Synthesis of an octamannosyled glycan chain, the key oligosaccharide structure in ER-associated degradation

Article abstract of DOI:10.1016/S0008-6215(03)00357-4

The high-mannose type decasaccharide (Man₈GlcNAc₂), the proposed ligand of ER residing mannosidase-like proteins (MLP), and its monoglycosylated homologue (α-Glc₁Man₈GlcNAc ₂) were synthesized. The oligosaccharide assembly was performed in a convergent and stereoselective manner, using three oligosaccharide components, a core trisaccharide having a β-mannoside bond, a liner mannotriose, and a branched mannotetraose.

Full text of DOI:10.1016/S0008-6215(03)00357-4

Carbohydrate Research 338 (2003) 2163Á2168
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Rapid Communication
Synthesis of an octamannosyled glycan chain, the key oligosaccharide
structure in ER-associated degradation
Ichiro Matsuo, Yukishige Ito*
RIKEN, The Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 Japan and CREST, JST, Kawaguchi 322-1102,
Japan
Received 29 May 2003; accepted 17 July 2003
Abstract
The high-mannose type decasaccharide (Man8GlcNAc2), the proposed ligand of ER residing mannosidase-like proteins (MLP),
and its monoglycosylated homologue (a-Glc1Man8GlcNAc2) were synthesized. The oligosaccharide assembly was performed in a
convergent and stereoselective manner, using three oligosaccharide components, a core trisaccharide having a b-mannoside bond, a
liner mannotriose, and a branched mannotetraose.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Synthesis; Octamannosyled glycan chain; Oligosaccharide structures
Oligosaccharide parts of glycoproteins play critical roles
in numerous biological events.1 In particular, important
roles of asparagine (Asn)-linked oligosaccharides in
glycoprotein quality control have attracted recent atten-
tion (Fig. 1). The central role of molecular chaperones,
calnexin (CNX) and calreticulin (CRT), has been well-
documented.2,3
proteins (MLPs), EDEM discovered from mammalian
cells6 and its yeast counterpart Mn11p/Htm1p,7 are
considered to have a lectin property and recognize
glycoproteins having M8 oligosaccharides (Man8-
GlcNAc2, B-isomer, 1a), which are formed by the action
of ER mannosidase-I from M9.8 Glycoproteins directed
to ERAD are dislocated to the cytosol,9 ubiquitinated,10
deglycosylated11 and degraded by the proteasome.12
Although the proposed role of MLPs seems reason-
able in a teleological sense, concrete evidence of their
M8 specific lectin property has been lacking. In addi-
tion, it is not clear if monoglucosylated M8 (G1M8, 2a)
is involved in the CNX/CRT cycle and/or MLP-
mediated ERAD. In order to gain a clear understanding
of the molecular mechanism of glycoprotein quality
control, access to the structurally defined oligosacchar-
ide is of critical importance. Recently, we achieved the
first chemical synthesis of monoglucosylated dodecasac-
charide (G1M9), which is a putative ligand of CNX/
CRT.13 As part of our program on the systematic
synthesis of high-mannose type oligosaccharides related
to protein quality control, we report herein the syntheses
of the proposed ligand of MLPs, Man8GlcNAc2 (M8,
1b) and its monoglucosylated homologue Glc1Man8Glc-
NAc2 (G1M8, 2b).
These compounds share a unique lectin property and
recognize
monoglucosylated
dodecasaccharide
(Glc1Man9GlcNAc2, G1M9) as a primary ligand, which
is formed by the stepwise removal of two Glc residues
from the tetradecasaccharide (Glc3Man9GlcNAc2,
G3M9) that is introduced to nascent polypeptides as a
common precursor of all Asn-linked glycans. After
removal of the innermost glucose by glucosidase II,
correctly folded proteins are transported to the Golgi
apparatus for further processing, while misfolded ones
are reglucosylated back to G1M9 by the action of UDP-
glucose:glycoprotein glucosyltransferase (UGGT).4 On
the other hand, ‘hopelessly misfolded’ glycoproteins are
delivered to the degradation pathway, the so-called ER-
associated degradation (ERAD).5 Mannosidase-like
* Corresponding author. Tel.: ꢀ
48-4624680.
E-mail address: yukito@postman.riken.go.jp (Y. Ito).
/
81-48-4679430; fax: ꢀ81-
/
For the convergent synthesis of target oligosacchar-
ides, fragments corresponding to Man1GlcNAc2 (4),
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00357-4

