First chemical synthesis of triglucosylated tetradecasaccharide (Glc 3Man9GlcNAc2), a common precursor of asparagine-linked oligosaccharides

Article abstract of DOI:10.1016/j.tetlet.2005.04.056

Triglucosylated high-mannose-type tetradecasaccharide (Glc ₃Man₉GlcNAc₂), the oligosaccharide part of the donor substrate of oligosaccharyl transferase (OST) complex, and diglucosylated tridecasaccharide (Glc₂Ma

Full text of DOI:10.1016/j.tetlet.2005.04.056

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 4197–4200
First chemical synthesis of triglucosylated tetradecasaccharide
(Glc3Man9GlcNAc2), a common precursor of asparagine-linked
oligosaccharides
Ichiro Matsuo, Toshinori Kashiwagi, Kiichiro Totani and Yukishige Ito*
RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
CREST, JST Kawaguchi 322-1102, Japan
Received 7 March 2005; revised 12 April 2005; accepted 13 April 2005
Available online 29 April 2005
Abstract—Triglucosylated high-mannose-type tetradecasaccharide (Glc3Man9GlcNAc2), the oligosaccharide part of the donor sub-
strate of oligosaccharyl transferase (OST) complex, and diglucosylated tridecasaccharide (Glc2Man9GlcNAc2) were synthesized.
These oligosaccharides were assembled in a convergent and stereoselective manner. Undecasaccharide 5 was employed as the
common intermediate, and coupling with trisaccharide (4) and disaccharide (3) donor afforded fully protected tetradeca-(17) and
tridecasaccharide (16), respectively. These oligosaccharides were deprotected to give Glc3Man9GlcNAc2 and Glc2Man9GlcNAc2,
respectively.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Addition of asparagine (Asn)-linked oligosaccharide
(N-glycosylation) is an important and highly conserved
modification of secretory and membrane-bound eukaryo-
tic proteins.1 The key step along this pathway is the
transfer of tetradecasaccharide (Glc3Man9GlcNAc2),
which occurs in the lumen of the endoplasmic reticulum
(ER).2 This oligosaccharide is assembled on dolichyl
pyrophosphate (Glc3Man9GlcNAc2-pp-Dol, 1b) and
transferred en bloc to the Asn-X-Ser/Thr sequence of
nascent polypeptide chains (Fig. 1). This transformation
is catalyzed by oligosaccharyltransferase (OST), a hetero-
oligomeric membrane-bound protein complex.3 After
being transferred, protein-bound Glc3Man9GlcNAc2
(1a) is trimmed by glucosidase-I (to Glc2Man9GlcNAc2,
2a) and then by glucosidase-II (to Glc1Man9GlcNAc2
and Man9GlcNAc2).
Since Glc3Man9GlcNAc2-pp-Dol is the preferred sub-
strate of OST, the presence of glucotriose (Glca1!2-
Glca1!3Glca; Glc3) has been inferred to be important
for OST recognition.4 It has been suggested that this tri-
Figure 1. Transfer of tetradecasaccharide from dolichyl pyrophos-
saccharide part has a well-defined conformation and,
phate to protein.
possibly, functions as the recognition epitope.5 How-
ever, due to the poor availability of the full length
substrate such as Glc3Man9GlcNAc2-pp-Dol,6,7 most
in vitro studies have been carried out with truncated
derivatives.8 Although Glc3Man9GlcNAc2 is the key
Keywords: Oligosaccharyl transferase; Synthesis; N-Glycan.
*
Corresponding author. Tel.: +81 48 4467 9430; fax: +81 48 462
4680; e-mail: yukito@postman.riken.go.jp
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.04.056

