Synthesis of monoglucosylated high-mannose-type dodecasaccharide, a putative ligand for molecular chaperone, calnexin, and calreticurin

Article abstract of DOI:10.1021/ja021288q

Convergent and stereoselective synthetic routes to Man₉GlcNAc₂ (1b), α-Glc₁M₉GlcNAc₂ (2b), and its stereoisomer β-Glc₁M₉GlcNAc₂ (3) were established. Interaction analy

Full text of DOI:10.1021/ja021288q

Published on Web 02/28/2003
Synthesis of Monoglucosylated High-Mannose-Type Dodecasaccharide, a
Putative Ligand for Molecular Chaperone, Calnexin, and Calreticurin
Ichiro Matsuo,† Megumi Wada,† Shino Manabe,† Yoshiki Yamaguchi,‡ Keisuke Otake,‡
Koichi Kato,‡ and Yukishige Ito*,†
RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan,
Graduate School of Pharmaceutical Sciences, Nagoya City UniVersity, Mizuho-ku, Nagoya 467-8603, Japan, and
CREST, JST Kawagucghi 332-1102, Japan
Received October 21, 2002 ; E-mail: yukito@postman.riken.go.jp
Addition of the asparagine (Asn)-linked (N-linked) glycan chain
is a prominent posttranslational modification of proteins.1 Because
of the discoveries of critical roles of high-mannose-type oligo-
saccharides in various aspects of protein quality control, their
biological significance is attracting renewed attention.2 Particularly
important in this respect is the calnexin/calreticulin-glucosyl-
transferase cycle. Calnexin (CNX) and carleticulin (CRT) are
homologous chaperones that reside in the lumen of endoplasmic
reticulum (ER). Recent investigations have clarified that the
interaction of glycoprotein with CNX/CRT is primarily mediated
by the terminally monoglucosylated high-mannose-type glycan
chain, most probably Glc1Man9GlcNAc2 (2a).3,4 On the other hand,
UDP-glucose:glycoprotein glucosyltransferase (UGGT) is believed
to function as a “folding sensor”, which detects incompletely folded
glycoproteins carrying Man9GlcNAc2 (1a) and reglucosylates them
back to 2a.5 However, the precise role of the glycan chain in
chaperone recognition is still controversial.6 We report herein the
first chemical syntheses of the putative ligand of CNX/CRT,
Glc1Man9GlcNAc2 (2b) (Figure 1). Additionally, the stereoisomeric
dodecasaccharide carrying â-linked Glc (3) was synthesized.
Furthermore, the first NMR-based evidence for the specific binding
of CRT to R-Glc1Man9GlcNAc2 was obtained.
Figure 1. Structures of high-mannose-type glycans.
Scheme 1. Preparation of Oligosaccharide Blocksa
Undecasaccharides 2b and 3 were retrosynthetically disconnected
to fragments 6, 7, and 8 (Figure 1). For the construction of
â-mannoside containing fragment 6, p-methoxybenzyl assisted
intramolecular aglycon delivery7 was used as the key reaction
(Scheme 1). Thus, â-mannosylation of glucosamine derivative 108
using 9 as a donor7a was performed by way of the mixed acetal 11,
according to the standard protocol to afford disaccharide 12a in
82% yield as a single stereoisomer. Corresponding acetate 12b was
then used as the glycosyl donor and coupled with 139 to give 14
which was converted to diol 6. Tri- and pentamannoside fragments
7 and 8 were synthesized from 1510 and 19,11 respectively (Scheme
1).
With all fragments in hand, the undecasaccharide skeleton was
assembled as depicted in Scheme 2. Coupling of 6 with 7 was
achieved by the action of MeOTf12 to afford 3-O-glycosylated
hexasaccharide 22a, together with its regioisomer in 77 and 13%
yield, respectively. Subsequent acetylation and removal of the
cyclohexylidene group afforded diol 22c, which in turn was reacted
with pentasaccharide donor 8 to give undecasaccharide 23a in 87%
yield which was converted to acetate 23b. Removal of the TBDPS
group was performed under high-pressure conditions as described
before13 to give 23c in 86% yield. For the incorporation of R-linked
glucose residue, thioglucoside 4 was proven to be highly satisfactory
as the glycosyl donor and provided desired dodecasaccharide 24
a Reagents and yields: (1) DDQ; (2) MeOTf, DTBMP, ClCH2CH2Cl,
83% (two steps); (3) Ac2O, pyridine, DMAP, 96%; (4) Cp2HfCl2, AgOTf,
