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Chemical glycobiology: why now?

Peter H Seeberger
Understanding the structure and function of carbohydrates remains a key challenge for chemical biologists. Developments in carbohydrate synthesis and analysis together with the advent of high-throughput methods such as carbohydrate microarrays have helped shed light on the function of glycoconjugates. Similarly, consortia have provided technology platforms and focus to a burgeoning field. Now, recruitment of scientists from related fields and further integration of chemistry and biology to achieve technical goals are needed for rapid advancements.
The structure of carbohydrateshexoses to be preciseposed one of the earliest chemical mysteries. Emil Fischer, a grandfather of chemical biologists, relied on phenyl hydrazine to elucidate the stereochemistry and constitution of these cyclic molecules in the 1880s (ref. 1). Despite this promising beginning, at the turn of the new millennium the situation in glycomics compared to genomics and proteomics was bleak. While other communities had set and communicated clear goals, were using mature and enabling technologies and had a common language in place, these features were for the most part absent in the sugar arena. These shortcomings were definitely not due to a lack of desire to better understand the role of carbohydrates in biology and medicine. Indeed, throughout the twentieth century it became increasingly clear that glycoconjugates were involved in important signaling and recognition events throughout biology2. Oligosaccharides on the cell surface determine human blood groups and serve as receptors during bacterial and viral infections. Carbohydrate-based vaccines entered routine vaccination schedules to prevent devastating bacterial diseases such as Haemophilus influenza type B. Low-molecular-weight heparin became the anticoagulant of choice, while altered versions of human glycoproteins such as erythropoietin fueled the pipelines of biotech companies3. The biological and medical relevance of carbohydrates motivated chemists
Peter H. Seeberger is at the Max-PlanckInstitute of Colloids and Interfaces, Department of Biomolecular Systems, Potsdam, Germany, and Freie Universitt Berlin Institute of Chemistry and Biochemistry, Berlin, Germany. e-mail: peter.seeberger@mpikg.mpg.de

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and biologists alike to study glycan structure and function. With such a strong impetus for an increased understanding of carbohydrates, why has the field struggled to advance in comparison with other disciplines? In this commentary, I outline what I see as the past and current challenges where the field is making tremendous progress, but I also call for improved infrastructure and focused efforts for the next generation of carbohydrate research. The complexity of biomolecules One stumbling block in glycomics is simply defining the problem at hand. For organic chemists, natural products have provided discrete targets with the added benefit that a complete synthesis confirms or corrects structural assignments and also advances our understanding of synthetic chemistry more broadly. For genomics, the number of parts was generally thought of as the size of the human genome, similarly providing a finite endpoint. Sequencing the genome in turn revealed the number of genes and thereby the number of proteins. These finite numbers of macromolecules establish a target for setting community challenges, as in structural proteomics, and the progress toward these goals can be measureda particularly attractive feature for the milestone-based culture of granting agencies. Measuring such successes in the areas of glycochemistry and glycobiologynow typically summarized under the term glycomicsis currently not possible. Identifying the number of glycan structures in a cell or organism, or the glycome, is complicated by the multitude of biomolecules that contain sugars, the variability in sugar identity and the connectivity between them. For example, the term car-

bohydrate includes several classes of natural products that contain sugar chains as part of a glycoconjugate, where sugars are attached to another molecule4. Glycosaminoglycans such as heparin are long, often highly sulfated, linear molecules that are attached to proteins. Most human proteins are glycosylated, yielding glycoproteins; many of these constructs are heavily glycosylated with significantly branched and often heterogeneous structures. Glycolipids are glycan chains such as the human blood group determinants that are anchored in the outside of the plasma membrane via the lipidic tails. Finally, glycosylphosphatidylinositol (GPI) anchors are glycolipids that anchor proteins such as prions into the plasma membrane and remain very poorly understood. These major classes of glycoconjugates can be distinguished into further subclasses and give a flavor of the structural complexity that make carbohydrates much more difficult to handle than oligonucleotides and peptides. Beyond this first level of complexity, the carbohydrate moieties themselves are complex. In mammalian systems, only ten monosaccharides are used, but in bacteria many more building blocks can be connected in a dazzling array of branched structures. Each glycosidic linkage that connects two sugars constitutes a new stereogenic center, which means that the next ring can reside above or below the first ring (Fig. 1)5. Given this potential structural freedom, how complex is the glycome really? Currently, no comprehensive database of structures exists, although efforts to construct them reach back as far as the 1980s. Estimates based on the number of transferases responsible for the biosynthesis of complex carbohydrates and analyses of collections of carbohydrates