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I. Matsuo et al. / Tetrahedron Letters 46 (2005) 4197–4200
intermediate in glycoprotein biosynthesis, its chemical
synthesis has yet to be achieved.
quence either required a large excess of glucosyl bromide
or resulted in incomplete selectivity.7 We planned to use
3,4,6-O-acylated glucosyl donors (11, 14/15) for stepwise
elongation. It was envisaged that these donors were suf-
ficiently deactivated to exhibit the desired a-selectivity11
and to minimize premature decomposition during acti-
vation. These components were prepared from 2-OH
derivative 7 and hemiacetal 8, which in turn were pre-
pared as described by Suzuki et al. from glucosyl bro-
mides 9a/b via cyclic acetals 10a/b.12 Hemiacetal 8 was
then converted to the fluoride 11 using diethylaminosul-
fur trifluoride (DAST) in 92% yield. Glycosylation of 7
with 11 (2 equiv) using Cp2HfCl2 and AgOTf13 in
CH2Cl2 afforded disaccharide 12 as a single isomer in
90% yield.14 Removal of the p-methoxybenzyl (PMB)
group using DDQ afforded hemiacetal 13 (73%) that
was converted to imidate 14 (quant.) and chloride 15
(quant.). When imidate 14 (1.3 equiv) was used as the
glycosyl donor15 to react with 6, under activation with
TMSOTf in toluene, trisaccharide 4 was obtained in
45% yield as a single stereoisomer.14 The same product
was obtained with chloride 15 (1.5 equiv) in slightly
higher yield, however, with reduced selectivity (57%,
a:b = 8.5:1). On the other hand, coupling of fluoride
11 (1.5 equiv) with 6 provided 63% yield of disaccharide
3 as a pure a-siomer [dH 5.73 (d, J 3.60 Hz); dC 95.56
(1JC–H 172 Hz, C-1)].14
As part of our effort toward comprehensive synthesis
of glycan chains9 related to glycoprotein processing,10
we wish to report herein the first chemical synthesis
of Glc3Man9GlcNAc2 (1c) as well as its glucosidase-I
product Glc2Man9GlcNAc2 (2b).
The target sugar chains were divided into di/trisaccharide
and undecasaccharide fragments as depicted in Scheme 1.
Because, we previously observed that, when thioglyco-
side was used as a donor, the use of 4,6-O-benzylidene
protected donor was important for a-selective glucosyl-
ation at 3-position of mannose,9a Glc2 and Glc3 donors
were designed as 3 and 4, respectively. Synthesis of the
undecasaccharide fragment 5 was described previously,9a
which can now be performed in multigram scale.
Preparation of 3 and 4 was conducted as shown in
Scheme 2. Previously reported synthesis of the Glc3 se-
Although yields of glycosylation steps toward 3 and 4
were modest, these di/triglucosyl fragments were ob-
tained with high stereochemical purity and can be used
directly for the next coupling without further manipul-
ation of the anomeric position.
With all oligosaccharide components in hand, fragment
condensation was undertaken as shown in Scheme 3.
Thus, tridecasaccharide skeleton was constructed using
3 (1.7 equiv) and 5, through thioglycoside activation
Scheme 1. Oligosaccharide fragments 3 and 4 for the synthesis of
Glc3Man9GlcNAc2 1 and Glc2Man9GlcNAc2 2.
Scheme 2. Reagents and conditions: (1) DAST, CH2Cl2, ꢀ40 ꢁC, 3 h,
92%; (2) Cp2HfCl2, AgOTf, MS 4A, CH2Cl2, ꢀ40 ꢁC, 7 h, 90%; (3)
DDQ, CH2Cl2/H2O, rt, 12 h, 73%; (4) CCl3CN, DBU, 0 ꢁC, 1 h,
quant.; (5) SOCl2, DMF (cat.), CH2Cl2, 0 ꢁC, 4 h, quant.; (6) 14,
TMSOTf, MS-AW300, toluene, ꢀ40 ꢁC, 2 h, 45%; (7) 15, AgOTf, di-
Scheme 3. Reagents and conditions: (1) MeOTf, MS 4A, DTBMP,
ClCH2CH2Cl/cyclohexane = 1:4, 50 ꢁC, 12 h, 85% (14), 57% (15;
a:b = 8.5:1); (2) ethylenediamine, n-BuOH, 90 ꢁC; (3) Ac2O, pyridine,
DMAP, rt; (4) NaOMe/MeOH, 40 ꢁC; (5) Pd(OH)2, H2, MeOH/
AcOH/H2O = 5:1:1, rt.
tert-butyl-4-methylpyridine
(DTBMP),
MS
4A,
toluene/
ClCH2CH2Cl = 2:1, ꢀ20 ꢁC–rt, 12 h, 51% (b-isomer 6%); (8)
Cp2HfCl2, AgOTf, MS 4A, ether/toluene = 2:1, ꢀ40 ꢁC, 7 h, 63%.

