Synthesis of modular building blocks using glycosyl phosphate donors for the construction of asymmetric N-glycans

Article abstract of DOI:10.1016/j.tet.2018.08.039

Glycosyl phosphates are known as versatile donors for the synthesis of complex oligosaccharides both chemically and enzymatically. Herein, we report the stereoselective construction of modular building blocks for the synthesis of N-glycan using glycosyl p

Full text of DOI:10.1016/j.tet.2018.08.039

Accepted Manuscript
Synthesis of modular building blocks using glycosyl phosphate donors for the
construction of asymmetric N-Glycans
Supriya Dey, Sumit O. Bajaj, Tsung-I. Tsai, Hong-Jay Lo, Kevin Wu, Chi-Huey Wong
PII:
S0040-4020(18)31005-6
10.1016/j.tet.2018.08.039
TET 29759
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 17 July 2018
Revised Date: 20 August 2018
Accepted Date: 23 August 2018
Please cite this article as: Dey S, Bajaj SO, Tsai T-I, Lo H-J, Wu K, Wong C-H, Synthesis of modular
building blocks using glycosyl phosphate donors for the construction of asymmetric N-Glycans,
Tetrahedron (2018), doi: 10.1016/j.tet.2018.08.039.
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Synthesis of Modular Building Blocks using Glycosyl Phosphate Donors for the Construction of
Asymmetric N-Glycans
Supriya Deya, Sumit O. Bajaja,b,Tsung-I Tsaia, Hong-Jay Loa, Kevin Wua and Chi-Huey Wong*a,c

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Synthesis of Modular Building Blocks using Glycosyl Phosphate Donors for the
Construction of Asymmetric N-Glycans
Supriya Deya, Sumit O. Bajaja,b, Tsung-I Tsaia, Hong-Jay Loa, Kevin Wua, and Chi-Huey Wong*a,c
a The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA, USA, 92037
b Corden Pharma Colorado Inc., 2075 55th Street, Boulder, CO, USA, 80301
c The Genomics Research Center, Academia Sinica, No. 128, Academia Rd., Section 2, Nankang District, Taipei, 115, Taiwan.
ARTICLE INFO
ABSTRACT
Glycosyl phosphates are known as versatile donors for the synthesis of complex
oligosaccharides both chemically and enzymatically. Herein, we report the stereoselective
construction of modular building blocks for the synthesis of N-glycan using glycosyl phosphates
as donors. We have synthesized four trisaccharide building blocks with orthogonal protecting
groups, namely, Manβ2GlcNAc(OAc)3β6GlcNAc (9), Manβ2GlcNAc-β6GlcNAc(OAc)3 (15),
Manβ2GlcNAc(OAc)3β4GlcNAc (18) and Manβ2GlcNAcβ4GlcNAc(OAc) (22) for further
selective elongation using glycosyltransferases. The glycosylation reaction using glycosyl
phosphate was found to be high yielding with shorter reaction time. Initially, The phthalimide
protected glucosamine donor was exploited to ensure the formation of β-glycosidic linkage and
later converted to the N-acetyl group before the enzymatic synthesis. The selective deprotection
of O-benzyl group was performed prior to enzymatic synthesis to avoid its negative interference.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
N-Glycans
Glycosyl phosphate donors
Chemo-enzymatic
Oligosaccharides
Orthogonal protecting groups
Modular building blocks
1. Introduction
Glycans are composed of a number of monosaccharides linked
together via glycosidic bond(s) and constitute the carbohydrate
portion of glycoproteins, glycolipids or other glycoconjugates. In
glycoproteins, glycans are broadly divided into O-glycans and N-
glycans. They affect several biological processes in the cell such
as protein folding, cell signaling, embryogenesis etc.1 N-Glycans
are assembled during protein biosynthesis through attachment to
the amide nitrogen of asparagine in a defined peptide chain. The
synthesis and processing of N-glycans generally takes place in
the endoplasmic reticulum and Golgi complex involving different
stages of glycosylation and deglycosylation.2 The glycan
precursor Glc3Man9GlcNAc2 is first transferred from a lipid
pyrophosphate derivative to a growing polypeptide in the
endoplasmic reticulum (ER) and further trimmed by ER
glucosidases before travelling to the Golgi complex for further
processing3 down to Man5GlcNAc2, which is further glycosylated
under the catalysis of N-acetylglucosamine transferase (GnT) to
convert it to a complex or hybrid-type glycan. Next, a series of
enzymes of the GnT family modify the glycoform with the
addition of GlcNAc moieties to the D1 arm of the glycan.4 Then,
the Golgi-generated glycoforms is converted to highly diverse
glycan species through a series of sequential galactosylation,
sialylation and fucosylation. The naturally occurring glycans can
only be isolated in minor quantities and as complex mixtures,
difficult to separate, and cannot provide a reliable source for
detailed biological studies. More than twenty thousand structures
of N-glycans exist depending on the linkages of
monosaccharides, and many of which are difficult to sequence
due to their isomeric nature. In order, to study the effect of
individual N-glycans on the function of a particular glycoprotein
would ideally require a structurally diverse collection of synthetic
N-glycans with systematic variations in compositions and
branching, terminal sugars, and core modifications. The chemical
synthesis to access diverse and complex N-glycan structures is
challenging due to step-wise synthesis and multiple purification
processes. However, over the last two decades, chemical and
chemo-enzymatic synthesis methods have been reported for the
synthesis of complex and hybrid type N-glycans.5,6 In 2013, we
reported a convergent synthesis of multi-antennary N-glycans
and their application to the study of HIV-antigenicity.7a Recently,
we also developed a modular chemo-enzymatic approach for the
synthesis of high-mannose, hybrid- and complex-type N-glycan
structures for the development of glycan arrays for the rapid
screening and identification of epitopes on HIV-1 recognized by
the broadly neutralizing antibodies isolated from HIV-1
patients.7b
In the last 100 years, synthetic chemists have been developing
efficient glycosyl donors for the synthesis of complex
oligosaccharides. Numerous versatile glycosyl donors have been
developed, among which glycosyl trichloroacetimidates8 and
thioglycosides9 are more commonly utilized. However, the
application of glycosyl donors, such as glycosyl sulfoxides,10 n-
pentenyl glycosides,11 glycosyl phosphites,12 glycosyl halides,13
anhydrosugars14, glucals15, has been also widely documented for
the synthesis of complex oligosaccharide. Since mid-1990’s,
glycosyl phosphates have become an attractive class of
glycosylating donors due to their stability, ease of activation and
high reactivity at low temperature. The use of glycosyl phosphate

2
Tetrahedron
ACCEPTED MANUSCRIPT
glucosamine donor 2 and acceptor 1 were coupled using
NIS/TMSOTf to furnish disaccharide 3 with 1,2-β-glycosidic
linkage in 86% yield. Selective opening of the 4,6-O-benzylidene
ring in the presence of BH3-THF/n-Bu2BOTf led to 6-hydroxy
mannosyl acceptor 4 in 82%. On the other hand, the glycosyl
phosphate donor 6 was prepared from thioglycoside donor 2,
which upon deacetylation under Zemplén condition followed by
benzyl protection using BnBr/NaH provided O-benzyl protected
thioglycoside 5. The anomeric phosphate group was installed
using dibutyl phosphate in the presence of NIS/TfOH to generate
6 with excellent yield. Next, disaccharide 4 and phosphate donor
6 were coupled using TMSOTf at -50 oC to obtain trisaccharide 7
in 80% yield. Later, removal of phthalimide protecting groups
using ethylene diamine/n-BuOH followed by N-acetylation using
Ac2O/Py generated trisaccharide 8. In order to avoid any negative
interference of the bulky -OBn groups during the enzymatic
reaction, we planned to remove the benzyl groups using
Pd(OH)2/C-H2 to furnish 9 (Scheme 1). The C4 hydroxyl group at
the GlcNAc (β6 arm) of trisaccharide 9 can be elongated by
enzymatic glycosylation to form the desired N-glycan structure.
donors for enzymatic syntheses is also well known. It is known
that glycosyltransferases use nucleotide 5′-phosphosugars
(NDPs)
for
the
biosynthesis
of
complex
oligosaccharides.16 Seeberger and co-workers reported the
synthesis of glycosyl phosphates for the synthesis of O-
glycosides,17 C-glycosides18, and complex oligosaccharides.19
Previously, we have reported the synthesis of glycosyl
phosphates and their application in the chemical and chemo-
enzymatic synthesis of oligosaccharides.12 In the current context,
we extend our chemo-enzymatic modular approach using
glycosyl phosphates and glycosyltransferases to construct several
modular building blocks which are useful for the assembly of
symmetric and asymmetric N-glycans.
2. Result and Discussion
The synthesis of the targeted trisaccharide 9 was initiated from
orthogonally protected glycosyl donor 2 and acceptor 1, which
were synthesized from commercially available D-mannose and D-
glucosamine, respectively, using known procedures.7b Initially,
Scheme 1: Synthesis of Manβ2GlcNAc(OAc)3β6GlcNAc
Scheme 2: Synthesis of Manβ2GlcNAcβ6GlcNAc(OAc)3
2

3
Scheme 3: Synthesis of Manβ2GlcNAc(OAc)3
A
β4
C
G
C
lcN
EPTED MANUSCRIPT
A
c
Scheme 4:
Synthesis of Man
β2GlcN-β4GlcN(OAc)
To further investigate the application of the phosphate donor
towards the synthesis of diverse branching units, we installed tri-
O-acetyl glucosamine entity on the β6 arm of the trisaccharide
motif. Glycosyl phosphate 6 was coupled with mannosyl
acceptor 1 using TMSOTf to furnish disaccharide 10 in good
yield. The presence of doublet at 5.27 ppm with a coupling
With the success towards the β2-β6 linked trisaccharide unit,
we were further intrigued to investigate the reactivity of
phosphate donor towards the systematic synthesis of β2-β4 linked
modules. In this regard, the benzylidene protected disaccharide 3
was treated Et3SiH-TFA to provide C-4 hydroxy derivative 16,
selectively. Next, glycosylation of the phosphate donor 6 with
acceptor 16 in the presence of TMSOTf led to the trisaccharide
with required β2-β4 linkage. Further, the phthalimide protecting
groups of trisaccharide were deprotected followed by N-
acetylation using Ac2O/Py generated 17 in 77%. Finally, removal
of the O-benzyl group using Pd(OH)2/C-H2 led to the formation
of trisaccharide 18 in 90% yield (Scheme 3). To show the
diversity of phosphate donors towards various trisaccharide
modules, we also focused on the synthesis of the trisaccharide
building block containing C-4-OAc N-acetyl glucosamine unit
attached with β4 glycosidic linkages to the -OPMP-mannose unit.
