Glycochemistry: Principles: Synthesis, and Applications

ISBN: 0-8247-0538-6 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-261-8482; fax: 41-61-261-8896 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright 䉷 2001 by Marcel Dekker, Inc.

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Preface

Ever since Emil Fischer established the stereochemical configuration of D-(⫹)-glucose at the end of the nineteenth century, carbohydrate chemistry has become an important branch of organic chemistry. Steady advances in carbohydrate synthetic and analytical methods have been made over the past hundred years. However, it is the past two decades that have seen remarkable new discoveries in the biology of carbohydrates and provided renewed impetus to the synthesis of complex glycoconjugates and pursuit of their medicinal and industrial applications. Glycobiology has emerged as an internationally recognized field of study and a potentially promising route to the discovery of novel medicines. Critical biological processes—including regulation of the growth and mobility of cells, immune responses, and reactions of cells to hormones and growth factors—all depend on carbohydrates. In addition, many viruses and bacteria use cell surface carbohydrates to get into cells and initiate infections. The diversity of roles played by carbohydrates makes them exciting new targets for the development of novel treatments for cancer, infectious diseases, inflammation, and cardiovascular disorders. The spur in glycobiology research resulted in the appearance of several enormously successful carbohydrate-based drugs including but certainly not limited to heparin (an anticoagulant) and Relenza (an antiviral). Increasing numbers of organic chemists are venturing into the field of glycochemistry to bring in their own chemical expertise and to expand the area into a truly multidisciplinary field. A major motivation among carbohydrate chemists is to develop tools for probing the biological functions of glycosylation. Genome sequencing projects present a vast repertoire of biosynthetic enzymes that may be targeted by selective carbohydrate-based drugs. Advances in automated oligosaccharide synthesis are highly significant as complex structures will soon be widely attainable and exploited in studies of biological phenomena. Their integration into complex assemblies (polymers, dendrimers, and liposomes) will provide cell surface mimics of unparalleled fidelity. Studies of glycobiology will benefit from the convergent application of chemical and biological techniques. Thus, in the past few years, glycochemistry has advanced from studies of chemical glycosylation reactions and synthesis of oligosaccharides to iii

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Preface

chemo-enzymatic and solid-phase synthesis of glycoconjugates, carbohydrate combinatorial libraries, multivalent glycoconjugates, carbohydrate–peptide hybrids, and much more. It is the aim of this book to capture the essence of these latest developments and to provide a comprehensive review of modern glycochemistry in one collected volume. In recognition of the great potential applications of glycochemistry in the pharmaceutical and chemical industries, we solicited chapters that contain pertinent examples of the development of carbohydrate-based pharmaceuticals and commercial polymers. Each chapter is written by experts in their respective fields. Many are young scientists who have just begun research programs in carbohydrate chemistry. Although some chapters include relatively detailed experimental procedures for novel synthetic methodology, the book is intended to provide a reference framework for the latest developments in the field of glycochemistry. Recent trends in glycochemistry are presented in three parts: synthesis, principles and applications. The first six chapters provide a comprehensive, up-to-date review on the chemical synthesis of complex carbohydrates for their potential use in biological systems. The following seven chapters reveal some fundamental principles that are used to design and exploit carbohydrates for their effects in biological settings. The remaining five chapters examine the applicability of enzymes towards the chemo-enzymatic synthesis and modification of carbohydrates and polysaccharides. In Chapter 1, Peter Seeberger reflects on the promising strategies for the solid support synthesis of oligosaccharides and glycoconjugates. The chapter puts in focus the glycal assembly method and reveals protecting group strategies for carbohydrate components, synthesis of biologically important oligosaccharides, and selective glycosylation strategies. Chapters 2 and 3 bring to light the potential of strategies for stereoselective glycosylation. David Gin examines a number of methodologies used in the glycosylation of 1-hydroxy donors and points out that in the direct dehydrative coupling with an appropriate dehydrating agent, the formation of undesired by-products of hemiacetal self-condensation can be minimized even when only a slight excess of the glycosyl acceptor components is employed. David Crich examines the glycosyl triflates as extremely reactive glycosyl donors. They are prepared from anomeric sulfoxides or thioglycosides upon activation with triflic anhydride or benzenesulfenyl triflate and provide access to the elusive ␤-mannopyranoside, which can be found in many biologically important oligomers. Hydrolytic vulnerability of O-linked glycosides makes the C-glycosides attractive sugar mimics and stable drug candidates. Maarten Postema and Daniel Calimente (Chapter 4) report on the many methods for the preparation of both ␣- and ␤-Cglycosides that have been developed, as well as some new approaches currently in the literature. One of the key issues surrounding the synthesis of C-glycosides concerns the stereochemistry of the C-glycosidation step, which has seen a remarkable improvement over the years. As C-glycosides make their way to the forefront of carbohydrate synthesis, many mild conditions will be examined, and these sophisticated synthetic methods will facilitate their production. Todd Lowary (Chapter 5) focuses on two major polysaccharides containing Darabinofuranose, namely, arabinogalactan and lipoarabinomannan, in a quest to identify new antibiotics. These glycopolymers are found as important components of the cell wall of the Actinomycete family. Although there is now an understanding of the