I. Matsuo et al. / Tetrahedron Letters 46 (2005) 4197–4200
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References and notes
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3402–3403; (b) Matsuo, I.; Ito, Y. Carbohydr. Res. 2003,
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Figure 2. 1H NMR spectra (400 MHz, D2O, 23 ꢁC, referenced to HOD
adjusted to 4.65 ppm) of tridecasaccharide 2b (A) and tetradecasac-
charide 1c (B). Anomeric signals derived from a-Glc are indicated by
arrows.
by MeOTf16 to afford 16 as a single isomer in 85% yield.
On the other hand, glycosylation of 5 with trisaccharide
donor 4 (1.6 equiv) gave 57% yield of tetradecasaccha-
ride 17, which, unfortunately, contaminated with a
detectable amount of the b-isomer (a/b = 19:1). Since
no chromatographic conditions were found to separate
these isomers in a preparative scale, the mixture was
carried on further. Complete deprotection of 16 was
performed as follows: (1) conversion of phthalimide
groups to acetamide, (2) Pd(OH)2 catalyzed debenzyl-
ation, and (3) deacetylation, to give tridecasaccharide
2b17 in 48% overall yield. The presence of 2 a-linked
Glc was confirmed by 1H NMR [dH 5.212 (d, J
3.66 Hz), 5.121 (d, J 3.66 Hz)]. Tetradecasaccharide 17
was deprotected in the same manner, and final purifica-
tion by HPLC using Fluorex column (conditions; 20/
· 250 mm, eluent; H2O, flow rate: 10 ml/min, detection
UV 214 nm) allowed the isolation of anomerically pure
1c [dH 5.381 (d, J 2.2 Hz), 5.122 (d, J = 3.17 Hz, 1H),
5.034 (d, J = 2.9 Hz, 1H)] in 56% yield.18 The 1H
NMR spectrum of 1c and 2b were in excellent agreement
with those reported for closely related compounds (Fig.
2).19
10. (a) Spiro, R. G. Cell. Mol. Life Sci. 2004, 61, 1025–1041;
(b) Helenius, A.; Aebi, M. Science 2001, 291, 2364–2369;
(c) Ellgaard, L.; Helenius, A. Nature Rev. Mol. Cell Biol.
2003, 4, 181–191.
11. Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155–
173; Mootoo, D. R.; Konradsson, P.; Udodong, U.;
Fraser-Reid, B. J. Am. Chem. Soc. 1988, 110, 5583–5584.
12. (a) Suzuki, K.; Mizuta, T.; Yamaura, M. J. Carbohydr.
Chem. 2003, 22, 143–147; (b) Suzuki, K.; Nonaka, H.;
Yamaura, M. Tetrahedron Lett. 2003, 44, 1975–1977.
13. Suzuki, K.; Maeta, H.; Matsumoto, T. Tetrahedron Lett.
1989, 30, 4853–4854.
1
14. 12: H NMR (400 MHz, CDCl3) d 6.03 (t, J 9.6 Hz, 1H),
5.82 (t, J 9.6 Hz, 1H), 5.41 (t, J 9.6 Hz, 1H,), 5.29 (t, J
9.6 Hz, 1H), 5.14 (d, J 3.6 Hz, 1H, H-1a-Glc), 4.93 (d, J
3.2 Hz, 1H, H-1a-Glc); 13C NMR (CDCl3) d 96.91 (1JC–H
168 Hz, C-1a-Glc), 94.98 (1JC–H 170 Hz, C-1a-Glc); MS
(MALDI-TOF) C72H66O21Na: Calcd 1289.1. Found:
In conclusion, convergent and stereoselective synthetic
routes to Glc3Man9GlcNAc2 (1c) and Glc2Man9Glc-
NAc2 (2b) were established. These oligosaccharides will
be valuable standards to reveal protein–oligosaccharide
interactions involved in glycoprotein biosynthesis. Fu-
ture studies are going to be directed to the development
of practical methodology to convert them to oligosac-
charide–dolichyl pyrophosphate conjugates.
1
1290.3 (M+Na)+. 4: H NMR (400 MHz, CDCl3) d 8.00–
6.63 (m, Ar), 6.14 (t, J 10 Hz, 1H) 5.86 (t, J 10 Hz, 1H), 5.82
(d, J 3.6 Hz, 1H, H-1a-Glc), 5.45, (t, J 10 Hz, 1H), 5.40 (s,
1H, PhCH), 5.27, (t, J 10 Hz, 1H), 4.93 (d, J 3.2 Hz, 1H,
H-1a-Glc), 3.83, 3.81, 3.50 (s · 3, OMe), 2.28 (s, 3H, SMe);
13C NMR (100 MHz, CDCl3) d 95.95 (1JC–H 173 Hz,
C-1a-Glc), 95.03 (1JC–H 175 Hz, C-1a-Glc), 86.05 (1JC–H 154 Hz,
C-1b-Glc); MS (MALDI-TOF) C85H80O24Na: Calcd 1540.5.
Found: 1540.2 (M+Na)+. 3: 1H NMR (400 MHz, CDCl3) d
8.02–6.69 (m, Ar), 6.00 (t, J 9.6 Hz, 1H), 5.73 (d, J 3.60 Hz,
1H, H-1a-Glc), 5.47 (s, 1H, PhCH), 5.41 (t, J 9.9 Hz, 1H),
4.51 (d, J 9.6 Hz, 1H, H-1b-Glc), 2.29 (s, 3H, SMe); 13C
NMR (CDCl3) d 102.21, 95.56 (1JC–H 172 Hz, C-1a-Glc),
86.09 (1JC–H 158 Hz, C-1b-Glc); MS (MALDI-TOF)
C55H52O13NaS: Calcd 975.3. Found: 975.7 (M+Na)+.
15. Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem.
Biochem. 1994, 50, 21–123.
Acknowledgements
We thank Ms. Akemi Takahashi for technical assis-
tance. Financial support from Ministry of Education,
Culture, Sports, Science and Technology [Grant-in-Aid
for Scientific Research (B) No. 16390011] is
acknowledged.

