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Combinatorial Chemistry for the Synthesis of Carbohydrate Libraries.

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Combinatorial Chemistry for the Synthesis of Carbohydrate Libraries
Prabhat Arya* and Robert N. Ben*
The use of combinatorial libraries in the identification and
elucidation of structure-activity relationships has become a
powerful tool in the pharmaceutical sector.[’] Traditionally,
novel lead compounds were obtained as natural products from
a number of sources including extracts from plants, animals,
insects, or microorganisms. When an extract shows a desired
biological activity, the active compound is identified, isolated,
and then subjected to further biological testing. Optimization of
the chemical structure to enhance biological activity is a laborintensive, time-consuming process, which dictates that each new
structure be independently synthesized. This overall approach
has made the development of new therapeutics a very lengthy
and expensive process.
In contrast, combinatorial chemistry has provided an attractive alternative to these traditional synthetic approaches since it
allows for the synthesis of a large number of structurally diverse
compounds within a short period of time. The approach utilizes
a large array of building blocks that are systematically assembled in such a way that all possible combinations are represented. Typically, a solution or solid-phase approach may be used in
conjunction with either a “split” or “parallel” synthetic strategy. Although the technology required to assemble a small molecule library is not new, combinatorial chemistry was not fully
exploited until recently, since efficient methods for screening such
libraries were virtually nonexistent. Many of these screening
strategies, as well as technical aspects of combinatorial chemistry, have been summarized in several well-written reviews.[’]
Unlike for protein -protein and nucleotide- protein interactions, progress in understanding the role of cell-surface carbohydrates in biological and pathological processes has been
Although comparatively little is known about these
weak, noncovalent interactions between cell-surface carbo[*I
Dr. P. Arya, Dr. R. Ben
Steacie Institute for Molecular Sciences
National Research Council of Canada
100 Sussex Drive. Ottawa KlAOR6 (Canada)
Fax: Int. code +(613)952-0068
e-rnail: prabhat arya(u? and rben(u.ned1
Q VCH Verlagsgesellschaft mbH, 0-69451 Weinheim. 1997
hydrate ligands and various protein receptors, they form the
basis of recognition events that are fundamental to a vastly
diverse range of biological and pathological processes. For instance, interactions of this nature have been implicated in cellto-cell communication, bacterial and viral infections, chronic
inflammation, cancer/metastasis formation, and rheumatoid
Oligosaccharides are very complex and diverse, which makes
their synthesis both labor-intensive and expensive. As a result,
the discovery of new biologically active oligosaccharide ligands
is a complicated problem. This aside, even when a promising
compound has been identified, optimization to enhance activity
is difficult and time-consuming. The synthesis of oligosaccharide libraries by a combinatorial approach offers a feasible solution to these problems.
In contrast to peptide and nucleotide libraries, preparation of
an oligosaccharide library is not a facile process. It is complicated by the issues of stereochemistry at the anomeric position and
the fact that multiple hydroxyl groups are present. Traditionally, these groups would be dealt with using a less than elegant
orthogonal protection-deprotection strategy. As an alternative, Hindsgaul et al[4a1demonstrated a random glycosylation
approach for forming small di- and trisaccharide libraries. This
strategy utilizes a glycosyl donor, which is protected with only
one type of protecting group, and a glycosyl acceptor in which
all hydroxyl groups are unprotected (Scheme 1). Hindsgaul
et al. coupled the benzylated glycosyl donor 1 to the disaccharide 2, which has six free hydroxyl groups. After three hours at
room temperature, a complex mixture was obtained in which
about 30% of acceptor 2 was fucosylated. Separation of the
mixture with reverse-phase chromatography furnished individual trisaccharides, which were analyzed by NMR spectroscopy.
Analysis confirmed that all six expected products were present
in yields of 8-23 YO.Ideally, a statistical mixture would contain
17 YOof each product.
In an alternate solution-phase approach, a latent - active glycosylation method was developed by Boons and co-workers
(Scheme2).14blThis strategy uses a glycosyl donor (4) and a
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1. BF3 Et20 (0.2 equiv)
2. H$Pd-C
six trisaccharides (major)
&linked fucosylated trisaccharides (minor)
Scheme 1. Random glycosylation accordlng to Hindsgdul et a1
( = 2,2’di-pava-nitrophenyl-5,5’-diphenyl-3,3’-(3,3’-dimethoxy-
n O
BnO Brio
Scheme 2. Latent-active glycosylation approach by Boons et a1
glycosyl acceptor (5); the two are derived from a common building block (3). Coupling of 4 and 5 produced a disaccharide ( 6 ) ,
which could be deacetylated and allowed to react with other
glycosyl donors in a combinatorial manner. Boons et al. demonstrated the feasibility of such a strategy by synthesizing a small
trisaccharide library containing anomeric mixtures that were
purified by gel-filtration column chromatography.