Preface

v

structure of mycobacterial arabinan, the biosynthetic pathway is yet to be understood. These investigations will depend critically on the access to synthetic oligosaccharides and their analogs, which are reviewed extensively. In Chapter 6, Biao Yu and Yongzheng Hui discuss the relevance of saponins and their chemical synthesis. This important class of glycosidic steroids is found mainly in plants used as herbal medicines. However, a major issue surrounding the saponins is their ambiguous mechanism of action. The authors discuss two important strategies for the construction of saponins, each involving protective group manipulation and various glycosylation methodologies. Increasing interest in traditional herbal medicine and in ‘‘carbohydrate drugs’’ should bring further attention to this group of natural glycosides. In Chapters 7–13, the focus shifts to the biochemistry of carbohydrates and the exploration of fundamental principles related to cell signaling and protein–carbohydrate interactions on a molecular level. Sialic acids are a class of nine-carbon monosaccharides found at the termini of oligosaccharides in many mammalian cellular systems. Randall Halcomb and Mark Chappell (Chapter 7) summarize the most current sialylation technology. Both chemical and enzymatic methodologies for coupling sialic acid to various carbohydrate moieties are examined while providing the reader with chemical detail that one requires to work in this field. In Chapter 8, David Mann and Laura Kiessling explore a number of fundamental binding principles such as the energetics surrounding hydrogen bonding, metal chelation, hydrophobic effects, coulombic interactions, and the role of water in protein–carbohydrate interactions. The information provides a prelude to the concept of multivalency in understanding the principles, methodologies, and techniques used to increase an overall binding affinity between carbohydrates and proteins. One technique used to achieve multivalency revolves around linking carbohydrates to polymeric supports so as to increase the number of binding sites as well as obtain optimal distal properties for effective binding. Rene´ Roy (Chapter 9), another pioneer in the application of multivalency effects in glycobiology, discusses some novel multivalent glycotools for biochemical investigations related to sialic acid. Roy describes methodology to produce sialyloligosaccharides, functionalized sialosides, sialoside clusters, N- and O-linked glycopeptides, amphiphilic calix[4]arene nanostructures, glycopolymers, and glycodendrimers. Hybrid dendrimer–polymer glycoclusters are also introduced as new and exciting complexes to show increased binding affinity. In Chapters 10 and 11, Jalal Haddad and Shahriar Mobashery, Lakshmi Kotra, and Mei-Zheng Liu examine the diversity of aminoglycosides and their significant role as antibiotics. They reveal the specificity of aminoglycosides for binding to RNA constructs derived from the HIV–RRE and TAR RNA activator regions and examine the varying inhibitory effects of aminoglycans on different microorganisms. They also introduce methodologies in the synthesis of aminoglycoside antibiotics by examining the chemical strategies that have been developed. In Chapter 12, Bryan Yeung, Pek Chong, and Peter Petillo survey the chemical preparation of the glycosaminoglycans, oligosaccharides of hyaluronan, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, and heparan sulfate. Despite the biological importance of these ubiquitous carbohydrate polymers, there are few reports of the chemical syntheses of glycosaminoglycan (GAG) oligosaccharides. The multiple functionality of GAGs provides an excellent scaffold on which structure–

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Preface

activity relationships can be studied, but their syntheses present an unparalleled challenge to the synthetic carbohydrate chemist. The authors recognize the difficulty in the construction of these highly functionalized carbohydrates and present newly developed protecting groups as well as glycosylation procedures to synthetically access GAG fragments. In Chapter 13, Jacquelyn Gervay-Hague and Thomas Weathers, Jr., discuss some of the more recent chemistry behind pyranosyl sugar amino acids and the important role they play in biological systems. The authors make a complex group of carbohydrates seem relatively simple and interesting by addressing the important issues and illustrating them with clear, concise pictorial schemes. Included are the synthesis of C-aminoglycosides, protecting groups, the chemistry of glycosidic linkage, strategies for block synthesis, and the introduction of solid-phase synthesis of mixed sugar amino acid conjugates. The emphasis of this chapter is twofold: to describe the synthesis of aminoglycans oligomers and to examine the stable secondary structural characteristics of amido-linked oligomers. Chapters 14–18 are meant to pioneer a chemo-enzymatic approach to the construction of carbohydrates. One of the underlying themes of this book is the exploitation of the complexity of carbohydrates and applying various methodologies to simplify their production. In Chapter 14, Xiangping Qian, Keiko Sujino, Monica Palcic, and Murray Ratcliffe discuss the potential of glycosyltransferases in oligosaccharide synthesis. The authors focus on the enzymes responsible for creating various glycosidic linkages and the sugar donor requirements for glycosyltransferases as well as giving some detailed information for applying this knowledge to largescale carbohydrate synthesis. Chapter 15, written by H. N. Cheng and Qu-Ming Gu, discusses the application of biotransformations to polysaccharides modification. Polysaccharides are natural materials ideally suited for enzymatic modifications. The chapter introduces lipasecatalyzed and ␤-galactosidase–catalyzed modifications of carbohydrate polymers. Peter Andreana, Wei Zhang, and Peng George Wang (Chapter 16) take a close look at the ␣-gal epitope as a case study. This carbohydrate sequence is responsible for the hyperacute organ rejection associated with xenotransplantation. Efficient production of this carbohydrate epitope can lead to increased studies directed toward solving the xenotransplantation conundrum. The authors examine both chemical and enzymatic routes for synthesizing the epitope and present some important physical properties associated with binding of the carbohydrate to the protein (epitope–antibody interaction). Bacterial glycosyltransferases, the topic of Chapter 17, by Przemyslaw Kowal, Xi Chen, and Peng George Wang, are enzymes responsible for the assembly of bacterial cell walls (e.g., succinoglycan) and lipopolysaccharides or polysaccharide structures attached to the lipids of the outer membrane of gram-negative bacterial cells. Lipopolysaccharides are at the forefront of bacterial interactions with the outside world. These structures have been found to be essential in processes ranging from root nodulation to human pathogenicity. Therefore studies on the corresponding glycosyltransferases are of great importance and interest. The authors mention an emerging technology using biosynthetic pathway engineering to produce carbohydrates on a large scale. We envision that such technology will be rapidly developed into commercial use.

