Convergent Synthesis of a Bisecting N-Acetylglucosamine (GlcNAc)-Containing N-Glycan

Article abstract of DOI:10.1002/asia.201800367

The chemical synthesis of a bisecting N-acetylglucosamine (GlcNAc)-containing N-glycan was achieved by a convergent synthetic route through [4+2] and [6+2] glycosylations. This synthetic route reduced the number of reaction steps, although the key glycosylations were challenging in terms of yields and selectivities owing to steric hindrance at the glycosylation site and a lack of neighboring group participation. The yields of these glycosylations were enhanced by stabilizing the oxocarbenium ion intermediate through ether coordination. Glycosyl donor protecting groups were explored in an effort to realize perfect α selectivity by manipulating remote participation. The simultaneous glycosylations of a tetrasaccharide with two disaccharides was investigated to efficiently construct a bisecting GlcNAc-containing N-glycan.

Full text of DOI:10.1002/asia.201800367

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Accepted Article
Title: Convergent synthesis of a bisecting GlcNAc-containing N-glycan
Authors: Yoshiyuki Manabe, Hiroki Shomura, Naoya Minamoto,
Masahiro Nagasaki, Yohei Takakura, Katsunori Tanaka, Alba
Silipo, Antonio Molinaro, and Koichi Fukase
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10.1002/asia.201800367
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Convergent synthesis of a bisecting GlcNAc-containing N-glycan
Yoshiyuki Manabe,[a, b] Hiroki Shomura,[a] Naoya Minamoto,[a] Masahiro Nagasaki,[a] Yohei Takakura,[a]
Katsunori Tanaka,[a, c] Alba Silipo,[d] Antonio Molinaro,[a, d] Koichi Fukase*[a, b]
Abstract: The chemical synthesis of
a
bisecting N-
Bisecting GlcNAc appears to provide diverse functions via
several mechanisms. For example, expression of bisecting
GlcNAc suppresses tumor metastasis.[8] GnT-III acts on the
same substrate with GnT-V and reduces the GnT-V product β1-
acetylglucosamine (GlcNAc)-containing N-glycan was achieved by a
convergent synthetic route via [4+2] and [6+2] glycosylations. This
synthetic route reduced the number of reaction steps, although the
key glycosylations were challenging in terms of yields and
selectivities due to steric hindrance at the glycosylation site and a
lack of neighboring group participation. The yields of these
glycosylations were enhanced by stabilizing the oxocarbenium ion
intermediate through ether coordination. Glycosyl donor protecting
groups were explored in an effort to realize perfect -selectivity by
manipulating remote participation. We investigated the simultaneous
6
GlcNAc branching structure, resulting in inhibition of
metastasis. A bisecting GlcNAc group on a synthetic IgG
glycoform significantly enhances binding between Fc and
FcγRIIIa, thereby activating the Fcγ receptor and increasing the
antibody-dependent cellular-cytotoxicity (ADCC) of IgG.[9]
Alzheimer’s disease (AD) patients have higher levels of
bisecting GlcNAc on the -site amyloid precursor protein
cleaving enzyme-1 (BACE1). In GnT-III-deficient mice, the
BACE1 lacking bisecting GlcNAc was localized to late
endosomes/lysosomes and lysosomal degradation of BACE1
was accelerated. Since GnT-III-deficient mice showed the
decrease in A plaques and improved cognitive function, GnT-III
may be a promising drug target for AD therapeutics.[10] Certain
lectins can directly recognize bisecting GlcNAc moieties. For
example, Calsepa and PHA-E lectins bound to the ‘back-fold’
conformation of the bisected N-glycan. The statistical
conformational analysis suggests that bisecting GlcNAc restricts
the conformations of branched structures in N-glycans.[11]
A sufficient supply of pure N-glycans containing bisecting
GlcNAc is needed for the further investigation of the biological
functions of these groups. Motivated by this need, we
investigated the chemical synthesis of bisecting GlcNAc-
containing N-glycans.
