98 (dd, 1
H, J2,3 = 10.9 Hz, J1,2 = 8.3 Hz, H-2), 4.35 (dd, 1 H, J3,4
4.5 Hz, J4,5 = 1.3 Hz, H-4), 4.91 (d, 1 H, J1,2 = 8.3 Hz, H-1),
6.91 (t, 1 H, Jm,p = 7.3 Hz, p-arom. H), 6.93 (d, 2 H, Jo,m
=
+
C16H23N2O6 339.1551, found 339.1562.
=
8.7 Hz, o-arom. H), 7.18 (dd, 2 H, Jo,m = 8.7 Hz, Jm,p = 7.3 Hz,
m-arom. H); 13C NMR (CD3OD, 100.62 MHz): δ 21.53 (q, 2-
NHCOCH3), 26.42 (q, 4-NHCOC (CH3)3), 38.83 (s, 4-
NHCOC (CH3)3), 50.31 (d, C-4), 53.30 (d, C-2), 60.84 (t,
C-6), 70.04 (d, C-3), 74.95 (d, C-5), 100.21 (d, C-1), 116.29
(d, o-arom. CH), 122.31 (d, p-arom. CH), 129.14 (d, m-arom.
CH), 157.64 (s, arom. C), 172.88 (s, 2-NHCOCH3), 181.40 (s,
4-NHCOC (CH3)3; HRMS (FAB) m/z [M + Na]+ calcd for
C19H28N2NaO6+ 403.1840, found 403.1849.
Phenyl
2-acetamido-4-benzamido-2,4-dideoxy-β-D-galacto
pyranoside (9)
The general procedure E with compound 7 (32 mg,
99.3 μmol, 1.0 equiv), 5% Pd-C (30 mg) and benzoyl
chloride (30 μL, 36.4 mg, 0.260 mmol, 2.6 equiv), follow-
ed by silica gel column chromatography eluting with 5% v/
v methanol-chloroform gave 9 (17 mg, 42.5 μmol, 43%) as
colorless crystals. Rf = 0.60 (20% v/v methanol-chloro-
Cyclohexyl
3,4,6-tri-O-aceyl-2-deoxy-2-(2,2,2-trichloroethoxy)
carbonylamino-β-D-glucopyranoside (12a)
1
form); H NMR (CD3OD, 400.13 MHz): δ 2.02 (s, 3 H,
2-NHCOCH3), 3.70 (d, 2 H, J5,6 = 6.2 Hz, H-6), 3.92 (td, 1
H, J5,6 = 6.2 Hz, J4,5 = 1.2 Hz, H-5), 4.04 (dd, 1 H, J2,3
11.0 Hz, J3,4 = 4.6 Hz, H-3), 4.28 (dd, 1 H, J2,3 = 11.0 Hz,
1,2 = 8.4 Hz, H-2), 4.72 (dd, 1 H, J3,4 = 4.6 Hz, J4,5
1.2 Hz, H-4), 5.08 (d, 1 H, J1,2 = 8.4 Hz, H-1), 7.03 (t, 1
H, Jm,p = 7.4 Hz, p-arom. H of 1-OPh), 7.07 (d, 2 H, Jo,m
8.5 Hz, o-arom. H of 1-OPh), 7.30 (dd, 2 H, Jo,m = 8.5 Hz,
m,p = 7.4 Hz, m-arom. H of 1-OPh), 7.50 (t, 2 H, Jm,p
7.5, Hz, Jo,m = 7.1 Hz, m-arom. H of 4-NHBz), 7.57 (t, 1 H,
m,p = 7.5, Hz, p-arom. H of 4-NHBz), 7.92 (d, 2 H, Jo,m
=
The general procedure F with compound 11 (2.000 g,
3.49 mmol, 1.0 equiv), cyclohexanol (0.55 mL, 0.521 g,
5.21 mmol, 1.5 equiv), N-iodosuccinimide (1.476 g,
6.56 mmol, 1.9 equiv), and MS4A (2.012 g) in dry dichloro-
methane (35 mL) and 1.0 M trifluoromethanesulfonic acid
(1.0 M in dichloromethane, 0.70 mL) at −10 °C for 1 h,
followed by silica gel column chromatography eluting with
a gradient from 15% v/v to 30% v/v ethyl acetate-hexane gave
12a (1.767 g, 3.14 mmol, 90%) as colorless crystals. Rf = 0.53
(50% v/v ethyl acetate-hexane); 1H NMR (CDCl3,
400.13 MHz): δ 1.18–1.23 (m, 1 H, H-4ax of cyclohexyl),
1.25–1.38 (m, 3 H, H-3ax, H-5ax, H-2ax of cyclohexyl),
1.41–1.44 (m, 1 H, H-6ax of cyclohexyl), 1.46–1.51 (m, 1
H, H-4 eq of cyclohexyl), 1.66–1.75 (m, 2 H, H-3 eq, H-
5 eq of cyclohexyl), 1.78–1.81 (m, 1 H, H-2 eq of cyclohexyl),
1.86–1.92 (m, 1 H, H-6 eq of cyclohexyl), 2.026 (s, 3 H, 4-
OCOCH3), 2.032 (s, 3 H, 3-OCOCH3), 2.08 (s, 3H, 6-
OCOCH3), 3.53 (ddd, 1 H, J2,3 = 9.9 Hz, J1,2 = 8.6 Hz,
J
=
=
J
=
J
=
7.1 Hz, o-arom. H of 4-NHBz); 13C NMR (CD3OD,
100.62 MHz): δ 21.55 (q, 2-NHCOCH3), 50.92 (d, C-4),
53.30 (d, C-2), 60.94 (t, C-6), 70.28 (d, C-3), 75.13 (d, C-
5), 100.28 (d, C-1), 116.34 (d, o-arom. CH of 1-OPh),
122.30 (d, p-arom. CH of 1-OPh), 127.39 (d, o-arom. CH
of 4-NHBz), 128.05 (d, m-arom. CH of 4-NHBz), 129.15
(d, m-arom. CH of 1-OPh), 131.40 (d, p-arom. CH of 4-
NHBz), 134.22 (s, arom. C of 4-NHBz), 157.70 (s, arom. C
of 1-OPh), 170.89 (s, CO of 4-NHBz), 172.95 (s, 2-
NHCOCH3); HRMS (APCI) m/z [M + H]+ calcd for
J2,NH = 8.1 Hz, H-2), 3.63 (tt, 1 H, JH-1ax,H-2ax = 9.1 Hz, JH-
+
C21H25N2O6 401.1707, found 401.1710.
