Chemical Synthesis of a Complex-Type N-Glycan Containing a Core Fucose

Article abstract of DOI:10.1021/acs.joc.6b02106

A chemical synthesis of a core fucose containing N-glycan was achieved. Asparagine was introduced at an early stage of the synthesis, and the sugar chain was convergently elongated. As for the fragment synthesis, we reinvestigated α-sialylation, β-mannosy

Full text of DOI:10.1021/acs.joc.6b02106

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Featured Article
Chemical Synthesis of a Complex-Type N-Glycan Containing a Core Fucose
Masahiro Nagasaki, Yoshiyuki Manabe, Naoya Minamoto,
Katsunori Tanaka, Alba Silipo, Antonio Molinaro, and Koichi Fukase
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02106 • Publication Date (Web): 24 Oct 2016
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Chemical Synthesis of a Complex-Type N-Glycan Containing a
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Core Fucose
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†
†
†
†, ‡
§
Masahiro Nagasaki , Yoshiyuki Manabe , Naoya Minamoto , Katsunori Tanaka , Alba Silipo , Antonio
†, §
*, †
Molinaro , and Koichi Fukase
†
Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama 1ꢀ1,
Toyonaka, Osaka, 560ꢀ0043, Japan
‡
Biofunctional Synthetic Chemistry Laboratory, RIKEN, Hirosawa 2ꢀ1, Wako, Saitama, 351ꢀ0198, Japan
§
Department of Chemical Science, University of Naples Federico II, Via Cinthia 4, 80126 Napoli, Italy
Corresponding Author
Eꢀmail: koichi@chem.sci.osakaꢀu.ac.jp
Abstract Graphic
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OAc
CO Me
AcO
2
OAc
O
O
Ac N
2
OBn
AcO
OAc
O
O
O
TrocHN
BzO
O
BnO
BzO
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AcO
OAc
BnO
AcO
OBn
O
O
BnO
OBn
OBn
O
O
O
O
OAc
HO
O
O
CO2Me
AcO
O
O
H
OAc
BnO
O
N
BnO
O
O
BnO
AcO
TrocHN
OAllyl
NHCbz
Ac N
2
TrocHN
O
AcO
OAc
BnO
O
O BnO
O
BzO
O
O
(4+8)
BnO
BzO
Glycosylations
TrocHN
(4+4)
Cleaving intermolecular hydrogen bonds of amides
Abstract
A chemical synthesis of a core fucose containing Nꢀglycan was achieved. The asparagine was
introduced at the earlyꢀstage of the synthesis and the sugar chain was convergently elongated. As for the
fragment synthesis, we reinvestigated αꢀsialylation, βꢀmannosylation, and Nꢀglycosylation to reveal that
precise temperature control was essential for these glycosylations. Intermolecular hydrogen bonds
involving acetamide groups were found to reduce the reactivity in glycosylations: the protection of
NHAc as NAc dramatically improved the reactivity. The dodecasaccharideꢀasparagine framework was
2
constructed via the (4+4) glycosylation and the (4+8) glycosylation using the tetrasaccharide donor and
the tetrasaccharideꢀasparagine acceptor. An etherꢀtype solvent enhanced the yields of these key
glycosylations between large substrates. After the whole deprotection of the dodecasaccharide, the target
Nꢀglycan was obtained.
Introduction
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Asparagineꢀlinked glycans (Nꢀglycans) in glycoproteins are oligosaccharides that are found in
eukaryotes and some prokaryotes and display a broad structural diversity. They are divided into three
types: high mannoseꢀtype, complexꢀtype, and hybridꢀtype. NꢀGlycans are generally heterogeneous, even
at one specific glycosylation site. This structural diversity, which is common in natural glycans, is called
glycoform.
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1
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Complexꢀtype Nꢀglycans play important roles in various biological events and diseases, including
1
,2
3
4,5
6,7
the regulation of glycoprotein dynamics, cell development, immunity, and cancer invasion. The
structures of the Nꢀglycans influence the function and dynamics of glycoproteins in vivo. For example,
Tanaka et al. conducted in vivo PET and fluorescence imaging of glycoproteins and glycoclusters to
reveal the remarkable dependence of the in vivo dynamics and bioꢀdistributions of these compounds on
the glycan structure. They found that trimming the nonꢀreducing end structure or linking sialic acid to
8,9
the galactose 3ꢀOH or 6ꢀOH positions affected the distribution of the glycoproteins.
Fucose residues linked to the reducing end GlcNAc through an α linkage form a core fucose
structure that comprises one of the major modifications of the complexꢀtype Nꢀglycans. Mammalian
core fucose is transferred to complexꢀtype Nꢀglycans by fucosyl transferase 8 (FUT8) to form an α(1ꢀ6)
1
0
fucosyl linkage. The core fucose has been shown to play an important role in various physiological and
pathological events. Fut8 knockout mice exhibited severe growth retardation, with a mortality rate 70%
11
during the 3 postnatal days.
Core fucosylated immunoglobulin G (IgG) showed the
antibodyꢀdependent cellꢀmediated cytotoxicity (ADCC) 100ꢀfold weaker than that without a core fucose
1
2
structure. The functional significance of the core fucosylation has been noted in a variety of the
1
3
pathophysiological steps involved in carcinogenesis and tumor progression. Human colon cancers with
reduced levels of core fucosylation were found to be resistant to TRAILꢀinduced apoptosis and escaped
immune surveillance. Core fucosylated αꢀfetoprotein levels were found to be significantly elevated in
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hepatocellular carcinoma and are useful as a liver cancer marker. The upꢀregulation of core
fucosylation on growth factor receptors, such as epidermal growth factor receptor (EGFR) and fibroblast
1
1,15,16
growth factor receptor (FGFR), has been found to activate these receptors.
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The functions of core fucose and its mechanisms of action have not yet been elucidated well. Core
fucose binding lectin has not been identified in mammals, although several core fucoseꢀbinding lectins
1
7ꢀ19
20,21
22
are found in plants,
fungi,
and bacteria.
Homogeneous preparations of core fucosylated
Nꢀglycans are needed to elucidate the biological functions of these structures, including the core
fucoseꢀdependent biodynamics and the identification of lectins responsible for core fucose recognition.
The isolation of core fucosylated Nꢀglycans from natural sources has proven to be difficult, although
2
3ꢀ25
certain series of Nꢀglycans are available from natural sources.
Motivated by this unmet need, we
investigated the chemical synthesis of Nꢀglycans containing core fucose.
Several syntheses of Nꢀglycans have been studied in an effort to investigate their biological
functions. Danishefsky et al. succeeded in synthesizing various types of Nꢀglycans, including a
2
6ꢀ28
coreꢀfucosylated glycan and a triantennary glycan.
Ito et al. synthesized highꢀmannose/complexꢀtype
29ꢀ31
glycans.
Unverzagt et al. reported the syntheses of Nꢀglycans with various structures, including core
3
2ꢀ35
fucose.
Chemoenzymatic approaches using a variety of glycosidases or glycosyltransferases have
3
6
37
38
been employed for the synthesis of Nꢀglycan libraries by Ito et al., Boons et al., Wang et al., and
3
9,40
Wong et al.
Schmidt et al. carried out the synthesis of complexꢀtype Nꢀglycans not only in liquid
4
1,42
phase but also on solid phase.
Our research group has reported the solidꢀphase synthesis of a sialic
43
acid containing glycan. Convergent synthetic strategies have been employed for the liquid phase
syntheses of glycans, and stepwise strategies have been employed for the solid phase syntheses. As for
the convergent Nꢀglycan syntheses, two strategies have been adopted. In path A, two donors possessing
a glucosamine residue at the reducing end were introduced to acceptors containing trimannosyl core
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(Figure 1). Stereoselective glycosylation was secured via neighboring group participation of a 2ꢀN–
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protecting group on the glucosamine residue. In path B, the glycan structure was constructed through
glycosylation at two branch positions (Figure 1). The reaction steps in path B were smaller than those
applied in path A, although neighboring group participation could not be used in glycosylations between
the stem and branch fragments in path B.