CH2Cl2, 85%; (5) TBAF/AcOH, DMF, 82%; (6) NaOMe/MeOH, 75%; (7)
AgOTf, CH2Cl2, 81%; (8) NaOMe/MeOH, quant.; (9) Cp2HfCl2, AgOTf,
CH2Cl2, 91%; (10) AgOTf, CH2Cl2, 72%; (11) NBS, DAST, 85%.
as a single isomer in 85% yield.14 The stereoisomeric dodeca-
saccharide carrying â-linked glucose residue 25 was synthesized
using glycosyl donor 5 in 81% yield. Finally, complete deprotection
† RIKEN and CREST.
‡ Nagoya City University and CREST.
9
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J. AM. CHEM. SOC. 2003, 125, 3402-3403
10.1021/ja021288q CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Convergent Synthesis of Undecasaccharide and
Introduction of Glucose Residuea
Figure 2. (A) Anomeric region of 1D 1H NMR spectra acquired of
R-GlcMan9GN2 (2b) in 2H2O, 10 mM Tris-HCl buffer, 10 mM CaCl2 at
pH 7.3. CRT was added to the sample stepwise; the final ratio of 2b and
CRT was 1:1. (B) Anomeric region of 1D 1H NMR spectra acquired of 2b
+ 3 (1:1) without (a) and with (b) 0.5 equiv of CRT.
assistance. Financial support from the Mizutani Foundation for
Glycoscience and Ministry of Education, Culture, Sports, Science
and Technology [Grant-in-Aid for Scientific Research (B) No.
13480191] is acknowledged.
1
Supporting Information Available: Preparative methods and H
and 13C NMR spectra of new compounds and procedure for the
expression of CRT (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
a Reagents and yields: (1) MeOTf, 77%; (2) Ac2O, pyridine, DMAP;
(3) p-TosOH, 56% (two steps); (4) Cp2HfCl2, AgOTf, toluene, 87%; (5)
Ac2O, pyridine, DMAP, 98%; (6) 10% HF/pyridine, DMF, 1 GPa, 86%;
(7) MeOTf, ClCH2CH2Cl/cyclohexane, 85%; (8) MeOTf, ClH2CH2Cl, 81%;
(9) ethylenediamine, n-BuOH; (10) Ac2O, pyridine, DMAP; (11) Pd(OH)2,
H2, 80% AcOH; (12) NaOMe/MeOH.
References
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Nichita, N.; Negroiu, G.; Petrescu, A. J.; Dwek, R. A. Biochemistry 2000,
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of 23c, 24, and 25 afforded Man9GlcNAc2 (1b),15 Glc1Man9GlcNAc2
(2b), and â-Glc1Man9GlcNAc2 (3), respectively (Scheme 2).
Synthetic dodecasaccharide 2b was subjected to 1H NMR
measurements to observe the specific interaction with CRT. In the
presence of recombinant CRT,16 peak heights of all anomeric signals
decreased in proportion with the amount of CRT, suggesting that
2b binds tightly with CRT under these conditions (Figure 2A).17
These peaks eventually disappeared after the addition of ∼1 equiv
of CRT. That the CRT-ligand interaction is specific to the fine
structure of the oligosaccharide was supported by a similar
measurement using a 1:1 mixture of 2b and its stereoisomer 3. In
the presence of 0.5 equiv (with respect to the mixture of 2b and 3)
of CRT, peaks derived from R-linked 2b were strongly suppressed,
while those from 3 were barely affected under these conditions
(Figure 2B).
(4) (a) Leach, M. R.; Cohen-Doyle, M. F.; Thomas, D. Y.; Williams, D. B.
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(14) Reaction using tetra-O-benzyl thioglycoside under similar conditions
afforded a mixture of R- and â-isomers (R:â ) 3:1).
In conclusion, convergent and stereoselective synthetic routes
to Man9GlcNAc2 (1b), R-Glc1M9GlcNAc2 (2b), and its stereoisomer
(3) were established. Using 1H NMR, we observed the interaction
of 2b with CRT. A more systematic study is in progress and will
be reported in due course.
(15) This compound was demonstrated to bind with F-box protein Fbx2, a
constituent of ubiquitin ligase: Yoshida, Y.; Chiba, T.; Tokunaga, F.;
Kawasaki, H.; Iwai, K.; Suzuki, T.; Ito, Y.; Matsuoka, K.; Yoshida, M.;
Tanaka, K.; Tai, T. Nature 2002, 418, 438-442.
(16) For the expression of CRT, see Supporting Information.
(17) Siriwardena, A. H.; Tian, F.; Noble, S.; Prestegard, J. H. Angew. Chem.,
Int. Ed. 2002, 41, 3454-3457.
Acknowledgment. We thank Dr. Teiji Chihara for the operation
of the high-pressure equipment, Prof. Shunji Natori for providing
the GST-CRT construct, and Ms. Akemi Takahashi for technical
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