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I. Matsuo, Y. Ito / Carbohydrate Research 338 (2003) 2163Á2168
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Fig. 1. Glycoprotein processing and quality control in ER.
Man4 (5) and Man3 (6) were designed (Fig. 2). Fragment
4 that contains a b-mannoside linkage was to be
constructed by p-methoxybenzyl (PMB) assisted intra-
molecular aglycon delivery (IAD)14 as the key reaction,
while one-pot sequential glycosylation strategy was
adopted for the preparation of the tetrasaccharide
fragment 5 (vide infra). Preparation of trimannoside 6
was reported previously.13
Fig. 2. Synthetic plan for high-mannose type undecasaccharide.

I. Matsuo, Y. Ito / Carbohydrate Research 338 (2003) 2163Á
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Scheme 1. Reagents and conditions: (a) Cp2HfCl2, AgOTf, 4A MS, CH2Cl2, ꢁ
(two steps). (c) DDQ, 4A MS, CH2Cl2, rt, 2 h. (d) MeOTf, DTBMP, 4A MS, ClCH2CH2Cl, 45 8C, 12 h, 74% (two steps). (e) Ac2O,
pyridine, DMAP, rt, 12 h, quant. (f) HFÁpyridine, DMF, 1 GPa, rt, 12 h, 88%.
/
45 8C, 2 h. (b) H2O2, LiOH, THF, 0 8C, 2 h, 64%
/
As a precursor of fragment 4, previously reported
12b13 was prepared in a slightly modified manner as
shown in Scheme 1. The coupling of compounds 715 and
816 was performed by using Suzuki’s protocol17 to give
9a that was deacetylated under controlled conditions
uneventfully under standard conditions to give diol
13d in 77% yield (three steps). One-pot sequential
glycosylation was performed with chemoselective acti-
vation22 of thioglycoside 14 and 16. The thioglycosides
14 and 16 were synthesized from octaacetylmanno-
biose20 and pentaacetylmannose by treatment with
thiophenol and TMSOTf, respectively. The first glyco-
sylation with 1.1 equiv of mannobiose donor 14 was
initiated in the presence of NIS and 1 equiv of TfOH in
(H2O2, LiOHÁ
THF).18 Resultant 9b, obtained in 64%
/
yield (two steps), was then subjected to b-mannosylation
using 2-O-PMB-equipped thioglycoside 10 as a donor.14
The reaction was performed according to our standard
two-step procedure; DDQ-mediated formation of mixed
acetal 11, and subsequent IAD promoted by MeOTf
afforded trisaccharide 12a in 74% yield as a single
stereoisomer. Stereochemistry of the mannoside linkage
was confirmed to be b by 13C NMR spectroscopy (C-
dichloromethane at ꢁ40 8C. After 1 h, TLC and TOF-
/
MS analyses revealed the predominant formation of
monoglycosylated product (presumably C-6 glycosyl-
ated trisaccharide 15). Subsequent addition of the
monosaccharide donor 16 to the reaction mixture
provided the desired tetrasaccharide fragment 5 in 48%
yield (pentasaccharide derivative was obtained in 21%
yield). The stereochemistries of the newly formed a-
mannoside linkages were confirmed by their 1JCH values
(174 and 176 Hz).
With all fragments in hand, the decasaccharide
skeleton was constructed as shown in Scheme 3.
Coupling of 4 with 6 was achieved by the action of
MeOTf23 to afford 3-O-glycosylated hexasaccharide
17a, which was isolated as a single isomer in 76% yield.
Removal of the cyclohexylidene group afforded diol
17b, and regioselective glycosylation of 6-OH of the b-
mannoside residue with the tetrasaccharide donor 5 gave
decasaccharide 18a that was converted to acetate 18b.
Removal of the TBDPS group was performed under
high-pressure conditions as described before19 to give
18c in 70% yield. For the incorporation of the a-linked
glucose residue, glycosylation with the thioglucoside 3
proceeded satisfactorily as described before13 and pro-
1
1Man: 99.7 ppm, JCH 156 Hz). The product 12a was
converted into corresponding acetate 12b in quantitative
yield. Subsequent desilylation was achieved cleanly
under high pressure (1 GPa) with HFÁpyridine to give
/
trisaccharide acceptor 4 in 88% yield.19 By contrast, this
particular transformation under standard conditions
was problematic. For instance, treatment with TBAFÁ
/
AcOH resulted in an extensive migration of the acetyl
group to afford a mixture of 2-O-Ac and 3-O-Ac
products, while treatment with HFÁpyridine under
/
atmospheric pressure resulted in the complete recovery
of 12b.
Synthesis of tetramannoside fragment 5 was carried
out as depicted in Scheme 2. It was started with the
preparation the glycosyl fluoride 13d having two hy-
droxy groups at the C-3 and C-6 positions. The
thiophenyl glycoside 13a20 was acetylated to give 13b
that was converted to the fluoride 13c using NBSÁ
/
DAST.21 Removal of the acetyl groups proceeded

