Download Reactivity-Based One-Pot Synthesis of Oligosaccharides for the

Reactivity-Based One-Pot Synthesis of
Oligosaccharides for the Development of a
Photocleavable Sugar Array
Jinq-Chyi Lee1, Chung-Yi Wu1,2, Junefredo V. Apon3, Gary Siuzdak3, and
Chi-Huey Wong1,2*
Abstract
A major challenge in proteomics is to understand the functional impact of posttranslational modification, and protein glycosylation represents the most complex
post-translational event. More than 50% of human proteins are glycosylated; however,
the role of carbohydrates in glycoproteins is relatively unknown, due to the lack of
tools for study. Carbohydrate arrays provide a solution to this long-standing problem.
Using the programmable reactivity-based one-pot synthesis of oligosaccharides
developed by Chi-Huey Wong allows a rapid access to a large number of oligosaccharides which have been very difficult to obtain by other means. These saccharides
were then printed on silicon surface with a photocleavable linker for direct characterization by mass spectrometry and used for the high-throughput analysis of sugar-protein interaction.
A major challenge in proteomics is to understand the functional
impact of post-translational modification and of which protein glycosylation
represents the most complex event. At the same time, increasing numbers of
receptors are being characterized that operate through binding to specific
oligosaccharide sequences on glycoproteins, glycolipids and polysaccharides. Such receptors are involved in the folding of nascent
proteins, the subcellular targeting of enzymes, mechanisms
of infection (microbe-host interactions), inflammation, cancer metathesis, differentiation and development, and immunity. More than 50% of human proteins are glycosylated;
however, the role of carbohydrates in glycoproteins is relatively unknown, due to the lack of tools for study. The search
for carbohydrate ligands or receptors and their functions
remains, however, one of the most challenging areas in cell
Figure 1. Schematic representation of (a) solid-phase and (b) programmable one-pot approaches to oligosaccharide synthesis.
biology. The conventional approaches to carbohydrate ligand
discovery are cumbersome, and there is a great need for sen-
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, CA, U.S.A.
The Genomics Research Center, Academia Sinica, Taipei, Taiwan
3
Department of Molecular Biology and Chemistry and the Scripps Center for Mass Spectrometry, The Scripps
Research Institute, CA, U.S.A.
1
2
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sitive, high-throughput technologies that allow
rapid analyses of carbohydrate-protein interactions.
Development of carbohydrate microarrays would
provide a new tool for this study and for the identification of carbohydrate receptors or ligands associated with cancer metathesis, bacterial or viral infection, immune response, differentiation and many
other intercellular recognition processes.
Carbohydrate microarray technologies, analo-
Figure 2. Programmed one-pot synthesis of Globo H
oligosaccharide.
gous to those developed for DNA and being developed for proteins, are new developments at the fron-
hydrates but also elucidation of their ligands and
tiers of glycomics and ideal for addressing this
mechanisms.
need. Only small amounts of product are required
Unlike peptides and nucleic acids, oligosac-
for fabricating microarrays, and many compounds
charides are difficult to synthesize chemically. This
can be screened in parallel in a single operation.
is because some oligosaccharide chains are linear,
Since the introduction of gene arrays, genomic
others are branched, the glycosidic linkages are in
research has progressed rapidly and various genetic
α or β anomeric configurations, and adjacent
variations related to diseases have been identified.
monosaccharides are linked via different hydroxyl
However, the result of gene array analysis may not
groups in their sugar rings. The possibility of form-
be directly correlated with the function at the pro-
ing a tetrasaccharide from the nine common mono-
tein level. Understanding the relationship between
saccharides found in humans is in the order of 15
genetic changes and disease states represents a new
millions, significantly higher than the correspon-
challenge, and functional genomics or proteomics
ding tetramers of peptides or nucleic acids, though
has thus become an important subject for study.
nature probably only uses a small fraction of these
Results of 'proof of concept' experiments with car-
tetramers. This degree of diversity makes the syn-
bohydrate-binding proteins of the immune system –
thesis of oligosaccharides very difficult, and multi-
antibodies, selectins, cytokines and chemokines –
ple selective protection and deprotection steps are
and several plant lectins indicate that microarrays of
required for the hydroxyl groups of monosaccha-
carbohydrates (glycoconjugates, oligosaccharides
rides during chemical synthesis of oligosaccharides.
and monosaccharides) will greatly facilitate not
An automated solid-phase method has been devel-
only the discovery of proteins that recognize carbo-
oped to facilitate the synthesis with elimination of
intermediate isolation and purification, but the
requirement of protecting group manipulation still
exists.
An alternative approach to the synthesis of
oligosaccharides is a programmable reactivitybased ‘one-pot’ approach. The approach is a newly
developed strategy which eliminates protecting
group manipulation completely to further simplify
the synthetic procedures. In this approach, an
Figure 3. Methods for covalent immobilization of sugars on
N-hydroxy succinimide ester-coated glass slides.