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Scarlett Goon and Carolyn Bertozzi close the book with a chapter discussing metabolic substrate engineering as a tool for glycobiology. In this approach, metabolic pathways are intercepted with unnatural monosaccharide substrates, leading to their incorporation into cell surface oligosaccharides. This provides an innovative method for studying the functions of the surface sugar structures. Modified substrates might also block biosynthetic enzymes, producing phenotypes similar to those induced by glycosyltransferase inhibitors. This chapter focuses on the flux of carbohydrate precursors and synthetic carbohydrate analogs through the metabolic pathways of the cell and the information that can be gained from investigating such processes. This book is intended for graduate students and researchers in carbohydrate chemistry, biochemistry, medicinal chemistry, and glycobiology in both academic and industrial laboratories. We feel privileged to have attracted such a distinguished group of investigators and express our sincerest gratitude for their time and effort in making this endeavor a meaningful contribution. Peng George Wang Carolyn R. Bertozzi

Contents

Preface Contributors Introduction: New Challenges in Carbohydrate Research

Chi-Huey Wong

1. Solid Phase Oligosaccharide Synthesis Peter H. Seeberger

iii xi xv 1

2. Dehydrative Glycosylation with 1-Hydroxy Donors David Gin

33

3. Chemistry of Glycosyl Triflates: Synthesis of ␤ -Mannopyranosides David Crich

53

4. C-Glycoside Synthesis: Recent Developments and Current Trends Maarten H. D. Postema and Daniel Calimente

77

5.

D-Arabinofuranosides from Mycobacteria: Synthesis and Conformation Todd L. Lowary

133

6. Chemical Synthesis of Bioactive Steroidal Saponins Biao Yu and Yongzheng Hui

163

7. Recent Developments in Technology for Glycosylation with Sialic Acid Randall L. Halcomb and Mark D. Chappell

177

8. The Chemistry and Biology of Multivalent Saccharide Displays David A. Mann and Laura L. Kiessling

221

9. Designing Novel Multivalent Glycotools for Biochemical Investigations Related to Sialic Acid Rene´ Roy 10. Aminoglycoside Antibiotics: Structures and Mechanisms of Action Jalal Haddad, Lakshmi P. Kotra, and Shahriar Mobashery

277 307 ix

x

Contents

11. Methodologies in Syntheses of Aminoglycoside Antibiotics Jalal Haddad, Mei-Zheng Liu, and Shahriar Mobashery

353

12. Synthesis of Glycosaminoglycans Bryan K. S. Yeung, Pek Y. C. Chong, and Peter A. Petillo

425

13. Pyranosyl Sugar Amino Acid Conjugates: Their Biological Origins, Synthetic Preparations, and Structural Characterization Jacquelyn Gervay-Hague and Thomas M. Weathers, Jr.

493

14. Glycosyltransferases in Oligosaccharide Synthesis Xiangping Qian, Keiko Sujino, R. Murray Ratcliffe, and Monica M. Palcic

535

15. Biotransformation of Polysaccharides H. N. Cheng and Qu-Ming Gu

567

16. From a Carbohydrate Sequence to Extensive Research Programs: The ␣-gal Epitope as a Case Study Peter R. Andreana, Wei Zhang, and Peng George Wang

581

17. Microbial Glycosyltransferases Przemyslaw Kowal, Xi Chen, and Peng George Wang

625

18. Metabolic Substrate Engineering as a Tool for Glycobiology Scarlett Goon and Carolyn R. Bertozzi

641

Index

675

Contributors

Peter R. Andreana Michigan

Department of Chemistry, Wayne State University, Detroit,

Carolyn R. Bertozzi Departments of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, and University of California, Berkeley, California Daniel Calimente Michigan

Department of Chemistry, Wayne State University, Detroit,

Mark D. Chappell Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado Xi Chen

Department of Chemistry, Wayne State University, Detroit, Michigan

H. N. Cheng

Hercules Incorporated, Wilmington, Delaware

Pek Y. C. Chong Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois David Crich Illinois

Department of Chemistry, University of Illinois at Chicago, Chicago,

Jacquelyn Gervay-Hague son, Arizona

Department of Chemistry, University of Arizona, Tuc-

David Gin Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois Scarlett Goon Departments of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, and University of California, Berkeley, California Qu-Ming Gu Jalal Haddad igan

Hercules Incorporated, Wilmington, Delaware Department of Chemistry, Wayne State University, Detroit, Mich-

Randall L. Halcomb Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado xi

xii

Contributors

Yongzheng Hui State Key Laboratory of Bio-organic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China Laura L. Kiessling Departments of Chemistry and Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin Lakshmi P. Kotra Michigan

Department of Chemistry, Wayne State University, Detroit,

Przemyslaw Kowal Michigan

Department of Chemistry, Wayne State University, Detroit,

Mei-Zheng Liu Michigan

Department of Chemistry, Wayne State University, Detroit,

Todd L. Lowary Ohio

Department of Chemistry, The Ohio State University, Columbus,

David A. Mann Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin Shahriar Mobashery Michigan Monica M. Palcic Alberta, Canada

Department of Chemistry, Wayne State University, Detroit,

Department of Chemistry, University of Alberta, Edmonton,

Peter A. Petillo Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois Maarten H. D. Postema Detroit, Michigan Xiangping Qian Alberta, Canada

Department of Chemistry, University of Alberta, Edmonton,

R. Murray Ratcliffe Rene´ Roy Canada

Department of Chemistry, Wayne State University,

SYNSORB Biotech Inc., Calgary, Alberta, Canada

Department of Chemistry, University of Ottawa, Ottawa, Ontario,

Peter H. Seeberger Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts Keiko Sujino Canada

Department of Chemistry, University of Alberta, Edmonton, Alberta,

Peng George Wang Michigan

Department of Chemistry, Wayne State University, Detroit,

Thomas M. Weathers, Jr. son, Arizona

Department of Chemistry, University of Arizona, Tuc-

Contributors

xiii

Bryan K. S. Yeung Department of Chemistry, University of Illinois at UrbanaChampaign, Urbana, Illinois Biao Yu State Key Laboratory of Bio-organic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China Wei Zhang