Selective opening of the 4,6-O-benzylidene ring of 10 in the
presence of Et3SiH/TFA led to 4-hydroxy disaccharide acceptor
20 in 74% yield. Next, the phosphate donor 19 was coupled with
disaccharide acceptor 20 using TMSOTf to generate trisaccharide
21. The removal of phthalimide protecting groups, followed by
N-acetylation and hydrogenolysis provided trisaccharide 22 with
GlcNAc(OAc) at the β4 arm in 86 % yield (Scheme 4).
1
constant (J = 8.4 Hz) in H NMR spectrum along with the signal
at 96.9 ppm in 13C NMR indicated the formation of 1,2-β-linked
disaccharide 10. Selective opening of the 4,6-O-benzylidene ring
in the presence of BH3-THF/n-Bu2BOTf led to the 6-hydroxy
disaccharide acceptor 11 in 78% yield. The absence of signal at
5.42 ppm and 101.5 ppm in 1H and 13C NMR spectrum,
respectively, indicated the cleavage of 4,6-O-benzylidene ring.
Next, the disaccharide acceptor 11 was coupled with phosphate
donor 12 using TMSOTf to furnish the β2-β6 linked trisaccharide
13. The presence of two doublet signals at 5.20 (d, J = 8.5 Hz,
1
1H) and 5.10 (d, J = 8.4 Hz, 1H) in H NMR confirmed that the
two glucosamine units are β2-β6 linked with mannose in 13.
Removal of the phthalimide protecting group using ethylene
diamine at 90 °C, followed by N-acetylation with Ac2O/Py
generated 14. Finally, Pd(OH)2/C-H2 mediated hydrogenation
furnished the desired trisaccharide 15 in 87% (Scheme 2).
Scheme 5: Stepwise Enzymatic elongation
β2
β4
OPMP
α2,3
β2
β4
OPMP
Reaction conditions: (i) β-1,4-GalT (15 U), UDP-Gal, Tris buffer (50 mM), MnCl2 (5mM), 37 oC, 12 h, 75%. (ii) α-2,3-ST (15U),
CMK (5 U), CSS (10 U), ATP (0.05 eq.), CTP (0.05 eq.), phosphoenolpyruvate (2.4 eq.), 37 oC, 54 h, 83%.

4
Tetrahedron
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The structural patterns of all these trisaccharides 9, 15, 18 and
General procedure for the synthesis of glycosyl phosphates: A
solution of thioglycoside donor (1.2 equiv.) and molecular sieves
4 Å (1.0 equiv.) in CH2Cl2 was stirred for 1 h under N2-
atmosphere at room temperature and then cooled to -40 °C.
Dibutyl phosphate (1.0 equiv.), NIS (1.2 equiv.) and TfOH (0.3
equiv.) were added to the mixture, and stirring was continued
until the complete conversion of the donor. The reaction mixture
was quenched with Et3N (2 equiv.) and filtered through a Celite
pad. The mixture was diluted with CH2Cl2 (2 × 30 mL), washed
with sat. NaHCO3 (25 mL), Na2S2O3 (25 mL) and brine (20 mL).
The crude product was dried over MgSO4 and concentrated in
vacuo, and the residue was purified through a silica gel column
chromatography to afford the corresponding glycosylated
product.
22 provide a platform for the selective elongation at the GlcNAc
unit using enzymes to create libraries of asymmetric branching
motifs. We utlized one of the trisaccharide, 18 to demonstrate the
applicabilty of enzymatic elongation. Initially, trisaccharide 18
was treated with β-1,4-galactosyltransferase (GalT) using UDP-
Gal in the presence of MnCl2 to furnish tetrasaccharide 23 with
complete stereospecificity in 75% yield. To further extend the
chain length and complexity, tetrasaccharide 23 was treated with
α-2,3-sialyltransferase (ST) along with ATP (0.05 eq.), CTP
(0.05 eq.), phosphoenolpyruvate (2.4 eq.), cytidine
monophosphate kinase (CMK, 5 units) and CMP-sialic acid
synthetase (CSS, 10U) in Tris buffer (50mM, pH 7.5) to
generate pentasaccharide 24 in 83% yield (Scheme 5).
In conclusion, we have developed a new and highly efficient
chemo-enzymatic strategy for the synthesis of modular building
blocks using glycosyl phosphate as donors for the synthesis of
complex/hybrid N-glycans. The phosphate donors were
synthesized from the corresponding thioglycosides. Overall, the
glycosylation reactions using phosphate donors were found to be
high yielding and highly stereoselective. One of the trisaccharide
building block 18 was further exploited for the enzymatic
synthesis. With the aid of purified enzymes β-1,4-GalT and α-
2,3-ST, we were able to elongate the β4 arm of the trisaccharide
even in the presence of three O-acetyl moieties at the β2 arm.
This result indicates that the presence of multiple OAc protecting
groups can be tolerated by the enzymes during the enzymatic
reactions. This work emphasizes the use of glycosyl phosphate
donors and enzymes to access complex and diverse N-glycan
structures.
General procedure for 4,6-O-benzylidene ring opening
using BH3-THF: A solution of compound (0.50 mmol) in
anhydrous THF (2mL) was mixed with BH3-THF complex (1M
solution in THF), followed by n-Bu2BOTf (1M solution in
CH2Cl2) at 0 °C under N2-atmosphere. After the complete
conversion of the starting material to product, Et3N (2 equiv.)
was added. The reaction mixture was further quenched with
dropwise addition of MeOH. When no more hydrogen was
evolved, the reaction mixture was concentrated in vacuo and the
residue was purified by flash column chromatography on silica
gel to afford the corresponding 6-hydroxy product.
General procedure for 4,6-O-benzylidene ring opening
using Et3SiH/TFA: To a solution of 4,6-O-benzylidene protected
compound (1.0 mmol) in anhydrous CH2Cl2 (20 mL) was added
triethyl silane (10 equiv.) followed by trifluroacetic acid (10
equiv.) at 0 oC. After 2 h, the reaction mixture was slowly
quenched with sodium bicarbonate solution (50 mL). The
aqueous layer was further extracted with CH2Cl2 (3 x 50 mL),
and the combined organic layer was washed with brine solution
(50 mL), dried over MgSO4, filtered and concentrated in vacuo.
The crude reaction mixture was purified by flash column
chromatography on silica gel to afford the corresponding 4-
hydroxy product.
Experimental Section:
General procedures. All chemicals were purchased as
reagent grade and used without further purification. All
anhydrous solvents were purchased from commercial source and
further dried in accordance with standard procedures. Molecular
sieves (MS 4 Å) were purchased from Aldrich, which were
ground into powdered form and activated before use. Reactions
were performed under an inert atmosphere and strictly anhydrous
conditions. Reactions were monitored with analytical thin-layer
chromatography (TLC) on silica gel 60 F254 plates and
visualized under UV (254 nm) and/or by spraying with 20%
General procedure for phthalimide deprotection and N-
acetylation: A protected trisaccharide (1 mmol) was dissolved
n
in a mixture of ethylene diamine: BuOH (1:4) and stirred at 90
oC for 12 h. Solvents were evaporated in vacuo and the reaction
mixture was dried for 2 h on high vacuum. The crude product
was treated with a mixture of Ac2O/pyridine (1: 2) at 0 oC. Then,
the reaction mixture was stirred at room temperature for 12 h.
After complete conversion of the starting material, solvents were
evaporated in vacuo. The mixture was diluted with EtOAc (2 ×
30 mL), washed with sat. NaHCO3 (25 mL), and brine (20 mL). It
was dried over MgSO4 and concentrated in vacuo. The crude
residue was purified through a silica gel column chromatography
to afford the corresponding N-acetyl amine derivative.
anisaldehyde in ethanol or with
a
solution of
(NH4)6Mo7O24·4H2O 25 g/L. H and 13C NMR were recorded on
600/500 MHz and 151 MHz spectrometers, respectively, with the
residual solvent signal acting as the internal standard. Chemical
shift (in ppm) was determined relative to tetramethylsilane in
deuterated chloroform (δ 0 ppm). Coupling constant(s) in hertz
(Hz) were measured from one-dimensional spectra. High-
resolution mass spectra were obtained from a Q-TOF instrument
by the electrospray ionization (ESI) technique.
1
General procedure for glycosyl phosphate coupling: To a
solution of glycosyl phosphate donor (1.2 equiv.) and acceptor
(1.0 eq) in CH2Cl2 was mixed with molecular sieves (4 Å). After
stirring for 1 h under N2-atmosphere at room temperature, the
mixture was cooled to -50 °C and was added TMSOTf (1.0
equiv.). After the reaction mixture was stirred for additional 1 h
at the same temperature, triethylamine (2 equiv.) was added and
the mixture was filtered through a pad of Celite and concentrated
in vacuo. The crude residue was purified through a silica gel
column chromatography to afford the corresponding glycosylated
product.
General procedure for hydrogenolysis:
A
protected
trisaccharide derivative (0.5 mmol) was dissolved in 10 mL
MeOH : HCOOH (9 : 1), and 20%-Pd(OH)2/C was added. The
mixture was placed under an atmosphere of hydrogen gas
bubbling through a needle. After 12h, the reaction mixture was
filtered through a pad of Celite and washed with a mixture of
CH3OH and H2O (1/1, v/v, 6 mL). Solvents were evaporated in
vacuo and the crude mixture was purified using silica gel column
chromatography to furnish the protecting group free
trisaccharide.
