A short synthesis of the trisaccharide building block of the N-linked glycans

Article abstract of DOI:10.1016/S0040-4039(03)00102-3

An efficient preparation of the core trisaccharide of N-linked glycoproteins containing β-azido functionality at the reducing terminus is described. In the synthesis, triflate-mediated direct β-mannosylation was employed for the formation of the β-D-Man-(1→4)-GlcNAc linkage; the anomeric azide installation was achieved through oxazoline ring opening.

Full text of DOI:10.1016/S0040-4039(03)00102-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1787–1789
A short synthesis of the trisaccharide building block of the
N-linked glycans
Vadim Y. Dudkin* and David Crich*
Department of Chemistry, University of Illinois at Chicago, 845 West Taylor St., Chicago, IL 60607, USA
Received 12 December 2002; revised 9 January 2003; accepted 10 January 2003
Abstract—An efficient preparation of the core trisaccharide of N-linked glycoproteins containing b-azido functionality at the
reducing terminus is described. In the synthesis, triflate-mediated direct b-mannosylation was employed for the formation of the
b-D-Man-(1“4)-GlcNAc linkage; the anomeric azide installation was achieved through oxazoline ring opening. © 2003 Elsevier
Science Ltd. All rights reserved.
In natural N-linked glycoproteins, the side chain of Asn
in a segment of the peptide containing the Asn-Xxx-
Ser(Thr) unit is glycosylated with a highly conserved
saccharide core. All types of N-linked glycans include a
trisaccharide comprising b-mannosylated chitobiose.1
The mannose can be further di- or tri-glycosylated with
various carbohydrate chains. The efficient assembly of
this core structure opens the way for the development
of practical synthesis of N-linked glycoproteins.2
exposable 3-, 4-, and 6-OH groups of the mannose
residue, whereas the anomeric azido functionality
would serve as a point of the peptide attachment either
through the Staudinger-type ligation9,10 or through
reduction to the corresponding amine.8 Azides have
been demonstrated to be convenient precursors in the
preparation of both natural and unnatural glycan/pep-
tide linkages, and, recently, in direct cell-surface
modification.11,12
Since the first synthesis of an N-linked glycan fragment
was disclosed by Jeanloz in 1976,3 there have been
many attempts targeting efficient preparations of these
structures.4–8 The ever increasing need for practically
useful quantities of glycopeptide samples comprising
well-defined single glycoforms demands simplified pro-
cedures for their chemical synthesis.2 Such methodology
would ultimately include a number of common building
blocks together with unambiguous coupling methods,
which have potential for automation. The appropriately
protected trisaccharide core containing a convenient
handle for peptide conjugation would undoubtedly be
useful as one of these building blocks. The design of
such compounds (Fig. 1) would include orthogonally
Despite the significant amount of research devoted to
the synthesis of core structures, the problem of the
formation of the more difficult b- -Man(1“4)GlcNAc
D
linkage which is central to any N-linked glycans, still
lacks a direct and universally accepted approach.1
Novel glycosylations based on substitution of anomeric
triflates, developed in this laboratory, proved to give
excellent results in the formation of some of the most
challenging b-mannosides.13–15 In a recent study in this
group, a number of GlcNAc surrogates were tested as
acceptors in the synthesis of the b- -Man(1“
D
4)GlcNAc bond: 2-azido-2-deoxyglucose derivatives,
providing both good yields and complete anomeric
selectivity, were found to be optimal.16 A readily avail-
able alcohol 517 was therefore the acceptor of choice in
the present synthesis. 4,6-Benzylidene protection of the
donor molecule provides torsional rigidity to the
pyranose ring necessary for high glycosylation stereose-
lectivity and is readily removed under acidic conditions.
A silyl group would be the optimal orthogonal protec-
tion for the mannose 3-OH; however, these adversely
affect the stereochemical outcome of the coupling reac-
tion.18 Consequently, a p-methoxybenzyl ether serves as
a less sterically demanding temporary substitute for the
OTBS group (Scheme 1). Various methods for the
Figure 1.
* Corresponding authors.
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(03)00102-3