Department of Chemistry, Wayne State University, Detroit, Michigan

Introduction: New Challenges in Carbohydrate Research

Over the past 30 years, most of the important discoveries in the biological sciences have been associated with proteins and nucleic acids, and thus chemists have been most active in conducting their research to address the relationship between the structure and function of these macromolecules as well as to develop small molecules as mechanistic probes or drug candidates to target these two types of biopolymers. This rapid progress of development is undoubtedly due to—other than the importance of proteins and nucleic acids in biological functions—the availability of various powerful tools (such as the polymerase chain reaction, solid-phase synthesizers, sequence analysis, and sensitive assay systems) to both chemists and biologists, and the close interplay between these two disciplines. Carbohydrates, especially oligo- and polysaccharides and their conjugates with proteins and lipids, have been neglected. Although carbohydrates display enormous structural and functional diversity—fulfilling roles as energy sources and as elements for recognition and for the control of structure, function, and dynamics of proteins —their precise functions at the molecular level are poorly understood. This slow pace of development in carbohydrate research is due mainly to the lack of tools available to the biologist interested in carbohydrates, and to a lesser extent to the chemist. No amplification method is available to prepare functionally important oligo- or polysaccharides in quantities large enough for structural analysis; no general method is available for the synthesis of glycoproteins with well-defined carbohydrate structures to investigate the roles that carbohydrates play in glycoprotein structure and function; and no solid-phase synthesizer is available for the synthesis of oligoand polysaccharides. From a medicinal chemistry point of view, carbohydrates are an uninteresting class of molecules for drug development because they are too difficult to synthesize on a large scale and too hydrophilic to have good bioavailability, and they are generally orally inactive and unstable. Perhaps the most fundamental problem is that carbohydrates bind their receptors or enzymes with weak affinity, usually with dissociation constants in the millimolar range. Polyvalent interactions are, however, xv

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Introduction

often used in sugar-mediated intercellular recognitions to increase affinity and specificity and to control or regulate function (such as signaling and cell surface protein shedding). The weak affinity in sugar–receptor interactions is attributed to a few causes: 1) there is a lack of hydrophobic groups in sugars which are often dominant in high-affinity receptor–ligand interactions; 2) the energy associated with multiply hydrogen-bond and other electrostatic interactions in sugar–receptor recognitions is significantly diminished by competition from bulk solvent; and 3) the flexible nature of many hydrogen-bond interactions results in a significant entropic penalty when they become constrained in space upon binding. In addition, it appears that not all functional groups of a carbohydrate ligand are essential for binding. Despite these bottlenecks, it is important to develop new strategies and methods to study carbohydrate-mediated biological recognition processes and to investigate their mechanisms of action, since understanding the mechanism of carbohydrate function may lead to the development of carbohydrate-based therapeutics. Recent advances in carbohydrate chemistry and biology have helped to solve some of the problems. Of particular significance are, for example, the development of new methods for the synthesis of complex carbohydrates, glycolipids, and glycoproteins, the design of carbohydrate mimetics including polyvalent mimetics and glycosyltransfer enzyme inhibitors, the manipulation of cell surface carbohydrates to probe intercellular interactions, the development of carbohydrate-based antibiotics targeting RNA and cell surface peptidoglycans, the study of sulfation in carbohydrate recognition, the study of carbohydrate effect on protein structure and function, and the development of carbohydrate-based vaccines. Future efforts perhaps may be directed to: 1) determine the structures of glycosyltransferases and postmodification enzymes such as sulfotransferases and carbohydrate receptors involved in metastasis, inflammation, and immunoresponse (e.g., CD1-glycolipid–TCR complex); 2) understand carbohydrate-mediated intercellular interaction and signaling; 3) design small molecules targeting carbohydrate–receptor and carbohydrate–enzyme interactions; and 4) develop efficient and rapid methods for the synthesis of oligosaccharides and glycoproteins and for their structural and functional analyses. In this new era of functional genomics, it is anticipated that more tools will be developed to facilitate discovery in glycobiology and that carbohydrate-based therapeutic breakthroughs will be realized. Chi-Huey Wong The Scripps Research Institute La Jolla, California

1 Solid Phase Oligosaccharide Synthesis Peter H. Seeberger Massachusetts Institute of Technology, Cambridge, Massachusetts

I.

INTRODUCTION

Polysaccharides are the structurally most complex of the major classes of biopolymers. While nucleic acids and proteins are linear assemblies, polysaccharides are structurally and stereochemically more diverse. Additional complexity is added to the polysaccharide structure by the formation of glycoconjugates such as glycolipids and glycoproteins [1]. Oligosaccharides in the form of glycoconjugates mediate a variety of events, including inflammation, immunological response, metastasis, and fertilization [2,3]. Cell surface carbohydrates act as biological markers of various tumors and as binding sites for other substances, including pathogens [4]. The increased understanding of the important roles oligosaccharides and glycoconjugates play in fundamental life-sustaining processes has stimulated a need for access to usable quantities of these materials. Glycoconjugates commonly exist as microheterogeneous mixtures and are difficult to isolate in homogeneous form, therefore yielding only small amounts of the desired material. The problems associated with isolation from natural sources give rise to opportunities for chemical synthesis [5]. The developments in synthetic carbohydrate chemistry have centered on finding solutions to two important challenges: the need to differentiate similar functionality (hydroxyl or amino) contained on each monosaccharide and the need to induce the selective formation of a variety of glycosidic linkages. Unlike linkages found in the other two classes of repeating biopolymers, each glycosidic bond to be fashioned in a growing oligosaccharide ensemble constitutes a new locus of stereogenicity. In response to these two key challenges, a wide variety of protecting group strategies 1