General procedure for enzyme expression and
purifications: Recombinant enzymes β-1,4 galactosyltransferase
4

5
(β-1,4-GalT), α-2,3 sialyltransferase, pyruvate kinase (PK),
pyrophosphatase (PPA), cytidine monophosphate kinase (CMK)
and CMP-sialic acid synthetases (CSS) were over expressed and
purified from E. coli BL21(DE3) using Ni-NTA column as our
previous protocol.7b
Disaccharide 4 was prepared from 3 (0.7 g, 0.774 mmol)
ACCEPTED MANUSCRIPT
using the general procedure for 4,6-O-benzylidene ring opening
using BH3-THF, followed by purification using silica gel column
chromatography (PhCH3: EtOAc = 8:2). Yield (0.575 g, 82 %).
1H NMR (600 MHz, CDCl3) δ 7.88 (dt, J = 7.2, 3.6 Hz, 2H), 7.79
(dd, J = 5.5, 3.0 Hz, 2H), 7.45 – 7.41 (m, 2H), 7.34 – 7.28 (m,
5H), 7.28 – 7.23 (m, 3H), 6.70 – 6.64 (m, 2H), 6.52 – 6.48 (m,
2H), 5.97 (dd, J = 10.8, 9.0 Hz, 1H), 5.44 (d, J = 8.5 Hz, 1H),
5.22 (dd, J = 10.2, 9.0 Hz, 1H), 5.01 (d, J = 2.2 Hz, 1H), 4.87 (d,
J = 10.8 Hz, 1H), 4.81 (d, J = 11.1 Hz, 1H), 4.62 (d, J = 11.2 Hz,
1H), 4.58 – 4.50 (m, 2H), 4.35 (dd, J = 12.2, 5.3 Hz, 1H), 4.26 –
4.22 (m, 2H), 4.03 (dd, J = 9.1, 3.1 Hz, 1H), 3.93 (ddd, J = 10.2,
5.3, 2.4 Hz, 1H), 3.81 (t, J = 9.4 Hz, 1H), 3.73 (s, 3H), 3.47 (ddd,
J = 10.0, 4.6, 2.6 Hz, 1H), 3.38 (ddd, J = 12.1, 5.1, 2.6 Hz, 1H),
3.29 (ddd, J = 12.3, 8.2, 4.6 Hz, 1H), 2.05 (app d, J = 3.4 Hz,
6H), 1.92 (s, 3H);13C NMR (151 MHz, CDCl3) δ 170.6, 170.2,
169.5, 154.8, 149.5, 138.3, 138.0, 128.4, 128.3, 128.2, 128.0,
127.8, 127.7, 116.8, 114.5, 97.5, 95.6, 77.5, 75.2, 74.5, 73.9,
72.5, 72.1, 71.4, 70.2, 69.0, 62.4, 62.1, 55.6, 54.8, 20.7, 20.6,
20.5; m/z (HRMS) calcd for C47H49NO16 [M+Na]+: 906.2649,
found: 906.2655.
General procedure for enzymatic β-1, 4-galactosylation: A
trisaccharide (1 equiv.) and UDP-Gal (2 equiv.) were dissolved
in Tris buffer (50 mM, pH 7.5) containing MnCl2 (5 mM) in a
small capped glass tube with β-1,4-GalT (15 units). The resulting
reaction mixture was incubated at 37 oC for 12 h. The protein was
removed by heating and centrifugation followed by purification
using C18 column chromatography. The pure fractions were
pooled and concentrated to give the corresponding product.
General procedure for enzymatic α-2,3-sialylation:
Trisaccharide (1 eq.), Neu5Ac (2 equiv.), ATP (0.05 equiv.),
CTP (0.05 equiv.), phosphoenolpyruvate (2.4 equiv.), cytidine
monophosphate kinase (CMK, 5U), CMP-sialic acid synthetases
(CSS, 10U), pyruvate kinase (PK, 5 units), pyrophosphatase
(PPA, 2.5U) and α-2,3 sialyltransferase (15 units) were dissolved
in Tris buffer (50 mM, pH 7.5) and transferred to a small glass
tube. The reaction was incubated at 37 °C and monitored by TLC
until completion. Heating and centrifugation was performed to
remove the protein and followed by purification using C18
column chromatography. The pure fractions were pooled and
concentrated to give the respective product.
p-Tolyl-3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-
D
-glucopyranoside (5)
Compound 2 (0.6 g, 1.09 mmol) was dissolved in anhydrous
methanol (8 mL) and sodium methoxide (0.15 g) was added. The
reaction mixture was stirred at room temperature for 3 h, and was
neutralized using acid resin (IR-120), filtered and concentrated in
vacuo, and later co-evaporated with toluene to remove traces of
waters. A solution of the tri hydroxy intermediate (0.5 g, 1.20
mmol) in DMF (10 mL) was added tetrabutylammonium iodide
(TBAI, 40 mg, 0.12 mmol) and benzyl bromide (0.49 mL, 4.21
mmol) under N2 atmosphere. The solution was cooled to 0 oC and
then NaH (60% dispersion in mineral oil, 0.24 g, 6.0 mmol) was
p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl)-4,6-O-benzylidene-3-O-
benzyl-α- -mannopyranoside (3)
D
Glycosyl donor 2 (0.5 g, 0.924 mmol, 1.2 equiv. based on
acceptor) and acceptor 1 (0.36 g, 0.775 mmol) in dry CH2Cl2 was
added 4 Å molecular sieves and the mixture was stirred under N2-
o
atmosphere for 1h, then cooled to -50 C. NIS (0.249 g, 1.10
o
added slowly. The reaction mixture was stirred at 0 C for 1 h
mmol) and TMSOTf (25 ꢀL, 0.272 mmol) were added, and the
reaction mixture was stirred for another 1 h. After complete
conversion of the starting materials, the reaction mixture was
quenched with Et3N (2 equiv.) and filtered through a Celite pad.
The mixture was diluted with CH2Cl2 (2 × 30 mL), washed with
sat. NaHCO3 (25 mL), Na2S2O3 (50 mL) and brine (50 mL). It
was then dried over MgSO4 and concentrated for column
chromatography purification on silica gel (PhCH3: EtOAc, 9:1) to
furnish 3 (0.7 g, 86 %) as yellow oil. Rf 0.52 (PhCH3/EtOAc 9:1);
1H NMR (600 MHz, CDCl3) δ 7.89 – 7.85 (m, 2H), 7.78 – 7.73
(m, 2H), 7.41 (td, J = 7.1, 6.5, 3.8 Hz, 4H), 7.34 – 7.31 (m,
4H), 7.31 – 7.28 (m, 2H), 6.76 – 6.72 (m, 2H), 6.67 – 6.63 (m,
2H), 5.89 (dd, J = 10.9, 9.1 Hz, 1H), 5.52 (d, J = 8.5 Hz, 1H),
5.46 (s, 1H), 5.22 (dd, J = 10.1, 9.1 Hz, 1H), 5.13 (d, J = 2.0
Hz, 1H), 4.79 – 4.71 (m, 2H), 4.52 (dd, J = 11.0, 8.5 Hz, 1H),
4.34 – 4.31 (m, 1H), 4.27 (dd, J = 3.2, 1.9 Hz, 1H), 4.23 (dd, J
= 12.2, 2.4 Hz, 1H), 4.04 (dd, J = 10.0, 3.1 Hz, 1H), 3.97 (t, J
= 9.5 Hz, 1H), 3.90 (ddt, J = 9.4, 4.6, 2.3 Hz, 1H), 3.76 (s,
3H), 3.68 – 3.60 (m, 2H), 3.18 – 3.11 (m, 1H), 2.06 (s, 3H),
2.05 (s, 3H), 1.91 (s, 3H).13C NMR (151 MHz, CDCl3) δ 170.8,
170.3, 169.5, 155.1, 149.5, 138.4, 137.5, 134.4, 133.9, 131.7,
130.1, 128.9, 128.3, 128.2, 127.7, 127.6, 126.1, 123.5, 117.0,
114.6, 101.6, 97.0, 96.24, 78.1, 77.3, 77.1, 76.9, 75.6, 73.7, 72.3,
71.9, 70.4, 69.1, 68.2, 64.4, 62.2, 55.7, 54.6, 20.8, 20.7, 20.6; m/z
(HRMS) calcd for C47H47NO16 [M+Na]+: 904.2793, found:
904.2789.
and then stirred at room temperature for additional 7 h. The
reaction mixture was quenched with aqueous NH4Cl and was
diluted with CH2Cl2 (2 × 30 mL), washed with sat. NH4Cl (25
mL), and brine (50 mL). It was dried over MgSO4 and
concentrated in vacuo. The crude mixture was purified using
silica gel column chromatography (hexane: EtOAc, 9:1) to
furnish 5 (0.57 g, 69 %) as colorless oil.1H NMR (600 MHz,
CDCl3) δ 7.74 (d, J = 7.3 Hz, 1H), 7.65 – 7.53 (m, 3H), 7.34 –
7.32 (m, 4H), 7.32 – 7.29 (m, 6H), 7.25 – 7.21 (m, 5H), 6.96 –
6.92 (m, 2H), 6.85 – 6.82 (m, 1H), 6.82 – 6.78 (m, 1H), 5.46
(dd, J = 10.0, 2.7 Hz, 1H), 4.78 (d, J = 10.9 Hz, 1H), 4.73 (d, J
= 12.1 Hz, 1H), 4.64 – 4.57 (m, 2H), 4.53 – 4.49 (m, 1H), 4.39
(d, J = 12.1 Hz, 1H), 4.32 (dd, J = 10.4, 8.5 Hz, 1H), 4.21 (td, J =
10.3, 3.4 Hz, 1H), 3.77 (td, J = 12.2, 11.6, 3.2 Hz, 2H), 3.74 –
3.67 (m, 1H), 3.62 (ddd, J = 9.9, 4.4, 2.1 Hz, 1H), 2.22 (s,
3H);13C NMR (151 MHz, CDCl3) δ 168.2, 167.5, 141.1, 138.3,
138.2, 138.0, 137.8, 134.0, 133.9, 133. 5, 131.7, 131.5, 129.7,
129.7, 128.7, 128.7 (2), 128.5, 128.5, 128.4, 128.2, 128.1, 128.1
(2), 127.9, 127.8, 127.7, 127.7(2), 127.6, 127.6(2), 127.5, 127.0,
123.5(2), 123.4, 83.5, 80.3, 79.5, 79.4, 75.1, 75.0, 73.5, 68.9,
55.1, 21.2.; m/z (HRMS) calcd for C42H39NSO6 [M+Na]+:
708.2396, found: 708.2399.