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In this study, we selected path B to synthesize a core fucose containing complexꢀtype Nꢀglycan 1,
which has an asparagineꢀlinked dodecasaccharide structure (Figure 1). We employed a new synthetic
strategy based on the earlyꢀstage introduction of asparagine into the glycan part. Previous reports of the
syntheses of Nꢀglycans have generally introduced asparagine into the glycan after preparing the
4
4ꢀ46
deprotected glycan.
The advantage of our method is the facile preparation of various Nꢀglycans
possessing an asparagine residue because any conversion is not necessary after the deprotection and the
protected glycosyl asparagines are easy to handle. NꢀGlycan 1 was synthesized by the successive
coupling of two branched tetrasaccharide donors 3 with the tetrasaccharideꢀAsn fragment 2, followed by
the global deprotection. Hydroxy groups at the 3ꢀ and 6ꢀpositions of the branched mannose of 2 were
orthogonally protected: 3ꢀOH was protected by 4ꢀazidoꢀ3ꢀchlorobenzyl (ClAzb) developed by our
4
7,48
group,
whereas 6ꢀOH was protected by a 4,6ꢀbenzylidene acetal. The key intermediates 2 and 3 were
synthesized by coupling 4 with 5 and 6 with 7, respectively.
The other important key to our synthesis involved the application of an amide protection strategy in
which 5ꢀNHAc of the sialic acid residue was protected with another acetyl group. We recently reported
that protection of the amide group of 5ꢀNHAc sialic acid significantly improved the reactivity of
4
9
glycosylation at a position away from the sialic acid residues. This strategy enabled efficient
glycosylation using sialic acid containing fragments, which sometimes have a low reactivity.
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OH
HO
CO H
2
OH
Path A
Path B
O
O
HO
AcHN
OH
(This work)
OH
O
O
O
AcHN
HO
O
HO
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HO
HO
OH
Core
OH Fucose
HO
HO
O
O
HO
OH
OH
O
O
O
HO
OH
O
O
CO2H
O
O
O
H
HO
O
HO
OH
N
HO
HO
HO
AcHN
OH
O
O
HO
AcHN
AcHN
O
HO
O
NH2
OH
O
HO
O
O
1
HO
HO
O
HO
AcHN
OAc
AcO
OAc
AcO
CO2Me
O
OAc
OBn
O
O
Ac2N
OBn
OBn
O
OAc AcO
BzO
OBn
O
O
Ph
O
O
O
O
O
O
+
O
O
O
H
N
BnO
O
O
BnO
O
BzO
BnO
TrocHN
TrocHN
OAllyl
TrocHN
BnO
AcO
O
NHCbz
3
NPh
CF3
N3
2
BnO
O
Cl
(ClAzb)
OBn
O
AcO
OAc
OAc
AcO
CO2Me
O
OBn
OAc
HO
OBn
O
O
OBn
O
BnO
O
O
Ph
O
O
Ac2N
O
TrocHN
NPh
+
+
OAc AcO
ClAzbO
O
NPh
BnO
AcO
BnO
O
O
O
O
H
BzO
TrocHN
O
CF3
HO
BnO
N
BnO
O
CF3
OAllyl
NHCbz
BzO
TrocHN
5
7
6
OAllyl
O
4
F
IGURE 1. Structure and synthetic strategy for achieving the N-glycan 1. Troc =
,2,2-trichloroethoxycarbonyl.
2
Results and Discussion
The tetrasaccharideꢀAsn fragment 2 was prepared as shown in Scheme 1. We first prepared the
asparagineꢀlinked disaccharide 10 through chemical Nꢀglycosylation. NꢀGlycosylation of asparagine was
5
0
51
first developed by Kahne and then improved by Tanaka and Takahashi. Tanaka and Takahashi found
that nitromethane was a suitable solvent for Nꢀglycosylation, whereas the reaction in CH Cl afforded
2
2
lower yields. Nitromethane, however, is highly flammable. We therefore investigated the
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Nꢀglycosylation in CH Cl and established efficient Nꢀglycosylation conditions under integrated
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1
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2
2
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6
microfluidic/batch conditions to obtain the asparagineꢀlinked monoꢀ and disaccharide fragments in high
5
2
yields. NꢀPhenyltrifluoroacetimidate glycosyl donors were mixed with an asparagine acceptor using a
micromixer at room temperature. The mixture was then transferred to a flask. The reaction mixture was
stirred until the reaction had reached completion to give the desired 10 in 84% yield (Scheme 1, entry 2).
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2
The yield was low under batch conditions, as reported by Tanaka and Takahashi (Scheme 1, entry 1) ;
however, the key to obtaining Nꢀglycosylation was the removal of the reaction heat, predominantly the
neutralization heat, during the addition of the Lewis acid. The disaccharideꢀAsn was obtained in 75%
yield under batch conditions through careful addition of diluted TMSOTf in CH Cl to the reaction
2
2
mixture containing the disaccharide donor 8 with the protected asparagine 9 at low temperatures
(Scheme 1, entry 3). The yield was much better than that obtained using the batch procedure reported in
our previous study, but was still lower than the yield obtained from the integrated microfluidic/batch
procedure. The Fmoc group of disaccharideꢀAsn 11 was then removed using 15% Et N to give the
3
disaccharide acceptor 4.
S
CHEME 1. Synthesis of the disaccharide acceptor 4 via N-glycosylation.
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a
Ref. 52. Reagents and conditions: (b) TMSOTf, MS4A, CH Cl ; (c) 15% Et N/CH Cl , rt, 2 h, 90%.
2
2
3
2
2
Stereoselective βꢀmannosylation using the donor 11 and the acceptor 12 posed another challenge
because neighboring group participation is unavailable, and the αꢀisomer is thermodynamically favored.
βꢀMannosylation has been achieved using a variety of methods, including intramolecular aglycon
5
3
54,55
delivery, S 2ꢀlike substitution of an αꢀglycosyl triflate intermediate,
and the use of a bulky Lewis
N
5
6
57ꢀ60
acid, among others.
We employed the glycosyl Nꢀphenyltrifluoroacetimidate 11 as a donor and
TMSOTf as an activator. The glycosylation was expected to proceed via the αꢀglycosyl triflate. The
reaction must be carried out under low temperatures in order to obtain a high βꢀselectivity; however, this
step was not easy in the context of a largeꢀscale synthesis. We developed a method for obtaining
integrated microfluidic/batch conditions that provided the βꢀproduct 13 in 77% yield on the gram scale
6
1
(
Scheme 2, entry 2). We also found that the key to the βꢀmannosylation was the removal of the
reaction heat during the addition of TMSOTf. The yield under batch conditions improved, as observed
with the Nꢀglycosylation through the careful addition of diluted TMSOTf to the reaction mixture. The
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desired stereoisomer was thus obtained in 77% yield (Scheme 2, entry 3). The allyl group of 12 was then
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
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3
3
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5
5
5
5
5
6
6
2
isomerized using an Ir complex, and the resulting compound was oxidatively cleaved using I and
2
63
64
H O to give the 1ꢀOH compound, which was converted to the Nꢀphenyltrifluoroacetimidate 5.
2
0
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The glycosyl donor 5 and acceptor 4 were then coupled using TMSOTf as a catalyst to give the
tetrasaccharideꢀAsn fragment 2 in good yield.
S
CHEME 2. Synthesis of the tetrasaccharide-Asn fragment 2.
OBn
O
OBn
O
OBn
O
OBn
O
Ph
O
Ph
O
b
NPh
O
O
+
HO
BnO
O
ClAzbO
ClAzbO
BnO
O
CF3
TrocHN
TrocHN
11
OAllyl
13
OAllyl
1
2
method to add
TMSOTf
OBn
O
OBn
O
entry
1a
temp
yield (β isomer)
61%
β/α
Ph
O
c, d
O
NPh
CF3
ClAzbO
O
syringe
–78 °C
4.9/1
5/1
BnO
O
5
TrocHN
Comet Xꢀ01
micromixer
2a
3
–90 °C to –50 °C
77%
77%
cannulation of
cooled solution
–78 °C
4.2/1
AcO
OAc
OBn
O
OBn
O
e
OBn
O
O
4
+
5
Ph
O
O
O
ClAzbO
O
O
H
BnO
O
BnO
N
TrocHN
OAllyl
NHCbz
TrocHN
2
O
a
Ref. 61. Reagents and conditions: (b) TMSOTf, MS4A, CH Cl ; (c) [Ir(cod)(PPh Me) ]PF , H , THF, rt, 30 min; then
2
2
2
2
6
2
I , H O, rt, 15 min, 97%; (d) Nꢀphenyltrifluoroacetimidoyl chloride, K CO , acetone, rt, 5 h, 88%; (e) TMSOTf, MS4A,
2
2
2
3
CH Cl , −20 °C, 20 min, 98%. cod = 1,5ꢀcyclooctadiene.