1ax,H-2eq = 3.8 Hz, H-1ax of cyclohexyl), 3.70 (ddd, 1 H, J4,5
=

Glycoconj J
9.8 Hz, J5,6b = 4.8 Hz, J5,6a = 2.4 Hz, H-5), 4.11 (dd, 1 H,
6a,6b = 12.2 Hz, J5,6a = 2.4 Hz, H-6a), 4.28 (dd, 1 H, J6a,6b
12.2 Hz, J5,6b = 4.8 Hz, H-6b), 4.68 (d, 1 H, Jgem = 11.9 Hz,
Ha of Troc), 4.76 (d, 1 H, Jgem = 11.9 Hz, Hb of Troc), 4.79 (d,
(0.10 mL, 98 mg, 0.813 mmol, 5.2 equiv) and pyridine
(0.18 mL, 2.23 mmol, 5.2 equiv) in dichloromethane (2 mL,
78 mM) at 0 °C for 3 h to afford, after silica gel chromatog-
raphy eluting with 20% v/v ethyl acetate-hexane, compound
14a (67 mg, 0.111 mmol, 71%) as colorless crystals. Rf = 0.40
(30% v/v ethyl acetate-hexane); 1H NMR (CDCl3,
400.13 MHz): δ 1.20 (s, 9 H, 3-OCOC (CH3)3), 1.22, (s, 9
H, 6-OCOC (CH3)3), 1.20–1.28 (m, 3 H, H-3ax, H-4ax, H-
5ax of cyclohexyl), 1.30–1.37 (m, 1 H, H-2ax of cyclohexyl),
1.41–1.43 (m, 1 H, H-6ax of cyclohexyl), 1.46–1.49 (m, 1 H,
H-4 eq of cyclohexyl), 1.66–1.73 (m, 2 H, H-3 eq, H-5 eq of
cyclohexyl), 1.77–1.80 (m, 1 H, H-2 eq of cyclohexyl), 1.86–
1.89 (m, 1 H, H-6 eq of cyclohexyl), 3.31 (br s, 1 H, 4-OH),
3.50 (td, 1 H, J4,5 = 9.3 Hz, J3,4 = 9.2 Hz, J4,OH = 4.8, H-4),
3.58–3.67 (m, 3 H, H-1ax of cyclohexyl, H-5, H-2), 4.29 (dd,
1 H, J6a,6b = 11.8 Hz, J5,6a = 6.3 Hz, H-6a), 4.43 (dd, 1 H,
J
=
1 H, J1,2 = 8.6 Hz, H-1), 5.05 (t, 1 H, J4,5 = 9.8 Hz, J3,4
9.6 Hz, H-4), 5.18 (d, 1 H, J2, N H = 8.1 Hz, 2-
NHCOOCH2CCl3), 5.37 (t, 1 H, J2,3 = 9.9 Hz, J
=
=
3,4
9.6 Hz, H-3); 13C NMR (CDCl3, 100.62 MHz): δ 20.59 (q,
4-OCOCH3), 20.63 (q, 3-OCOCH3), 20.70 (q, 6-OCOCH3),
23.65 (t, C-3 of cyclohexyl), 23.77 (t, C-5 of cyclohexyl),
25.43 (t, C-4 of cyclohexyl), 31.58 (t, C-2 of cyclohexyl),
33.23 (t, C-6 of cyclohexyl), 56.62 (d, C-2), 62.25 (t, C-6),
68.95 (d, C-4), 71.58 (d, C-5), 71.76 (d, C-3), 74.43 (t, 2-
NHCOOCH2CCl3), 77.96 (d, C-1 of cyclohexyl), 95.38 (s,
2-NHCOOCH2CCl3), 98.94 (d, C-1), 153.87 (s, 2-
NHCOOCH2CCl3), 169.46 (s, 4-OCOCH3), 170.55 (s, 3-
OCOCH3), 170.66 (s, 6-OCOCH3); HRMS (FAB) m/z [M +
J6a,6b = 11.8 Hz, J5,6b = 1.7 Hz, H-6b), 4.64 (d, 1 H, Jgem =
Na]+ calcd for C21H30Cl3NNaO10 584.0828, found
11.9 Hz, Ha of Troc), 4.66 (d, 1H, J1,2 = 6.5 Hz, H-1), 4.74 (d,
1 H, Jgem = 11.9 Hz, Hb of Troc), 5.20 (t, 1 H, J2,3 = 10.0 Hz,
+
584.0842.
J3,4 = 9.2 Hz, H-3), 5.63 (d, 1 H, J2,NH = 9.2 Hz, 2-NHTroc);
Cyclohexyl 2-deoxy-2-(2,2,2-trichloroethoxy)
carbonylamino-β-D-glucopyranoside (13a)
13C NMR (CDCl3, 100.62 MHz): δ 23.64 (t, C-3 of
cyclohexyl), 23.73 (t, C-5 of cyclohexyl), 25.47 (t, C-4 of
cyclohexyl), 26.97 (q, 3-OCOC (CH3)3), 27.09 (q, 6-OCOC
(CH3)3), 31.58 (t, C-2 of cyclohexyl), 33.27 (t, C-6 of
cyclohexyl), 38.84 (s, 6-OCOC (CH3)3), 38.94 (s, 3-OCOC
(CH3)3), 56.18 (d, C-2), 63.65 (t, C-6), 70.04 (d, C-4), 73.77
(d, C-5), 74.52 (t, 2-NHCOOCH2CCl3), 74.84 (d, C-3), 77.73
(d, C-1 of cyclohexyl), 95.42 (s, 2-NHCOOCH2CCl3), 99.47
(d, C-1), 154.19 (s, 2-NHCOOCH2CCl3), 179.00 (s, 6-OCOC
(CH3)3), 179.72 (s, 3-OCOC (CH3)3); HRMS (APCI) m/z
Following general procedure D, compound 12a (0.767 g,
1.36 mmol, 1.0 equiv) was treated with 1.0 M NaOMe-
MeOH (0.27 mL, 0.27 mmol, 0.20 equiv) at 0 °C for 1.5 h
to give 13a (0.591 g, 1.35 mmol, 99%) as a colorless solid.
1
Rf = 0.28 (20% v/v methanol-chloroform); H NMR (D2O,
400.13 MHz): δ 1.12–1.25 (m, 5 H, H-4ax, H-3ax, H-5ax,
H-2ax, H-6ax of cyclohexyl), 1.34–1.36 (m, 1 H, H-4 eq of
cyclohexyl), 1.53–1.54 (m, 2 H, H-3 eq, H-5 eq of
cyclohexyl), 1.69–1.75 (m, 2 H, H-6 eq, H-2 eq of
cyclohexyl), 3.24–3.31 (m, 3 H, H-2, H-5, H-4), 3.41–3.42
(m, 1 H, H-3), 3.62 (d, 1 H, J6a,6b = 11.3 Hz, H-6a), 3.61–3.66
(m, 1 H, H-1ax of cyclohexyl), 3.79 (d, 1 H, J6a,6b = 11.3 Hz,
+
[M + H]+ calcd for C25H41Cl3NO9 604.1841, found
604.1838.
Cyclohexyl
2-acetamido-2-deoxy-3,6-di-O-pivaloyl-β-D-glucopy
H-6b), 4.53 (d, 1 H, J1,2 = 7.8 Hz, H-1), 4.61 (d, 1 H, Jgem
=
ranoside (15a)
12.7 Hz, Ha of Troc), 4.80 (d, 1 H, Jgem = 12.7 Hz, Hb of
Troc); 13C NMR (D2O, 100.62 MHz): δ 23.02 (t, C-3 of
cyclohexyl), 23.24 (t, C-5 of cyclohexyl), 24.95 (t, C-4 of
cyclohexyl), 30.95 (t, C-2 of cyclohexyl), 32.54 (t, C-6 of
cyclohexyl), 57.58 (d, C-2), 60.71 (t, C-6), 69.89 (d, C-4),
73.83 (d, C-3), 74.18 (t, 2-NHCOOCH2CCl3), 75.78 (d, C-
5), 78.28 (d, C-1 of cyclohexyl), 94.93 (s, 2-
NHCOOCH2CCl3), 99.12 (d, C-1), 156.78 (s, 2-
NHCOOCH2CCl3); HRMS (APCI) m/z [M + H]+ calcd for
C15H25Cl3NO7+ 436.0691, found 436.0688.