2

Seeberger

[6] and increasingly powerful and selective glycosylating agents have been developed [7]. Still, the synthesis of complex oligosaccharides and glycoconjugates remains a difficult and time-consuming task. The introduction of solid support synthesis methods for preparation of structurally defined oligopeptides [8] and oligonucleotides [9] has led to tremendous improvements in terms of synthesis speed and efficiency. The advantages of solid phase synthesis, such as maximized yields by use of excess reagents, ease of purification, and synthesis automation, are now well appreciated. The biopolymers obtained by these rapid synthetic methods significantly impacted the fields of peptide and nucleic acid biochemistry. Glycobiology would benefit in many ways from straightforward synthetic methods, which could be used by nonexperts to prepare oligosaccharides. Since the level of complexity associated with the synthesis of oligosaccharides on a polymer support is much greater than that associated with the other two classes of repeating biooligomers, this task seemed for a long time too difficult to tackle. The development of protocols for the solid support synthesis of oligosaccharides and glycopeptides requires the scientists in the field to find solutions to several problems: (1) selection of an overall synthetic strategy and development of methods for attachment of the carbohydrate to the polymeric support through the ‘‘reducing’’ or the ‘‘nonreducing’’ end, (2) choice of a solid support material, (3) selection of a linker (‘‘support-bound protecting group’’) that is stable during the synthesis but can be easily cleaved when desired, (4) a highly flexible protecting group strategy, (5) stereospecific and high-yielding coupling reactions, and (6) ‘‘on-resin’’ methods to monitor chemical transformations. Pioneering efforts in the area of solid support oligosaccharide synthesis were undertaken in the early 1970s by several research groups, utilizing the limited set of glycosylating agents available at the time. While some fundamental issues were explored, the lack of glycosylation reactions compatible with polymeric supports impeded further progress. This early work has been reviewed [10] and is not covered in this chapter. Since these initial attempts, a great deal of progress has been achieved in assembling relatively complex carbohydrate ensembles through simplification and refinement of protecting group strategies and the development of new and powerful glycosylation methodology. This progress is a result of the efforts that have focused on solid support oligosaccharide synthesis since the early 1990s. The different approaches to solid support oligosaccharide synthesis by chemical rather than enzymatic methods are summarized in this chapter. Much effort has been focused on the use of soluble, polyethylene glycol (PEG) based polymeric supports for the synthesis of oligosaccharides. Reaction development using these soluble polymers has proven quite facile, since the reactions are run under homogeneous conditions and conventional analytical methods may be employed [11]. A number of glycosylation strategies have been explored by Krepinsky and coworkers [12] and van Boom et al. [13]. An orthogonal glycosylation strategy in conjunction with soluble polymeric supports has been developed [14]. In 1996 a very innovative ‘‘gatekeeper’’ approach [15] to the synthesis of oligosaccharides containing ␤-mannosidic linkages was introduced. This work, which used a PEG polymer [16], was reviewed in 1998 [17] and is not covered in this chapter.

Solid Phase Oligosaccharide Synthesis

II.

3

SYNTHETIC STRATEGIES

Three major strategies have been explored for the solid support synthesis of oligosaccharides and glycoconjugates. In one variation, the first carbohydrate is anchored to the support via its ‘‘reducing’’ end (see Scheme 1, Case 1). The carbohydrate bound to the solid support functions as an acceptor in the coupling event to a solution-based donor (D). In the cycle, a unique acceptor hydroxyl must be exposed

Scheme 1

Glycosyl acceptor (Case 1) and donor (Case 2) bound to the solid support, and bidirectional synthesis (Case 3): S, solid support; P, unique protecting group; X, activating group; asterisk, uniquely differentiated hydroxyl group.

4

Seeberger

in the now elongated, resin-bound carbohydrate construct. In Case 1, this strategy virtually demands for the donor (D) employed in the preceding glycosidation step a uniquely removable blocking group at the site of the next proposed elongation. The need to expose the unique hydroxyl group in the context of the polymer support will necessitate multiple functional group manipulations in synthesizing D. Alternatively, the carbohydrate that is to undergo elongation may be mounted to the support somewhere in a ‘‘nonreducing’’ region, thereby making the reducing end available as a glycosyl donor for coupling with solution based acceptor A (Case 2). The use of A, of course, demands that the precise acceptor site be properly identified. In anticipation of the next coupling event, the reducing end of acceptor A is so functionalized that a new donor capability can be installed at the anomeric carbon of the elongated construct. This approach necessitates in the acceptor an anomeric group that does not serve as a glycosyl donor itself but rather may be converted in a straightforward manner into a glycosyl-donating moiety. A hybrid of both strategies is the bidirectional synthesis approach (Case 3), which is based on the orthogonal glycosylation concept. The first monosaccharide (A/D) may serve as glycosyl donor as well as glycosyl acceptor and is attached to the support matrix through a position other than the anomeric center. This glycoside contains an anomeric leaving group (as for Case 2), and in addition exhibits an uniquely distinguished acceptor site (analogous to Case 1). During the initial phase of the synthesis the support-bound sugar functions as a glycosyl acceptor A without compromising the chemical integrity of the anomeric moiety. In this fashion, oligosaccharide synthesis following the Case 1 paradigm may be carried out. If an orthogonal set of glycosylating conditions is used, the support-bound sugar may be activated to serve as a glycosyl donor in chain elongation to follow a Case 1 synthetic scheme. This chapter discusses the different approaches to solid phase oligosaccharide synthesis developed to date, grouped by the overall synthetic paradigm they obey.

III.

THE GLYCOSYL DONOR BOUND STRATEGY: THE GLYCAL ASSEMBLY APPROACH

A.

Overview of the Glycal Assembly Approach

While the donor-bound paradigm (Case 2) minimizes the number of protecting group manipulations that have to be carried out on the solid support, it mandates the presence of a latent glycosyl donor moiety in the solution-based glycosyl acceptor. Glycal building blocks fulfill these requirements, since they serve as glycosyl acceptors but may be converted into powerful glycosylating agents. Danishefsky and coworkers successfully explored the application of the glycal assembly method to the solid support synthesis of oligosaccharides and glycoconjugates [18]. The general principle of the glycal assembly method is outlined in Scheme 2. Attachment of a glycal to a solid support through the nonreducing end results in the presence of a terminal glycal function that may be readily converted into donor 2. Since glycals are employed as the solution-based acceptor, the protecting group scheme used for the identification of hydroxyls predestined for glycosylation remains relatively simple. Compound 2 could be a 1,2-anhydro sugar [19], derived by reaction with an epoxidizing agent, or it could be a transient species, derived by activation with an

Solid Phase Oligosaccharide Synthesis

5

Scheme 2 Use of the glycal assembly method in a general strategy for the synthesis of oligosaccharides on a solid support.