2
2
6
5
αꢀSelective sialylation is a critical issue for the synthesis of sialylated glycans because no
neighboring participation is available, the formation of unnatural βꢀsialoside is thermodynamically more
favorable, and glycals are readily formed as byꢀproducts. Recent advances in αꢀsialylation have featured
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substituent effects at the 5ꢀposition, since Boons et al. found that the 5ꢀNAc donor showed a high
2
6
6,67
68,69
70
71
reactivity.
Glycosylation using NHꢀTFA,
NHTroc, and N donors provided higher reactivities
3
with improved αꢀselectivity compared to the NHAc donor. The 4,5ꢀoxazolidinone sialyl donor, in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
2ꢀ75
particular, provided almost perfect αꢀselectivity for various glycosyl acceptors.
Our research group
7
6
developed the 5ꢀNPhth donor, which provided excellent yields and selectivities. We deployed the
5
ꢀNPhth and 5ꢀN donors in the microflow reactor to obtain the disaccharides in almost quantitative
3
7
7,78
yield and perfect αꢀselectivity.
The αꢀorienting solvent effect of nitrile has been used for sialylation,
except in the context of an oxazolidinone sialyl donor. The lower reaction temperature needed for
sialylation affords better αꢀselectivity in general, because αꢀsialylation is a kinetically controlled
reaction. The key to successful microflow sialylation was, therefore, the rapid removal of the reaction
heat to maintain the reaction temperature at −78 °C.
These substituents were converted into natural forms, such as NHAc, after sialylation. By contrast,
the 5ꢀNHAc donors were readily derived from commercially available sialic acids and did not require
Nꢀderivatization after sialylation. The critical point is that the reaction should be carried out around
−80 °C to obtain a high αꢀselectivity. Although NHAc sialyl donors have a lower reactivity than the
corresponding 5ꢀNꢀmodified sialyl donors, Yu’s Nꢀphenyltrifluoroacetimidate donor 14 had a reactivity
79
high enough to allow the reaction to proceed around −80 °C. We thus investigated the practical
αꢀsialylation using the 5ꢀNHAc donor 14 with the galactose acceptor 15 to obtain the disaccharide 16
(Table 1). Previously, we reported that a reaction of 14 and 15 gave 16 with moderate selectivity (α/β =
76
7
7/23) (entry 1); however, we then realized that the reaction temperature might not have been
wellꢀcontrolled, because TMSOTf was added in a single step using a micropipette. The careful addition
of TMSOTf using a microsyringe required substoichiometric amounts of TMSOTf, and the selectivity
8
0
was quite high (entry 2).
The largeꢀscale αꢀsialylation was achieved using the integrated
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microfluidic/batch procedure to afford the disaccharide 16 in 89% yield with a high selectivity (α/β =
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
0
94/6) in the gram scale (entry 3). Because the key to the integrated microfluidic/batch procedure was
the removal of the reaction heat during the addition of TMSOTf, similar results were obtained under
batch conditions by delivering a preꢀcooled TMSOTf solution via cannula to the substrate solution at
−78 °C (entry 4). These results indicated that efficient removal of the reaction heat was important for the
αꢀselective sialylation. Thus, the αꢀsialyl disaccharide 16 was obtained efficiently on the multiꢀgram
scale, even in a flask, although the microflow system offered better reproducibility and scalability.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TABLE 1. Investigation of the α-sialylation.
entry
scale
50 mg
30 mg
1 g
TMSOTf (eq)
apparatus
flask
method to add TMSOTf
yield
93%
86%
89%
α/β
77/23
93/7
94/6
a
1
0.2
1.0
1.0
micropipette
microsyringe
b
2
flask
b
3
Comet Xꢀ01
microfluidic
cannulation of cooled
TMSOTf solution
4
4.5 g
0.9
flask
85%
95/5
a
b
Ref. 76. Ref. 80.
Tetrasaccharides containing a sialic acid residue were then synthesized by coupling the disaccharide
acceptor 6 and the disaccharide donors 23 and 7 via a (2+2) pathway. The acceptor 6 and donors 23 and
43
7
were prepared as shown in Scheme 3. The Fmoc group of the protected mannose 18 was removed,
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
43
and the resulting alcohol 19 was glycosylated with the GlcNAc donor 17 to obtain the disaccharide 20.
The Fmoc group in 20 was cleaved to give the disaccharide acceptor 6. Two types of donor, 23 and 7,
were prepared from the αꢀsialyl disaccharide 16 by acetylation under two sets of conditions. Acetic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
6
anhydride in pyridine gave the 4ꢀOꢀacetate 21 whereas isopropenyl acetate with pꢀTsOH gave the
4ꢀOꢀacetyl diacetylimide 22. Both 21 and 22 were converted into the glycosyl imidates 23 and 7 via
twoꢀstep reactions.
S
CHEME 3. Preparation of the disaccharide acceptor 6 and the disaccharide donors 23/7.
OBn
2
O
OBn
O
OR1
R O
BnO
O
BnO
O
b
AcO
NPh
+
TrocHN
FmocO
BnO
BnO
BnO
AcO
O
O
CF3
OAllyl
a
TrocHN
BnO
1
17
18: R = Fmoc
OAllyl
2
2
0: R = Fmoc
1
19: R = H
c
2
6
: R = H
OAc
OAc
R3
CO Me
CO Me
AcO
2
AcO
2
OAc
OAc
O
O
O
O
AcHN
d for 21
e for 22
HO
AcO
OAc
OAc
O
O
BzO
BzO
16
BzO
21: R = NHAc BzO
3
OAllyl
AcO
OAllyl
3
22: R = NAc
2
OAc
CO Me
2
OAc
R3
O
O
f, g
AcO
OAc
NPh
CF3
O
BzO
O
3
BzO
2
3: R = NHAc
3
7
: R = NAc
2
Reagents and conditions: (a) 15% Et N/CH Cl , rt, 3 h, quant; (b) TMSOTf, MS4A, CH Cl , −78 °C to rt, overnight,
3
2
2
2
2
96%; (c) 15% Et N/CH Cl , rt, 5.5 h, quant; (d) Ac O, pyridine, 0 °C to rt, overnight; (e) isopropenyl acetate, pꢀTsOH,
3
2
2
2
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5 °C, quant; (f) [Ir(cod)(PPh Me) ]PF , H , THF, rt, then I , H O, rt; (g) Nꢀphenyltrifluoroacetimidoyl chloride, K CO ,
2 2 6 2 2 2 2 3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acetone, rt, 95% for 23 from 16, 75% for 7 from 22.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The syntheses of the tetrasaccharides via (2+2) glycosylation were investigated using the donors
2
3 and 7 (Table 2). The donor 7, with a NAc group, showed good reactivity, and the desired
2
tetrasaccharide 25 was obtained in 96% yield. On the other hand, the donor 23, with a NHAc group,
showed a much lower reactivity. The reaction required a greater quantity of TMSOTf and a higher
temperature to give tetrasaccharide 24 in 52% yield.
TABLE 2. Comparison of the two donors 23 and 7 in the context of the (2+2) glycosylation.