1788
V. Y. Dudkin, D. Crich / Tetrahedron Letters 44 (2003) 1787–1789
Scheme 1.
tion of the a-trichloroacetimidate were needed for the
coupling. In the event, treatment of the reagent mixture
with BF3·OEt2 in toluene afforded the desired b-glu-
coside in 76% yield. Attempts at utilizing the ‘nitrile
effect’ to achieve stereochemical control failed in this
case, as the reaction was poorly reproducible and typi-
cally afforded only 1/5 mixture of a/b anomers. Follow-
ing the glycosylation, the two azides in the trisaccharide
10 were converted into acetamides through a conve-
nient (if not particularly pleasant) one-step reaction
with potassium thioacetate/thioacetic acid.24
generation of the triflate 4 were investigated. Among
these, the benzenesulfinylpiperidine (BSP)/triflic anhy-
dride combination19 was found to be the most practical
method to activate the thiomannoside donor 2,20
despite providing the desired disaccharide 6 in slightly
lower yield than the AgOTf/PhSCl couple21 (64% ver-
sus 74%). The latter procedure suffers from low stabil-
ity of starting materials as well as difficulty in product
isolation, which detract from its applicability in large-
scale syntheses.22
Sulfoxide 320 was also used in the preparation of triflate
4 but was somewhat inferior to the above methods in
terms of both yield and product purification.23 Com-
plete stereocontrol was achieved with all three activa-
tion protocols. After glycosylation, the PMB protection
was easily exchanged for TBS in 94% overall yield.
Introduction of the anomeric azido group into the
chitobiose unit is usually achieved by multi-step
sequences such as developed in the Danishefsky labora-
tory.25 We envisioned a much shorter route capitalizing
on the recent discovery of an efficient oxazoline ring
opening with a combination of trimethylsilyl azide and
tetrabutylammonium fluoride.26 Toward this end, the
terminal n-pentenyl group in 11 was activated with
NIS/TESOTf in anhydrous dichloromethane to afford
oxazoline 12 which was converted into azide 13 imme-
diately following purification. The mannose 3-OH was
simultaneously liberated in this step. Compound 13
To complete the chitobiose moiety at the reducing
terminus, the disaccharide 7 was converted into the
anomeric a-trichloroacetimidate 9 and then coupled
with a second aliquot of acceptor 5 (Scheme 2). Since
the azido substituent in the donor is incapable of aiding
trans-glycosylation, conditions favoring SN2 substitu-
Scheme 2. Reagents and conditions: a: NIS, MeCN/H2O; b: CCl3CN, DBU, CH2Cl2; c: BF3·OEt2, toluene; d: AcSK/AcSH, DMF;
e: NIS, TESOTf, CH2Cl2; f: TMSN3/TBAF, THF; g: 80% aq. AcOH.

V. Y. Dudkin, D. Crich / Tetrahedron Letters 44 (2003) 1787–1789
1789
itself can be viewed as a valuable building block in the
synthesis of N-linked glycans as it provides an isolated
a-mannose attachment point. Additional glycosylation
can be performed after reductive cleavage of the benzyl-
idene acetal, or its complete removal. The latter is easily
achieved by treatment with 80% aqueous AcOH which,
in the case of 13, provided triol 1 in 92% yield.
10. Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett.
2001, 3, 9–12.
11. Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357–
2364.
12. Hang, H. C.; Bertozzi, C. R. Acc. Chem. Res. 2001, 34,
727–736.
13. Crich, D.; Sun, S. X. J. Am. Chem. Soc. 1997, 119,
11217–11223.
In conclusion, a short synthesis of the N-glycan trisac-
charide building block with a terminal azido group is
reported. The procedure can readily be adopted to
access a wide range of glycans with any substitution
pattern at the central mannose residue of the core
region.
14. Crich, D.; Sun, S. Tetrahedron 1998, 54, 8321–8348.
15. Crich, D.; Dudkin, V. Org. Lett. 2000, 2, 3941–3943.
16. Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123,
6819–6825.
17. Olsson, L.; Jia, Z. J.; Fraser-Reid, B. J. Org. Chem. 1998,
63, 3790–3792.
18. Crich, D.; Dudkin, V. Tetrahedron Lett. 2000, 41, 5643–
5646.
19. Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123,
9015–9020.
Acknowledgements
20. Crich, D.; Li, H. M.; Yao, Q. J.; Wink, D. J.; Sommer,
R. D.; Rheingold, A. L. J. Am. Chem. Soc. 2001, 123,
5826–5828.
We thank the NIH (GM 62160) for support of this
work.
21. Crich, D.; Sun, S. X. J. Am. Chem. Soc. 1998, 120,
435–436.
22. Mannosylations were conducted in the presence of a
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