Dibutyl-3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-
glucopyranoside phosphate (6)
Dibutyl phosphate derivative 6 was prepared from 5 (0.57 g,
0.774 mmol) using the general procedure for the synthesis of
glycosyl phosphates, followed by purification using silica gel
column chromatography (hexane: EtOAc = 7:3). Yield (0.564 g,
88 %).1H NMR (600 MHz, CDCl3) δ 7.70 – 7.64 (m, 4H), 7.37 –
7.33 (m, 3H), 7.33 – 7.27 (m, 3H), 7.26 – 7.22 (m, 3H), 6.99 (dt,
p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl)-3,4-di-O-benzyl-α-D-
mannopyranoside (4)

6
Tetrahedron
ACCEPTED MANUSCRIPT
J = 6.2, 1.4 Hz, 2H), 6.90 – 6.86 (m, 2H), 6.86 – 6.82 (m, 2H),
5.82 (dd, J = 8.5, 7.2 Hz, 1H), 4.84 (d, J = 10.8 Hz, 1H), 4.80 (d,
J = 12.2 Hz, 1H), 4.66 (dd, J = 11.5, 9.2 Hz, 2H), 4.54 (d, J =
12.0 Hz, 1H), 4.50 – 4.42 (m, 2H), 4.27 (dd, J = 10.8, 8.5 Hz,
1H), 3.99 – 3.95 (m, 1H), 3.95 – 3.90 (m, 1H), 3.89 – 3.85 (m,
1H), 3.85 – 3.81 (m, 1H), 3.78 (d, J = 1.9 Hz, 1H), 3.77 – 3.75
(m, 1H), 3.75 – 3.72 (m, 1H), 3.72 – 3.69 (m, 1H), 3.69 – 3.66
(m, 1H), 1.54 – 1.48 (m, 2H), 1.27 (ddt, J = 14.5, 11.9, 6.3 Hz,
4H), 1.07 – 1.00 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H), 0.69 (t, J = 7.4
Hz, 3H); 13C NMR (151 MHz, CDCl3) δ 167.7, 138.0, 138.0(2),
137.9, 133.8, 131.6, 128.5, 128.4, 128.1, 128.0, 127.9, 127.8,
127.8(2), 127.7, 127.4, 123.3, 94.2, 94.2(2), 79.0, 78.7, 75.5,
75.1, 74.1, 73.6, 68.3, 68.0, 68.0 (2), 67.8, 67.8, 56.3, 56.3, 32.1,
32.1, 31.9, 31.8, 18.6, 18.4, 13.6, 13.5.; m/z (HRMS) calcd for
C43H50NPO10 [M+Na]+: 794.3070, found: 794.3078.
4.49 (m, 3H), 4.37 (dd, J = 3.5, 2.0 Hz, 1H), 4.25 (dd, J = 12.2,
5.0 Hz, 1H), 4.14 (dd, J = 9.1, 3.5 Hz, 1H), 4.10 (dd, J = 12.3,
2.3 Hz, 1H),3.92 – 3.86 (m, 2H), 3.84 (t, J = 9.4 Hz, 1H), 3.72
(app s, 4H), 3.67 (d, J = 3.3 Hz, 2H), 3.62 – 3.54 (m, 2H), 3.53 –
3.49 (m, 1H), 3.42-3.39 (m, 2H), 3.33 (dt, J = 10.0, 3.3 Hz, 1H),
2.03 (s, 3H), 1.99 (s, 3H), 1.97 (s, 3H), 1.92 (s, 3H), 1.82 (s,
3H);13C NMR (151 MHz, CDCl3) δ 173.1, 171.2, 170.7, 170.5,
169.7, 154.8, 149.8, 139.5, 138.5(2), 138.4, 138.1, 137.96, 128.5-
127.6, 116.8, 114.7, 101.6, 97.8, 95.1, 80.6, 79.1, 78.1, 75.0,
74.9, 74.6, 73.4, 73.4, 72.5, 71.7, 71.6, 71.1, 70.9, 70.1, 69.8,
68.7, 67.3, 62.5, 55.7, 23.9, 23.8, 20.7, 20.7(2), 20.6; m/z
(HRMS) calcd for C70H80N2O20 [M+Na]+: 1291.5202, found:
1291.5212.
p-Methoxyphenyl-2-O-(2-acetamido-2-deoxy-β-
glucopyranosyl)-6-O-(2-acetamido-2-deoxy-β-
glucopyranosyl)-α- -mannopyranoside (9)
D-
D-
p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-
D
phthalimido-β-
deoxy-2-phthalimido-β-
-mannopyranoside (7)
D
-glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-2-
Trisaccharide 9 was prepared from trisaccharide 8 (0.09 g,
0.07 mmol) using the general procedure for hydrogenolysis and
followed by purification using silica gel column chromatography
(MeOH: CHCl3 = 1:20). Yield (0.05 g, 91 %).1H NMR (600
MHz, MeOD) δ 8.10 (s, 2H), 7.06 – 6.96 (m, 2H), 6.90 – 6.84
(m, 2H), 5.55 (td, J = 10.3, 3.7 Hz, 1H), 5.38 (s, 1H), 4.47 – 4.41
(m, 1H), 4.45–4.40 (m, 1H), 4.32 (ddd, J = 12.3, 4.4, 1.8 Hz,
1H), 4.17 – 4.11 (m, 1H), 4.10 (dd, J = 12.4, 2.4 Hz, 1H), 4.06
(d, J = 10.7 Hz, 1H), 3.94 – 3.87 (m, 1H), 3.86-3.84 (m, 1H),
3.76 (s, 3H), 3.73 – 3.68 (m, 2H), 3.68 – 3.59 (m, 2H), 3.66-3.60
(m, 2 H), 3.48 (ddd, J = 10.3, 8.4, 1.7 Hz, 1H), 3.35 – 3.33 (m,
1H), 3.30 – 3.22 (m, 2H), 2.04 (s, 3H), 2.01 (s, 3H), 1.99 (s, 6H),
1.87 (s, 3H); 13C NMR (151 MHz, MeOD) δ 174.6, 173.9, 172.3,
171.9, 171.2, 164.7, 156.6, 151.9, 119.1, 119.1, 115.7, 103.1,
99.5, 97.8, 78.5, 77.8, 75.9, 73.9, 72.9, 72.8, 72.1, 71.3, 70.3,
69.7, 68.5, 63.1, 62.7, 57.3, 56.3, 56.1, 23.7, 23.7(2), 23.4,
23.4(2), 20.6, 20.5; m/z (HRMS) calcd for C35H50N2O10
[M+Na]+: 841.2855, found: 841.2860.
D-glucopyranosyl)-3,4-di-O-benzyl-α-
D
Trisaccharide 7 was prepared from glycosyl phosphate donor 6
(0.104 g, 0.135 mmol) and disaccharide acceptor 4 (0.1 g, 0.113
mmol) using the general procedure for glycosyl phosphate
coupling and followed by purification using silica gel column
chromatography (PhCH3: EtOAc = 8:2). Yield (0.130 g, 80
%).1H NMR (600 MHz, CDCl3)
δ 7.84 – 7.54 (m, 4H), 7.52 (dd,
J = 5.5, 3.0 Hz, 4H), 7.33 – 7.21 (m, 16H), 7.10 – 7.07 (m,
3H), 6.98 – 6.95 (m, 2H), 6.91 – 6.81 (m, 4H), 6.65 – 6.58 (m,
4H), 5.83 (dd, J = 10.8, 9.0 Hz, 1H), 5.45 (d, J = 8.5 Hz, 1H),
5.16 (dd, J = 10.2, 9.0 Hz, 1H), 4.96 (dd, J = 5.5, 2.9 Hz, 2H),
4.80 (d, J = 10.9 Hz, 1H), 4.74 (d, J = 12.0 Hz, 1H), 4.62 (dd,
J = 11.2, 5.9 Hz, 2H), 4.50 – 4.45 (m, 3H), 4.43 – 4.42 (m,
1H), 4.42 – 4.37 (m, 2H), 4.27 (dd, J = 12.2, 5.0 Hz, 1H), 4.22
(dd, J = 10.7, 8.6 Hz, 1H), 4.19 (d, J = 2.4 Hz, 1H), 4.18 –
4.14 (m, 1H), 4.12 (t, J = 2.9 Hz, 1H), 3.99 (dd, J = 10.7, 8.4
Hz, 1H), 3.85 (dd, J = 8.2, 3.2 Hz, 1H), 3.82 (ddd, J = 10.2,
5.1, 2.4 Hz, 1H), 3.74 (s, 3H), 3.73 – 3.71 (m, 1H), 3.65 (dd, J
= 11.4, 2.3 Hz, 2H), 3.63 – 3.58 (m, 1H), 3.58 – 3.54 (m, 1H),
3.48 (ddd, J = 9.9, 4.6, 1.9 Hz, 1H), 3.38 (dd, J = 9.5, 8.2 Hz,
1H), 3.10 (dd, J = 11.1, 6.2 Hz, 1H), 2.04 (s, 3H), 2.02 (s,
3H), 1.88 (s, 3H);13C NMR (151 MHz, CDCl3) δ 170.8, 170.3,
169.6, 154.9, 150.3, 138.3, 138.3(2), 138.1, 138.1, 138.0, 134.1,
131.5, 128.5, 128.5(2), 128.4, 128.3, 128.3, 128.2, 128.1,
128.1(2), 128.0, 128.0(2), 127.9, 127.8, 127.7, 127.6, 127.5,
127.4, 117.9, 114.4, 98.7, 97.3, 96.6, 79.8, 79.3, 77.9, 75.1, 74.9,
74.9, 73.5, 72.2, 71.7, 71.7, 70.6, 69.4, 69.2, 68.8, 62.4, 55.8,
55.7, 54.6, 20.8, 20.8, 20.6; m/z (HRMS) calcd for C82H80N2O22
[M+Na]: 1467.5100, found: 1467.5116.