iodonium source [20]. Support-bound 2 acts as a glycosyl donor when treated with a solution of acceptor glycal 3, along with any necessary agents to promote the glycosylation, thereby forming 4. The process can be repeated to assemble the desired oligosaccharide, followed by retrieval from the support and purification by chromatographic methods. All studies on the solid support synthesis of oligosaccharides by the glycal assembly approach employed a polystyrene 1% divinylbenzene copolymer, which is commonly used in solid support peptide synthesis because of its high loading capacity, compatibility with a wide range of reaction conditions, and low price. The first glycal was linked to the solid support through a disilane linkage that could be cleaved rapidly and completely by treatment with fluoride [21]. The validity of the approach was first demonstrated by the synthesis of a linear tetrasaccharide [22] and a hexasaccharide 13 [23] as outlined in Scheme 3. Polymerbound galactal 5 was converted to the 1,2-anhydro sugar 6 by epoxidation with 3,3dimethyldioxirane [24]. Polymer-bound 6 acted as a glycosyl donor when reacted with a solution of 7 in the presence of zinc chloride, resulting in the formation of disaccharide 8a. Upon repetition, this glycosylation procedure accommodated the secondary alcohol glycosyl acceptor 10 as well as disaccharide acceptor 12. Fluoridolysis with tetrabutylammonium fluoride (TBAF) was used to cleave the desired products from the polymeric support and furnish hexasaccharide 13 in 29% overall yield from 5 [16]. B.

Solid Phase Synthesis of Blood Group Determinants

Carbohydate blood group determinants in the form of glycoproteins or glycolipids were found to play key roles in cell adhesion and other binding phenomena [25,26]. Furthermore, glycoconjugates related to these blood group substances have been recognized as markers for the onset of various tumors. These tumor-associated antigens are currently being studied in vaccines for cancer immunotherapy [27,28]. The

6

Seeberger

Solid Phase Oligosaccharide Synthesis

Scheme 3

Solid phase synthesis of a hexasaccharide by the glycal assembly method.

7

8

Seeberger

glycal assembly approach outlined above found its first applications in the synthesis of a H-type 2 tetrasaccharide (Scheme 4) [29,30]. Treatment of polymer-bound 1,2-anhydro sugar 6 with a solution of glucal acceptor 14 provided disaccharide 15. Upon opening of a 1,2-anhydro sugar during glycosylation, a C2 hydroxyl group is exposed, which may in turn serve as a glycosyl acceptor to form branched oligosaccharides [31]. Compound 15 was fucosylated using a solution of fucosyl donor 16 [32] to furnish trisaccharide 17. Treatment of 17a with TBAF provided trisaccharide glycal 17b in 50% overall yield from 5. Because of the lack of solid support methodology to fashion glycosidic linkages bearing C2-acylamino functions, solution phase chemistry had to be employed to access the H-type 2 blood group determinant glycal 18. Ready functionalization at the reducing end was achieved through the terminal glycal handle. The Lewisb blood group antigen (Leb) is of particular interest because it has been identified as a mediator for the binding of Helicobacter pylori to human gastric epithelium [33]. Clinical studies have identified H. pylori as a causative agent in gastric and duodenal ulcers [34], and antimicrobial treatments are an effective means to combat infection [35]. Since bacterial attachment is a prerequisite for infection [36], analogs of the Leb oligosaccharide may serve as therapeutic alternatives to broad spectrum antibiotics. The first approach to Leb addressed the synthesis of the core tetrasaccharide 22, which was assembled on the polymer support as depicted in Scheme 5 [37]. Polymer-bound galactal 19 was epoxidized with dimethyldioxirane and then reacted with a solution of glucal derivative 20 to give disaccharide diol 21. This reaction proceeded in highly regioselective fashion wherein glycosylation occurred at the allylic position at C3 of 20. Bisfucosylation of 21 using donor 16 provided polymerbound tetrasaccharide glycal 22a. Treatment of 22a with TBAF gave 22b, which

Scheme 4

Solid phase synthesis of an H-type blood group determinant by the glycal assembly method.

Solid Phase Oligosaccharide Synthesis

Solid phase synthesis of the Lewisb blood group determinant pentasaccharide glycal.

9

Scheme 5

10

Seeberger

was obtained in a 40% overall yield from 19. Initially, solution chemistry was used to further convert glycal 34b into a hexasaccharide of the Leb system, whereupon 34b was conjugated with human serum albumin to provide the desired neoglycoprotein. These approaches toward the synthesis of blood group determinants on a solid support were hampered by a serious shortcoming in the methodological arsenal of the glycal assembly method. While this strategy permitted rapid and concise access to ␤-glycosidic linkages, solution phase methodology solution had to be used for construction of N-acetylamine glucosidic linkages prevalent in biologically important blood group determinants, gangliosides, and N-linked glycopeptides [37]. To install the appropriate functionality at C2, solution phase chemistry has relied on a trans-diaxial addition of an iodonium electrophile in the presence of an amine to form an iodosulfonamide. Displacement of iodine proceeds presumably through an aziridine intermediate and may be induced by a thiolate nucleophile to fashion thioethyl 2-amidoglycosyl donors [38]. Successful transfer of this method to the solid support allowed polymer-bound glycals to be converted into thioethyl glycosyl donors. These donors were in turn coupled with a variety of glycosyl acceptors, including glycals [39]. After an efficient coupling protocol for the synthesis of ␤-2-amidoglucosidic linkages had been established, the difficulties encountered during the earlier synthesis of the Lewisb pentasaccharide glycal could be overcome. Branched tetrasaccharide 22a was converted into the thioethyl donor 23. Coupling to galactal acceptor 24 yielded 71% of the desired pentasaccharide 25a (Scheme 5). TBAF was used to effect retrieval of the pentasaccharide, affording 25b in 20% overall yield from 19 [32]. C.

