Synthesis of pentaantennary N-glycans with bisecting GlcNac and core fucose

Article abstract of DOI:10.1002/anie.200604788

(Chemical Equation Presented) Always use protection? The omission of a single protecting group led to the convergent synthesis of the highly branched dodecasaccharide N-glycan that contains two neighboring tetrasubstituted mannoside groups. A serendipitou

Full text of DOI:10.1002/anie.200604788

Angewandte
Chemie
DOI: 10.1002/anie.200604788
Carbohydrates
Synthesis of Pentaantennary N-Glycans with Bisecting GlcNAc and
Core Fucose**
Steffen Eller, Ralf Schuberth, Gislinde Gundel, Joachim Seifert, and Carlo Unverzagt*
Dedicated to Professor Joachim Thiem on the occasion of his 65th birthday
Recombinant therapeutic glycoproteins contain mainly
asparagine-linked oligosaccharides (N-glycans), which
are often essential for the proper function of the
glycoprotein. The heterogeneity of the glycans in
natural glycoproteins is a large obstacle for the entire
research in the field of glycobiology,[1] and despite
recent advances in chemical synthesis of N-glycans,[2–10]
the majority of N-glycans for biological studies are still
isolated from natural sources.[11] We have developed
modular building blocks for the most abundant complex
N-glycans,[12] which after fortuitous results from the test
of this modular system allowed the synthesis of complex
N-glycans with the maximum number of branches and
core substitutions (glycan F; Scheme 1).
Previously we have developed modular building
blocks for the synthesis of complex N-glycans with up to
four antennae,[12] which contained a bisecting GlcNAc
moiety[4,13,14] or a core fucose moiety.[15] As a result of
the steric hindrance, bisected N-glycans with three or
four antennae are especially difficult to obtain.[4]
Encouraged by recent improvements[14] we investigated
the complex N-glycan F (Scheme 1). A particularly high
substitution pattern is found at the Mana1,6Manb unit
of F where each mannose contains a total of four
glycosidic partners. Pentaantennary N-glycans are
found in ovomucoid,[16] fish hyosophorin,[17] CHO
cells,[18] and HepG2 cells.[19]
Scheme 1. Retrosynthesis of pentaantennary N-glycans with bisecting GlcNAc
and core fucose groups. Ac=acetyl, Bn=benzyl, Pht=phthalimido, PMB=
p-methoxybenzyl, TFAc=trifluoroacetyl.
First, unsubstituted pentaantennary N-glycans were
assembled to reduce the synthetic complexity of F. The
synthesis of tetrasaccharide donor D began with the 3-O-
allylation of benzylmannoside (2) via a stannylene acetal to
give 3 (Scheme 2).[20,21] Threefold glycosylation of triol 3 with
donor 1 (6 equiv) gave the tetrasaccharide 4 (77%). After
deallylation of 4, the acetylation of alcohol 5 required
catalytic amounts of DMAP. After catalytic hydrogenation
of 6 to remove the benzyl group, the hemiacetal was
converted into imidate D and coupled with the hexasacchar-
ide 12 to give the decasaccharide 13 in 65% yield after
optimization (Scheme 3).
The high reactivity of donor D prompted us to incorporate
a bisecting GlcNAc moiety and a core fucose residue. Thus,
the branched trisaccharide B[12] was coupled to the core
trisaccharide A (Scheme 4).[15] The resulting hexasaccharide
(80%) was acetylated and the benzylidene acetal was cleaved
(75% over 2 steps). After the selective chloroacetylation of
14, the hexasaccharide 15 was coupled with thioglycoside C[4]
to give the bisected heptasaccharide 16. Dechloroacetylation
yielded the acceptor 17, which was coupled with the
disaccharide 18 (77%). The nonasaccharide 19 was depro-
tected and fucosylated to give the triantennary decasacchar-
ide 21 in 93% yield.
Next we investigated the coupling of the heptasaccharide
17 with the donor tetrasaccharide D (5 equiv; Scheme 5).
After purification by flash chromatography and HPLC, only
9% of the bisected pentaantennary compound 22 was
obtained. To evaluate the activation using thioglycosides,
imidate D was converted into the thioglycoside 8. Reaction of
the donor 8 (3.3 equiv) with the acceptor 17 gave the product
[*] S. Eller, R. Schuberth, G. Gundel, J. Seifert, Prof. C. Unverzagt
Bioorganische Chemie, Gebäude NW1, Universität Bayreuth
95440 Bayreuth (Germany)
Fax: (+49)921-555-365