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in existing, rudimentary databases seem to suggest that only a rather limited set of the theoretically possible structures are actually present. However, it has yet to be shown how great the diversity of carbohydrate structures produced by nature really is. A second major barrier in glycomics is that of carbohydrate synthesis: until the late 1970s, tri- and tetrasaccharides constituted major synthetic challenges. The 1980s saw methodological improvements in the form of muchimproved leaving groups that enabled highly specialized laboratories to access many oligosaccharides of biological relevance when given sufficient time and resources6. However, each complex glycan remained a major research project that took sometimes years to complete. Futile attempts at streamlining carbohydrate synthesis in the 1970s were revisited in the mid 1990s, but general methods are not yet available. Finally, communication within the carbohydrate field about these various structures has also been difficult. Carbohydrate chemists and glycobiologists spokeand for a large part still do speakdifferent languages when it comes to describing carbohydrates. Chemists like to draw exact structural depictions, including connectivities to the different positions at the ring and the stereochemistry of the glycosidic linkages. Glycobiologists more routinely use three-letter abbreviations for each sugar, together with descriptions of the connectivity and stereochemistry at the anomeric carbon. Trivial as nomenclature issues may seem, they complicate interactions and collaborations. From projects to big science Though the early days of the glycosciences lagged behind their parallel disciplines, there were successes to celebrate, and some important foundations were laid. Collaborations between carbohydrate chemists and immunologists, cell biologists and developmental biologists (among others) were established, even given language barriers. Since Merrifields seminal work on solid-phase peptide synthesis, chemists had been making efforts to assemble oligosaccharides in an analogous manner. The sequence analysis of complex carbohydrates had progressed over decades of work. About ten years ago, however, advances in genomics and proteomics began to impact the glycosciences at various levels. With the impact of automated sequencing and synthesis technologies in the other two fields in plain view, scientists working toward advanced methodologies received a needed boost in motivation and support. The increased necessity for information exchange across an entire field

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Figure 1 Carbohydrate complexity is a result of branching and the stereochemistry of the glycosidic linkage. In this scheme, the two disaccharides (already isomers) can be further extended at any OH group, creating a huge amount of diversity. These modifications can proceed in a linear fashion (for example, at the 6 position, see red circle) or can create a branched structure (for example, from the 3 position, see blue circle).

promoted a common foundation through education, with the first comprehensive text, Essentials of Glycobiology4, both summarizing the state of the art in the glycosciences and showing the white spots on the map. Beyond these focused changes, leading glycoscientists began to consider a big science approach. They believed that the strategy that worked so well in genomics and proteomics would also fare well in glycomics: centralized technology platforms in core facilities should be available to provide services to the general community. The data generated in these core centers would be made available in curated databases for the scientific community to use as a reference and mining resource. These core centers could operate on a regional, national or even international basis, and indeed, large transdisciplinary and transnational collaborative efforts that would further bundle resources and foster exchange would further accelerate scientific progress. In response, national efforts sprang up. In the United States, the National Institutes of Health (NIH)-sponsored Consortium for Functional Glycomics (http:// www.functionalglycomics.org/) remains the most ambitious and best-funded effort. Japan, Switzerland and the United Kingdom also initiated smaller-scale operations. These glycomics centers set ambitious goals of providing support in several key technologies, including sequencing and analysis, synthesis and screening, the development of knockout mice and bioinformatics cores. The past decade has seen tremendous progress toward these goals. However, although advances in carbohydrate sequencing and automated oligosaccharide synthesis have