3
entry
donor
23
R
TMSOTf (eq)
0.2 + 0.2
0.2
temp
0 °C to rt
0 °C
time
yield
52%
96%
1
2
NHAc
NAc2
1.5 h + 1 h
20 min
7
The difference between the reactivities of the donors 23 and 7 was attributed to an intermolecular
hydrogen bond formed by the NHAc group. As mentioned above, the 5ꢀNHAc sialyl donor displayed a
lower reactivity than the 5ꢀNꢀmodified sialyl donors. The formation of a donor cluster via intermolecular
hydrogen bonds involving 5ꢀNHAc appeared to contribute to the low reactivity of the 5ꢀNHAc donor. In
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
81,82
fact, Kononov and coꢀworkers
reported that a 5ꢀNHAc sialic acid monosaccharide forms a dynamic
clusterꢀlike structure in solution that affects the reactivity and stereoselectivity of sialylation. We
hypothesized that this effect may arise from intermolecular hydrogen bonds formed by the NHAc group
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
in the disaccharide. This hypothesis was tested by collecting the H NMR spectra of the 5ꢀNHAc donor
23 at various concentrations (Figure 2a). The chemical shifts of the 5ꢀNHAc proton moved downfield at
higher substrate concentrations, indicating the formation of intermolecular hydrogen bonds. The
intermolecular hydrogenꢀbonding network may form a clusterꢀlike structure among the molecules
containing 5ꢀNHAc sialic acid (Figure 2b) and reduce the reactivity of the donor 23 by inhibiting
molecular motion and intermolecular reactions.
(a)
OAc
AcO
H
CO Me
2
O
O
1
N
H NMR
AcO
OAc
AcO
O
NPh
CF3
in CD Cl2
O
2
BzO
BzO
O
23
1
mM
(b)
OAc
AcO
H
CO Me
2
1
0 mM
0 mM
O
O
N
OAc
AcO
O
AcO MeO C
2
AcO
H
O
OAc
OAc
N
H
O
O
N
AcO
AcO
AcO
OAc
5
O
CO Me O
2
O
100 mM
PPM
5
.4
5.2
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1
F
IGURE 2. (a) H NMR of the disaccharide donor 23 at various concentrations. (b) Plausible intermolecular
hydrogen bonds formed by the NHAc groups in sialic acids.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Our research group recently observed similar hydrogenꢀbonding effects in the synthesis of a
4
9
disialylated tetrasaccharide motif. These results indicated that intermolecular hydrogen bonding in
sialic acid residues greatly affects the reactivity of glycosylation. Glycosylation between larger
oligosaccharide fragments should be more sensitive to intermolecular hydrogen bonds, considering the
lower mobility of the larger molecules. Therefore, the use of 5ꢀNAc sialic acid should improve the
2
efficiency of the large oligosaccharide synthesis.
With both the reducing and nonꢀreducing end tetrasaccharide fragments in hand, we next prepared
the tetrasaccharideꢀAsn acceptor 26 and the tetrasaccharide donor 3 for the synthesis of
dodecasaccharideꢀAsn (Scheme 4). The ClAzb group of the tetrasaccharideꢀAsn fragment 2 was cleaved
via formation of iminophosphorane with phosphine, followed by DDQ oxidation, to give the
tetrasaccharideꢀAsn acceptor 26. On the other hand, the 1ꢀallyl group of the tetrasaccharide fragment 25
was converted into glycosyl imidate to obtain the donor 3.
S
CHEME 4. Synthesis of the tetrasaccharide-Asn acceptor 26 and the tetrasaccharide donor 3.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: (a) PPh , CH Cl , rt, 1 h, then DDQ, AcOH, H O, rt, 20 min, 89%; (b) [Ir(cod)(PPh Me) ]PF ,
3
2
2
2
2
2
6
H , THF, rt, 1.5 h, then I , H O, rt, 10 min, 98%; (c) Nꢀphenyltrifluoroacetimidoyl chloride, K CO , acetone, rt, 1 h,
2
2
2
2
3
99%.
The synthesis of the octasaccharideꢀAsn 27 was carried out via (4+4) coupling using the
tetrasaccharide donor 3 and the tetrasaccharideꢀAsn acceptor 26 (Table 3). In previous studies of
Nꢀglycan synthesis, CH Cl or toluene was used as a solvent for the coupling of large fragments, and a
2
2
variety of glycosyl donors and activation methods have been investigated. We first carried out the
reaction at 0 °C in CH Cl to give the desired product in 56% yield with α/β = 3/1 (entry 1).
2
2
Glycosylation in MeCN at 0 °C resulted in a moderate yield and low selectivity (entry 2). In general,
coupling between large fragments is difficult in comparison to coupling of small fragments, because
both the mobility of the molecules and the accessibility of the glycosyl acceptor to oxocarbenium ion
intermediate are expected to be low. We postulated that the coordination of ether to the intermediate
oxocarbenium ion should stabilize the cationic intermediate and prolong its lifetime to enable the attack
of the large acceptor to the activated large donor before degradation of the activated donor. In fact,
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glycosylation in cyclopentylmethylether (CPME) provided an exceedingly high (91%) yield (entry 3).
The more strongly coordinating THF reduced the stereoselectivity (entry 4). The temperature had little
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
effect on the selectivity (entry 5). The selectivity was decreased when TMSClO was used as a promoter
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(entry 6). TMSI was not strong enough to activate the donor 3 (entry 7). DMF was added to the reaction
8
3,84
mixture to generate a DMF adduct in situ;
however, the donor degraded (entries 8, 9).
TABLE 3. Investigation of the (4+4) glycosylation.
entry
activator
solvent
CH Cl
temp
0 °C
0 °C
0 °C
additive (eq)
yield
56%
59%
91%
α/β
3/1
5/3
3/1
1
2
3
TMSOTf (0.5)
TMSOTf (1.0)
TMSOTf (0.5)
—
—
—
2
2
MeCN
CPME
CPME/THF
= 1/1
4
TMSOTf (0.5)
0 °C
—
68%
5/3
5
6
7
TMSOTf (0.5)
CPME
rt
—
—
—
63%
81%
3/1
5/3
—
a
TMSClO (0.5)
CPME
0 °C
4
b
TMSI (0.5 + 2.0)
CPME
0 °C to rt
0%
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c
c
8
9
TMSOTf (0.5)
TMSOTf (0.5)
CPME
CH Cl
0 °C to rt
0 °C to rt
DMF (5.0)
DMF (5.0)
0%
0%
—
—
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
c
generated in situ by TMSCl/AgClO . no reaction. hydrolysis and βꢀelimination of 3 occurred.
4
The octasaccharideꢀAsn 27 was treated with TFA to cleave benzylidene acetal. After purification by
column chromatography, the αꢀisomer 28 was isolated in 58% yield. Glycosylation of the donor 3 with
28 was carried out in CPME to obtain the dodecasaccharideꢀAsn 29 in a quite good 87% yield with α/β
=
1/1 (Scheme 5). The stereoisomers could be separated at a later stage in the synthesis. The use of the
5ꢀNAc –sialylated donor suppressed cluster formation by the donor via intermolecular hydrogen bonds.
2
Coupling of the large oligosaccharide fragments was thereby achieved with a high efficiency. The low
selectivity was attributable to the incompatibility between the donor and the acceptor. This issue is
expected to be addressed in the next generation synthesis.
S
CHEME 5. (4+8) glycosylation to provide the dodecasaccharide-Asn 29.
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reagents and conditions: (a) TFA, H O, CH Cl , 0 °C to rt, 1.5 h, 58% for α isomer (14% for β isomer); (b) 3,
2
2
2
TMSOTf, CPME, MS4A, 0 °C, 10 min, 87% as α/β mixture.
With the protected Nꢀglycan 29 in hand, global deprotection of 29 was carried out (Scheme 6).
8
5
Cleavage of the NꢀTroc group with aqueous LiOH was identified by our research group. This method
appeared to be applicable to the dodecasaccharideꢀAsn 29; however, our investigation suggested that
aspartimide formed under basic conditions. This side reaction was expected to occur via nucleophilic
attack by the nitrogen in the side chain on the allyl ester. This ester was selectively cleaved using a Pd
8
6
catalyst. The resulting carboxylic acid was treated with aqueous LiOH, and subsequent Nꢀacetylation
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
gave compound 30 without aspartimide formation. Compound 30 was purified by reverseꢀphase HPLC,
and the two stereoisomers generated in the (4+8) glycosylation were separated in this step. Finally, the
desired isomer 30 was hydrogenated to obtain the target Nꢀglycan 1.
0
1
2
3
4
5
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9
0
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7
8
9
0
S
CHEME 6. Deprotection of dodecasaccharide-Asn.