The syntheses of N-glycans have been extensively explored by
several groups. Danishefsky et al. achieved the syntheses of
various types of N-glycans, including N-glycans containing
bisecting GlcNAc, multi-antennary N-glycans, and others.[12]
Unverzagt et al. synthesized several N-glycans containing
bisecting GlcNAc or core fucose.[13] We have reported the
synthesis of N-glycan-containing core fucose and sialic acids.[14]
Ito et al.,[15] Boons et al.,[16] Wang et al.,[17] and Wong et al.[18]
reported the syntheses of N-glycan libraries using
chemoenzymatic approaches. Solid-phase syntheses of
complex-type N-glycans have been reported by Schmidt et al.[19]
and by our group.[20]
glycosylations of
a tetrasaccharide with two disaccharides to
efficiently construct a bisecting GlcNAc-containing N-glycan.
Introduction
Glycosylation on protein is the most common post-translational
modification. Asparagine-linked glycans (N-glycans) have a high
structural diversity and are categorized as high-mannose-type,
complex-type, or hybrid-type oligosaccharides. N-Glycans
provide diverse physiological functions. They control protein
quality,[1] protein dynamics,[2] cell development,[3] cancer
invasion[4] and immunity,[5] depending on their type, modification,
and position on a protein. N-acetylglucosaminyltransferase III
(GnT-III) is an N-glycan processing enzyme that transfers
GlcNAc to the branching mannose with a 1,4-linkage to form a
bisecting N-acetylglucosamine (GlcNAc).[6] The bisecting
GlcNAc moiety is a modification characteristic of complex-type
N-glycans and has a variety of unique biological functions.[7]
[a]
[b]
Dr. Y. Manabe, Dr. H. Shomura, Dr. N. Minamoto, Dr. M. Nagasaki,
Prof. K. Tanaka, Prof. A. Molinaro, Prof. K. Fukase
Department of Chemistry, Graduate School of Science
Osaka University
1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan
E-mail: koichi@chem.sci.osaka-u.ac.jp
Dr. Y. Manabe, Prof. K. Fukase
Core for Medicine and Science Collaborative Research and
Education, Project Research Center for Fundamental
Sciences, Graduate School of Science, Osaka University
Machikaneyama 1-1, Toyonaka, Osaka, 560-0043, Japan
Prof. K. Tanaka
Biofunctional Synthetic Chemistry Laboratory, RIKEN
2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
Prof. A. Silipo, Prof. A. Molinaro
The synthetic approach to obtain bisecting GlcNAc-containing N-
glycan 1 is shown in Figure 1. An octasaccharide was
constructed via two glycosylations at the 3- and 6-positions of
mannose in the tetrasaccharide 2 using the disaccharides 3–7.
One of the key steps in the synthesis of 1 involved the
introduction of a bisecting GlcNAc. Unverzagt et al. introduced
the bisecting GlcNAc at the final stage of glycan construction.[13b,
[c]
[d]
Department of Chemical Science, University of Naples Federico II
Via Cinthia 4, 80126 Napoli, Italy
13c, 13e]
They achieved the efficient glycosylation of a bisecting
GlcNAc at the 4-position of the branching mannose using
trifluoroacetamide-protected N-glycans.[13e] Here, we instead
elongated the bisecting GlcNAc to form a reducing end fragment
Supporting information for this article is given via a link at the end of
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prior to introducing the branching disaccharides 3–7. This
approach is similar to Danishefsky's route.[12b]
not necessary after deprotection, and the protected glycosyl
asparagines could be easily handled.
Other important steps involved the construction of the branching
structure. Previous studies explored two major strategies. The
first strategy involved the introduction of glucosaminyl donors to
Results and Discussion
acceptors containing
a trimannosyl core that could be
constructed via stereoselective -mannosylation using the
neighboring group participation of a 2-O-acyl-protected mannose
residue. The other strategy involved a convergent synthetic
route in which the branching structure was constructed via two
glycosylations at the branching mannose residue. Although the
latter strategy required fewer reaction steps than the former,
neighboring group participation was not available in the
glycosylations of the reducing end fragments and the branch
fragments. Here, we employed the latter strategy. After
thoroughly exploring the reaction conditions, the key
glycosylations of the large fragments were achieved in good to
moderate yields in the presence of ether as a solvent. The -
selectivity was improved by designing and synthesizing the
mannosyl donors 3–7. Per-acylated mannosyl donor 7 gave
perfect -selectivity by permitting remote group participation.[21]
We also demonstrated the stepwise and simultaneous
introduction of branching fragments at the 3- and 6-positions of
mannose. The stepwise method enabled the syntheses of
asymmetric N-glycans, whereas the simultaneous method
reduced the number of reaction steps needed.