p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-
phthalimido-β-
D
-glucopyranosyl)-(1→2)-3-O-benzyl-4,6-O-
benzylidene-α-
D
-mannopyranoside (10)
Disaccharide 10 was prepared from glycosyl phosphate donor
6 (0.6 g, 0.775 mmol) and acceptor 1 (0.4 g, 0.646 mmol) using
the general procedure for glycosyl phosphates coupling and
followed by purification using silica gel column chromatography
(PhCH3: EtOAc = 20:1). Yield (0.530 g, 80 %).1H NMR (600
MHz, CDCl3) δ 7.72 – 7.62 (m, 2H), 7.43 – 7.35 (m, 5H), 7.35
– 7.28 (m, 9H), 7.27 – 7.20 (m, 5H), 7.07 – 7.02 (m, 4H), 6.93
– 6.87 (m, 2H), 6.87 – 6.82 (m, 2H), 6.74 – 6.67 (m, 2H), 6.65
– 6.58 (m, 2H), 5.42 (s, 1H), 5.27 (d, J = 8.4 Hz, 1H), 5.06 (d,
J = 1.9 Hz, 1H), 4.87 (d, J = 10.8 Hz, 1H), 4.83 (d, J = 12.2
Hz, 1H), 4.78 (d, J = 12.3 Hz, 1H), 4.67 (dd, J = 16.5, 11.6
Hz, 2H), 4.60 (d, J = 12.1 Hz, 1H), 4.55 (d, J = 12.2 Hz, 1H),
4.48 (d, J = 12.2 Hz, 1H), 4.41 (dd, J = 10.8, 8.3 Hz, 1H),
4.35 (dd, J = 10.8, 8.3 Hz, 1H), 4.24 (dd, J = 3.1, 1.9 Hz, 1H),
4.12 (q, J = 7.2 Hz, 1H), 3.98 (dd, J = 10.0, 3.1 Hz, 1H), 3.96
– 3.91 (m, 1H), 3.82 – 3.76 (m, 2H), 3.74 (s, 3H), 3.72 – 3.68
(m, 1H), 3.59 – 3.53 (m, 2H), 3.21 (s, 1H), 3.06 – 2.99 (m,
1H);13C NMR (151 MHz, CDCl3) δ 176.4, 171.2, 155.0, 149.7,
138.5-137.6, 133.8, 132.0, 128.8- 127.4, 126.1, 123.1, 117.1,
114.6, 101.5, 96.9, 96.3, 79.8, 79.2, 78.1, 75.6, 75.2, 74.9,
74.9(2), 73.7, 73.4, 71.5, 69.3, 68.3, 64.3, 60.5, 59.9, 56.0, 55.7;
m/z (HRMS) calcd for C62H59N2O13 [M+Na]+: 1048.3884, found:
1048.3889.
p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-
deoxy-β-D-glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-2-
acetamido-2-deoxy-β-D-glucopyranosyl)-3,4-di-O-benzyl-α-D-
mannopyranoside (8)
Trisaccharide 8 was prepared from trisaccharide 7 (0.130 g,
0.09 mmol) using the general procedure for phthalimide
deprotection and N-acetylation. The crude mixture was purified
using silica gel column chromatography (PhCH3: EtOAc = 6:4).
Yield (0.09 g, 79 %).1H NMR (600 MHz, CDCl3) δ 7.82 – 7.72
(m, 2H), 7.45 – 7.42 (m, 2H), 7.39 – 7.35 (m, 2H), 7.35 – 7.31
(m, 4H), 7.31 – 7.25 (m, 9H), 7.25 – 7.23 (m, 3H), 7.23 – 7.16
(m, 3H), 6.90 – 6.87 (m, 2H), 6.81 – 6.78 (m, 2H), 5.83 (t, J =
10.0 Hz, 1H), 5.50 (d, J = 1.9 Hz, 1H), 5.46 – 5.36 (m, 1H), 5.09
– 5.04 (m, 1H), 4.89 (dd, J = 16.7, 11.5 Hz, 2H), 4.80 (dd, J =
11.3, 7.8 Hz, 2H), 4.67-4.63 (m 2H), 4.59 – 4.55 (m, 1H), 4.55 –
6

7
p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-
6.63 (m, 2H), 6.57 – 6.54 (m, 2H), 5.59 (dd, J = 10.7, 9.0 Hz,
ACCEPTED MANUSCRIPT
phthalimido-β-
D
-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-α-
D-
1H), 5.20 (d, J = 8.5 Hz, 1H), 5.10 (d, J = 8.4 Hz, 1H), 4.91 –
4.86 (m, 2H), 4.85 (d, J = 10.9 Hz, 1H), 4.79 (d, J = 12.1 Hz,
1H), 4.74 (d, J = 11.7 Hz, 1H), 4.63 (d, J = 10.9 Hz, 1H), 4.59 (d,
J = 10.9 Hz, 1H), 4.56 (d, J = 12.1 Hz, 1H), 4.51 (s, 1H), 4.49 (d,
J = 3.5 Hz, 1H), 4.48 – 4.43 (m, 2H), 4.40 (dd, J = 10.8, 8.3 Hz,
1H), 4.28 (dd, J = 10.8, 8.5 Hz, 1H), 4.19 (d, J = 11.0 Hz, 1H),
4.13 (t, J = 6.0 Hz, 1H), 4.05 (dd, J = 10.7, 8.4 Hz, 1H), 3.85 –
3.82 (m, 3H), 3.77 (s, 3H), 3.75 – 3.71 (m, 2H), 3.71 – 3.67 (m,
2H), 3.56 – 3.52 (m, 2H), 3.30 (dd, J = 9.7, 8.3 Hz, 1H), 3.10 –
3.04 (m, 1H), 1.99 (s, 3H), 1.95 (s, 3H), 1.91 (s, 3H), 1.84 (s,
3H), 1.82 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 170.7, 170.2,
169.6, 154.8, 150.4, 138.4, 138.3, 138.2, 138.0, 134.3, 133.6,
128.6-127.4, 123.6, 117.8, 114.3, 98.6, 97.5, 97.0, 79.7, 79.3,
75.2, 75.1, 74.8, 73.6, 71.5, 71.4, 70.9, 70.6, 69.4, 69.2, 62.3,
56.0, 55.7, 54.6, 31.1, 20.7, 20.7, 20.5; m/z (HRMS) calcd for
C70H80N2O20 [M+Na]+: 1291.5202, found: 1291.5208
mannopyranoside (11)
Disaccharide 11 was prepared from 10 (0.53 g, 0.687 mmol)
using the general procedure for 4,6-O-benzylidene ring opening
with BH3-THF, followed by purification using silica gel column
chromatography (PhCH3: EtOAc = 8:2). Yield (0.413 g, 78 %).
1H NMR (600 MHz, CDCl3) δ 7.78 – 7.51 (m, 5H), 7.35 – 7.32
(m, 2H), 7.28 – 7.21 (m, 8H), 7.21 – 7.12 (m, 9H), 7.00 – 6.97
(m, 2H), 6.83 (dd, J = 8.2, 6.8 Hz, 2H), 6.78 – 6.74 (m, 1H), 6.58
– 6.54 (m, 2H), 6.40 – 6.35 (m, 2H), 5.10 (d, J = 8.5 Hz, 1H),
4.83 (d, J = 2.2 Hz, 1H), 4.80 (s, 1H), 4.79 – 4.78 (m, 2H), 4.76
(dd, J = 11.8, 1.9 Hz, 2H), 4.58 (d, J = 10.9 Hz, 1H), 4.53 – 4.48
(m, 2H), 4.48 – 4.43 (m, 2H), 4.43 – 4.38 (m, 2H), 4.27 (dd, J =
10.8, 8.5 Hz, 1H), 4.11 (t, J = 6.0 Hz, 1H), 3.89 (dd, J = 9.2, 3.1
Hz, 1H), 3.72 – 3.68 (m, 1H), 3.68 (d, J = 4.0 Hz, 1H), 3.65 (app
s, 4H), 3.36 – 3.31 (m, 2H) 3.24 (dd, J = 12.7, 2.8 Hz, 1H); 13C
NMR (151 MHz, CDCl3) δ 168.8, 168.0, 154.8, 149.7, 138.5,
138.3, 138.3(2), 138.2, 138.1, 137.8, 134.2, 133.7, 128.6 -127.4,
116.9, 114.5, 97.6, 95.8, 79.7, 79.1, 75.3, 75.2, 74.8, 74.1, 73.7,
72.4, 70.9, 70.7, 69.5, 62.5, 56.3, 55.7; m/z (HRMS) calcd for
C62H59N2O13 [M+Na]+: 1048.3884, found: 1048.3889.