provided the foundation for a host of tools of great utility to glycoscientists, the techniques have not yet reached the ease that is commonplace for oligonucleotides and peptides. Similarly, though greatly improved mass spectrometry techniques and bioinformatics approaches (co-opted from proteomics efforts and applied to N-glycan, O-glycan and glycolipid sequencing) have led to important insights in proteomics studies, determining the sequence of glycosaminoglycans remains a serious challenge7,8. In particular, small differences in GAG structures (even in one stereocenter as with glucuronic and iduronic acid) or modifications such as sulfation require enzymatic steps to be distinguished in sequencing protocols. Efforts to further improve carbohydrate sequencing methods have been thwarted by the lack of pure, homogeneous glycans. Amplification techniques such as PCR for nucleic acids or bacterial expression to prepare large amounts of proteins are not available for carbohydrates. Oligosaccharide microheterogeneity means that typically several closely related glycans may be encountered that only differ in one sugar or (even worse) are just anomers or positional isomers. Pure samples of carbohydrate fragments would benefit analytical chemists as standards and to train the sequencing techniques. Complex glycoconjugates such as GPI anchors suffer from the difficulties associated with both isolation and sequencing. Less than a handful of laboratories can access such structures, and assignments are rarely definitive. Chemical synthesis is an attractive alterative for carbohydrate procurement given that the molecules are constructed

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from first principles, allowing definitive assignment of structure and preparation of ample (>5 mg) material. Access to pure carbohydrates by chemical synthesis clearly holds immense potential; the availability of standards and training molecules for sequencing is just one example of their value. Structural work on carbohydrates and particularly on the interaction of carbohydrates with carbohydrate-binding proteins has been hampered by the lack of defined carbohydrate ligands and substrates. The few protein structures in the Protein Data Bank that incorporate oligosaccharides reflect these difficulties in working with carbohydrate conjugates. Many other biochemical, medicinal chemistry and immunological research efforts also suffer from a shortage of materials. To address these problems, carbohydrate chemists have developed increasingly sophisticated solutions to the two fundamental problems of carbohydrate synthesis: differentiation of a host of hydroxyl and amine groups using protective groups in order to control the regioselectivity of reactions, and the need for high yielding and stereoselective glycosylation reactions to connect different sugars. In 2001, two complementary means to automate different aspects of oligosaccharide assembly were disclosed: automated solid-phase synthesis9 and programmed one-pot synthesis10 (Fig. 2). Both methods have since been improved, and other approaches have been advanced. Thus, while no single technology to satisfy all demands has emerged yet, the seeds for generally applicable methods have been planted. Screening, like sequencing, has made good progress in the last decade. Defining interactions of carbohydrates with proteins had traditionally been a laborious task carried out on a one-by-one basis. With the core centers and community support in place, high-throughput screens became a hallmark of the omics era. The placement of isolated and synthetic carOBn O BnO OPiv O O P OBu OBu

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bohydrates to create carbohydrate arrays in chip format was not a giant intellectual leap from their gene chip predecessors, but it was a breakthrough in practical terms11. Quickly, carbohydrate interactions with protein receptors, antibodies, RNA, bacteria and viruses were determined (Fig. 3). Detection of antibodies in blood began to associate the presence of anticarbohydrate antibodies with disease states. The screening core of the NIH CFG and other screening efforts helped to define carbohydrate-protein interactions for a host of important signaling molecules. At the same time it became clear that the screens would only be as good as the content that was placed on the chips; as with carbohydrate sequencing, synthesis and isolation of homogenous structures again represents the bottleneck to rapid, high-quality data. The central facilities have met other goals with greater success. Animal models of disease provide a functional correlation between the
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Figure 2 Strategies for carbohydrate synthesis. (a) General scheme for the workflow of an automated carbohydrate synthesizer, akin to that for DNA and peptides. (b) Optimer is a software program that selects the ideal building blocks to be combined in just one reaction vessel. Different building blocks with different reactivities are mixed and react in a predetermined way. Bn, benzyl ether; Bz, benzoate; Lev, levaloyl; Tol, toluyl; Piv, pivaloyl; Fmoc, 9-fluorenylmethoxycarbonyl; Bu, butyl; TCA, trichloroacetyl; NBz, 4-nitrobenzoate.