Another important discovery in the area of combinatorial
synthesis with di- and trisaccharides was made by Kahne and
co-workers at Princeton.‘4c1Their approach utilized solid-phase
technology to synthesize a saccharide library (Scheme 3). This
was especially challenging, since bonds between monomers
A n g m Chem I n / . Ed. EngI. 1997, 36, N,J I2
must be formed in high yields for a solid-phase approach to
be successful. This is not trivial, as most high-yielding coupling reactions in carbohydrate chemistry are not general in nature.”]
Kahne et al. employed a novel coupling procedure that utilized anomeric sulfoxides as glycosyl donors. Such compounds
are attractive intermediates, since they can be activated a t low
temperatures independent of other protecting groups, and give
nearly quantitative yields (about 90 Oh) in solid-phase syntheis.[^^] A sizable library with approximately 1300 di- and trisaccharides, possessing a diverse array of linkages, was synthesized
in three steps. The approach, which utilized a split and mix
synthesis, first involved the separate coupling of six different
monomers onto TentaGel resin beads. Next, twelve glycosyl
sulfoxide donors were coupled separately to mixtures of beads
containing the six monomers. The beads were then combined,
and the azido group on the glycosyl acceptor was reduced to an
amine. At this point, the beads were divided again into eighteen
groups and allowed to react with various acylating reagents.
Finally, all the protecting groups were removed.
To screen the library a colorimetric assay was performed
with a lectin. The library containing approximately 1300 compounds was exposed to biotinylated lectin, streptavidin-linked
alkaline phosphatase, and stained with “nitro blue tetrazolium”
4,4’-diphenylene)ditetrazoliumchloride). The beads that were
stained exhibited the greatest degree of binding and were removed with the aid of a simple light microscope. Remarkably,
only 0.3 YO(25 beads) were stained, and of these thirteen had the
same disaccharide core acylated with various hydrophobic
groups. The remaining twelve hits were discarded, since none of
these compounds exhibited any degree of commonality. Surprisingly, the natural ligand for the lectin was not identified as a hit
even though it was present in the library. Through separate
experiments it was proven that all of the thirteen hits were, in
fact, better ligands than the natural substrate.
As pointed out by Kahne et al. it is very interesting that the
lectin discriminates so well in its binding of certain di- and
trisaccharides. One of the paradoxes of carbohydrate binding is
that carbohydrate-binding proteins may bind different substrates in solution but function with remarkable specificity in
cell-to-cell recognition. This work also emphasized that the presentation of the saccharide on the surface of the bead is also
critical to lectin binding, since solution-affinity experiments
demonstrated that both the natural ligand and all thirteen compounds identified as hits bind the lectin in the solution phase.
This is indeed surprising, since presentation effects complicate
most on-bead screening techniques.
The random-glycosylation techniques by Hindsgaul et al., the
latent -active glycosylation by Boons et al., and solid-phase
methodology by Kahne et al. now make the synthesis of di- and
trisaccharide combinatorial libraries a feasible process. Furthermore, a colorimetric assay can make the screening of such libraries facile and efficient, since the same approach could be
applied to other lectins. There can be little doubt that these
recent advances in di- and trisaccharide combinatorial chemistry will have a great impact in the understanding of cell-surface
interactions and aid in the design of polyvalent compounds that
inhibit these interactions.
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Scheme 3. Di- and trisaccharide library from Kahne et a]. using split and mix synthesis and screening on a solid phase
Although these recent advances facilitate the synthesis of diand trisaccharide combinatorial libraries, the synthesis of
oligosaccharide libraries (such as tetrasaccharides and higher
derivatives) is still quite challenging. This is largely due to the
fact that the present glycosylation methods are not quantitative.
Therefore, more efficient, quantitative glycosylation methods as
well as improved separation techniques need to be developed
before the combinatorial synthesis of oligosaccharide libraries is
German version: Angew. Chem. 1997. 109. 1335-1337
Keywords: combinatorial chemistry
- glycosylations - carbo-
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0 VCH Verlagsgesellschaft mbH. 0-69451
Weinheim, 1997
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Angen,. Chem. In!. Ed. Engl. 1997, 36, No. 12
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chemistry, synthesis, libraries, carbohydrate, combinatorics
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