The synthesis of the asparagine-linked tetrasaccharide 2 was
20]
initiated from disaccharide 8,[14,
which was synthesized via
selective -mannosylation, as reported by our group (Scheme
1).-Selective mannosylation was achieved by activating 4,6-
benzyliden protected mannosyl donor at low temperature (-78
ºC). Precise temperature control was the key of this
glycosylation to achieve high -selectivity (/≈1/4). After
cleavage of the benzylidene group, an Fmoc group was
selectively introduced at the 6-position of mannose to afford 10.
Glycosylation of 10 with N-pheny-2,2,2-trifluoroacetimidate[24]
glucosaminyl donor 11 afforded trisaccharide 12 in 90% yield
with perfect -selectivity without formation of oxiazoline.[25]
Trisaccharide 12 was then converted to glycosyl donor 14 via
cleavage of the allyl group using an iridium complex, followed by
imidation with N-phenyl-2,2,2-trifluoroacetimidoyl chloride.[24]
23, 25]
Glycosylation between 14 and 15,[14,
synthesized by N-
glycosylation, afforded tetrasaccharide 16 in 68% yield. The
protecting group at the 6-position of mannose in 16 was
converted from Fmoc to TBS to give 2.
The other important feature of our synthesis involved the early-
stage introduction of asparagine.[14] An asparagine residue was
generally attached after deprotecting the glycan.[22] We achieved
N-glycosylation between the glucosaminyl donor and the
asparagine acceptor.[23] Here, The sugar chain was elongated
from glucosaminyl asparagine which is obtained by N-
glycosylation. Our method facilitated the preparation of an N-
glycan-processing asparagine residue because conversion was
Scheme 1. Synthesis of asparagine-linked tetrasaccharide 2.
Glycosyl donors 3 and 4 were synthesized to have different
leaving groups (Scheme 2). After cleavage of the Fmoc group of
18,[20] obtained 19 was glycosylated with glucosaminyl donor
20[20] to give disaccharide 21 in 96% yield.[25] Cleavage of the
Figure 1. Synthetic plan of bisecting GlcNAc-containing N-glycan 1.
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allyl group, followed by imidation or fluorination, afforded
glycosyl donors 3 or 4, respectively.
Table 1, entries 1–6). Although the reaction did not proceed in
CH2Cl2, THF, or EtCN (Scheme 3, Table 1, entries 1, 4, 5),
desired pentasaccharide 26 was obtained using Et2O, CPME or
toluene:dioxane = 10:1 (Scheme 3, Table 1, entries 2, 3, 4).
Et2O, in particular, gave higher yields compared to other
solvents. The ether solvent probably stabilized the
oxocarbenium ion intermediate generated from glycosyl donor 3
to give a better yield, as observed in previous studies of the
synthesis of a core fucose-containing N-glycan.[14] Although
ether solvent is generally expected to enhance -selectivity,
especially, in the case of mannosylation,[27] -selectivity was
poor in these glycosylations. The TBSOTf or TfOH activators in
Et2O gave lower yields compared to TMSOTf, although TfOH
provided a higher -selectivity (Scheme 3, Table 1, entries 2, 7,
8). The yields were improved dramatically at lower reaction
temperatures: a 90% yield was obtained using TMSOTf, a 66%
yield was obtained using TfOH (Scheme 3, Table 1, entries 2, 8,
9, 10). Next, glycosylation using glycosyl fluoride 4 was
investigated. As with the investigation using glycosyl imidate 3,
ether gave a good yield compared to the other solvents
(Scheme 3, Table 1, entries 11, 12). A variety of activators of
glycosyl fluoride were investigated; however, the yields
remained moderate to low (Scheme 3, Table 1, entries 12–17).
cheme 2. Syntheses of glycosyl donors 3 and 4.
Prior to investigating the glycosylations of tetrasaccharide
asparagine 2 with disaccharides 3 or 4, a model study using
trisaccharide 25 with 3 or 4 was carried out (Scheme 3, Table 1).