p-Methoxyphenyl-2-O-(2-acetamido-2-deoxy-β-
glucopyranosyl)-6-O-(2-acetamido-2-deoxy-β-
glucopyranosyl)-α- -mannopyranoside (15)
D-
D-
D
Trisaccharide 15 was prepared from trisaccharide 14 (0.3 g,
0.236 mmol) using the general procedure for hydrogenolysis and
followed by column chromatography on silica gel (MeOH:
CHCl3 = 1:20). Yield (0.168 g, 87 %).1H NMR (600 MHz,
MeOD) δ 7.88 – 7.80 (m, 1H), 7.11 – 7.00 (m, 2H), 6.93 – 6.81
(m, 2H), 5.34 – 5.30 (m, 1H), 5.24 (ddd, J = 10.5, 9.3, 1.1 Hz,
1H), 4.96 – 4.90 (m, 1H), 4.85-4.82 (m, 4H), 4.71 – 4.67 (m,
1H), 4.63 (dd, J = 8.2, 1.3 Hz, 1H), 4.16 (dd, J = 12.2, 4.9 Hz,
1H), 4.11 (dd, J = 3.4, 1.9 Hz, 1H), 4.05 (ddd, J = 12.4, 10.0, 2.2
Hz, 2H), 3.90 – 3.84 (m, 3H), 3.84 – 3.79 (m, 1H), 3.77 (s, 3H),
3.76 – 3.72 (m, 1H), 3.69 (dt, J = 11.8, 5.7 Hz, 1H), 3.61 – 3.56
(m, 1H), 3.55-3.48 (m, 2H), 3.20 (q, J = 7.3 Hz, 1H), 2.02 (app d,
6H), 2.01 (s, 3H), 1.98 (s, 3H), 1.89 (s, 3H);13C NMR (151 MHz,
MeOD) δ 174.3, 173.4, 172.3, 171.7, 171.2, 156.6, 152.0, 119.5,
115.6, 102.5, 100.9, 98.5, 79.3, 79.1, 78.9, 78.4, 77.9, 74.7, 73.9,
73.8, 72.6, 72.0, 71.4, 70.8, 70.2, 68.7, 63.2, 62.4, 57.7, 56.1,
55.6, 23.5, 23.1, 23.0, 20.7, 20.6; m/z (HRMS) calcd for
p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-
phthalimido-β-
acetyl-2-deoxy-2-phthalimido-β-
3,4-di-O-benzyl-α- -mannopyranoside (13)
D
-glucopyranosyl)-(1→2)-6-O-(3,4,6-tri-O-
D-glucopyranosyl)-(1→6)-
D
Trisaccharide 13 was prepared from glycosyl phosphate donor
12 (0.302 g, 0.483 mmol) and disaccharide acceptor 11 (0.413 g,
0.402 mmol) using the general procedure for glycosyl phosphates
coupling and followed by column chromatography on silica gel
(PhCH3: EtOAc = 8:2). Yield (0.471 g, 81 %).1H NMR (600
MHz, CDCl3) δ 7.61 (s, 2H), 7.49 (s, 3H), 7.31 – 7.12 (m,
21H), 7.06 – 7.02 (m, 2H), 6.98 – 6.94 (m, 2H), 6.85 – 6.81
(m, 2H), 6.80 – 6.76 (m, 1H), 6.61 – 6.53 (m, 2H), 6.52 – 6.41
(m, 2H), 5.59 (dd, J = 10.7, 9.0 Hz, 1H), 5.20 (d, J = 8.5 Hz,
1H), 5.10 (d, J = 8.4 Hz, 1H), 4.90 – 4.86 (m, 2H), 4.85 (d, J
= 10.9 Hz, 1H), 4.79 (d, J = 12.1 Hz, 1H), 4.74 (d, J = 11.7
Hz, 1H), 4.63-4.58 (m, 2H), 4.56 (d, J = 12.1 Hz, 1H), 4.51 (s,
1H), 4.49 (d, J = 3.5 Hz, 1H), 4.47 – 4.44 (m, 2H), 4.40 (dd, J
= 10.8, 8.3 Hz, 1H), 4.28 (dd, J = 10.8, 8.5 Hz, 1H), 4.19 (d, J
= 11.0 Hz, 1H), 4.14 – 4.12 (m, 1H), 4.05 (dd, J = 10.7, 8.4
Hz, 1H), 3.85 – 3.82 (m, 2H), 3.77 (s, 3H), 3.75 – 3.72 (m,
2H), 3.72 – 3.69 (m, 1H), 3.67 (dq, J = 9.9, 2.3 Hz, 1H), 3.56
– 3.51 (m, 2H), 3.30 (dd, J = 9.7, 8.3 Hz, 1H), 2.02 – 1.96 (m,
3H), 1.91 (s, 3H), 1.84 (s, 3H);13C NMR (151 MHz, CDCl3) δ
170.7, 170.2, 169.6, 154.8, 150.4, 138.4, 138.3, 138.2, 138.0,
134.3, 133.6, 128.6-127.4, 123.6, 117.8, 114.3, 98.6, 97.5, 97.0,
79.7, 79.3, 75.2, 75.1, 74.8, 73.6, 71.5, 71.4, 70.9, 70.6, 69.4,
69.2, 62.3, 56.0, 55.7, 54.6, 31.1, 20.7, 20.7, 20.5. m/z (HRMS)
calcd for C82H80N2O22 [M+Na]: 1467.5100, found:1467.5109.
C35H50N2O10 [M+Na]+: 841.2855, found: 841.2860
p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-β- -glucopyranosyl)-4,6-di-O-benzyl-α-
mannopyranoside (16)
.
D
D-
Disaccharide 16 was prepared from 3 (0.3 g, 0.34 mmol) using
the general procedure for 4,6-O-benzylidene ring opening using
Et3SiH/TFA, followed by purification using silica gel column
chromatography (PhCH3: EtOAc = 8:2). Yield (0.210 g, 70
%).1H NMR (600 MHz, CDCl3) δ 7.86 – 7.75 (m, 1H), 7.73 –
7.64 (m, 1H), 7.44 – 7.40 (m, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.32
– 7.28 (m, 2H), 7.28 – 7.25 (m, 2H), 7.25 – 7.22 (m, 2H), 7.09 –
7.03 (m, 2H), 6.79 – 6.75 (m, 2H), 6.75 – 6.69 (m, 2H), 5.83 (dd,
J = 10.8, 9.1 Hz, 1H), 5.54 (d, J = 8.5 Hz, 1H), 5.26 – 5.14 (m,
2H), 4.83 (d, J = 11.0 Hz, 1H), 4.53 (d, J = 11.0 Hz, 1H), 4.47
(dd, J = 10.8, 8.5 Hz, 1H), 4.31 – 4.28 (m, 1H), 4.27 (d, J = 4.9
Hz, 1H), 4.21 (dd, J = 12.2, 2.4 Hz, 1H), 4.07 (s, 2H), 3.89 (ddd,
J = 10.3, 5.0, 2.4 Hz, 1H), 3.84 (dd, J = 9.1, 3.1 Hz, 1H), 3.76 –
3.72 (m, 4H), 3.70 – 3.65 (m, 1H), 3.41 (dd, J = 10.7, 3.2 Hz,
1H), 3.04 (dd, J = 10.7, 6.4 Hz, 1H), 2.61 (dt, J = 7.3, 2.3 Hz,
1H), 2.03 (s, 3H), 2.03 – 2.00 (s, 3H), 1.87 (s, 3H);13C NMR (151
MHz, CDCl3) δ 170.7, 170.2, 169.5, 155.0, 149.9, 138.2, 137.9,
137.8, 134.2, 129.1-127.3, 125.3, 123.5, 117.5, 114.5, 96.8, 95.9,
77.0, 73.2, 72.9, 72.1, 71.7, 70.9, 70.5, 70.5(2), 69.0, 67.5, 62.2,
55.6, 54.5, 21.5, 20.7, 20.7, 20.5; m/z (HRMS) calcd for
C47H49NO16 [M+Na]+: 906.2649, found: 906.2653.
p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-acetamido-2-
deoxy-β-
acetamido-2-deoxy-β-
mannopyranoside (14)
D
-glucopyranosyl)-6-O-(3,4,6-tri-O-acetyl-2-
D-glucopyranosyl)-3,4-di-O-benzyl-α-D-
Trisaccharide 14 was prepared from trisaccharide 13 (0.471 g,
0.326 mmol) using the general procedure for phthalimide
deprotection and N-acetylation and followed by column
chromatography on silica gel (PhCH3: EtOAc = 6:4). Yield
(0.302 g, 73 %).1H NMR (600 MHz, CDCl3) δ 7.68 (s, 2H), 7.56
(s, 2H), 7.33 – 7.29 (m, 4H), 7.29 – 7.26 (m, 5H), 7.25 – 7.23 (m,
2H), 7.22 (dt, J = 6.4, 1.7 Hz, 3H), 7.13 – 7.10 (m, 2H), 7.04 –
7.01 (m, 2H), 6.92 – 6.88 (m, 2H), 6.87 – 6.84 (m, 1H), 6.65 –
p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-
deoxy-β-D-glucopyranosyl)-4-O-(3,4,6-tri-O-benzyl-2-

8
Tetrahedron
ACCEPTED MANUSCRIPT
acetamido-2-deoxy-β-
mannopyranoside (17)
D
-glucopyranosyl)-3,4-di-O-benzyl-α-
D-
= 10.9, 8.0 Hz, 1H), 4.32 – 4.26 (m, 2H), 3.80 – 3.74 (m, 4H),
3.73 (app s, 4H), 3.71 – 3.59 (m, 3H), 3.38 (dd, J = 10.7, 3.1
Hz, 1H), 2.92 (dd, J = 10.7, 6.4 Hz, 1H); m/z (HRMS) calcd
for C62H61NO13 [M+Na]+: 1050.4041, found: 1050.4043.