E-mail: carlo.unverzagt@uni-bayreuth.de
[**] We thank the Deutsche Forschungsgemeinschaft and the Fonds der
Deutschen Chemischen Industrie for funding.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Communications
Scheme 2. a) 1. Bu2SnO, MeOH, reflux; 2. AllBr, CsF, DMF, (1.–2.,
70%); b) BF3·OEt2, CH2Cl2, À108C, MS (4 Š), 77%; c) PdCl2, MeOH,
77%; d) Ac2O, pyridine, DMAP, 81%; e) 1. H2, PdO·H2O, AcOH,
MeOH, 82%; 2. Cl3CCN, DBU, CH2Cl2, 08C, 85%; f) 1. H2, PdO·H2O,
AcOH, MeOH, 77%; 2. Cl3CCN, K2CO3, CH2Cl2, 82%; g) 5-tert-butyl-2-
methyl-thiophenol, BF3·OEt2, CH2Cl2, 08C, MS (4 Š), 69%; h) 5-tert-
butyl-2-methyl-thiophenol, BF3·OEt2, CH2Cl2, À108C, MS (4 Š), 86%;
i) Ac2O, pyridine, DMAP, 58%; j) 1. ethylenediamine, nBuOH, 808C,
68%; 2. PfpOTFAc, NEt3, THF; 3. Ac2O, pyridine, (2.–3., 63%);
k) Ac2O, pyridine, 84%. All=allyl, DBU=1,8-diazabicyclo[5.4.0]-
undec(7)ene, DMAP=4-dimethylamino-pyridine, MS=molecular
sieves, Pfp=pentafluorophenyl, THF=tetrahydrofuran.
Scheme 4. a) A, B, BF3·OEt2, CH2Cl2, À258C, MS (4 Š), 80%;
b) 1. Ac2O, pyridine; 2. pTosOH·H2O, CH3CN, (1.–2., 75%); c) chloro-
acetic anhydride, pyridine, CH2Cl2, 08C, 77%; d) C, NIS, TfOH, CH2Cl2,
À308C, MS (4 Š), 61%; e) K2CO3, CH2Cl2/MeOH (10:1), À108C, 84%;
f) BF3·OEt2, CH2Cl2, À258C, MS (4 Š), 77%; g) CAN, CH3CN, toluene,
H2O, 89%; h) E, CuBr2, Bu4NBr, CH2Cl2, DMF, 93%. CA=chloroacetyl,
CAN=ceric ammonium nitrate, DMF=N,N-dimethylformamide,
NIS=N-iodosuccinimide, PMP=p-methoxyphenyl, TfOH=trifluorme-
thanesulfonic acid, Tos=toluene-4-sulfonyl.
lacked an acetyl group. Since the acceptor 17 was pure, we
examined the donor 8 by HPLC-MS, which revealed the
presence of a second compound, which lacked an acetyl group
(10%). The impurity was confirmed as compound 9, which
had resulted from an incompletely 3-O-acetylated batch of
donor D. The outcome of the glycosylation with the 9:1 mix-
ture of donor 8 and 9 indicated that the side product 9 must
serve as a very potent donor because the undecasaccharide 23
accounted for 50% of the product mixture. In contrast,
HPLC-MS analysis of the reaction mixture obtained with the
impure imidate D (which contained 10% of 7) revealed only
traces of the undecasaccharide 23 along with the undecasac-
charide 22.
Thus the pure donors 8 and 9 were synthesized from the
intermediate 5. Unexpectedly, formation of imidate 7 from 5
was only successful using K2CO3 as a base to prevent
additional formation of the imidate at the 3-OH position.[22,23]
Imidate 7 was converted into the thioglycoside 9, which was
acetylated to give 8.
Scheme 3. a) D, BF3·OEt2, CH2Cl2, À208C, MS (4 Š), 65%.
in 16% yield after preparative HPLC. However, analytical
HPLC-MS showed the product to be a 1:1 mixture of the
undecasaccharide 22 and a second undecasaccharide, which
Glycosylation experiments using the donors 8 and 9
showed marked differences. We were pleased to see that,
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Angewandte
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Removal of the PMP group (82%) of 24 and subsequent
core fucosylation (75%) finally gave the dodecasaccharide 26.
Global deprotection of the base labile groups and N-
acetylation was achieved in three steps and in a one-pot
reaction to furnish the dodecasaccharide 27. Compound 27 is
a derivative of F and contains an azide group for the
generation of neoglycoproteins or glycopeptides.
Herein we have reported the first chemical synthesis of
highly branched pentaantennary N-glycans and derivatives
with bisecting and core fucosyl modifications. The hindered
glycosylation reactions were systematically optimized after
the identification of key protecting groups which reduce the
proximal and peripheral crowding. The use of a universally
applicable modular set of building blocks is expected to
facilitate the general chemical synthesis of branched N-
glycans.
Received: November 24, 2006
Revised: January 23, 2007
Published online: April 20, 2007
Keywords: carbohydrates · glycoproteins · glycosylation ·
.
protecting groups · steric hindrance
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Angew. Chem. Int. Ed. 2007, 46, 4173 –4175
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