Following general procedure A, a solution of compound 14a
(0.252 g, 0.417 mmol, 1.0 equiv) in dichloromethane (6.0 mL)
and methanol (6.0 mL) was treated with activated zinc
(3.543 g, 54.17 mmol, 20.8 equiv) and acetic acid (1.19 mL)
at room temperature for 2 h, followed by acetylation with
acetic anhydride (0.82 mL, 0.886 g, 8.67 mmol, 20.8 equiv)
at room temperature for 18 h to give, after separated by silica
gel column chromatography eluting with 15% v/v ethyl ace-
tate-hexane, compound 15a (0.144 g, 0.305 mmol, 73%) as
colorless crystals. Rf = 0.30 (30% v/v ethyl acetate-hexane);
1H NMR (CDCl3, 400.13 MHz): δ 1.20 (s, 9 H, 3-OCOC
(CH3)3), 1.22 (s, 9 H, 6-OCOC (CH3)3), 1.19–1.27 (m, 3 H,
H-3ax, H-4ax, H-5ax of cyclohexyl), 1.29–1.31 (m, 1 H, H-
2ax of cyclohexyl), 1.38–1.42 (m, 1 H, H-6ax of cyclohexyl),
1.45–1.49 (m, 1 H, H-4 eq of cyclohexyl), 1.68–1.73 (m, 2 H,
H-3 eq, H-5 eq of cyclohexyl), 1.75–1.79 (m, 1 H, H-2 eq of
Cyclohexyl
2-deoxy-3,6-di-O-pivaloyl-2-(2,2,2-trichloroethoxy)
carbonylamino-β-D-glucopyranoside (14a)
Following general procedure B, compound 13a (68 mg,
0.156 mmol, 1.0 equiv) was reacted with pivaloyl chloride

Glycoconj J
cyclohexyl), 1.85–1.89 (m, 1 H, H-6 eq of cyclohexyl), 1.92
(s, 3 H, 2-NHCOCH3), 3.10 (d, 1 H, J4,OH = 5.4 Hz, 4-OH),
3.49 (td, 1 H, J4,5 = 9.7 Hz, J3,4 = 9.1 Hz, J4,OH = 5.4 Hz, H-4),
3.58 (ddd, 1 H, J4,5 = 9.5 Hz, J5,6a = 5.8 Hz, J5,6b = 2.5 Hz, H-
5), 3.58–3.61 (m, 1 H, H-1ax of cyclohexyl), 3.80 (dt, 1 H,
(CH3)3), 27.11 (q, 6-OCOC (CH3)3), 31.72 (t, C-2 of
cyclohexyl), 33.32 (t, C-6 of cyclohexyl), 38.74 (s, 6-OCOC
(CH3)3), 39.19 (s, 3-OCOC (CH3)3), 52.99 (d, C-2), 60.73 (d,
C-4), 62.72 (t, C-6), 70.45 (d, C-5), 71.27 (d, C-3), 77.72 (d,
C-1 of cyclohexyl), 98.80 (d, C-1), 170.03 (s, 2-NHCOCH3),
177.91 (s, 6-OCOC (CH3)3), 177.93 (s, 3-OCOC (CH3)3);
J
2,3 = 10.5 Hz, J2,NH = 8.8 Hz, J1,2 = 8.5 Hz, H-2), 4.33 (dd, 1
H, J6a,6b = 11.9 Hz, J5,6a = 5.8 Hz, H-6a), 4.40 (dd, 1 H,
6a,6b = 11.9 Hz, J5,6b = 2.5 Hz, H-6b), 4.67 (d, 1 H, J1,2
HRMS (APCI) m/z [M + H]+ calcd for C24H41N4O7
+
J
=
497.2970, found 497.2962.
8.5 Hz, H-1), 5.16 (dd, 1 H, J2,3 = 10.5 Hz, J3,4 = 9.1 Hz, H-
3), 5.78 (d, 1 H, J2,NH = 8.8 Hz, 2-NH); 13C NMR (CDCl3,
100.62 MHz): δ 23.25 (q, 2-NHCOCH3), 23.77 (t, C-3 of
cyclohexyl), 23.87 (t, C-5 of cyclohexyl), 25.52 (t, C-4 of
cyclohexyl), 27.05 (q, 3-OCOC (CH3)3), 27.17 (q, 6-OCOC
(CH3)3), 31.75 (t, C-2 of cyclohexyl), 33.33 (t, C-6 of
cyclohexyl), 38.90 (s, 6-OCOC (CH3)3), 39.01 (s, 3-OCOC
(CH3)3), 54.65 (d, C-2), 63.54 (t, C-6), 69.96 (d, C-4), 74.02
(d, C-5), 75.13 (d, C-3), 77.53 (d, C-1 of cyclohexyl), 99.38
(d, C-1), 169.85 (s, 2-NHCOCH3), 178.96 (s, 6-OCOC
(CH3)3), 179.89 (s, 3-OCOC (CH3)3); HRMS (FAB) m/z
[M + H]+ calcd for C24H42NO8+ 472.2905, found 472.2877.
Cyclohexyl
2-acetamido-4-azido-2,4-dideoxy-β-D-galacto
pyranoside (17a)
Following general procedure D, compound 16a (338 mg,
0.681 mmol, 1.0 equiv) in dry methanol (7 mL, 0.097 M)
was treated with 1.0 M NaOMe-MeOH (2.72 mL,
2.72 mmol, 4.0 equiv) at 0 °C for 17.5 h to provide, after
silica gel chromatography eluting with a gradient from
10% v/v to 20% v/v methanol-chloroform, compound
17a (208 mg, 0.633 mmol, 93%) as a colorless solid.
Rf = 0.55 (20% v/v methanol-chloroform); 1H NMR
(CD3OD, 400.13 MHz): δ 1.21–1.33 (m, 4 H, H-2ax, H-
3ax, H-4ax, H-5ax of cyclohexyl), 1.36–1.41 (m, 1 H, H-
6ax of cyclohexyl), 1.44–1.49 (m, 1 H, H-4 eq of
cyclohexyl), 1.69–1.72 (m, 2 H, H-3 eq, H-5 eq of
cyclohexyl), 1.77–1.81 (m, 2 H, H-2 eq, H-6 eq of
cyclohexyl), 1.96 (s, 3 H, 2-NHCOCH3), 3.55 (td, 1 H,
Cyclohexyl
2-acetamido-4-azido-2,4-dideoxy-3,6-di-O-pivaloyl-β-
D-galactopyranoside (16a)
Following general procedure C, compound 15a (0.603 g,
1.28 mmol, 1.0 equiv) in pyridine (1.03 mL) and dichloro-
methane (12 mL) was reacted with trifluoromethanesulfonic
anhydride (0.43 mL, 0.740 g, 2.62 mmol, 2.1 equiv) at −10 °C
for 1.5 h, followed by treatment with sodium azide (0.210 g,
3.23 mmol, 2.5 equiv) in DMF (12 mL) at room temperature
for 17 h to give, after silica gel column chromatography elut-
ing with 20% v/v ethyl acetate-hexane, compound 16a
(0.358 g, 0.720 mmol, 56%) as colorless crystals. Rf = 0.20
(30% v/v ethyl acetate-hexane); 1H NMR (CDCl3,
400.13 MHz): δ 1.21 (s, 9 H, 6-OCOC (CH3)3), 1.24 (s, 9
H, 3-OCOC (CH3)3), 1.19–1.28 (m, 4 H, H-3ax, H-4ax, H-
5ax, H-2ax of cyclohexyl), 1.35–1.45 (m, 1 H, H-6ax of
cyclohexyl), 1.47–1.50 (m, 1 H, H-4 eq of cyclohexyl),
1.67–1.72 (m, 2 H, H-3 eq, H-5 eq of cyclohexyl), 1.76–
1.78 (m, 1 H, H-2 eq of cyclohexyl), 1.86–1.90 (m, 1 H, H-
6 eq of cyclohexyl), 1.92 (s, 3 H, 2-NHCOCH3), 3.58 (tt, 1 H,
J
5,6 = 6.5 Hz, J4,5 = 0.9 Hz, H-5), 3.60–3.66 (m, 1 H, H-
1ax of cyclohexyl), 3.67 (d, 2 H, J5,6 = 6.5 Hz, H-6), 3.75
(dd, 1 H, J2,3 = 10.6 Hz, J1,2 = 8.4 Hz, H-2), 3.90 (dd, 1 H,
J
3,4 = 3.7 Hz, J4,5 = 0.9 Hz, H-4), 3.97 (dd, 1 H, J2,3 =
10.6 Hz, J3,4 = 3.7 Hz, H-3), 4.50 (d, 1 H, J1,2 = 8.4 Hz,
H-1); 13C NMR (CD3OD, 100.62 MHz): δ 23.08 (q, 2-
NHCOCH3), 24.56 (t, C-3 of cyclohexyl), 24.73 (t, C-5 of
cyclohexyl), 26.81 (t, C-4 of cyclohexyl), 32.61(t, C-2 of
cyclohexyl), 34.38 (t, C-6 of cyclohexyl), 55.18 (d, C-2),
62.22 (t, C-6), 64.04 (d, C-4), 72.99 (d, C-3), 74.80 (d, C-
5), 78.06 (d, C-1 of cyclohexyl), 101.22 (d, C-1), 173.94
(s, 2-NHCOCH3); HRMS (APCI) m/z [M + H]+ calcd for
+
C14H25N4O5 329.1820, found 329.1811.