Disaccharide acceptor 16 upon glycosylation with glysocyl
donor 6 using general procedure for glycosyl phosphates
coupling provided trisaccharide. The phthalimide groups of
trisaccharide were converted to NHAc using general procedure
for phthalimide deprotection and N-acetylation and followed by
column chromatography on silica gel (PhCH3: EtOAc = 6:4) to
provide trisaccharide 17. Yield (0.141 g, 77 %).1H NMR (600
MHz, CDCl3) δ 7.41 – 7.36 (m, 3H), 7.33 – 7.21 (m, 20H),
7.19 – 7.15 (m, 2H), 6.98 – 6.94 (m, 2H), 6.81 – 6.74 (m, 2H),
5.91 (s, 1H), 5.54 (s, 1H), 5.36 (d, J = 2.8 Hz, 1H), 5.30 (d, J
= 8.8 Hz, 1H), 5.11 (d, J = 8.4 Hz, 1H), 5.01 (t, J = 9.7 Hz,
1H), 4.77 (d, J = 6.9 Hz, 1H), 4.76 – 4.74 (m, 2H), 4.74 – 4.67
(m, 3H), 4.60 – 4.55 (m, 2H), 4.54 (d, J = 10.9 Hz, 1H), 4.48
(q, J = 12.2 Hz, 2H), 4.41 (d, J = 11.7 Hz, 1H), 4.26 (d, J =
2.8 Hz, 1H), 4.15 (dd, J = 12.2, 4.6 Hz, 1H), 4.13 – 4.08 (m,
3H), 3.89 – 3.85 (m, 1H), 3.84 – 3.81 (m, 1H), 3.75 (s, 3H),
3.73 – 3.70 (m, 1H), 3.68 – 3.64 (m, 1H), 3.64 – 3.59 (m, 1H),
3.58 (dd, J = 10.5, 4.6 Hz, 1H), 3.52 – 3.44 (m, 1H), 3.37
(ddd, J = 9.3, 4.7, 2.2 Hz, 1H), 1.98 (s, 3H), 1.92 (s, 3H), 1.89
(s, 3H), 1.70 (app s, 6H);13C NMR (151 MHz, CDCl3) δ 171.2,
170.7, 170.4, 170.2, 169.5, 155.0, 150.3, 138.6, 138.4, 138.4(2),
138.2, 138.0, 128.5-127.6, 117.6, 114.7, 100.5, 98.3, 96.7, 81.9,
78.4, 77.3, 74.9, 74.7, 74.6, 73.4, 73.1 72.3, 71.7, 71.5, 69.3,
69.0, 68.9, 62.1, 56.3, 55.7, 55.4, 23.5, 23.2, 20.7, 20.6, 20.6(2);
m/z (HRMS) calcd for C70H80N2O20 [M+Na]+: 1291.5202, found:
1291.5208.
p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-
phthalimido-β-
phthalimido-β-
mannopyranoside (21)
D
-glucopyranosyl)-4-O-(4-O-acetyl-2-deoxy-2-
-glucopyranosyl)-3,4-di-O-benzyl-α-
D
D
-
Trisaccharide 21 was prepared from glycosyl phosphate donor 19
(0.225 g, 0.291 mmol) and disaccharide acceptor 20 (0.250 g,
0.243 mmol) using the general procedure for glycosyl phosphate
coupling and followed by column chromatography on silica gel
(PhCH3: EtOAc = 8:2). Yield (0.292 g, 78 %).1H NMR (600
MHz, CDCl3) δ 7.63-7.56 (m, 6H), 7.43 – 7.33 (m, 1H), 7.29
(qd, J = 5.9, 2.8 Hz, 12H), 7.25 – 7.14 (m, 10H), 7.08 – 6.99
(m, 5H), 6.99 – 6.95 (m, 2H), 6.95 – 6.81 (m, 7H), 6.63 – 6.58
(m, 2H), 6.58 – 6.51 (m, 2H), 5.21 (d, J = 8.4 Hz, 1H), 4.93 –
4.86 (m, 3H), 4.84 (d, J = 10.9 Hz, 1H), 4.78 (d, J = 12.0 Hz,
1H), 4.70 (d, J = 11.5 Hz, 1H), 4.62 (d, J = 10.9 Hz, 1H), 4.55
(dd, J = 12.0, 9.5 Hz, 2H), 4.50 (d, J = 12.0 Hz, 2H), 4.45 (dd,
J = 11.7, 2.9 Hz, 2H), 4.38 (dd, J = 10.8, 8.4 Hz, 1H), 4.33 –
4.28 (m, 3H), 4.28 – 4.22 (m, 2H), 4.13 (t, J = 2.8 Hz, 1H),
4.10 (d, J = 11.0 Hz, 1H), 3.92 (dd, J = 10.7, 8.4 Hz, 1H),
3.82 (dd, J = 8.4, 3.2 Hz, 1H), 3.74 (app s, 4H), 3.72 – 3.68
(m, 1H), 3.66 (td, J = 7.2, 3.6 Hz, 2H), 3.55 (dd, J = 11.3, 2.3
Hz, 1H), 3.51 (ddd, J = 9.8, 5.7, 4.0 Hz, 1H), 3.33 – 3.27 (m,
2H), 3.18 (dd, J = 10.6, 5.6 Hz, 1H), 3.06 (dd, J = 11.2, 6.7
Hz, 1H), 2.36 (s, 1H), 1.89 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ 169.8, 154.8, 150.5, 138.4, 138.2, 138.2, 138.1,
138.0, 137.7, 133.8, 131.6, 129.1-127.4, 125.4, 117.8, 114.4,
98.7, 97.3, 96.4, 79.7, 79.4, 77.6, 75.2, 75.1, 74.9, 74.8, 73.9,
73.6, 73.5, 73.1, 72.9, 71.3, 71.1, 70.1, 69.9, 69.4, 55.9, 55.7,
55.4, 21.0; m/z (HRMS) calcd for C92H88N2O22 [M+Na]+:
1563.5822, found: 1563.5859.
p-Methoxyphenyl-2-O-(2-acetamido-2-deoxy-β-
glucopyranosyl)-4-O-(2-acetamido-2-deoxy-β-
glucopyranosyl)-α- -mannopyranoside (18)
D-
D-
D
Trisaccharide 18 was prepared from trisaccharide 17 (0.14 g,
0.110 mmol) using the general procedure for hydrogenolysis and
followed by column chromatography on silica gel (MeOH:
CHCl3 = 1:20). Yield (0.081 g, 90 %).1H NMR (600 MHz,
MeOD) δ 7.02 (d, J = 9.0 Hz, 2H), 6.90 – 6.77 (m, 2H), 5.47 –
5.36 (m, 1H), 5.31 – 5.20 (m, 1H), 4.87 – 4.77 (m, 1H),4.67 –
4.51 (m, 1H), 4.50 (d, J = 8.4 Hz, 1H), 4.33 (dp, J = 11.9, 6.8
Hz, 2H), 4.23 – 4.10 (m, 2H), 4.07 (tt, J = 6.4, 3.9 Hz, 1H),
3.99 – 3.86 (m, 2H), 3.87 – 3.75 (m, 2H), 3.74 (app s, 3H),
3.73 – 3.67 (m, 2H), 3.68 – 3.55 (m, 2H), 3.55 – 3.44 (m, 1H),
3.42 – 3.32 (m, 2H), 2.04 (app s, 3H), 1.99 (app s, 6H), 1.96
(app s, 6H);13C NMR (151 MHz, MeOD) δ 174.2, 173.8, 172.5,
171.9, 171.3, 164.7, 156.7, 151.7(2), 119.1, 119.1, 119.0, 115.6,
103.1, 101.2, 97.9, 78.5, 78.4, 78.0, 75.8, 73.9, 73.5, 72.9, 71.8,
71.8(2), 70.0, 69.7, 63.1, 62.5, 62.1, 62.0, 57.3, 56.1, 55.4, 23.3,
23.0, 20.7, 20.5; m/z (HRMS) calcd for C35H50N2O10 [M+Na]+:
841.2855, found:841.2859.
p-Methoxyphenyl-2-O-(2-acetamido-2-deoxy-β-
glucopyranosyl)-(4-O-acetyl-2-acetamido-2-deoxy-β-
glucopyranosyl)-α- -mannopyranoside (22)
D-
D-
D
Trisaccharide 21 (0.2 g, 0.13 mmol) was converted to
protecting group free trisaccharide 22 in a two steps sequence: (i)
deprotection of phthalimide groups and converted to NHAc using
general procedure for phthalimide deprotection and N-acetylation
(ii) catalytic hydrogenation in the presence of Pd(OH)2/C
removed all benzyl ethers. Finally, the crude trisaccharide was
purified by column chromatography on silica gel (MeOH: CHCl3
= 1:9) to furnish 24. Yield (0.059 g, 86 %).1H NMR (600 MHz,
MeOD) δ 7.11 – 7.02 (m, 2H), 6.95 – 6.85 (m, 2H), 5.36 (s,
1H), 4.87 – 4.81 (m, 3H), 4.78 (t, J = 9.2 Hz, 1H), 4.68 (d, J =
8.3 Hz, 1H), 4.54 (d, J = 7.6 Hz, 1H), 4.17 – 4.07 (m, 2H),
3.95 – 3.86 (m, 2H), 3.78 (s, 3H), 3.77 – 3.74 (m, 1H), 3.73 –
3.68 (m, 1H), 3.66 – 3.59 (m, 3H), 3.59 – 3.54 (m, 2H), 3.52
(dd, J = 12.2, 5.8 Hz, 1H), 3.45 (ddd, J = 8.9, 6.0, 2.5 Hz,
1H), 2.11 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H). 13C NMR (151
MHz, MeOD) δ 174.5, 172.1, 156.8, 152.0, 119.5, 115.7,
102.9, 98.7, 78.9, 78.0, 75.8, 74.8, 74.0, 73.3, 73.2, 72.1, 71.5,
70.1, 68.8, 62.5, 62.4, 57.8, 57.6, 56.1, 23.6, 23.3, 20.9. m/z
(HRMS) calcd for C31H46N2O18 [M+Na]+: 757.2638, found:
757.2647.
p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-
phthalimido-β-
mannopyranoside (20)
D-glucopyranosyl)-3,6-di-O-benzyl-α-D-
Disaccharide 20 was obtained from the disaccharide 10 (0.4 g,
0.39 mmol) using the general procedure for 4,6-O-benzylidene
ring opening using Et3SiH/TFA and followed by column
chromatography on silica gel (PhCH3: EtOAc = 9:1). Yield
(0.294 g, 74 %).1H NMR (500 MHz, CDCl3) δ 7.51 (dd, J =
5.5, 3.0 Hz, 2H), 7.42 – 7.37 (m, 2H), 7.33 – 7.21 (m, 18H),
7.05 – 6.98 (m, 4H), 6.90 – 6.85 (m, 2H), 6.85 – 6.81 (m, 1H),
6.77 – 6.72 (m, 2H), 6.71 – 6.67 (m, 2H), 5.29 (d, J = 8.3 Hz,
1H), 5.08 (d, J = 1.9 Hz, 1H), 4.86 (dd, J = 11.0, 8.2 Hz, 2H),
4.80 (d, J = 12.1 Hz, 1H), 4.64 (d, J = 10.9 Hz, 1H), 4.53 (d, J
= 11.9 Hz, 1H), 4.48 (s, 1H), 4.47 – 4.43 (m, 2H), 4.36 (dd, J
p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-
deoxy-β-D-glucopyranosyl)-4-O-(β-D-galactopyranosyl-2-
8

9
4. (a) Mizuochi, T.; Spellman, M. W.; Larkin, M.; Solomon, J.;
acetamido-2-deoxy-β-
mannopyranoside (23)
D-glucopyranosyl)-α-D-
ACCEPTED MANUSCRIPT
Basa, L. J.; Feizi, T. Biochem. J. 1988, 254, 599. (b) Pabst, M.;
Chang, M.; Stadlmann, J.; Altmann, F. Biol. Chem. 2012, 393,
719.