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Reagents and conditions: (a) Pd(OAc) , PPh , sodium 2ꢀethylhexanoate, acetone, rt, 2 h; (b) 1) 3 M LiOH aq, THF,
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dioxane, rt, overnight; 2) Ac O, NaHCO , H O, rt, 1 h × 2, then LiOH•H O, rt, 2 h, 27% for 30 (27% for β isomer)
2
3
2
2
from 29; (c) 20% Pd(OH) /C, H , tꢀBuOH, H O, AcOH, rt, overnight, 60%.
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In summary, the chemical synthesis of a core fucose containing Nꢀglycan 1 was achieved. We
developed a universal route to various asparagineꢀlinked Nꢀglycans based on several new synthetic
strategies. An asparagine residue was introduced by Nꢀglycosylation during the early step of the
synthesis, whereas asparagine was introduced during the final step of the synthesis by coupling glycosyl
amine with the aspartic acid residue in previous studies. Protection of 5ꢀacetamide in sialic acid using an
additional acetyl group dramatically increased the reactivity of glycosylation during fragment coupling
by avoiding intermolecular hydrogen bonding involving the 5ꢀNHAc group. The fragment coupling
strategy successfully reduced the total reaction steps. The stepwise coupling of the branch
tetrasaccharides at the 3ꢀ and 6ꢀpositions of mannose in the stem tetrasaccharide asparagine was the key
to this synthetic strategy. High yields of the fragment coupling reaction were obtained by stabilizing the
intermediate oxocarbenium ion through coordination of the ether solvent. The low selectivity of the
glycosylation during fragment coupling represents an issue that will be addressed in future work. The
present study enables the efficient synthesis of various Nꢀglycans and precise biological studies using
synthetic Nꢀglycans.
Experimental Section
1
13
General procedures. H and C NMR spectra were recorded in an indicated solvent with a 400
MHz spectrometer, a 500 MHz spectrometer, or a 600 MHz spectrometer equipped with a cryoprobe.
1
For H NMR analysis, the chemical shifts in CDCl are given δ values from tetramethylsilane (TMS) as
3
an internal standard. Acetone (δ = 2.22 ppm) is used as an internal standard for the measurement in D O.
2
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4
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5
5
5
5
5
5
5
5
6
CHD OD (δ = 3.30 ppm), CHD COCD (δ = 2.05 ppm), and CHDCl (δ = 5.32 ppm) are used as
2
2
3
2
references for the measurements in CD OD, acetoneꢀD , and CD Cl , respectively. Highꢀresolution mass
3
6
2
2
spectra were obtained on an ESIꢀOrbitrap (FTMS) or an ESIꢀTOF mass spectrometer. Unless otherwise
noted, reactions in anhydrous solvent were carried out under argon atmosphere. Distilled CH Cl was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
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5
6
7
8
9
0
1
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
distilled from calcium hydride. MS4A were activated with a microwave oven and dried in vacuo three
times before use. All other commercially available reagents and solvents were used as purchased.
α
γ
N -Benzyloxycarbonyl-N -(6-O-(3,4-di-O-acetyl-2-O-benzyl-
y-4-O-(9-fluorenylmethoxycarbonyl)-2-(2,2,2-trichloroethyloxycarbonylamino)-
sparagine allyl ester (10). Disaccharide
ꢀOꢀ(3,4ꢀdiꢀOꢀacetylꢀ2ꢀOꢀbenzylꢀαꢀ
α
-
L
-fucopyranosyl)-3-O-benzyl-2-deox
-glucopyranosyl)- -a
donor
β
-D
L
6
L
ꢀfucopyranosyl)ꢀ3ꢀOꢀbenzylꢀ2ꢀdeoxyꢀ4ꢀOꢀ(9ꢀfluorenylmethoxycar
5
2
bonyl)ꢀ2ꢀ(2,2,2ꢀtrichloroethoxycarbonylamino)ꢀDꢀglucopyranosyl Nꢀphenyltrifluoroacetimidate (8)
α
52
(
1.00 g, 0.863 mmol) and asparagine acceptor N ꢀbenzyloxycarbonylꢀLꢀasparagine allyl ester (9) (529
mg, 1.73 mmol) were lyophilized from benzene and activated MS4A powder was added. To the mixture
was added dist. CH Cl (17.3 mL). To the solution was added TMSOTf (31.3 µL, 0.173 mmol) at −
2
2
7
8 °C and the solution was stirred for 20 min at the same temperature. The solution was warmed up to
10 °C and stirred for 21 h. The reaction was quenched by sat. aqueous NaHCO and insoluble materials
3
were filtered. The filtrate was poured into sat. aqueous NaHCO and the aqueous layer was extracted
3
with CHCl . The organic layer was washed by brine, dried over Na SO , filtered, and concentrated in
3
2
4
vacuo to give a crude product. Silicaꢀgel column chromatography (toluene/EtOAc = 4/1 to 2/1) was
carried out to obtain product 10 (831 mg, 75%) as a white solid. For analytical data, see ref. 52.
α
γ
N -(Benzyloxycarbonyl)-N -(6-O-(3,4-di-O-acetyl-2-O-benzyl-
oxy-2-(2,2,2-trichloroethoxycarbonylamino)- -glucopyranosyl)-
solution of DisaccharideꢀAsn 10 (1.00 g, 0.784 mmol) in CH Cl (26.7 mL) was added Et N (4.7 mL).
α
-
L
-fucopyranosyl)-3-O-benzyl-2-de
β
-D
L-asparagine allyl ester (4). To a
2
2
3
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After stirring for 2 h at rt, the reaction mixture was diluted with toluene and concentrated in vacuo. The
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
residue was coꢀevaporated four times with toluene to give a crude product. Silicaꢀgel column
chromatography (toluene/EtOAc = 2/1 to 1/1) was carried out to obtain 4 (745 mg, 90%) as a white solid.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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2
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7
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H NMR (500 MHz, CDCl ): δ = 7.40ꢀ7.28 (m, 15H), 6.92 (d, 1H, J = 7.9 Hz), 5.91 (d, 1H, J = 8.7 Hz),
3
5.87ꢀ5.79 (m, 1H), 5.30 (dd, 1H, J = 10.4, 3.4 Hz), 5.28ꢀ5.24 (m, 2H), 5.18 (dd, 1H, J = 10.4, 1.1 Hz),
5
.13ꢀ5.07 (m, 2H), 4.84 (d, 1H, J = 3.6 Hz), 4.81ꢀ4.77 (m, 2H), 4.75 (dd, 1H, J = 11.1, 11.1 Hz), 4.71 (d,
H, J = 4.2 Hz), 4.67 (s, 1H), 4.63 (dd, 1H, J = 11.1, 11.1 Hz), 4.59ꢀ4.55 (m, 5H), 4.15 (q, 1H, J = 6.5
1
Hz), 3.90ꢀ3.85 (m, 2H), 3.83 (d, 2H, J = 4.7 Hz), 3.53ꢀ3.44 (m, 3H), 3.25 (dd, 1H, J = 10.2, 8.9 Hz),
.85 (dd, 1H, J = 16.6, 3.3 Hz), 2.68 (dd, 1H, J = 16.6, 3.9 Hz), 2.13 (s, 3H), 2.00 (s, 3H), 1.08 (d, 3H, J
2
1
3
= 6.5 Hz). C NMR (125 MHz, CDCl ); δ = 170.6, 170.4, 170.0, 156.1, 137.9, 137.6, 136.3, 131.6,
3
1
9
3
1
28.8, 128.6, 128.6, 128.5, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 127.9, 118.5,
8.2, 95.3, 80.1, 79.0, 74.8, 74.8, 73.9, 73.8, 73.8, 72.9, 71.5, 70.5, 68.5, 67.0, 66.2, 64.9, 55.1, 50.5,
+
7.7, 20.8, 20.7, 15.8. HR ESIꢀOrbitrap MS: m/z calcd for C H Cl N O [M+Na] 1074.2573, found
4
8
56
3
3
17
074.2589.