Benzyl
J
H-1ax,H-6ax = 9.1 Hz, JH-1ax,H-2eq = 3.9 Hz, H-1ax of
cyclohexyl), 3.76 (dt, 1 H, J2,3 = 11.0 Hz, J1,2 = 8.3 Hz,
2,NH = 7.9 Hz, H-2), 3.86 (td, 1 H, J5,6b = 6.7 Hz, J5,6a
6.5 Hz, J4,5 = 0.9 Hz, H-5), 3.96 (dd, 1 H, J3,4 = 3.7 Hz,
4,5 = 0.9 Hz, H-4), 4.17 (dd, 1 H, J6a,6b = 11.2 Hz, J5,6a
6.5 Hz, H-6a), 4.30 (dd, 1 H, J6a,6b = 11.2 Hz, J5,6b = 6.7 Hz,
3,4,6-tri-O-aceyl-2-deoxy-2-(2,2,2-trichloroethoxy)
carbonylamino-β-D-glucopyranoside (12b)
J
=
The general procedure F with compound 11 (2.001 g,
3.49 mmol, 1.0 equiv), benzyl alcohol (0.54 mL, 0.56 g,
5.22 mmol, 1.5 equiv), N-iodosuccinimide (1.4775 g,
6.57 mmol, 1.9 equiv), trifluoromethanesulfonic acid
(65 μL, 111 mg, 0.741 mmol, 0.21 equiv), and MS4A
(2.012 g) in dry dichloromethane (35 mL) at −10 °C for
23 h, followed by silica gel column chromatography elut-
ing with a gradient from 13% v/v to 27% v/v ethyl acetate-
J
=
H-6b), 4.88 (d, 1 H, J1,2 = 8.3 Hz, H-1), 5.54 (d, 1 H, J2,NH
7.9 Hz, 2-NHCOCH3), 5.55 (dd, 1 H, J2,3 = 11.0 Hz, J3,4
=
=
3.7 Hz, H-3); 13C NMR (CDCl3, 100.62 MHz): δ 23.31 (q, 2-
NHCOCH3), 23.77 (t, C-3 of cyclohexyl), 23.87 (t, C-5 of
cyclohexyl), 25.48 (t, C-4 of cyclohexyl), 27.04 (q, 3-OCOC

Glycoconj J
hexane, gave 12b (1.758 g, 3.08 mmol, 88%) as colorless
crystals. Rf = 0.44 (50% v/v ethyl acetate-hexane); 1H
NMR (CDCl3, 400.13 MHz): δ 2.02 (s, 3 H, 4-
OCOCH3), 2.03 (s, 3 H, 3-OCOCH3), 2.12 (s, 3 H, 6-
OCOCH3), 3.67 (ddd, 1 H, J4,5 = 9.7 Hz, J5,6b = 4.5 Hz,
Benzyl
2-deoxy-3,6-di-O-pivaloyl-2-(2,2,2-trichloroethoxy)
carbonylamino-β-D-glucopyranoside (14b)
Following general procedure B, compound 13b (0.709 g,
1.59 mmol, 1.0 equiv) was reacted with pivaloyl chloride
(0.585 mL, 0.573 g, 4.75 mmol, 3.0 equiv) in pyridine
(0.78 mL) and dichloromethane (5.9 mL) at −10 °C for 20 h
to afford, after silica gel chromatography eluting with a gra-
dient from 10% v/v to 30% v/v ethyl acetate-hexane, com-
pound 14b (0.841 g, 1.37 mmol, 86%) as colorless crystals.
J
5,6a = 2.2 Hz, H-5), 3.73 (dt, 1 H, J2,3 = 10.3 Hz, J2,NH
=
9.1 Hz, J1,2 = 8.8 Hz, H-2), 4.17 (dd, 1 H, J6a,6b = 12.3 Hz,
J
J
5,6a = 2.2 Hz, H-6a), 4.30 (dd, 1 H, J6a,6b = 12.3 Hz,
5,6b = 4.5 Hz, H-6b), 4.617 (d, 1 H, J1,2 = 8.8 Hz, H-1),
4.621 (d, 1 H, Jgem = 12.0 Hz, Ha of 1-OCH2C6H5), 4.72
(s, 2 H, CH2 of Troc), 4.92 (d, 1 H, Jgem = 12.0 Hz, Hb of
1-OCH2C6H5), 4.99 (d, 1 H, J2, NH = 9.1 Hz, 2-
1
Rf = 0.48 (30% v/v ethyl acetate-hexane); H NMR (CDCl3,
NHCOOCH2CCl3), 5.10 (t, 1 H, J4,5 = 9.7 Hz, J3,4
=
400.13 MHz): δ 1.19 (s, 9 H, 3-OCOC (CH3)3), 1.26 (s, 9 H,
6-OCOC (CH3)3), 2.91 (s, 1 H, 4-OH), 3.48–3.55 (m, 2 H, H-
9.6 Hz, H-4), 5.23 (dd, 1 H, J2,3 = 10.3 Hz, J3,4 = 9.6 Hz,
H-3), 7.26–7.37 (m, 5 H, arom. H); 13C NMR (CDCl3,
100.62 MHz): δ 20.64 (q, 4- or 3-OCOCH3), 20.66 (q, 3-
or 4-OCOCH3), 20.80 (q, 6-OCOCH3), 56.22 (d, C-2),
62.04 (t, C-6), 68.61 (d, C-4), 70.85 (t, 1-OCH2C6H5),
71.90 (d, C-3), 71. 90 (d, C-5), 74. 51 (t, 2-
NHCOOCH2CCl3), 95.36 (s, 2-NHCOOCH2CCl3), 99.24
(d, C-1), 128.06 (d, o-arom. CH), 128.19 (d, p-arom. CH),
128.57 (d, m-arom. CH), 136.50 (s, arom. C), 154.00 (s, 2-
NHCOOCH2CCl3), 169.46 (s, 4-OCOCH3), 170.67 (s, 3-
OCOCH3), 170.73 (s, 6-OCOCH3); HRMS (APCI) m/z
4, H-5), 3.75 (dt, 1 H, J2,3 = 10.2 Hz, J2,NH = 9.1 Hz, J1,2
=
8.4 Hz, H-2), 4.40 (dd, 1 H, J6a,6b = 12.0 Hz, J5,6a = 1.9 Hz, H-
6a), 4.44 (dd, 1 H, J6a,6b = 12.0 Hz, J5,6b = 3.5 Hz, H-6b), 4.47
(d, 1 H, J1,2 = 8.4 Hz, H-1), 4.61 (d, 1 H, Jgem = 12.3 Hz, 1-
OCH (H)C6H5), 4.62 (d, 1 H, Jgem = 12.0 Hz, Ha of Troc),
4.76 (d, 1 H, Jgem = 12.0 Hz, Hb of Troc), 4.90 (d, 1 H, Jgem
12.3 Hz, 1-OCH (H)C6H5), 4.96 (dd, 1 H, J2,3 = 10.2 Hz,
3,4 = 8.5 Hz, H-3), 5.00 (d, 1 H, J2,NH = 9.1 Hz, 2-
=
J
NHCOCH3), 7.29–7.36 (m, 5 H, arom. H); 13C NMR
(CDCl3, 100.62 MHz): δ 27.00 (q, 3-OCOC (CH3)3), 27.24
(q, 6-OCOC (CH3)3), 39.00 (s, 6-OCOC (CH3)3), 39.01 (s, 3-
OCOC (CH3)3), 55.84 (d, C-2), 63.05 (t, C-6), 69.64 (d, C-4),
70.37 (t, 1-OCH2C6H5), 74.36 (d, C-5), 74.50 (d, C-3), 74.65
(t, 2-NHCOOCH2CCl3), 95.27 (s, 2-NHCOOCH2CCl3),
99.49 (d, C-1), 127.90 (d, o-arom. CH), 128.04 (d, p-arom.