Generation and Use of Thioethyl Donors on the Solid Support

While the use of glycals on the solid support allowed for the construction of ␤galactosyl linkages with great efficiency even with hindered glycosyl acceptors, the analogous ␤-glucosidic linkages could not be prepared reliably. In the galactose series, the anhydride is relatively stable to very mild Lewis acids, particularly anhydrous zinc chloride, because a conformationally constraining cyclic carbonate protecting group is used. The stability allows for galactosylation of even hindered acceptors such as C4 hydroxyls flanked by protecting groups at C3 and C6. No analogous constrained glucosyl epoxy donor is available, and glucosyl systems in the presence of zinc chloride are highly reactive and thus are prone to donor deterioration. To overcome the problems associated with the formation of ␤-glucosidic linkages, an approach that allowed for the conversion of glycals into thioethyl glycosyl donors was developed in solution phase [40]. These thioethyl glycosyl donors constitute a class of extremely powerful glycosylating agents upon activation with thiophilic reagents [41]. The glycal-derived donors were equipped with a C2 pivaloyl neighboring group and coupled to glycal acceptors to fashion a variety of glycosidic linkages with high efficiency. Pivaloyl neighboring groups had been shown by Kunz and others to prevent the formation of orthoester products during glycosylations [42]. Conversion of 26 to the protected thioethyl glycosyl donors 27 was achieved through epoxidation with dimethyldioxirane to yield the 1,2-anhydro sugar, followed

Solid Phase Oligosaccharide Synthesis

11

by opening of this intermediate by ethanethiol in the presence of a trace of acid. Thioethyl glycoside 27 was obtained in 91% yield and transformed into fully protected thioethyl donor 28a by pivaloylation in near quantitative yield. Support-bound thioglycoside 28a was activated using methyl triflate as a thiophile, while the nonnucleophilic base di-tert-butylpyridine (DTBP) provided stability for the glycal linkage during coupling. In model studies the formation of ␤-glucosyl (1→4), ␤-glucosyl (1→3), and ␤-glucosyl (1→6) linked disaccharides was achieved in good yield and complete selectivity [43]. After an efficient coupling protocol involving support-bound thioethyl glucosyl donors had been established, this methodology was applied to the synthesis of tetrasaccharide 31 containing exclusively ␤-(1→4) glucosidic linkages. Transformation of disaccharide glycal 29a into the C2 pivaloyl thioethyl glycosyl donor was followed by coupling to provide trisaccharide 30a in 45% overall yield from 26. Conversion of 30a to the thioethyl glycosyl donor was followed by coupling to glycal acceptor 14 to yield the desired tetrasaccharide 31a. After cleavage from the support 31b was obtained from 26 in 20% yield over nine steps, corresponding to 84% average yield per step (Scheme 6). D.

Solid Phase Synthesis of N-Linked Glycopeptides

N-linked and O-linked glycoproteins are the two major subgroups of glycoproteins, with the former being the most abundant in nature [44]. The biosynthesis of these glycoproteins results from cotranslational glycosylation usually occurring in the endoplasmic reticulum. The sugars of N-linked glycoproteins are usually attached by an oligosaccharyltransferase to an asparagine having the glycosylation sequence AsnX-Ser/Thr. Advances in glycopeptide synthesis have been achieved by several groups [45–47]. The synthesis of N-linked glycopeptides [48] on the solid support using a terminal glycal of a synthetic oligosaccharide domain aimed at a highly convergent synthetic strategy [49]. Polymer-supported trisaccharide 32 was reacted with anthracenesulfonamide and I(sym-coll)2ClO4 to form intermediate 33 (Scheme 7). Reaction of the iodosulfonamide 33 with tetra-n-butylammonium azide, followed by acetylation, provided the anomeric azide 34. The anthracenesulfonamide linkage can be cleaved under mild conditions such as 1,3-propanedithiol and Hu¨nig’s base, which concomitantly effected the reduction of the azide. The resulting amine was coupled with pentapeptide 35 in the presence of IIDQ to afford the protected glycopeptide 36. Orthogonal protecting groups on the C- and N-termini of the peptide provided the opportunity to extend the peptide chain while the ensemble was bound to the solid support. Alternatively, after removal from the support, the liberated peptide terminus may provide a functionality for linking to a carrier molecule to generate other glycoconjugates. The C-terminus of 36 was deprotected to give the acid 37, which was coupled to tripeptide 38 with a free N-terminus to give glycopeptide 39. Retrieval from the solid support afforded trisaccharide-octapeptide 40 in 18% overall yield from 19 [50]. IV.

STRATEGIES USING SUPPORT-BOUND GLYCOSYL ACCEPTORS

The solid support synthesis of oligopeptides and oligonucleotides is routinely carried out by reacting a support-bound structure (acceptor) with an excess of a solution-

12 Seeberger

Using glycal-derived polymer-bound thioethyl glucosyl donors in the synthesis of a ␤-(1→4) linked tetrasaccharide.

Scheme 6

Solid Phase Oligosaccharide Synthesis

Synthesis of N-linked glycopeptides on a solid support.

13

Scheme 7

14

Seeberger

based reactive species (donor). While the glycal assembly method, which utilizes a support-bound reactive species, has been used very successfully for the synthesis of oligosaccharides and glycopeptides, all other synthetic approaches explored to date have followed an acceptor-bound strategy. The acceptor-bound paradigm allows for the glycosyl donor to be used in excess during elongation of the growing oligosaccharide from the reducing to the nonreducing end. Because the donor is used in excess, side reactions that usually affect the glycosyl donor, resulting in inactive degradation products, do not impact the overall yield. Each hydroxyl group to serve as a glycosyl acceptor needs to be distinguished by a unique protecting group, which may be removed before each coupling cycle. In this section synthetic strategies employing a variety of glycosylating agents are reviewed. A.

