Solid-Phase Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries Peter H. Seeberger* and Wilm-Christian Haase Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received April 12, 2000 Contents C. Synthesis of Deoxyglycosides 22 D. Synthesis of Thio-Oligosaccharides 22 I. Introduction 1 E. Miscellaneous Procedures 23 A. Challenges of Carbohydrate Chemistry 2 X. Synthesis of Complex Oligosaccharides 23 B. Advantages of Polymer-Supported Synthesis 3 A. Glycal Assembly Approach 23 C. Central Aspects of Solid-Phase 3 Oligosaccharide Synthesis B. Glycosyl Sulfoxides 24 II. Early Work 3 C. Glycosyl Trichloroacetimidates 25 III. Synthetic Strategies 7 D. Thioglycoside Donors 28 A. Donor-Bound Glycosylation Strategy 7 E. n-Pentenyl Glycosides 32 B. Acceptor-Bound Glycosylation Strategy 8 F. Glycosyl Phosphates 32 C. Bidirectional Strategy 8 XI. Synthesis of Combinatorial Carbohydrate 32 Libraries IV. Polymer Supports 8 A. Combinatorial Oligosaccharide Libraries 33 A. Insoluble Supports 8 B. Carbohydrates as Scaffolds for Combinatorial 36 B. Soluble Supports 10 Libraries V. Linker Systems 10 C. Special Methods for the Construction of 37 A. Silyl Ether Linkers 10 Carbohydrate-Related Libraries B. Acid- and Base-Labile Linkers 11 XII. Toward Automation of Oligosaccharide Synthesis 40 C. Thioglycoside Linkers 12 A. Solution-Phase Approaches 40 D. Linkers Cleaved by Oxidation 12 B. Automation of Solid-Phase Oligosaccharide 41 E. Linkers Cleaved by Hydrogenation 13 Synthesis F. Photocleavable Linkers 14 XIII. Conclusion and Outlook 42 G. Linkers Cleaved by Olefin Metathesis 14 XIV. List of Abbreviations 42 VI. Protecting Groups 16 XV. References 43 A. Benzyl Ethers 16 B. Base-Labile Protecting Groups 17 C. Acid-Labile Protecting Groups 17 I. Introduction D. Silyl Ether Protecting Groups 18 The transfer of information is a fundamental E. Other Protecting Groups 18 process of life and central to all cellular systems. VII. Glycosylating Agents Used for 18 From a biological perspective, information needs to Polymer-Supported Oligosaccharide Synthesis be transmitted intracellularly and intercellularly and A. Glycosyl Trichloroacetimidates 18 passed on from generation to generation. The three B. Glycosyl Sulfoxides 18 major biopolymers, proteins, nucleic acids, and gly- C. 1,2-Anhydrosugars 18 coconjugates, are mainly responsible for information D. Thioglycosides 19 transfer. While the biological importance of proteins E. Glycosyl Fluorides 19 and nucleic acids has been appreciated for a long F. n-Pentenyl Glycosides 19 time, oligosaccharides in the form of glycoconjugates are less well understood and have only more recently G. Glycosyl Phosphates 19 generated interest. Glycolipids and glycoproteins1 VIII. “On-Bead” Analytical Tools 19 play a major role in inflammation, immune response, A. High-Resolution Magic Angle Spinning NMR 19 metastasis, fertilization, and many other biomedi- Spectroscopy cally important processes.2,3 Specific carbohydrate 13 B. Gated-Decoupling C NMR Spectroscopy 20 structures have been identified as markers for certain C. FT-IR Microspectroscopy 21 types of tumors while others are binding sites for IX. Special Procedures for Polymer-Supported 21 bacterial and viral pathogens.4 Oligosaccharide Synthesis A major impediment to the rapidly growing field A. Orthogonal Glycosylations 21 of molecular glycobiology is the lack of pure, struc- B. “Gatekeeper Approach” 21 turally defined complex carbohydrates and glycocon- 10.1021/cr9903104 CCC: $36.00 © xxxx American Chemical Society Published on Web 00/00/0000 PAGE EST: 46 B Chemical Reviews Seeberger and Haase increased in recent years, the synthesis of these complex molecules remains time-consuming and is carried out by a few specialized laboratories. Oligo- nucleotides6 and oligopeptides,7 on the other hand, are now routinely prepared on automated synthesiz- ers, providing pure substances in a rapid and efficient manner. The effect of an automated oligosaccharide synthesizer on the field of glycobiology may be readily envisioned when considering the impact of automated solid-phase peptide and oligonucleotide synthesis on the biochemistry of these molecules. Solid-phase synthesis lends itself particularly well to automation and will be the focus of this review. This retrospective begins with a brief outline of the Peter H. Seeberger received his B.S. degree in 1989 from the Universita¨t central issues of carbohydrate chemistry. After re- Erlangen-Nu¨rnberg, where he studied chemistry as a Bavarian government viewing the early work in the field from 1970 to 1991, fellow. In 1990 he moved as a Fulbright Scholar to the University of different synthetic strategies will be discussed. The Colorado, where he earned his Ph.D. degree in