Protecting-group-free one-pot synthesis of glycoconjugates directly from reducing sugars

Article abstract of DOI:10.1002/anie.201406694

The conversion of sugars into glycomimetics typically involves multiple protecting-group manipulations. The development of methodology allowing the direct aqueous conversion of free sugars into glycosides, and mimics of oligosaccharides and glycoconjugates in a high-yielding and stereoselective process is highly desirable. The combined use of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate and the Cu-catalyzed Huisgen cycloaddition allowed the synthesis of a range of glycoconjugates in a one-step reaction directly from reducing sugars under aqueous conditions. The reaction, which is completely stereoselective, may be applied to the convergent synthesis of triazole-linked glycosides, oligosaccharides, and glycopeptides. The procedure provides a method for the one-pot aqueous ligation of oligosaccharides and peptides bearing alkyne side chains.

Full text of DOI:10.1002/anie.201406694

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Chemie
DOI: 10.1002/anie.201406694
Carbohydrates
Protecting-Group-Free One-Pot Synthesis of Glycoconjugates Directly
from Reducing Sugars**
David Lim, Margaret A. Brimble, Renata Kowalczyk, Andrew J. A. Watson, and
Antony John Fairbanks*
Abstract: The conversion of sugars into glycomimetics
typically involves multiple protecting-group manipulations.
The development of methodology allowing the direct aqueous
conversion of free sugars into glycosides, and mimics of
oligosaccharides and glycoconjugates in a high-yielding and
stereoselective process is highly desirable. The combined use of
2-azido-1,3-dimethylimidazolinium hexafluorophosphate and
the Cu-catalyzed Huisgen cycloaddition allowed the synthesis
of a range of glycoconjugates in a one-step reaction directly
from reducing sugars under aqueous conditions. The reaction,
which is completely stereoselective, may be applied to the
convergent synthesis of triazole-linked glycosides, oligosac-
charides, and glycopeptides. The procedure provides a method
for the one-pot aqueous ligation of oligosaccharides and
peptides bearing alkyne side chains.
T
he number of chemical processes by which completely
deprotected carbohydrates may be selectively converted into
other materials is reasonably limited,[1] as differentiation
between multiple hydroxy groups is difficult without the aid
of either protecting groups or enzymatic catalysis. However
exploitable differences do exist, and under certain circum-
stances, selective reactions of free sugars are feasible,[2]
though very rarely in water.
In 2009, Shoda et al. reported the use of the reagent 2-
chloro-1,3-dimethylimidazolinium chloride (DMC) for the
Table 1: One-pot synthesis of glycosyl triazoles directly from the
corresponding reducing sugars in water.
Entry Sugar
Product
Yield[a] a/b
1
2
GlcNAc
86% b only
GalNAc
glucose
75% b only
73% b only
3
4
mannose
73% a only
Scheme 1. Direct synthesis of glycosyl azides using ADMP. Reaction
conditions: a) 1, Et3N, D2O/MeCN (4:1), 90%; b) 1, Et3N, D2O/MeCN
(4:1), 50% of 5, 50% of 6; b) 1, Et3N, D2O/MeCN (4:1), then HCl to
pH 2, 86% of 5.
5
isomaltose
86% b only
[*] D. Lim, Dr. A. J. A. Watson, Prof. A. J. Fairbanks
Department of Chemistry, University of Canterbury
Private Bag 4800, Christchurch 8140 (New Zealand)
E-mail: antony.fairbanks@canterbury.ac.nz
Prof. M. A. Brimble, Dr. R. Kowalczyk
School of Chemical Sciences and School of Biological Sciences, The
University of Auckland
6
isomaltotriose
88% b only
23 Symonds Street, Auckland 1142 (New Zealand)
[**] We thank Dr. Marie Squire for technical assistance, and the Royal
Society of New Zealand Marsden Fund (Grant UOC0910), the
Biomolecular Interaction Centre, the University of Canterbury (Ph.D
Scholarship to D.L.), and the Freemasons Roskill Foundation
(Postdoctoral Fellowship to R.K.) for financial support.
[a] Sugar, Et3N (5 equiv), ADMP (3 equiv) in D2O/MeCN (4:1, 270 mm)
at 08C. After 3 h, alkyne (2 equiv), CuSO4·5H2O (1.5 mol%), and l-
ascorbic acid (0.2 equiv) were added and the mixture heated to 508C for
14 h.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201406694.
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direct synthesis of glycosyl oxazolines from reducing sugars in
water.[3] Subsequently, the use of DMC and derivatives was
applied to the synthesis of glycosyl azides,[4] pyridyl thioglyco-
sides,[5] peptides linked to carbohydrates through a sulphur
atom,[6] and sugar nucleoside diphosphates.[7]
One of the most powerful and widely applied reactions of
recent times is the modified Huisgen cycloaddition.[8] This
high-yielding transformation may be performed in water,
leading to numerous applications, as reaction conditions are
compatible with biological systems.
A logical development is the combination of these two
processes, that is, selective reaction of the anomeric center
and elaboration by “click” chemistry.[9] A single-step process
would be particularly attractive, as it would allow the direct
conjugation of free sugars with any material containing
alkyne functionality, without the need for protecting groups,
and using water as the reaction solvent.
The reported route to glycosyl azides using DMC involves
the addition of ten or more equivalents of sodium azide, and
the use of D2O as the solvent.[10] Seeking a route that did not
require a large excess of azide, we reasoned that 2-azido-1,3-
by Kitamura et al.[13] The reaction of ADMP with glucose 2 in
a mixture of D2O and MeCN[14] (4:1), with an excess of Et3N,