After TBS protection of 9, the resulting 23 was glycosylated with
11 to give 24. Cleavage of the 4-azido-3-chlorobenzyl (ClAzb)
group[26] in 24 via the Staudinger reaction, followed by DDQ
treatment, afforded 25. The glycosylations of 25 with 3 or 4 were
then investigated. The solvents were examined using imidate 3
as a glycosyl donor and TMSOTf as an activator (Scheme 3,
To summarize these investigations, glycosylation with
a
sterically hindered acceptor gave good yields in Et2O at low
temperatures (Scheme 3, Table 1, entry 9), although -
selectivity was not satisfactory. After cleavage of the TBS group
of 26, the desired -isomer was isolated to give pure 27.
Scheme 3. Model study of the glycosylations of 25 with 3 and 4.
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A model study of the second mannosylation was conducted
(Scheme 4). Glycosylation at the 6-position of mannose of 27
with glycosyl imidate 3 was carried out using TMSOTf as an
activator in Et2O to give desired octasaccharide 28 in 91% yield
with perfect -selectivity.
Scheme 4. Model study of glycosylation between 27 and 3.
In these model studies, a branching structure was constructed in
good yield; however, the stereoselectivity of glycosylation
between 25 and 3 was low. The low selectivity was considered
to be attributed to remote participation, as reported by Kim et
al.[21] (Figure 2). To improve the -selectivity of these key
mannosylations, we designed new glycosyl donors 5 and 6,
which did not include an acyl group at the 4-position, to avoid
Scheme 5. Syntheses of glycosyl donors and 6.
remote participation. Compound
5
enabled further sugar
mannosyl donors 3, 5, and 6 (Scheme 6). Cleavage of the ClAzb
group of 2 afforded 38 and the [4+2] glycosylations between 38
and 3, 5, or 6 were investigated. The use of 3, which was
expected to have undesired remote participation, provided
desired hexasaccharide 39 in 70% yield with low -selectivity
(/ = 1.5/1, Scheme 6, Table 2, entry 1). Glycosylations using
newly designed 5 or 6 showed moderate yields and improved -
selectivities (5: 38%,  only, 6: 53%, / = 3/1, Scheme 6, Table
2, entries 2, 3). After cleavage of the TBS group with
HF·pyridine, the [6+2] glycosylations of 42, 43, and 44 were
investigated. In these reactions, glycosyl donors 3 and 5 both
provided perfect -selectivity (42+3: 34%,  only, 43+5: 52%, 
only, Scheme 6, Table 3, entries 1, 2). On the other hand,
glycosylation using 6 did not give the desired octasaccharide 47
because the oxocarbenium ion generated from 6 decomposed
smoothly, even at low temperatures, due to its high reactivity
(Scheme 6, Table 3, entry 3). The employment of milder
conditions using TBSOTf provided 47 in 55% yield with a low -
selectivity (/ = 1/1, Scheme 6, Table 3, entry 4). In this case,
SN2-like reaction from the -isomer of glycosyl imidate 6
probably competed to afford a substantial amount of the
undesired -product.
The global deprotection of 47 was carried out (Scheme 7). Allyl
cleavage of 47 with palladium gave carboxylic acid 48.[28] After
removal of all acyl and Troc groups under basic conditions,[29]
acetyl groups were introduced to the amino groups.
Hydrogenation, followed by Fmoc protection of the amino group
of asparagine afforded desired bisecting GlcNAc-containing N-
glycan 1 in 53% yield in 4 steps. Fmoc group was introduced to
purify the deprotected octasaccharide by HPLC using an ODS
column.
elongation after cleavage of the Fmoc group, whereas
compound 6 was readily synthesized and required a simpler
deprotection process compared to 5.
Figure 2. The -selectivity of the key mannosylation was low, motivating the
design of new mannosyl donors 5 and 6.
Newly designed mannosyl donors 5 and 6 were synthesized
(Scheme 5). After PMB protection of 29,[20] reductive cleavage of
the benzylidene of 30 was followed by benzylation to afford 31.
The PMB group was cleaved to give 32 for further glycosylation.
Glycosylation between 32 and 20 gave the disaccharide 33 in
86% yield.[25] Cleavage of the allyl group of 33, followed by
imidation, afforded glycosyl imidate 5. Glycosyl imidate 6 was
synthesized in the same manner. After glycosylation between 32
and 11, the allyl group was converted to the imidate to give 6.
The two glycosylations at the 3- and 6-positions of the branching
mannose constructed a bisecting GlcNAc-containing N-glycan
skeletal structure. These glycosylations were investigated using
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Scheme 7. Global deprotection of bisecting GlcNAc-containing N-glycan 47.