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I. Matsuo et al. / Tetrahedron Letters 46 (2005) 4197–4200
16. Lo¨nn, H. J. Carbohydr. Chem. 1987, 6, 301–306.
17. 2b: 1H NMR (400 MHz, D2O) d 5.256 (s, 1H, H-1Man),
5.212 (d, J 3.66 Hz, 1H, H-1Glc), 5.186 (s, 1H, H-1Man),
5.159 (s, 1H, H-1Man), 5.121 (d, J 3.66 Hz, 1H, H-1Glc),
4.994 (s, 1H, H-1Man), 4.906 (s, 1H, H-1Man), 4.888 (s, 2H,
H-1Man), 4.439 (d, J 7.0 Hz, 1H, H-1GlcNAc), 4.350 (d, J
7.0 Hz, 1H, H-1GlcNAc), 1.917 (s, 3H, Ac), 1.876 (s, 3H,
Ac), 1.391 (m, 2H), 0.709 (t, 3H); MS (MALDI-
TOF) C91H154N2O71Na: Calcd 2433.7. Found: 2432.8
(M+Na)+.
(s, 1H, H-1Man), 5.122 (d, J 3.2 Hz, 1H, H-1Glc), 5.034 (d,
J 2.9 Hz, 1H, H-1Glc), 4.992 (s, 1H, H-1Man), 4.904 (s, 1H,
H-1Man), 4.886 (s, 2H, H-1Man), 4.436 (d, J 7.3 Hz, 1H,
H-1GlcNAc), 4.350 (d, J 7.3 Hz, 1H, H-1GlcNAc), 1.919 (s,
3H, Ac), 1.878 (s, 3H, Ac), 1.394 (m, 2H), 0.711 (t,
7.07 Hz, 3H); MS (MALDI-TOF) C85H144N2O66Na:
Calcd 2272.9. Found: 2273.6 (M+Na)+.
19. (a) Wormald, M. R.; Wooten, E. W.; Bazzo, R.; Edge, C.
J.; Feinstein, A.; Rademacher, T. W.; Dwek, R. A. Eur.
J. Biochem. 1991, 198, 131–139; (b) Woods, R. J.;
Pathiaseril, A.; Wormald, M. R.; Edge, C. J.; Dwek, R.
A. Eur. J. Biochem. 1998, 258, 372–386.
1
18. 1c: H NMR (400 MHz, D2O) d 5.381 (d, J 2.2 Hz, 1H,
H-1Glc), 5.254 (s, 1H, H-1Man) 5.183 (s, 1H, H-1Man), 5.156