gave the desired b-azide 3 in 93% yield (Scheme 1). However,
when the reaction was applied to GlcNAc (4) a mixture of the
b-azide 5 and oxazoline 6 was formed. Oxazoline 6 was not
converted into azide 5 under the reaction conditions, indicat-
Table 2: Scope of alkyne substrate.
Entry Sugar
Alkyne
Product[a]
Yield
95%
1
2
3
GlcNAc
glucose
glucose
97%
81%
dimethylimidazolinium
hexafluorophosphate
(1,
ADMP),[11,12] could be used as both activating agent for the
anomeric hydroxy group and as the source of azide. ADMP
was prepared by conversion of the hexafluorophosphate salt
of DMC to ADMP by reaction with sodium azide, as reported
4
GlcNAc
78%
[a] In all cases, only pure b products were formed.
Scheme 2. Direct linking of deprotected carbohydrates to access mimics of oligosaccharides. Reaction conditions: a) sugar, ADMP, Et3N, D2O,
MeCN, 08C, 3 h, then add alkyne, CuSO4, ascorbic acid, and heat to 508C for 14 h.
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ing that this material is not an intermediate on the pathway
from reducing sugar to glycosyl azide in the Shoda procedure.
Given the preponderance for competitive oxazoline forma-
tion, the requirement for a large excess of azide (10 equiv-
alents or more) becomes evident, as in the Shoda process
intermolecular nucleophilic attack by azide must outcompete
oxazoline formation. As a large excess of azide may
complicate the click reaction, conditions were sought under
which the glycosyl azide 5 was formed in high yield using only
three equivalents of 1. A simple solution involved changing
the pH value of the reaction mixture,[15] which effected the
complete conversion of 4 into 5 as the sole product. Using this
procedure, GlcNAc was converted into azide 5 in 86% yield.
We next turned our attention to the development of a one-
pot click reaction. After the formation of the glycosyl azide
was complete, the addition of propargyl alcohol, copper
sulphate, and sodium ascorbate, and heating the mixture to
508C led to the formation of the glycosyl triazole in excellent
yield with complete stereoselectivity (Table 1). This simple
procedure was applied to a number of mono-, di-, and
trisaccharides (Table 1). The stereoselectivity of reactions was
complete; all sugars except mannose gave the b product
exclusively, while mannose gave only the a-glycosyl triazole.
Other alkynes were investigated to assay the generality of
the process. Simple alkyl-substituted alkynes reacted in high
yield. The efficacy of the reaction was not reduced by the use
of carboxylic acid or tertiary alcohol substitution, and the
process was equally applicable to more complex and poten-
tially biologically interesting substrates (Table 2).
Triazole-linked oligosaccharides have been demonstrated
to act as glycomimetics,[16] though published routes involve
multiple-step syntheses and other difficulties, for example, the
Scheme 3. Synthesis of MUC1 glycopeptides in water through direct reaction with reducing sugars. Reaction conditions: a) sugar, ADMP, Et3N,
D2O, MeCN, RT, 6 h, then add peptide, CuSO4, ascorbic acid, and heat to 508C for 14 h.
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problematic removal of benzyl protecting groups.[17] The
direct linking of carbohydrates through click reactions would
allow rapid and efficient access to a wide range of such
materials without the need for any protecting-group manip-
ulations. The applicability of the approach was therefore
assessed for the assembly of oligosaccharide structures. The
conjugation of free reducing sugars to a variety of other
carbohydrate materials containing an alkyne functionality
gave rise to a variety of di-, tri-, and pentasaccharide mimics
in a single operation (Scheme 2). In all cases, the reactions
were high yielding and completely stereoselective. From these
preliminary studies it would appear that there is little
limitation as to the oligosaccharides structures that can be
linked by this process.
Click chemistry has been applied to access glycopepti-
des[18,19] and glycoproteins,[20] though all of the reported
procedures require multiple-step manipulations of the carbo-
hydrate component. As further exemplification, this
approach was applied to the direct synthesis of glycopeptides.
Propargyl glycine (Pra) is readily incorporated into synthetic
peptides using solid-phase peptide synthesis (SPSS). Glyco-
sylated versions of the tandem repeat domain of the cancer-
associated mucin MUC1[21] have recently shown significant
potential as components of synthetic anticancer vaccines.[22]
Two synthetic MUC1 peptides incorporating Pra were
selected as alkyne-bearing peptides for glycoconjugation.
Fragment 26, corresponding to residues 11–20 in which T16
was replaced by Pra, and the full-length tandem repeat
domain 27, in which both T4 and T16 were replaced by Pra,
were assembled as previously described.[23]
In conclusion, methodology has been developed that
allows the direct conjugation of reducing sugars to alkynes
under aqueous conditions in a single reaction vessel. The
completely stereoselective procedure appears to have wide
applicability, both in terms of carbohydrate and alkyne
coupling partner. Reducing sugars may be linked directly to
peptides and other carbohydrates, giving facile access to
mimics of glycopeptides and oligosaccharides, respectively.
Received: July 2, 2014
Published online: September 8, 2014
Keywords: carbohydrates · click chemistry · glycopeptides ·
.
oligosaccharide mimetics · triazoles
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