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oligosaccharide of interest is generated by the
sequential addition of building blocks (thioglyco-
sides) that are either fully protected or have one
hydroxyl group exposed, and each building block
has defined relative reactivity values (RRVs). It has
been shown that the RRV of a thioglycoside building block in the glycosidation reaction can be tuned
in the presence of protecting groups; more than 400
building blocks, with RRVs ranging from 1 to 105,
have been designed and synthesized, and stored as a
reactivity database. A computer programme called
‘Optimer’ has thus been developed to guide the
selection of building blocks for the one-pot synthesis of a given oligosaccharide. Based on this database and simple chemistry logic, an appropriate
Figure 4. Binding of monoclonal antibodies Mbr1 (b) and VK-9 (c) to Globo H
and truncated sequences.
combination of thioglycoside building blocks can
be identified from the database for any given
oligosaccharide structure and used in a sequential
manner for the one-pot synthesis. If RRVs differ by
more than 102, the desired glycosidic bonds can be
formed by the sequential addition of building
blocks in the order of the RRVs. Different from the
qualitative one-pot synthesis, the reactivity of each
thioglycoside building block is determined quantitatively. These RRVs form a reactivity database
that, together with the “Optimer” computer program, provides an automatic methodology for the
reactivity-based one-pot synthesis of oligosaccharides. Once the required building blocks with pro-
Figure 5. Representative glycan structures on the array.
tecting groups are prepared, oligosaccharides can
be synthesized in a short period of time (in minutes
or hours, instead of days or months using traditional
methods) using this programmable one-pot
approach. We have thus successfully prepared a
small oligosaccharide library, the breast cancer antigenic determinant Globo H, the colon cancer antigen Lewis Y, and the lung cancer antigen fucosylGM1.
Desorption/ionization on silicon mass spectrometry (DIOS-MS) is an ionization method that
uses a porous silicon surface to generate gas-phase
ions of small molecules (<3000 Da) without a
matrix. With this technique, the process of sample
Figure 6. Glycan microarray analyses of Viet 04 and DK 97 avian influen-
manipulation is minimized and it only requires very
za virus.
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biomedical interest become available for investigation, the evaluation of their carbohydrate binding
specificity will be greatly helped by carbohydrate
microarrays.
In summary, the work reported by Wong and
postdoctoral associates Chung-Yi Wu and others
has solved a long-standing problem in carbohydrate
array development. The one-pot synthesis allows a
Figure 7. Preparation and analysis of a photocleavable
rapid access to a large number of oligosaccharides
DIOS-MS sugar array.
which have been very difficult to obtain by other
small volumes for analysis. The carbohydrates syn-
means. In addition, with attachment of a photo-
thesized are then printed on the surface of modified
cleavable linker between the sugar moiety and glass
porous silicon with a photocleavable linker, which
surface, the array can be characterized directly by
could be cleaved by a laser (=337 nm) in the mass
mass spectrometry, as a mass spectrometer is
spectrometric analysis to detect the carbohydrates
equipped with a 337-nm laser which cleaves the
on the porous silicon.
sugar from the glass surface for direct characteriza-
The characterizable carbohydrate microarrays
tion. According to the method, one milligram of
described in this report hold great promise as a
saccharides can be used for 100-million spots on the
high-throughput means for detecting the interac-
surface and each glass slide contains thousands of
tions of proteins with diverse oligosaccharide
spots, each with different saccharides. This new
sequences of glycoproteins, glycolipids and poly-
breakthrough — the rapid synthesis of oligosaccha-
saccharides. The development should find use in
rides and attachment of them to glass slides through
detailed characterizations of carbohydrate-protein
a photosensitive linker — is expected to replace the
interaction and would lead to or be accompanied by
traditional ELISA method for diagnosis and to facil-
structural and functional studies of such recogni-
itate carbohydrate-based drug discovery.
tions. As more and more recombinant proteins of
The original paper was published in Angewandte Chemie International Edition 45 (2006): 2753-2757.
References:
1. Z. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat T. Baasov, C.-H. Wong, (1999) J. Am. Chem. Soc. 121, 734-753.
2. P. Sears, C.-H. Wong, (2001) Science 291, 2344-2350.
3. K.-K.T. Mong, C.-H. Wong, (2002) Angew. Chem. Int. Ed. 41, 4087-4090.
4. T. K-K. Mong, H. K. Lee, S.G. Duron, C.-H. Wong, (2003) Proc. Natl. Acad. Sci. USA 100, 797-802.
5. T. Feizi, F. Fazio, W. Chai, C.-H. Wong, (2003) Curr. Opin. Struct. Biol. 13, 637-645 (2003).
6. H.-K. Lee, C. N. Scanlan, C.-Y. Huang, A. Y. Chang, D. A. Calarese, R. A. Dwek, P. M. Rudd, I. A. Wilson, D. R. Burton, C.-H.
Wong, (2004) Angew. Chem. Int. Ed. 43, 1000-1003.
7. M. C. Bryan, F. Fazio, H.-K. Lee, C.-Y. Huang, A. Chang, M. D. Best, D. A. Calarese, O. Blixt, J. C. Paulson, D. Burton, I. A.
Wilson, C.-H. Wong, (2004) J. Am. Chem. Soc. 126, 8640-8641 (2
8. O. Blixt, S. Head, T. Mondala, C. Scanlan, M. E. Huflejt, R. Alvarez, M. C. Bryan, F. Fazio, D. Calarese, J. Stevens, N. Razi, I. van
Die, D. Burton, I. A. Wilson, R. Cummings, N. Bovin, C.-H. Wong, J. C. Paulson, (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 1703317038.
9. S. R. Hanson, M. D. Best, M. C. Bryan, C.-H. Wong, (2004) Trends Biochem. Sci. 29, 656-663.
10. C.-Y. Huang, D. A. Thayer, A. Y. Chang, M. D. Best, J. Hoffman, S. Head, C.-H. Wong, (2006) Proc.Natl. Acad. Sci. U.S.A. 103(1),
15-20.
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