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HO HO O Reactive group O HO Synthetic glycans O Spacer Nu O O

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Figure 3 Carbohydrate arrays help to quickly screen carbohydrate-protein interactions.

absence of a particular glycosyl transferase and a phenotype12. As such, the production of knockout animals and evaluation of relevant phenotypes in core facilities has become an important service to the community. The cost and time associated with producing such animals of course requires choices to be made, as not all ideas can be pursued. The generation of tremendous amounts of information from these phenotypic experiments and screening approaches necessitates database creation and information management. Each of the national efforts has included the formation of an appropriate database: the European Glycoscientists Carb-Bank includes structural data from the literature, efforts in the United States have focused on data from the analysis and screening cores, and the Japanese consortium has mainly collected sequencing data13. Unfortunately, as each database has focused on a different type of information, there is no single database that integrates all information relevant to glycoscientists, in stark contrast to the situation for DNA and proteins. Looking to the future Ten years after concerted efforts in different countries began to bring a big science approach to the carbohydrate arena, the question has to be asked: was this approach successful? In my opinion, large-scale efforts in the glycosciences have helped to advance the field tremendously. Key technological challenges were identified, and with the creation of core centers, tasks that would be too technically difficult or expensive for many single laboratories were brought to the general community. Additionally, laboratories that came across carbohydrate-related problems while pursuing scientific problems not connected to carbohydrates were given the chance to enter into the glyco arena. For example, newly identified proteins that turned out to bind carbohydrates could be screened to find out what the specific glycan recognition motifs werea previously difficult task that has been reduced to one microarray experiment.

With this success to build on, we now need to look to the future of the field. Where do we go from here? What are the challenges? Where do we need to improve? Identifying these challenges and opportunities, and setting appropriate/relevant individual and community goals, will serve as an impetus for further conceptual and technological development. Similarly, establishing measurable milestones toward a final target will enhance our ability to communicate the importance of our work to the public and to funding agencies. Enabling technologies will be at the heart of further developments. We need to be able to analyze glycans found in biological samples of interest more quickly and successfully. Therefore, analysis techniquesmost importantly, mass spectrometrymust be further improved, the expertise needs to be disseminated and costs must be decreased. Currently, too few experts around the world are available to analyze and sequence carbohydrates; more training and education programs should be established to address this problem. As discussed, carbohydrate synthesis has made great strides, but the goal of a general automated method that is broadly available has not yet been achieved. Our own laboratory is beginning to furnish automated synthesizers to some carbohydrate centers around the world, but the technology remains unproven outside our laboratories. As we continue to move forward, we must look beyond the concerns raised by automation critics, who argue that too many equivalents of expensive building blocks are consumed, that purification and quality control of the final product are impossible to achieve, and that automated synthesis does not provide access to all possible structures. The achievements in peptide and DNA synthesis14 have taught us that the prices for building blocks will fall with demand. HPLC and other new methods will enable purification. And yes, access to all members of a class of biomolecules may not be possible, but that should not preclude the ready synthesis of the bulk of these compounds.

The chemistry community should accept the challenge not just to collect total syntheses of particular carbohydrates like trophies, but also to develop generally applicable, reproducible methods accessible to non-experts. Further improvements of the solid-phase technology but also other approaches to rapid and reliable carbohydrate synthesis are sorely needed. This requires more work in fundamental carbohydrate chemistry. Enzymatic means to prepare oligosaccharides will complement chemical ways to produce molecules of interest. In my view, trying to automate and generalize these synthetic protocols does not mean the end of carbohydrate chemistry; on the contrary, it will make synthesis more important than ever before. Additionally, performing this service to the community will pay dividends to biologically minded chemists, as improved sequencing techniques will identify new, biomedically relevant targets. If we cannot take on this challenge within academic circles, funding needs to be made available for more centralized efforts. Access to many different carbohydrates and glycoconjugates will be key to the further development of tools that are based on pure glycans. Carbohydrate microarrays, surface plasmon resonance experiments with carbohydrates, affinity columns and fluorescently tagged glycans all require improved synthetic access. Involving an increasing number of synthetic groups will produce a larger pool of structures to be used by biologists. In addition, methods to procure large (kilogram) amounts of specific carbohydrates for medical applications will be needed more and more. Finally, core facilities must build on their current successes in biological models and bioinformatics. Additional funding must be used to extend knockout animal production and the development and analysis of disease models. Databases across the globe must be integrated to create a common resource and knowledge base. To accomplish this, the community must first agree on standards regarding minimal acceptable data representation and experimental reporting.