Biochemistry under the linkers used to connect the first monosaccharide to guidance of Marvin H. Caruthers in 1995. After a postdoctoral fellowship the polymeric support will be covered as will be with Samuel J. Danishefsky at the Sloan-Kettering Institute for Cancer special protecting groups that were developed for use Research in New York City, he began his independent career at the Massachusetts Institute of Technology in January 1998. Since 1999 he on the solid support. Efforts to apply different gly- has been the Firmenich Assistant Professor of Chemistry. His research cosylating agents to the assembly of oligosaccharides interests focus on the interface of chemistry and biology and in particular on a polymer matrix will be reviewed, followed by a on the role of complex carbohydrates and glycoconjugates in information description of on-resin analytical methods. Next, a transfer in biological systems. His group has developed new methods for brief summary of special procedures for the assembly the automated solid-phase synthesis of complex carbohydrates and of unusual or difficult structures will be followed by glycosaminoglycans that serve as molecular tools. Other interests include synthetic methodology, total synthesis, immunology, and biochemical and an extensive review of a range of complex oligosac- biophysical studies of carbohydrates. charides prepared on polymer support. Finally, the efforts of different groups directed at the preparation of carbohydrates will be covered. The focus of all discussions will be on chemical methods, while enzymatic approaches will be mentioned where ap- propriate. The review will conclude with a compari- son of the currently available methods and the prospects for the development of an automated solid- phase oligosaccharide synthesizer. A. Challenges of Carbohydrate Chemistry Organic chemists have been intrigued by the synthesis of complex oligosaccharides for over 100 years. The assembly of these natural products pre- sents two crucial challenges. A multitude of func- tional groups (amino and hydroxyl) of similar reac- Wilm-Christian Haase, born 1969, received his chemistry Diplom from tivity on each monomer emphasize the need for the Rheinische Friedrich-Wilhelms-Universita¨t Bonn, Germany, in 1996, where he joined the group of Professor Dr. K. H. Do¨tz. During his effective differentiation to allow for access to branched dissertation work he explored the synthesis and application of Fischer- structures. Furthermore, a new stereogenic center is type glycosylidene complexes as novel organometallic reagents for the created each time a glycosidic linkage is formed and assembly of C-glycosidic linkages. As a DAAD/NATO-postdoctoral fellow, complicates matters far beyond the synthetic situa- he then joined the group of Professor Dr. P. H. Seeberger at the tion encountered with peptides and nucleic acids. The Massachusetts Institute of Technology, Cambridge, MA, in 1999, where need to purify the reaction products by chromato- he focused on the development of methods for the combinatorial solid- phase synthesis of human milk oligosaccharides. His research interests graphy after each step makes oligosaccharide syn- include developing novel transition-metal-catalyzed as well as solid- thesis a laborious, time-consuming, and expensive supported methods for the synthesis of C-saccharides. task. jugates. Besides the fact that these molecules are Over the years a host of protective groups for the often found only in low concentrations in nature, the masking of amino and hydroxyl groups has been identification and isolation of complex carbohydrates introduced by taking advantage of the reactivities of from natural sources is greatly complicated by their the respective protective groups. Even more impor- microheterogeneity. Detailed biophysical and bio- tantly, a variety of anomeric groups that allow for chemical studies of carbohydrates require sufficient the high-yielding, selective, and reliable formation of quantities of defined oligosaccharides. The procure- many glycosidic linkages have been developed. While ment of synthetic material presents a formidable much progress has been made, some linkages still challenge to the synthetic chemist.5 While the need remain difficult to install. In particular, the synthesis for chemically defined oligosaccharides has steadily of large, branched oligosaccharides presents multiple Solid-Phase Oligosaccharide Synthesis Chemical Reviews C difficulties. Steric and electronic changes in either linkages in the product was assumed based on the of the coupling partners can make each new glyco- comparison of the optical rotation with model struc- sidic bond to be created a challenge. tures from solution-phase synthesis. These