Allyl
4
-O-(3-O-(4-azido-3-chlorobenzyl)-2-O-benzyl-4,6-O-benzylidene-
β
-
D
-mannopyranosyl)-3,6-di-O-benzy
(13). Mannose donor
ꢀmannopyranosyl
(2.00 g, 2.88 mmol) and GlcN acceptor allyl
l-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-
D
-glucopyranoside
3
ꢀOꢀ(4ꢀazidoꢀ3ꢀchlorobenzyl)ꢀ2ꢀOꢀbenzylꢀ4,6ꢀOꢀbenzylideneꢀ
D
61,78
Nꢀphenyltrifluoroacetimidate (11)
5
6
3
,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀ2ꢀ(2,2,2ꢀtrichloroethoxycarbonylamino)ꢀ
D
ꢀglucopyranoside (12)
(1.66 g,
2
.88 mmol) were coꢀevaporated with toluene three times and activated MS4A powder was added. To the
mixture was added dist. CH Cl (40 mL), and the mixture was cooled to −80 °C. A solution of TMSOTf
2
2
(
156 µL, 0.864 mmol) in dist. CH Cl (18 mL) was dried over activated MS4A pellets and cooled to −
2
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2
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5
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7
8 °C. To the solution of 11 and 12 was added the solution of TMSOTf via cannula and the mixture was
stirred for 2.5 d at −80 °C. The reaction was quenched by Et N (1.0 mL) and insoluble materials were
3
filtered. The filtrate was concentrated in vacuo to give a crude product. Silicaꢀgel column
chromatography (toluene/EtOAc = 15/1 to 10/1) was carried out to obtain product 13 (2.41 g, 77%) as a
yellow solid. For analytical data, see ref. 61, 78.
0
1
2
3
4
5
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8
9
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8
9
0
4
-O-(3-O-(4-azido-3-chlorobenzyl)-2-O-benzyl-4,6-O-benzylidene-
benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)- -glucopyranose.
Ir(cod)(PPh Me) ]PF (157 mg, 0.185 mmol) in anhydrous THF (24 mL) was stirred under H
2
β
-D
-mannopyranosyl)-3,6-di-O-
D
A
suspension of
[
2
2
6
atmosphere for 5 min to give a yellow solution. The solution was added to a solution of disaccharide
allyl glycoside 13 (4.00 g, 3.70 mmol) in anhydrous THF (50 mL) and the mixture was stirred for 30
min at rt. To the reaction solution was added H O (20 mL) and I (1.88 g, 7.40 mmol) and the mixture
2
2
was stirred for additional 15 min. The reaction was quenched by 20% aqueous Na S O and the aqueous
2
2
3
layer was extracted by EtOAc. The organic layer was washed with sat. aqueous NaHCO and brine,
3
dried over Na SO , filtered, and concentrated in vacuo to give a crude product. Silicaꢀgel column
2
4
chromatography (toluene 100% to toluene/EtOAc = 3/1) was carried out to give the 1ꢀOH product (3.72
1
g, 97%) as a brown solid of α/β mixture. H NMR (500 MHz, CDCl ) of major isomer: δ = 7.45ꢀ7.41 (m,
3
4H), 7.39ꢀ7.34 (m, 4H), 7.34ꢀ7.27 (m, 12H), 7.25ꢀ7.22 (m, 3H), 7.18 (dd, 1H, J = 8.2, 1.9 Hz), 7.05 (d,
1
H, J = 8.2 Hz), 5.48 (s, 1H), 5.32 (t, 1H, J = 3.7 Hz), 5.05 (d, 1H, J = 7.2 Hz), 5.03 (d, 1H, J = 11.5 Hz),
.84 (s, 2H), 4.72 (d, 1H, J = 11.7 Hz), 4.67ꢀ4.60 (m, 3H), 4.48ꢀ4.46 (m, 2H), 4.40 (d, 1H, J = 12.0 Hz),
4
4.08 (dd, 1H, J = 10.5, 4.8 Hz), 4.05 (dd, 1H, J = 9.5, 9.5 Hz), 3.97 (d, 1H, J = 3.0 Hz), 3.96 (s, 1H),
3
1
.90 (td, 1H, J = 9.7, 3.0 Hz), 3.70 (td, 1H, J = 9.2, 3.2 Hz), 3.62ꢀ3.56 (m, 2H), 3.50 (dd, 1H, J = 10.3,
0.3 Hz), 3.33 (dd, 1H, J = 9.8, 2.9 Hz), 3.10 (td, 1H, J = 9.6, 4.9 Hz), 2.90 (d, 1H, J = 2.7 Hz).HR
+
ESIꢀOrbitrap MS: m/z calcd for C H Cl N O [M+Na] 1061.2077, found 1061.2091.
5
0
50
4
4
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-O-(3-O-(4-azido-3-chlorobenzyl)-2-O-benzyl-4,6-O-benzylidene-β-D-mannopyranosyl)-3,6-di-O-
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-
D
-glucopyranosyl N-phenyltrifluoroacetimidate
solution of
ꢀmannopyranosyl)ꢀ3,6ꢀdiꢀOꢀbenz
ꢀglucopyranose (100 mg, 0.0961 mmol) in acetone
1.9 mL) were added Nꢀphenyltrifluoroacetimidoyl chloride (39.9 mg, 0.192 mmol) and K CO (39.8
(5).
To
a
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
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8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
ꢀOꢀ(3ꢀOꢀ(4ꢀazidoꢀ3ꢀchlorobenzyl)ꢀ2ꢀOꢀbenzylꢀ4,6ꢀOꢀbenzylideneꢀβꢀ
D
ylꢀ2ꢀdeoxyꢀ2ꢀ(2,2,2ꢀtrichloroethoxycarbonylamino)ꢀ
D
(
2
3
mg, 0.288 mmol). After stirring for 5 h at rt, insoluble materials were filtered and the filtrate was
concentrated in vacuo to give a crude product. Silicaꢀgel column chromatography (toluene/EtOAc =
1
3
0/1 to 10/1) was carried out to obtain 5 (103 mg, 88%) as a yellowish solid of α/β mixture. H NMR
(
500 MHz, CDCl ) of major isomer: δ = 7.45 (dd, 2H, J = 7.6, 1.9 Hz), 7.42 (dd, 2H, J = 7.6, 1.0 Hz),
3
7
.39ꢀ7.35 (m, 4H), 7.32 (q, 8H, J = 6.9 Hz), 7.27 (d, 7H, J = 8.4 Hz), 7.20 (dd, 1H, J = 8.2, 1.8 Hz), 7.10
(tt, 1H, J = 7.4, 1.1 Hz), 7.06 (d, 1H, J = 8.2 Hz), 6.78 (d, 2H, J = 7.7 Hz), 6.32 (br s, 1H), 5.50 (s, 1H),
5.03 (d, 1H, J = 11.6 Hz), 4.88 (d, 1H, J = 11.9 Hz), 4.82 (d, 1H, J = 11.9 Hz), 4.75 (d, 1H, J = 12.1 Hz),
4.71 (d, 1H, J = 8.1 Hz), 4.67 (d, 1H, J = 3.3 Hz), 4.64 (d, 1H, J = 2.6 Hz), 4.63ꢀ4.60 (m, 1H), 4.51 (d,
1H, J = 12.7 Hz), 4.49 (s, 1H), 4.49 (s, 1H), 4.38 (d, 1H, J = 12.1 Hz), 4.12 (dd, 1H, J = 10.5, 4.8 Hz),
4.07 (dd, 1H, J = 9.5, 9.5 Hz), 4.07 (d, 1H, J = 8.9 Hz), 4.03 (s, 1H), 3.72 (d, 2H, J = 2.8 Hz), 3.65 (dd,
1H, J = 9.8, 9.8 Hz), 3.59ꢀ3.51 (m, 3H), 3.35 (dd, 1H, J = 9.9, 2.9 Hz), 3.13 (td, 1H, J = 9.7, 4.8 Hz). HR
+
ESIꢀOrbitrap MS: m/z calcd for C H Cl F N O [M+Na] 1232.2373, found 1232.2384.