CH), 128.51 (d, m-arom. CH), 136.80 (s, arom. C), 154.12 (s,
2-NHCOOCH2CCl3), 179.16 (s, 6-OCOC (CH3)3), 179.65 (s,
3-OCOC (CH3)3); HRMS (APCI) m/z [M + H]+ calcd for
C26H37Cl3NO9+ 612.1528, found 612.1524.
+
[M + H]+ calcd for C22H27Cl3NO10 570.0695, found
570.0679.
Benzyl 2-deoxy-2-(2,2,2-trichloroethoxy)
carbonylamino-β-D-glucopyranoside (13b)
Following general procedure D, compound 12b (0.947 g,
1.66 mmol, 1 equiv) in dry methanol (23.7 mL, 0.070 M)
was treated with 1.0 M NaOMe-MeOH (0.30 mL,
0.30 mmol, 0.18 equiv) at −5 °C for 2.5 h to provide
13b (0.719 g, 1.62 mmol, 97%) as a colorless solid.
Rf = 0.18 (10% v/v methanol-chloroform); 1H NMR
(CD3OD, 400.13 MHz): δ 3.27 (ddd, 1 H, J4,5 = 9.7 Hz,
Benzyl
2-acetamido-2-deoxy-3,6-di-O-pivaloyl-β-D-gluco
pyranoside (15b)
J
5,6a = 5.9 Hz, J5,6b = 2.2 Hz, H-5), 3.34 (t, 1 H, J3,4 =
10.0 Hz, J4,5 = 9.7 Hz, H-4), 3.41–3.49 (m, 2 H, H-3, H-
4), 3.71 (dd, 1 H, J6a,6b = 11.9 Hz, J5,6a = 5.9 Hz, H-6a),
3.91 (dd, 1 H, J6a,6b = 11.9 Hz, J5,6b = 2.2 Hz, H-6b), 4.48
(d, 1 H, J1,2 = 7.8 Hz, H-1), 4.61 (d, 1 H, Jgem = 11.9 Hz,
Ha of 1-OCH2C6H5), 4.65 (dd, 1 H, Jgem = 12.0 Hz, Ha of
Troc), 4.87 (dd, 1 H, Jgem = 12.0 Hz, Hb of Troc), 4.91 (d,
1 H, Jgem = 12.0 Hz, Hb of 1-OCH2C6H5), 7.23–7.33 (m,
5 H, arom. H); 13C NMR (CD3OD, 100.62 MHz): δ59.37
(d, C-2), 62.97 (t, C-6), 71.82 (t, 1-OCH2C6H5), 72.34 (d,
C-4), 75.69 (t, 2-NHCOOCH2CCl3), 75.90 (d, C-3), 78.14
(d, C-5), 97.32 (s, 2-NHCOOCH2CCl3), 102.17 (d, C-1),
128.75 (d, p-arom. CH), 128.90 (d, o-arom. CH), 129.44
(d, m-arom. CH), 139.24 (s, arom. C), 157.23 (s, 2-
NHCOOCH2CCl3); HRMS (APCI) m/z [M + H]+ calcd
Following general procedure A, a solution of compound 14b
(0.803 g, 1.31 mmol, 1.0 equiv) in dichloromethane (9.4 mL)
and methanol (9.4 mL) was treated with activated zinc
(10.379 g, 158.7 mmol, 121 equiv) and acetic acid
(3.56 mL) at rt. for 4 h, followed by acetylation with acetic
anhydride (0.89 mL, 0.96 g, 9.4 mmol, 8.3 equiv) at rt. for
16 h to give, after separated by silica gel column chromatog-
raphy eluting with 15% v/v ethyl acetate-hexane, compound
15b (0.427 g, 0.305 mmol, 68%) as colorless crystals. Rf =
1
0.20 (40% v/v ethyl acetate-hexane); H NMR (CDCl3,
400.13 MHz): δ 1.19 (s, 9 H, 3-OCOC (CH3)3), 1.26 (s, 9
H, 6-OCOC (CH3)3), 1.90 (s, 3 H, 2-NHCOCH3), 2.86 (d, 1
H, J4,OH = 4.2 Hz, 4-OH), 3.49 (ddd, 1 H, J4,5 = 9.7 Hz,
J
5,6b = 4.5 Hz, J5,6a = 2.3 Hz, H-5), 3.53 (ddd, 1 H, J4,5
=
+
for C16H21Cl3NO7 444.0378, found 444.0370.
9.7 Hz, J3,4 = 8.4 Hz, J4,OH = 4.2 Hz, H-4), 4.06 (ddd, 1 H,

Glycoconj J
m/z [M + H]+ calcd for C25H37N4O7 505.2657, found
+
J2,3 = 10.8 Hz, J2,NH = 9.4 Hz, J1,2 = 8.5 Hz, H-2), 4.38 (dd, 1
H, J6a,6b = 12.2 Hz, J5,6a = 2.3 Hz, H-6a), 4.43 (d, 1 H, J1,2
=
505.2658.