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Beyond technology Glycosciences have seen a boom over the past ten years. There is no question that the glycome is key to virtually all aspects of human health and disease processes, as glycans on the surface of cells are key to interactions and information transfer. The technologies that are needed have been identified, and first cores have crystallized in different countries. Now technologies such as analysis and sequencing need to be fundamentally improved by encouraging and funding basic science in the areas of analytical and synthetic chemistry. To fund coordinated largescale research efforts, funding agencies such as the NIH in the United States, the European Union and national funding agencies around the globe should work together not just to bundle resources in one country but to create transnational collaborations. In addition to coordinating efforts across country boundaries, we must seek scientists beyond the boundaries of traditional disciplines to help in the next phase of scientific development. Structural biologists can help by providing three-dimensional maps of these complex biomolecules. Imaging experts can help track down the location and density of glycoconjugates. Editors and editorial boards, in combination with community policing by attentive referees, can help enforce standards in reporting analytical data. Building a strong team will be an important part of future growth. It will also be important to involve the biotech industry and create markets. It cannot, of course, solely be the task of public agencies and journal editors to advance the glycosciences for the sake of fundamental science. The role of glycans in human disease is already beginning to open market opportunities in the areas of diagnostics, preventive medicine and therapeutics. Large pharmaceutical companies have initially been slow in warming to the glycomics field, which is perhaps not really surprising when we consider the lack of reliable technologies. However, despite the current economic situation, the glycosciences are primed to be a rapidly expanding field of the biotech industry. As technologies continue to improve and become more standardized, we are already beginning to see the appearance of startup companies in the glycosciences arena. Some first successes with carbohydrate-based companies such as GlycoFi, GlycArt and other role models exist. The development of carbohydrate-based biotechnology will bring important financial incentives, jobs for those trained in the field, and a return on public investment. This development will automatically draw scientists from other fields into the glycosciences and will get the interest of larger companies. We are currently building on a strong foundation of synthetic and analytical chemistry knowledge and critical questions in immunology and neurobiology, to name just a few relevant fields. The time for carbohydrate chemists and glycobiologists to stimulate this progress even further by increasing their interactions, developing consistent nomenclature to facilitate a common language, and developing and improving enabling technologies is now. Fundamental discoveries wait to be made and products have to be developed. It is a great time to join forces and advance chemical glycobiology.
COMPETING INTERESTS STATEMENT The author declares competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/ naturechemicalbiology/.
1. Kunz, H. Angew. Chem. Int. Ed. 41, 44394451 (2002). 2. Varki, A. Glycobiology 3, 97130 (1993). 3. Seeberger, P.H. & Werz, D.B. Nature 446, 10461051 (2007). 4. Varki, A. et al. Essentials of Glycobiology (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1999). 5. Boons, G.J. Carbohydrate Chemistry (Blackie Academic & Professional, Glasgow, Scotland, 1998). 6. Schmidt, R.R. Angew. Chem. Int. Edn Engl. 25, 212 235 (1986). 7. Dell, A. & Morris, H.R. Science 291, 23512356 (2001). 8. Venkataraman, G., Shriver, Z., Raman, R. & Sasisekharan, R. Science 286, 537542 (1999). 9. Plante, O.J., Palmacci, E.R. & Seeberger, P.H. Science 291, 15231527 (2001). 10. Sears, P. & Wong, C.-H. Science 291, 23442350 (2001). 11. Paulson, J.C., Blixt, O. & Collins, B.E. Nat. Chem. Biol. 2, 238248 (2006). 12. Ohtsubo, K. & Marth, J.D. Cell 126, 855867 (2006). 13. von der Lieth, C.-W. J. Carbohydr. Chem. 23, 277297 (2004). 14. Caruthers, M.H. Science 230, 281285 (1985).

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