5
8
54
4
3
5
12
α
γ
N -benzyloxycarbonyl-N -(6-O-(3,4-di-O-acetyl-2-O-benzyl-
azido-3-chlorobenzyl)-2-O-benzyl-4,6-O-benzylidene- -mannopyranosyl)-3,6-di-O-benzyl-2-deoxy-2-(
,2,2-trichloroethoxycarbonylamino)- -glucopyranosyl)-3-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy
carbonylamino)- -glucopyranosyl)- -asparagine allyl ester (2). Disaccharide donor 5 (1.26 g, 1.04
α-L-fucopyranosyl)-4-O-(4-O-(3-O-(4-
β
-D
2
β-D
β
-D
L
mmol) and disaccharideꢀAsn acceptor 4 (1.00 g, 0.949 mmol) were lyophilized from benzene and
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4
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5
5
5
5
5
6
activated MS4A powder was added. To the mixture was added dist. CH Cl (19 mL). To the mixture was
2
2
added TMSOTf (34.4 µL, 0.190 mmol) at −20 °C and the mixture was stirred for 20 min at the same
temperature. The reaction was quenched by sat. aqueous NaHCO at 0 °C and insoluble materials were
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
filtered. The filtrate was poured into sat. aqueous NaHCO and the aqueous layer was extracted by
3
CHCl . The organic layer was washed by brine, dried over Na SO , filtered, and concentrated in vacuo
3
2
4
to give a crude product. Silicaꢀgel column chromatography (CHCl /acetone = 20/1 to 8/1) was carried
3
1
out to obtain product 2 (1.94 g, 98%) as a yellowish solid. H NMR (500 MHz, CDCl ): δ = 7.43ꢀ7.38
3
(m, 6H), 7.37ꢀ7.27 (m, 16H), 7.24ꢀ7.12 (m, 16H), 7.03 (d, 1H, J = 8.2 Hz), 6.91 (d, 1H, J = 7.4 Hz),
6.15 (d, 1H, J = 8.1 Hz), 5.90 (d, 1H, J = 8.8 Hz), 5.85ꢀ5.77 (m, 1H), 5.45 (s, 1H), 5.37 (dd, 1H, J = 10.5,
3.3 Hz), 5.24 (dddd, 1H, J = 17.2, 1.3, 1.3, 1.3 Hz), 5.19 (dd, 1H, J = 3.1, 0.8 Hz), 5.16 (dddd, 1H, J =
10.5, 1.3, 1.3, 1.3 Hz), 5.12 (d, 1H, J = 12.2 Hz), 5.04 (d, 1H, J = 6.0 Hz), 5.02 (d, 1H, J = 5.2 Hz), 4.98
(d, 1H, J = 2.3 Hz), 4.89ꢀ4.76 (m, 5H), 4.73ꢀ4.64 (m, 4H), 4.61ꢀ4.48 (m, 6H), 4.42 (d, 1H, J = 12.3 Hz),
4
.39 (d, 1H, J = 7.0 Hz), 4.38 (d, 1H, J = 12.3 Hz), 4.34 (d, 1H, J = 11.5 Hz), 4.25 (d, 1H, J = 12.1 Hz),
.23 (dd, 1H, J = 13.6, 5.5 Hz), 4.08 (dd, 1H, J = 12.1, 3.4 Hz), 4.06 (dd, 1H, J = 9.2, 9.2 Hz), 4.00 (dd,
4
1H, J = 9.8, 9.8 Hz), 3.98 (dd, 1H, J = 10.2, 5.2 Hz), 3.86ꢀ3.80 (m, 3H), 3.69 (d, 1H, J = 3.2 Hz), 3.68
dd, 1H, J = 8.6, 2.5 Hz), 3.63 (d, 1H, J = 9.5 Hz), 3.54 (dd, 2H, J = 10.9, 2.9 Hz), 3.50 (dd, 2H, J = 9.8,
.5 Hz), 3.43ꢀ3.39 (m, 3H), 3.32 (dd, 1H, J = 9.5, 9.5 Hz), 3.25 (dd, 1H, J = 9.8, 3.1 Hz), 3.04 (td, 1H, J
(
2
= 9.6, 4.9 Hz), 2.83 (dd, 1H, J = 16.7, 3.4 Hz), 2.62 (dd, 1H, J = 16.7, 4.0 Hz), 2.05 (s, 3H), 1.78 (s, 3H),
13
1
.00 (d, 3H, J = 6.4 Hz). C NMR (100 MHz, CDCl ): δ = 170.5, 170.5, 170.3, 169.9, 156.2, 156.1,
3
154.3, 139.1, 138.3, 138.1, 137.5, 137.1, 136.7, 136.2, 136.2, 131.6, 129.4, 129.0, 128.9, 128.8, 128.6,
1
1
28.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8, 127.2, 127.1, 126.7, 126.0,
24.9, 119.4, 118.5, 103.6, 102.0, 101.4, 97.2, 95.6, 95.2, 81.4, 81.0, 80.4, 78.5, 78.4, 78.0, 77.7, 77.3,
76.2, 75.9, 74.9, 74.8, 74.7, 74.7, 74.1, 73.7, 73.6, 73.4, 73.4, 71.7, 71.0, 69.6, 68.9, 68.4, 67.3, 67.0,
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6.2, 64.9, 57.5, 55.9, 50.4, 37.6, 20.8, 20.6, 15.5. HR ESIꢀOrbitrap MS: m/z calcd for C H Cl N O
98 104 7 7 28
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
[
M+Na] 2094.4647, found 2094.4646.
Allyl
2,3-di-O-benzoyl-6-O-(Methyl
-galacto-2-nonulopyranosylonate)- -g
acid donor methyl
ꢀgalactoꢀ2ꢀnonulopyranosylonate
(4.50 g, 6.80 mmol) and galactose acceptor allyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
alactopyranoside (16). Sialic
ꢀacetamideꢀ4,7,8,9ꢀtetraꢀOꢀacetylꢀ3,5ꢀdideoxyꢀ ꢀglyceroꢀD
D
-glycero-
α-D
α-D
5
D
7
6
Nꢀphenyltrifluoroacetimidate (14)
,3ꢀdiꢀOꢀbenzoylꢀαꢀ ꢀgalactopyranoside (15) (4.39 g, 10.2 mmol) were lyophilized from benzene and
7
6
2
D
activated MS4A powder was added. To the mixture was added dist. EtCN (120 mL), and the mixture
was cooled to −78 °C. A solution of TMSOTf (1.11 mL, 6.12 mmol) in dist. EtCN (16 mL) was dried
over activated MS4A pellets and cooled to −78 °C. The solution of TMSOTf was added to the solution
of 14 and 15 via cannula and the mixture was stirred for 6 h at −78 °C. The reaction was quenched by
Et N (5.0 mL) and insoluble materials were filtered. The filtrate was concentrated in vacuo to give a
3
crude product. Silicaꢀgel column chromatography (toluene/EtOAc = 1/1 to 1/5) was carried out to obtain
1
6 (85%, α/β = 95/5) as a white solid. For analytical data, see ref. 80.
Allyl 4-O-acetyl-3,6-di-O-benzyl- -mannopyranoside (19). To a solution of protected mannose
allyl 4ꢀOꢀacetylꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀOꢀ(9ꢀfluorenylmethoxycarbonyl)ꢀαꢀ
g, 2.33 mmol) in CH Cl (79 mL) was added Et N (14 mL). The mixture was stirred for 3 h at rt and
α
-D
4
3
D
ꢀmannopyranoside (18) (1.50
2
2
3
diluted with toluene. The resulting solution was concentrated in vacuo and coꢀevaporated four times
with toluene to give a crude product. Silicaꢀgel column chromatography (toluene/EtOAc = 5/1 to 3/1)
1
was carried out to obtain product 19 (1.03 g, quant) as a colorless oil. H NMR (500 MHz, CDCl ): δ =
3
7.36ꢀ7.24 (m, 10H, aromatic), 5.95ꢀ5.87 (m, 1H), 5.29 (dddd, 1H, J = 17.2, 1.5, 1.5, 1.5 Hz), 5.24 (dd,
1
H, J = 9.8, 9.8 Hz), 5.20 (dddd, 1H, J = 10.5, 1.5, 1.5, 1.5 Hz), 4.95 (d, 1H, J = 1.8 Hz), 4.66 (d, 1H, J
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=
12.0 Hz), 4.53 (d, 1H, J = 12.0 Hz), 4.53 (s, 2H), 4.21 (dddd, 1H, J = 13.0, 5.2, 1.5, 1.5 Hz), 4.05 (m,
1
1
H), 4.01 (dddd, 1H, J = 13.0, 6.3, 1.5, 1.5 Hz), 3.86 (m, 1H), 3.81 (dd, 1H, J = 9.8, 3.5 Hz), 3.57 (dd,
1
3
H, J = 10.8, 5.5 Hz), 3.53 (dd, 1H, J = 10.8, 3.5 Hz), 2.60 (d, 1H, J = 2.0 Hz, 1H), 1.89 (s, 3H). C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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5
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7
8
9
0
NMR (125 MHz, CDCl ): δ = 169.9, 138.0, 137.7, 133.6, 128.5, 128.3, 128.3, 127.9, 127.7, 127.6, 127.5,
3
117.6, 98.2, 77.1, 73.5, 71.8, 69.7, 69.6, 68.3, 68.2, 68.1, 20.8. HR ESIꢀTOF MS: m/z calcd for C H O
7
2
5
30
+
[
M+Na] 465.1884, found 465.1893.