8.5 Hz, H-1), 4.46 (dd, 1 H, J6a,6b = 12.2 Hz, J5,6b = 4.5 Hz, H-
6b), 4.60 (dd, 1 H, Jgem = 12.3 Hz, 1-OCH (H)C6H5), 4.88
(dd, 1 H, Jgem = 12.3 Hz, 1-OCH (H)C6H5), 4.91 (dd, 1 H,
Benzyl 2-acetamido-4-azido-2,4-dideoxy-β-D-galacto
pyranoside (17b)
J
2,3 = 10.8 Hz, J3,4 = 8.4 Hz, H-3), 5.32 (d, 1 H, J2,NH
9.4 Hz, 2-NHCOCH3), 7.29 (dd, 2 H, Jo,m = 7.6 Hz, o-arom.
H), 7.28–7.33 (m, 1 H, p-arom. H), 7.35 (ddd, 2 H, Jm,p
=
Following general procedure D, compound 16b (109.1 mg,
0.216 mmol, 1.0 equiv) in dry methanol (1.08 mL) was treated
with 1.0 M NaOMe-MeOH (0.86 mL, 0.86 mmol, 4.0 equiv)
at 0 °C for 22.5 h to give, after separated by reversed phase
chromatography eluting with a gradient from 30% v/v to 25%
v/v water-methanol, compound 17b (50.7 mg, 0.151 mmol,
70%) as a colorless solid. Rf = 0.44 (10% v/v methanol-chlo-
roform); 1H NMR (CD3OD, 400.13 MHz): δ 1.95 (s, 3 H, 2-
NHCOCH3), 3.58 (dt, 1 H, J5,6b = 6.7 Hz, J5,6a = 6.2 Hz,
=
8.4 Hz, Jo,m = 7.6 Hz, m-arom. H); 13C NMR (CDCl3,
100.62 MHz): δ 23.31 (q, 2-NHCOCH3), 27.03 (q, 3-OCOC
(CH3)3), 27.24 (q, 6-OCOC (CH3)3), 39.02 (s, 3,6-OCOC
(CH3)3), 53.64 (d, C-2), 63.05 (t, C-6), 69.27 (d, C-4), 70.07
(t, 1-OCH2C6H5), 74.41 (d, C-5), 74.79 (d, C-3), 99.55 (d, C-
1), 128.01 (d, p-arom. CH), 128.04 (d, o-arom. CH), 128.47
(d, m-arom. CH), 137.07 (s, arom. C), 169.80 (s, 2-
NHCOCH3), 179.29 (s, 6-OCOC (CH3)3), 179.77 (s, 3-
OCOC (CH3)3); HRMS (APCI) m/z [M + H]+ calcd for
C25H38NO8+ 480.2592, found 480.2599.
J4,5 = 0.9 Hz, H-5), 3.70 (dd, 1 H, J6a,6b = 11.0 Hz, J5,6a =
6.2 Hz, H-6a), 3.75 (dd, 1 H, J6a,6b = 11.0 Hz, J5,6b = 6.7 Hz,
H-6b), 3.90–3.93 (m, 3 H, H-2, H-3, H-4), 4.42–4.44 (m, 1 H,
H-1), 4.57 (d, 1 H, Jgem = 12.5 Hz, Ha of 1-OCH2C6H5), 4.84
(d, 1 H, Jgem = 12.5 Hz, Hb of 1-OCH2C6H5), 7.24–7.34 (m, 5
H, arom. H); 13C NMR (CD3OD, 100.62 MHz): δ 23.15 (q, 2-
NHCOCH3), 54.74 (d, C-2), 62.41 (t, C-6), 64.06 (d, C-4),
71.70 (t, 1-OCH2C6H5), 73.20 (d, C-3), 75.10 (d, C-5), 102.22
(d, C-1), 128.84 (d, p-arom. CH), 128.99 (d, o-arom. CH),
129.48 (d, m-arom. CH), 139.24 (s, arom. C), 174.16 (s, 2-
NHCOCH3); HRMS (APCI) m/z [M + H]+ calcd for
C15H21N4O5+ 337.1507, found 337.1500.
Benzyl
2-acetamido-4-azido-2,4-dideoxy-3,6-di-O-pivaloyl-β-
D-galactopyranoside (16b)
Following general procedure C, compound 15b (0.520 g,
1.08 mmol, 1.0 equiv) in pyridine (1.6 mL) and dichlorometh-
ane (4.7 mL) was reacted with trifluoromethanesulfonic anhy-
dride (0.45 mL, 0.774 g, 2.74 mmol, 2.5 equiv) at −10 °C for
2.5 h, followed by treatment with sodium azide (0.231 g,
3.55 mmol, 3.3 equiv) in DMF (3.7 mL) at rt. for 17.5 h to
give, after silica gel column chromatography eluting with 15%
v/v ethyl acetate-hexane, compound 16b (0.2704 g,
0.536 mmol, 49%) as colorless crystals. Rf = 0.50 (30% v/v
ethyl acetate-hexane); 1H NMR (CDCl3, 400.13 MHz): δ 1.23
(s, 9 H, 3-OCOC (CH3)3), 1.24 (s, 9 H, 6-OCOC (CH3)3),
1.90 (s, 3 H, 2-NHCOCH3), 3.81 (dt, 1 H, J5,6a = 6.6 Hz,
Sulfotransferase activity assay
GalNAc4S-6ST activity was assayed by the radioisotope la-
beling method using [35S] PAPS (Perkin Elmer, Boston, MA)
as described previously [20, 40]. The reaction mixture in a
final volume of 50 μL at pH 6.8 consisted of 50 mM sodium
potassium phosphate, 2 mM dithiothreitol (DTT), [35S] PAPS
(1 nmol, 0.1 μCi), 10 μg of CS-A from whale cartilage
(Seikagaku, Tokyo, Japan), and the recombinant human
GalNAc4S-6ST [41] in the absence or presence of the synthe-
sized GalNAc derivatives (0.1–5.0 mM). The reaction mixture
was incubated at 37 °C for 30 min and stopped by heating at
100 °C for 1 min. The radiolabeled products were isolated by
gel filtration using a Superdex Peptide HR 10/300 column and
quantified by liquid scintillation counting.
J
5,6b = 6.5 Hz, J4,5 = 1.0 Hz, H-5), 3.92 (dd, 1 H, J3,4
=
3.6 Hz, J4,5 = 1.0 Hz, H-4), 4.12 (dt, 1 H, J2,3 = 11.1 Hz,
J
2,NH = 8.8 Hz, J1,2 = 8.4 Hz, H-2), 4.22 (dd, 1 H, J6a,6b
=
=
11.3 Hz, J5,6a = 6.6 Hz, H-6a), 4.35 (dd, 1 H, J6a,6b
11.3 Hz, J5,6b = 6.5 Hz, H-6b), 4.57 (d, 1 H, Jgem = 12.1 Hz,
1-OCH (H)C6H5), 4.59 (d, 1 H, J1,2 = 8.4 Hz, H-1), 4.87 (d, 1
H, Jgem = 12.1 Hz, 1-OCH (H)C6H5), 5.22 (d, 1 H, J2,NH
8.8 Hz, 2-NHCOCH3), 5.30 (dd, 1 H, J2,3 = 11.1 Hz, J3,4
=
=
3.6 Hz, H-3), 7.26–7.36 (m, 5 H, arom. H); 13C NMR (CDCl3,
100.62 MHz): δ 23.35 (q, 2-NHCOCH3), 27.04 (q, 3-OCOC
(CH3)3), 27.18 (q, 6-OCOC (CH3)3), 38.81 (s, 6-OCOC
(CH3)3), 39.24 (s, 3-OCOC (CH3)3) 51.71 (d, C-2), 60.63
(d, C-4), 62.60 (t, C-6), 70.33 (t, 1-OCH2C6H5), 70.72 (d,
C-5), 71.59 (d, C-3), 99.36 (d, C-1), 128.07 (d, p-arom.
CH), 128.13 (d, o-arom. CH), 128.48 (d, m-arom. CH),
136.95 (s, arom. C), 169.88 (s, 2-NHCOCH3), 177.97 (s, 6-
OCOC (CH3)3), 178.23 (s, 3-OCOC (CH3)3); HRMS (APCI)
Funding This work was supported by the Joint Usage/Research Center
on Tropical Disease, Institute of Tropical Medicine, Nagasaki University
(to K.I.P.J.H, 2016-Ippan-11).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.