Glycan 23 was prepared from 18 using the general procedure for
enzymatic β-1, 4-galactosylation. The crude compound was
purified using C18 column chromatography using MeOH/H2O as
eluent. Fractions containing the product were combined,
evaporated, and lyophilized to give the tetrasaccharide 23 (9 mg,
75 %). 1H NMR (600 MHz, D2O) δ 7.16 – 7.12 (m, 2H), 7.03 –
6.99 (m, 2H), 5.49 (d, J = 2.1 Hz, 1H), 5.28 (dd, J = 10.7, 9.2 Hz,
1H), 5.12 (dd, J = 10.1, 9.3 Hz, 1H), 4.91 (d, J = 8.4 Hz, 1H),
4.58 (d, J = 8.3 Hz, 1H), 4.50 (d, J = 7.9 Hz, 1H), 4.45 (dd, J =
12.6, 3.4 Hz, 1H), 4.35 (dd, J = 3.4, 2.1 Hz, 1H), 4.26 – 4.20 (m,
2H), 4.17 – 4.11 (m, 1H), 4.06 – 4.03 (m, 1H), 4.04 – 3.99 (m,
1H), 3.95 (d, J = 3.5 Hz, 1H), 3.90 – 3.86 (m, 1H), 3.83 (s, 3H),
3.82 – 3.79 (m, 1H), 3.81 – 3.76 (m, 1H), 3.76 (ddd, J = 5.0, 3.6,
2.0 Hz, 2H), 3.75 – 3.71 (m, 1H), 3.71 – 3.68 (m, 1H), 3.62 –
3.57 (m, 1H), 3.57 – 3.54 (m, 1H), 2.15 (s, 3H), 2.11 (s, 3H),
2.08 (s, 3H), 2.06 – 2.04 (m, 3H), 1.99 (s, 3H), 13C NMR (151
MHz, D2O) δ 177.2, 177.0, 176.2, 175.7, 175.4, 157.3, 152.3,
121.2, 117.6, 105.4, 104.0, 102.1, 98.9, 80.6, 79.8, 79.2, 77.9,
77.4, 75.0, 74.9, 74.7, 73.7, 73.5, 71.1, 71.0, 70.7, 65.0, 64.3,
63.5, 63.2, 62.5, 58.3, 57.7, 56.0, 25.8, 24.7, 24.6, 22.6, 22.6(2),
22.5; m/z (HRMS) calcd for C41H60N2O23 [M+Na]+: 1003.3383,
found:1003.3384.
5
(a) Wang, G.; Zhang, W.; Lu, Z.; Wang, P.; Zhang, X.; Li, Y. J.
Org. Chem. 2009, 74, 2508. (b) Nagasaki, M.; Manabe, Y.;
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J. J. Am. Chem. Soc. 2012, 134, 16430. (d) Walczak, M. A.;
Hayashida, J.; Danishefsky, S. J. J. Am. Chem. Soc. 2013, 135,
4700. (e) Eller, S.; Schuberth, R.; Gundel, G.; Seifert, J.;
Unverzagt, C. Angew. Chem., Int. Ed. 2007, 46, 4173. (f)
Unverzagt, C.; Gundel, G.; Eller, S.; Schuberth, R.; Seifert, J.;
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(a) Wang, Z,; Chinoy, Z. S.; Ambre, S. G.; Peng, W.; McBride,
R.; de Vries, R. P.; Glushka, J.; Paulson, J. C.; Boons, G.-J.
Science, 2013, 341, 379-383. (b) Fujikawa, K.; Koizumi, A.;
Hachisu, M.; Seko, A.; Takeda, Y.; Ito, Y. Chem. Eur. J. 2015, 21,
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J. Am. Chem. Soc. 2017, 139, 1011. (d) Ting, C.-Y.; Lin, Y.-W.;
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(a) Shivatare, S. S.; Chang, S. H.; Tsai, T.-I.; Ren, C. T.; Chuang,
H.Y, Hsu, L.; Lin, C. W.; Li, S. T.; Wu, C. Y.; Wong. C.-H. J.
Am. Chem. Soc. 2013, 135, 2605. (b) Shivatare, S. S.; Chang, S.-
H.; Tsai, T.-I.; Susan Yu Tseng, S. Y.; Shivatare, V. S.; Lin, Y.-S.;
Cheng, Y.-Y.; Ren, C.-T.; Lee, C.-C. D, Pawar, S.; Tsai, C.-S.;
Shih, H.-W.; Zeng, Y.-F.;Liang, C.-H.; Kwongs, P. D.; Wu, C. -
6
7
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8. Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem.
1994, 50, 21.
9. Garegg, P. J. Adv. Carbohydr. Chem. Biochem. 1997, 52, 179.
10. Kahne, D.; Walker, S.; Cheng, Y.; Van Engen, D. J. Am. Chem.
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p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-
deoxy-β-
D
-glucopyranosyl)-(1→2)-O-(5-Acetamido-3,5-
-glycero-α- -galacto-2-nonulopyrano-sylonate-
-galactopyranosyl-β-(1→4)-2-acetamido-2-deoxy-β-
-glucopyranosyl)-(1→4)-α- -mannopyranoside (24)
dideoxy-
(2→3)-α-
D
D
D
11. Fraser-Reid, B.; Konradsson, P.; Mootoo, D. R.; Udodong, U. J.
Chem. Soc., Chem. Commun. 1988, 823.
D
D
12. (a) Kondo, H.; Aoki, S.; Ichikawa, Y.; Halcomb, R. L.; Ritzen,
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115, 2260.
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17. (a) Plante, O. J.; Palmacci, E. R.; Andrade, R. B.; Seeberger, P. H.
J. Am. Chem. Soc. 2001, 123, 9545. (b) Plante, O. J, Andrade, R.
B, Seeberger, P. H. Org. Lett. 1999, 1, 211. (c) Alessandra Ravida,
A.; Liu, X.; Kovacs, L.; Seeberger, P. H. Org. Lett. 2006, 8, 1815.
18. Palmacci, E. R.; Seeberger, P. H. Org. Lett. 2001, 3, 1547.
19. (a) Love, K. R.; Andrade, R. B.; Seeberger, P. H. J. Org. Chem.
2001, 66, 8165. (b) Werz, D. B.; Castagner, B.; Seeberger, P. H. J.
Am. Chem. Soc. 2007, 129, 2770. (c) Oberli, M. A.; Bindschäldler,
P.; Werz, D. B.; Seeberger, P. H. Org. Lett. 2008, 10, 905. (d)
Schütte, O. M.; Ries, A.; Orth, A.; Patalag, L. J.; Römer, W.;
Steinem, C.; Werz, D. B. Chem. Sci. 2014, 5, 3104.
Glycan 24 was prepared from 23 using the general procedure for
enzymatic α-2, 3-sialylation. The crude compound was purified
using C18 column chromatography using MeOH/H2O as eluent.
Fractions containing the product were combined, evaporated, and
lyophilized to give the pentasaccharide 24 (11.8 mg, 83 %). 1H
NMR (600 MHz, D2O) δ 7.06 (d, J = 8.3 Hz, 2H), 6.93 (d, J =
8.2 Hz, 2H), 5.40 (s, 1H), 5.19 (t, J = 10.0 Hz, 1H), 5.04 (t, J =
9.7 Hz, 1H), 4.82 (d, J = 8.3 Hz, 1H), 4.74-4.78 (m, 1H), 4.50 (d,
J = 8.3 Hz, 1H), 4.41 (d, J = 7.5 Hz, 1H), 4.37 (d, J = 12.7 Hz,
1H), 4.27 (s, 1H), 4.18 – 4.10 (m, 2H), 3.98 – 3.90 (m, 3H), 3.86
(s, 1H), 3.83 – 3.76 (m, 1H), 3.75 (d, J = 2.1 Hz, 3H), 3.72 – 3.63
(m, 12H), 3.63 – 3.54 (m, 2H), 3.54 – 3.44 (m, 2H), 2.97 – 2.87
(m, 1H), 2.07 (app s, 3H), 2.04 – 1.98 (app m, 9H), 1.97 (s, 3H),
1.93 (s, 3H), 1.29 – 1.18 (m, 1H); 13C NMR (151 MHz, D2O) δ
174.7, 174.5, 173.6, 173.2, 172.8, 154.7, 149.7, 118.6, 115.1,
102.9, 101.5, 99.5, 96.3, 78.1, 77.3, 76.7, 75.3, 74.8, 72.5, 72.4,
72.1, 72.0, 71.1, 70.9, 68.5, 68.4, 68.2, 62.5, 61.8, 61.0, 59.9,
59.5, 55.8, 55.1, 53.5, 30.2, 23.2, 22.1, 22.1, 20.1, 20.1(2), 19.9.
Acknowledgments
The National Institute of Health (AI072155), the Kwang Hua
Foundation and National Science Foundation (1664283)
supported this work.
Supplementary Material
References
1
Supplementary material is included for the H and 13C NMR
spectra of all intermediates.
1. Hart, G. W.; Copeland, R. J. Cell. 2010, 143, 672.
2.
3.
Helenius, A.; Aebi, M. Science. 2001, 291, 2364.
Gagneux, P.; Varki, A. Glycobiology. 1999, 9, 747. (b) Kwong, P.
D.; Wyatt, R.; Majeed, S.; Robinson, J.; Sweet, R. W.; Sodroski,
J.;Hendrickson, W. A. Structure. 2000, 8, 1329.
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