Allyl
4
-O-acetyl-3,6-di-O-benzyl-2-O-(3,6-di-O-benzyl-4-O-(9-fluorenylmethyloxycarbonyl)-2-deoxy-2-(2,2,2-
-glucopyranosyl)- -mannopyranoside (20). GlcNAc donor
trichloroethoxycarbonylamino)-
β
-D
α-D
3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀ4ꢀOꢀ(9ꢀfluorenylmethoxycarbonyl)ꢀ2ꢀ(2,2,2ꢀtrichloroethoxycarbonylamino)ꢀDꢀ
43
glucopyranosyl Nꢀphenyltrifluoroacetimidate (17) (659 mg, 0.710 mmol) and mannose acceptor 19
(377 mg, 0.852 mmol) were lyophilized from benzene and activated MS4A powder was added. To the
mixture was added dist. CH Cl (7.1 mL). To the mixture was added TMSOTf (25.0 µL, 0.142 mmol) at
2
2
−
78 °C and the mixture was stirred for 15 min at rt. The reaction was quenched by sat. aqueous NaHCO3
and insoluble materials were filtered. CH Cl was evaporated and the residual mixture was poured into
2
2
sat. aqueous NaHCO . The aqueous layer was extracted by EtOAc and the organic layer was washed
3
with H O and brine, dried over Na SO , filtered, and concentrated in vacuo. Silicaꢀgel column
2
2
4
chromatography (toluene/EtOAc = 20/1 to 5/1) was carried out to give 20 (805 mg, 96%) as a white
1
solid. H NMR (500 MHz, CDCl ): δ = 7.74 (dd, 2H, J = 7.6, 3.2 Hz, Fmoc aromatic), 7.56 (ddd, 2H, J
3
=
22.1, 7.6, 0.8 Hz, Fmoc aromatic), 7.39ꢀ7.14 (m, 24H, aromatic), 5.92ꢀ5.84 (m, 1H, ꢀCH ꢀCH=CH ),
2 2
5
.42 (br s, 1H, NHTroc), 5.26 (dd, 1H, J = 9.5, 9.5 Hz, Hꢀ4), 5.23 (dddd, 1H, J = 17.3, 1.5, 1.5, 1.5 Hz,
ꢀCH ꢀCH=CH ), 5.18 (dddd, 1H, J = 10.5, 1.5, 1.5, 1.5 Hz, ꢀCH ꢀCH=CH ), 5.07 (br d, 1H, J = 6.5 Hz,
2
2
2
2
Hꢀ1’), 4.83 (s, 1H, Hꢀ1), 4.81 (dd, 1H, J = 9.5, 9.5 Hz, Hꢀ4’), 4.71 (d, 1H, J = 12.0 Hz, ꢀCH Ph), 4.66 (d,
2
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H, J = 12.0 Hz, ꢀCH Ph), 4.59 (d, 1H, J = 12.0 Hz, ꢀCH Ph), 4.58 (s, 2H, ꢀCH Ph), 4.52 (s, 2H,
2 2 2
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ꢀ
CH Ph), 4.47 (d, 1H, J = 12.0 Hz, ꢀCH Ph), 4.45 (d, 1H, J = 12.0 Hz, ꢀNHCO CH CCl ), 4.42 (d, 1H, J
2 2 2 2 3
=
12.0 Hz, ꢀNHCO CH CCl ), 4.33 (br s, 1H, Hꢀ3’), 4.29 (d, 2H, J = 7.4 Hz, ꢀCOCH ꢀfluorenyl), 4.18
2 2 3 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
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7
8
9
0
1
2
3
4
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7
8
9
0
(dd, 1H, J = 3.2, 3.2 Hz, Hꢀ2), 4.16 (dddd, 1H, J = 13.0, 6.3, 1.5, 1.5 Hz, ꢀCH ꢀCH=CH ), 4.10 (t, 1H, J
2 2
= 7.4 Hz, Fmoc fluorenyl), 3.94 (dddd, 1H, J = 13.0, 6.0, 1.5, 1.5 Hz, ꢀCH ꢀCH=CH ), 3.84 (dd, 1H, J =
2
2
9
.5, 3.2 Hz, Hꢀ3), 3.79 (m, 1H, Hꢀ5), 3.71 (m, 1H, Hꢀ5’), 3.60ꢀ3.51 (m, 4H, Hꢀ6, Hꢀ6’), 3.14 (br s, 1H,
1
3
Hꢀ2’), 1.92 (s, 3H, Ac). C NMR (125 MHz, CDCl ): δ = 169.7, 154.3, 153.9, 143.3, 143.1, 141.3,
3
1
1
41.2, 138.2, 138.0, 137.8, 133.6, 129.0, 128.3, 128.2, 127.9, 127.8, 127.7, 127.6, 127.6, 127.6, 127.1,
25.3, 125.1, 125.0, 120.0, 117.5, 95.5, 75.1, 74.2, 73.6, 73.4, 73.0, 72.8, 71.2, 70.5, 70.0, 69.8, 68.7,
+
68.3, 57.8, 46.7, 20.9. HR ESIꢀTOF MS: m/z calcd for C H Cl NO [M+Na] 1202.3234, found
6
3
64
3
15
1
202.3234.
Allyl
4-O-acetyl-3,6-di-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-
glucopyranosyl)- -mannopyranoside (6). To a solution of protected disaccharide 20 (200 mg, 0.169
mmol) in CH Cl (5.8 mL) was added Et N (1.0 mL) and the solution was stirred for 5.5 h at rt. The
β-D-
α-D
2
2
3
reaction solution was concentrated in vacuo and coꢀevaporated three times with toluene to give a crude
product. Silicaꢀgel column chromatography (toluene/EtOAc = 4/1 to 3/1) was carried out to give product
1
6
(162 mg, quant) as a colorless solid. H NMR (400 MHz, acetoneꢀD ): δ = 7.37ꢀ7.19 (m, 20H), 6.96 (d,
6
1
H, J = 8.6 Hz), 5.99ꢀ5.89 (m, 1H), 5.25 (dddd, 1H, J = 17.3, 1.6, 1.6, 1.6 Hz), 5.15 (dd, 1H, J = 9.8, 9.8
Hz), 5.13 (dddd, 1H, J = 10.9, 1.6, 1.6, 1.6 Hz), 4.99 (d, 1H, J = 1.4 Hz), 4.89ꢀ4.76 (m, 5H), 4.60 (d, 1H,
J = 13.7 Hz), 4.57 (d, 1H, J = 10.2 Hz), 4.53 (s, 4H), 4.46 (d, 1H, J = 12.0 Hz), 4.30 (t, 1H, J = 2.3 Hz),
4
.19 (dddd, 1H, J = 13.2, 5.2, 1.6, 1.6 Hz), 3.98 (dddd, 1H, J = 13.2, 5.8, 1.6, 1.6 Hz), 3.89 (dd, 1H, J =
10.7, 2.0 Hz), 3.83 (dd, 1H, J = 9.4, 3.3 Hz), 3.82 (ddd, 1H, J = 12.9, 6.2, 3.3 Hz), 3.77ꢀ3.69 (m, 2H),
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