Glycoconj J
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Chem. 283(49), 34294–34304 (2008). https://doi.org/10.1074/jbc.
M806015200
12. Sato, Y., Nakanishi, K., Tokita, Y., Kakizawa, H., Ida, M., Maeda,
H., Matsui, F., Aono, S., Saito, A., Kuroda, Y., Hayakawa, M.,
Kojima, S., Oohira, A.: A highly sulfated chondroitin sulfate prep-
aration, CS-E, prevents excitatory amino acid-induced neuronal cell
death. J. Neurochem. 104(6), 1565–1576 (2008). https://doi.org/10.
1111/j.1471-4159.2007.05107.x
13. Basappa, M.S., Sugahara, K.N., Lee, C.M., ten Dam, G.B., van
Kuppevelt, T.H., Miyasaka, M., Yamada, S., Sugahara, K.:
Involvement of chondroitin sulfate E in the liver tumor focal for-
mation of murine osteosarcoma cells. Glycobiology. 19(7), 735–
742 (2009). https://doi.org/10.1093/glycob/cwp041
14. Kobayashi, T., Yan, H., Kurahashi, Y., Ito, Y., Maeda, H., Tada, T.,
Hongo, K., Nakayama, J.: Role of GalNAc4S-6ST in astrocytic
tumor progression. PLoS One. 8(1), e54278 (2013). https://doi.
org/10.1371/journal.pone.0054278
15. Seko, A., Yamase, T., Yamashita, K.: Polyoxometalates as effective
inhibitors for sialyl- and sulfotransferases. J. Inorg. Biochem.
103(7), 1061–1066 (2009). https://doi.org/10.1016/j.jinorgbio.
2009.05.002
16. Yoneyama, H., Shibazaki, Y., Fujii, S.: Therapeutic agents contain-
ing specified 3-nitropyridine derivatives for treatment of chronic
inflammatory disease. Jpn. In: Kokai Tokkyo Koho JP
2012062286 A 20120329 (2012)
17. Yoneyama, H., Shibazaki, Y., Fujii, S.: Therapeutic agents contain-
ing specified hydrazone compounds for treatment of chronic in-
flammatory disease. Jpn. In: Kokai Tokkyo Koho JP 2012046453
A 20120308 (2012)
18. Cheung, S.T., Miller, M.S., Pacoma, R., Roland, J., Liu, J.,
Schumacher, A.M., Hsieh-Wilson, L.C.: Discovery of a small-
molecule modulator of glycosaminoglycan sulfation. ACS Chem.
Biol. 12(12), 3126–3133 (2017). https://doi.org/10.1021/
acschembio.7b00885
19. Sawada, T., Fujii, S., Nakano, H., Ohtake, S., Kimata, K., Habuchi,
O.: Synthesis of sulfated phenyl 2-acetamido-2-deoxy-D-
galactopyranosides. 4-O-sulfated phenyl 2-acetamido-2-deoxy-β-
D-galactopyranoside is a competitive acceptor that decreases
sulfation of chondroitin sulfate by N-acetylgalactosamine 4-sulfate
6-O-sulfotransferase. Carbohydr. Res. 340(12), 1983–1996 (2005).
https://doi.org/10.1016/j.carres.2005.06.010
20. Nozaki, H., Tomoyama, Y., Takagi, H., Yokoyama, K., Yamada, C.,
Kaio, K., Tsukimori, M., Nagao, K., Itakura, Y., Ohtake-Niimi, S.,
Nakano, H., Habuchi, O.: Inhibition of N-acetylgalactosamine 4-
sulfate 6-O-sulfotransferase by β-D-4-O-sulfo-N-
acetylgalactosaminides bearing various hydrophobic aglycons.
Glycoconj. J. 27(2), 237–248 (2010). https://doi.org/10.1007/
s10719-009-9272-7
21. Nishiyama, T., Ichikawa, Y., Isobe, M.: Glycocinnasperimicin D
synthetic studies: synthesis of cinnamoyl glycosides via
iodination-heck reaction sequence starting from phenyl glycosides.
Synlett. (1, 1), 89–92 (2004). https://doi.org/10.1055/s-2003-43376
22. Greig, I.R., Macauley, M.S., Williams, I.H., Vocadlo, D.J.: Probing
synergy between two catalytic strategies in the glycoside hydrolase
O-GlcNAcase using multiple linear free energy relationships. J.
Am. Chem. Soc. 131(37), 13415–13422 (2009). https://doi.org/
10.1021/ja904506u
23. van Wijk, X.M., Lawrence, R., Thijssen, V.L., van den Broek, S.A.,
Troost, R., van Scherpenzeel, M., Naidu, N., Oosterhof, A.,
Griffioen, A.W., Lefeber, D.J., van Delft, F.L., van Kuppevelt,
T.H.: A common sugar-nucleotide-mediated mechanism of inhibi-
tion of (glycosamino) glycan biosynthesis, as evidenced by 6F-
GalNAc (Ac3). FASEB J. 29(7), 2993–3002 (2015). https://doi.
org/10.1096/fj.14-264226
References
1. Ohtake-Niimi, S., Kondo, S., Ito, T., Kakehi, S., Ohta, T., Habuchi,
H., Kimata, K., Habuchi, O.: Mice deficient in N-
acetylgalactosamine 4-sulfate 6-O-sulfotransferase are unable to
synthesize chondroitin/dermatan sulfate containing N-
acetylgalactosamine 4,6-bissulfate residues and exhibit decreased
protease activity in bone marrow-derived mast cells. J. Biol.
Chem. 285(27), 20793–20805 (2010). https://doi.org/10.1074/jbc.
M109.084749
2. Nishimura, K., Ishii, M., Kuraoka, M., Kamimura, K., Maeda, N.:
Opposing functions of chondroitin sulfate and heparan sulfate dur-
ing early neuronal polarization. Neuroscience. 169(4), 1535–1547
(2010). https://doi.org/10.1016/j.neuroscience.2010.06.027
3. Koike, T., Izumikawa, T., Tamura, J., Kitagawa, H.: Chondroitin
sulfate-E fine-tunes osteoblast differentiation via ERK1/2, Smad3
and Smad1/5/8 signaling by binding to N-cadherin and cadherin-
11. Biochem. Biophys. Res. Commun. 420(3), 523–529 (2012).
https://doi.org/10.1016/j.bbrc.2012.03.024
4. Deepa, S.S., Umehara, Y., Higashiyama, S., Itoh, N., Sugahara, K.:
Specific molecular interactions of oversulfated chondroitin sulfate
E with various heparin-binding growth factors. Implication as phys-
iological binding partner in the brain and other tissues. J. Biol.
Chem. 277(46), 43707–43716 (2002). https://doi.org/10.1074/jbc.
M207105200
5. Monzavi-Karbssi, B., Stanley, J.S., Hennings, L., Jousheghany, F.,
Artaud, C., Shaaf, S., Kieber-Emmons, T.: Chondroitin sulfate gly-
cosaminoglycans as major P-selectin ligands on metastatic breast
cancer cell lines. Int. J. Cancer. 120(6), 1179–1191 (2007). https://
doi.org/10.1002/ijc.22424
6. Purushothaman, A., Fukuda, J., Mizumoto, S., ten Dam, G.B., van
Kuppevelt, T.H., Kitagawa, H., Mikami, T., Sugahara, K.:
Functions of chondroitin sulfate/dermatan sulfate chains in brain
development. Critical roles of E and iE disaccharide units recog-
nized by a single chain antibody GD3G7. J. Biol. Chem. 282(27),
19442–19452 (2007). https://doi.org/10.1074/jbc.M700630200
7. Ishii, M., Maeda, N.: Oversulfated chondroitin sulfate plays critical
roles in the neuronal migration in the cerebral cortex. J. Biol. Chem.