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I. Matsuo, Y. Ito / Carbohydrate Research 338 (2003) 2163Á2168
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Scheme 2. Reagents and conditions: (a) Ac2O, pyridine, rt, 7 h. (b) NBS, DAST, CH2Cl2, ꢁ
/
10 8C, 12 h. (c) NaOMe, MeOH, 0 8C, 4
h, 77% (three steps). (d) NIS, TfOH, 4A MS, CH2Cl2, ꢁ40 8C, 1 h, then 16, ꢁ40 8C, 0.5 h, 48% (ꢀ
/
/
/
21% pentasaccharide).
vided the desired undecasaccharide 19 as a single isomer
in 74% yield. Finally, complete deprotection of 18c and
19 afforded Man8GlcNAc2 (1b) and Glc1Man8GlcNAc2
as molecular probes to analyze glycoprotein-MLP
recognition.
1
(2b), respectively. The H NMR spectrum of 1b and 2b
were in good agreement with the ones reported for
closely related compounds (Fig. 3).24
Acknowledgements
In conclusion, convergent and stereoselective syn-
thetic routes to Man8GlcNAc2 (1b), and a-
Glc1M8GlcNAc2 (2b) were established. By employing
high-pressure assisted desilylation and one-pot glycosyl-
ation, the target sugar chains were synthesized in a
concise manner. These oligosaccharides could be useful
We thank Megumi Wada for her contribution at the
exploratory stage of this work, Dr Teiji Chihara and his
staff for elemental analyses and high-pressure equip-
ment, and Akemi Takahashi for her technical assistance.
This work was partially supported by Japan Society for
the Promotion of Science [Grant-in-Aid for Scientific
Scheme 3. Reagents and conditions: (a) MeOTf, 4A MS, ClCH2CH2Cl, 40 8C, 12 h, 76%. (b) p-TosOH×
Cp2HfCl2, AgOTf, 4A MS, toluene, ꢁ40 to ꢁ20 8C, 10 h, 72%. (d) Ac2O, pyridine, DMAP, rt, 72 h, 82%. (e) 10% HFÁ
DMF, 1 GPa, rt, 18 h, 70%. (f) MeOTf, DT BMP, ClCH2CH2ClÁcyclohexane, 4A MS, 30 8C, 60 h, 74%. (g) ethylenediamine, n-
BuOH, 70 8C, 48 h. (h) Ac2O, pyridine, DMAP. (i) Pd(OH)2, H2, 80% AcOH, rt, 4 days. (j) NaOMeÁMeOH, 40 8C, 12 h (1b: 87%
from 18c, 2b: 58% from 19).
/
H2O, MeCN, 56%. (c)
/
/
/pyridine,
/
/

I. Matsuo, Y. Ito / Carbohydrate Research 338 (2003) 2163Á
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2167
Fig. 3. 1H NMR spectra (400 MHz, D2O, 23 8C, referenced to HOD adjusted to 4.65 ppm) of undecasaccharide 2b (A) and
decasaccharide 1b (B).
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