Figure 3. Redesign of glycosyl donor 7.
Scheme 6. Investigations of the [4+2] glycosylations and [6+2] glycosylations.
Although we achieved the chemical synthesis of bisecting
GlcNAc-containing N-glycan 1, the /-selectivities of the key
[4+2] and [6+2] glycosylations were unsatisfactory. To achieve
-selective mannosylation, we redesigned new mannosyl donor
7 (Figure 3). The acyl groups introduced at the 3- and 6-
positions of 7 were expected to enhance -selectivity by remote
participation.[21]
Compound 7 was synthesized, as shown in Scheme 8. Selective
benzylation at the 3-position of 49[20] using Me2SnCl2 afforded
50.[30] Glycosylation between 50 and 11 gave disaccharide 51.
After converting the protecting groups from benzylidene to acetyl
groups, allyl deprotection of 52, followed by imidation of 53,
gave 7.
Scheme 8. Synthesis of glycosyl donor 7.
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Scheme 9. Investigation of simultaneous glycosylation between 55 and 6 or 7
In addition to utilizing newly designed mannosyl donor 7, we
investigated the simultaneous glycosylations at the 3- and 6-
positions of branching mannose to achieve the efficient
construction of a bisecting GlcNAc-containing N-glycan skeletal
structure (Scheme 9). After cleavage of the ClAzb and Fmoc
groups of 16, glycosylations between 55 and 6 or 7 were
investigated. When 6 was used as a glycosyl donor, relatively
harsh conditions gave good results (Scheme 9, Table 4, entries
1–3). Activation by TfOH at 0 ºC gave desired octasaccharide 47
in 62% yield (Scheme 9, Table 4, entry 3). In this case, the
undesired -isomer at the 6-position was also obtained (/ =
3/1). Activation of 7 with TfOH provided octasaccharide 56 in
52% yield with perfect -selectivity (Scheme 9, Table 4, entry 4).
realized by stabilizing the oxocarbenium ion intermediate
through coordination of the ether solvent. -Selective
mannosylations were achieved by manipulating the mannosyl
donor protection pattern to ensure remote participation. This
study provides a universal synthetic strategy for the efficient
synthesis of various types of N-glycans in an effort to accelerate
functional studies of synthetic N-glycans.
Experimental Section
The synthetic procedures and characterization of the compounds studied
herein can be found in the Supporting Information.
Activation of
7
by TMSOTf gave excellent results:
octasaccharide 56 was obtained in 78% yield with perfect -
selectivity (Scheme 9, Table 4, entry 5). As shown here, an -
selective mannosylation was achieved by manipulating the
protecting group of the glycosyl donor.
Acknowledgements
This work was financially supported in part by JSPS KAKENHI
Grant Number 15H05836 in Middle Molecular Strategy, JSPS
KAKENHI Grant Number 16H01885, JSPS KAKENHI Grant
Number 16H05924, JSPS KAKENHI Grant Number 17K19201,
Naito research grant, and Nikki Saneyoshi research grant.
Conclusions
In summary, the chemical synthesis of a bisecting GlcNAc-
containing N-glycan was achieved. We developed a convergent
synthetic route in which a branching structure was constructed
via two glycosylations at the 3- and 6-positions of a branching
mannose. This synthetic strategy reduced the number of
reaction steps. High yields of the key glycosylations were
Keywords: N-glycan • oligosaccharides • bisecting GlcNAc •
glycosylation
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10.1002/asia.201800367
Chemistry - An Asian Journal
FULL PAPER
FULL PAPER
Synthesis of a bisecting GlcNAc-
containing N-glycan: The convergent
Yoshiyuki Manabe, Hiroki Shomura,
Naoya Minamoto, Masahiro Nagasaki,
Yohei Takakura, Katsunori Tanaka, Alba
Silipo, Antonio Molinaro, Koichi Fukase*
synthesis of
a bisecting GlcNAc-
containing N-glycan was achieved via
two glycosylations at branching
positions. In these key glycosylations,
high yields were obtained by
stabilizing the oxocarbenium ion
intermediate in ether, and perfect -
Page No. – Page No.
Convergent synthesis of bisecting
GlcNAc containing N-glycan
selectivity
was
achieved
by
manipulating the protecting groups.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.