283(47), 32610–32620 (2008). https://doi.org/10.1074/jbc.
M806331200
8. Mizumoto, S., Watanabe, M., Yamada, S., Sugahara, K.:
Expression of N-acetylgalactosamine 4-sulfate 6-O-
sulfotransferase involved in chondroitin sulfate synthesis is respon-
sible for pulmonary metastasis. Biomed. Res. Int. 2013(656319),
9–9 (2013). https://doi.org/10.1155/2013/656319
9. Salgueiro, A.M., Filipe, M., Belo, J.A.: N-acetylgalactosamine 4-
sulfate 6-O-sulfotransferase expression during early mouse embry-
onic development. Int. J. Dev. Biol. 50(8), 705–708 (2006). https://
doi.org/10.1387/ijdb.062168as
10. ten Dam, G.B., van de Westerlo, E.M., Purushothaman, A., Stan,
R.V., Bulten, J., Sweep, F.C., Massuger, L.F., Sugahara, K., van
Kuppevelt, T.H.: Antibody GD3G7 selected against embryonic gly-
cosaminoglycans defines chondroitin sulfate-E domains highly up-
regulated in ovarian cancer and involved in vascular endothelial
growth factor binding. Am. J. Pathol. 171(4), 1324–1333 (2007).
https://doi.org/10.2353/ajpath.2007.070111
11. Li, F., Ten Dam, G.B., Murugan, S., Yamada, S., Hashiguchi, T.,
Mizumoto, S., Oguri, K., Okayama, M., van Kuppevelt, T.H.,
Sugahara, K.: Involvement of highly sulfated chondroitin sulfate
in the metastasis of the Lewis lung carcinoma cells. J. Biol.
24. Cocinero, E.J., Stanca-Kaposta, E.C., Dethlefsen, M., Liu, B.,
Gamblin, D.P., Davis, B.G., Simons, J.P.: Hydration of sugars in

Glycoconj J
the gas phase: regioselectivity and conformational choice in N-ace-
Chem. Lett. 21(9), 1755–1758 (1992). https://doi.org/10.1246/cl.
1992.1755
tyl glucosamine and glucose. Chem. Eur. J. 15(48), 13427–13434
(2009). https://doi.org/10.1002/chem.200901830
34. Boullanger, P., Jouineau, M., Bouammali, B., Lafont, D., Descotes,
G.: The use of N-alkoxycarbonyl derivatives of 2-amino-2-deoxy-
D-glucose as donors in glycosylation reactions. Carbohydr. Res.
202, 151–164 (1990). https://doi.org/10.1016/0008-6215(90)
84077-8
25. Macauley, M.S., Stubbs, K.A., Vocadlo, D.J.: O-GlcNAcase cata-
lyzes cleavage of thioglycosides without general acid catalysis. J.
Am. Chem. Soc. 127(49), 17202–17203 (2005). https://doi.org/10.
1021/ja0567687
26. Vocadlo, D.J., Withers, S.G.: Detailed comparative analysis of the
catalytic mechanisms of β-N-acetylglucosaminidases from families
3 and 20 of glycoside hydrolases. J. Biol. Chem. 44(38), 12809–
12818 (2005). https://doi.org/10.1021/bi051121k
27. Zemlyakov, A.E., Tsikalov, V.V.: Kur'yanov, V.O., Chirva, V.Y.,
Bovin, N.V.: synthesis of N-acetylmuramyl-L-alanyl-D-
isoglutamine aryl β-glycosides. Russian J. Bioorg. Chem. 27(6),
390–394 (2001). https://doi.org/10.1023/A:1012940803366
28. Roy, R., Tropper, F.D.: Carbohydrate-protein interactions.
Syntheses of agglutination inhibitors of wheat germ agglutinin by
phase-transfer catalysis. Canadian J. Chem. 69(5), 817–821 (1991).
https://doi.org/10.1139/v91-121
29. Grathe, S., Thygesen, M.B., Larsen, K., Petersen, L., Jensen, K.J.:
Glucosamine derived DISAL donors for stereoselective glycosyla-
tions under neutral conditions. Tetrahedron Asymmetry. 16(8),
1439–1448 (2005). https://doi.org/10.1016/j.tetasy.2005.02.029
30. Matsubara, K., Mukaiyama, T.: Catalytic stereoselective synthesis
of 2-amino-2-deoxy-α-D-glucopyranosides and galactosides.
Chem. Lett. 22(12), 2145–2148 (1993). https://doi.org/10.1246/cl.
1993.2145
35. Pertel, S.S., Kononov, L.O., Zinin, A.I., Chirva, V.J., Kakayan,
E.S.: Synthesis of some 2-alkoxy glyco-[2,1-d]-2-oxazolines and
evaluation of their glycosylation reactivity. Carbohydr. Res. 356,
172–179 (2012). https://doi.org/10.1016/j.carres.2012.03.026
36. Kurosu, M., Li, K.: Mild and selective O-glycosylations of primary
alcohols with the thioglucosaminide derivative promoted by N-
iodosuccinimide and HBF4-adsorbed on silica gel. Heterocycles.
80(1), 115–123 (2010). https://doi.org/10.3987/COM-09-S(S)24
37. Bennett, C.S., Dean, S.M., Payne, R.J., Ficht, S., Brik, A., Wong,
C.H.: Sugar-assisted glycopeptide ligation with complex oligosac-
charides: scope and limitations. J. Am. Chem. Soc. 130(36),
11945–11952 (2008). https://doi.org/10.1021/ja8010513
38. Qin, X., Liu, Y., Jia, J.: Process for synthesis of N-
acetylgalactosamine from N-acetylglucosamine. (Tianjin
Ingenochem technology co., ltd., Peop. Rep. China), (2016) CN
105524124 a, Apr 27, 2016
39. Malleron, A., Benjdia, A., Berteau, O., Le Narvor, C.: Chondroitin-
4-O-sulfatase from Bacteroides thetaiotaomicron: exploration of
the substrate specificity. Carbohydr. Res. 353, 96–99 (2012).
https://doi.org/10.1016/j.carres.2012.03.033
31. Matsubara, K., Mukaiyama, T.: An efficient method for the
stereoselective synthesis of 2-amino-β-D- and α-D-glycosides
from peracylated sugars using active acidic species. Polish J.
Chem. 68(11), 2365–2382 (1994)
32. Mukaiyama, T., Matsubara, K. (Asahi Chemical Ind.): Preparation
of 2-deoxy-2-amino sugars by glycosidation. Jpn. Kokai Tokkyo
Koho JP H0656868 A, 19940301(1992)
33. Mukaiyama, T., Matsubara, K.: Stereoselective synthesis of 2-ami-
no-2-deoxy-β-D-glucopyranosides and galactopyranosides by
using a catalytic amount of tin (II) trifluoromethanesulfonate.
40. Sugiura, N., Shioiri, T., Chiba, M., Sato, T., Narimatsu, H., Kimata,
K., Watanabe, H.: Construction of a chondroitin sulfate library with
defined structures and analysis of molecular interactions. J. Biol.
Chem. 287(52), 43390–43400 (2012). https://doi.org/10.1074/jbc.
M112.412676
41. Ohtake, S., Ito, Y., Fukuta, M., Habuchi, O.: Human N-
acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is relat-
ed to human B cell recombination activating gene-associated gene.
J. Biol. Chem. 276(47), 43894–43900 (2001). https://doi.org/10.
1074/jbc.M104922200