Synthesis of Oligosaccharides Using Glycosyl Sulfoxides

The use of anomeric sulfoxides as glycosylating reagents was reported by Kahne and coworkers in 1989 [51]. Upon activation with triflic anhydride at low temperatures, anomeric sulfoxides are transformed into extremely reactive glycosyl donors, which can glycosylate very hindered acceptors. The excellent reactivity of the sulfoxide donors coupled with the selectivity achieved by use of a participating group in the C2 position prompted their use in the synthesis of oligosaccharides on the solid support [52]. Merrifield’s resin (polystyrene cross-linked with 1% divinylbenzene) was chosen as a polymer matrix, and an anomeric hydroxy thiophenyl ether moiety was selected as linker to the solid support (Scheme 8). The thiophenyl linkage is stable to all coupling and deprotection conditions but may be cleaved readily with mercuric trifluoroacetate. The first monosaccharide 42 was selectively deprotected and coupled by addition of 4 equiv of galactosyl sulfoxide 44, in the presence of DTBP as a base, and triflic anhydride as activator. Coupling at ⫺60⬚C for 30 min, filtration, and washing were followed by deprotection of the acetyl group. One further coupling and cleavage from the polymeric support by reaction with mercuric trifluoride furnished trisaccharide 50 in 52% overall yield. It was estimated that each coupling proceeded in 94–95% yield based on a detachment efficiency of 70–75% (as determined by solution phase studies). The glycosyl sulfoxide strategy successfully accommodated secondary hydroxyl acceptor groups, and ␣-fucosidic linkages were generated by the use of fucosyl sulfoxides [52]. These advances in the solid support synthesis of oligosaccharides using glycosyl sulfoxides were applied to the preparation of a combinatorial library of approximately 1300 disaccharides and trisaccharides [53]. A polystyrene–polyethylene glycol (PS-PEG) copolymer that swells in a wide range of solvents, including water, was employed in the syntheis of these libraries. Screening of the carbohydrate structures against a bacterial lectin from Bauthinia purpurea was performed with the carbohydrate structures still attached to the resin beads. Use of a chemical tagging system developed by Still and coworkers [54] allowed for rapid structure determination of the hits in the screening assay. Alternate binding structures with higher affinity than the natural ligand for the lectin of interest were identified in this manner. The glycosyl sulfoxide method has proven very efficient and flexible in constructing a variety of glycosidic linkages on the solid support. Combinatorial carbohydrate libraries prepared by this strategy hold great potential for the identification

Solid Phase Oligosaccharide Synthesis

Scheme 8

15

Solid phase synthesis of a trisaccharide by the glycosyl sulfoxide method.

of natural and nonnatural lectin ligands. While the synthesis of larger oligosaccharide structures using the glycosyl sulfoxide method still needs to be demonstrated, this strategy holds great potential for many applications. B.

Synthesis of Oligosaccharides Using Glycosyl Trichloroacetimidates

Among the multitude of glycosylating agents now at the disposal of the synthetic chemist, glycosyl trichloroacetimidates have become the most widely used building blocks. These donors have been used to prepare a large number of very complex oligosaccharides and glycoconjugates in solution phase [55]. High coupling yield, versatility, and excellent selectivity are hallmarks of this approach to oligosaccharide synthesis. Recently the first syntheses of oligosaccharides using trichloroacetimidate donors on the solid support have been reported. Schmidt and coworkers initially used Merrifield’s resin and explored a variety of ether and thioether linkers [56]. Attach-

16

Seeberger

ment of the first residue via a thioether linkage proved most reliable, and cleavage was successfully effected by reaction with a nucleophile. Reaction with water or methanol in the presene of N-bromosuccinimide yielded the lactol or the methyl glycoside, respectively. A two-step coupling cycle (Scheme 9) was used to assemble a series of (1→2) linked mannosides. Removal of the temporary acetyl-protecting group by sodium methoxide in methanol was followed by coupling of the trichloroacetimidate mannosyl donor 53 to the exposed axial C2 hydroxyl group. Repetition of this cycle and cleavage of the thioether linker resulted in assembly of the desired tetrasaccharide 56 in 34% overall yield. Penta- and hexamannosides obtained by using this method were characterized by mass spectrometry, but no yields were reported [57]. In addition to 1,2-trans-glycosides, disaccharides containing the synthetically more challenging 1,2-cis-glycosidic linkage were prepared. Fucosyl trichloroacetimidate was coupled to the C2 position of a support-bound mannosyl acceptor under the agency of trimethylsilyl triflate (TMSOTf) to yield 54% of the desired disaccharide [57]. Promising results were also obtained for the synthesis of a (1→2) trimannoside, which was prepared on a mercaptoethyl-functionalized controlled pore glass (CPG) solid support. This nonswelling support performed well under the described coupling and deprotection conditions [58]. More recently, the trichloroacetimidate strategy has been applied to the synthesis of a branched pentasaccharide unit common to most complex N-glycan structures (Scheme 10). Reaction of mannosyl donor 57 with thiol-functionalized resin resulted in attachment to the polymer matrix through the reducing end of the sugar 58. Removal of the benzoyl protecting groups on the C3 and C6 positions was effected by treatment with sodium methoxide to furnish diol 59. Dimannosylation was accomplished by reaction with trichloroacetimidate mannosyl donor 53 to afford solid support bound trisaccharide 60 in 38% overall yield. Cleavage of the acetyl protecting groups of the axial C2 hydroxyl functionalities revealed support-bound trisaccharide diol 61. Conversion of 61 to pentasaccharide 63 was achieved by reaction with glucosamine donor 62. Cleavage of the thioether linker with N-bromosuccinimide in the presence of methanol furnished the desired pentasaccharide methyl glycoside in 20% overall yield. Adinolfi et al. [59] explored the use of different solid supports for the synthesis of disaccharides employing trichloroacetimidate donors. The glycosyl acceptor was connected through the C2, C3, or C6 hydroxyl group to amino-functionalized solid support via a succinimide linker. Polystyrene, PS-PEG copolymer (TentaGel), and CPG supports were tested for their performance with trichloroacetimidate donors. Best coupling yields (95%) were obtained in these studies when the donor was reacted with acceptor bound to CPG or polystyrene support. PEG-containing copolymers performed significantly poorer in these glycosylations. Hunt and Roush [60] used a solid phase method to prepare 6-deoxy di- and trisaccharides. A sulfonate linker was employed to connect the first monosaccharide, a glycal, via its C6 position to the polymer resin. A galactosyl trichloroacetimidate donor was used in the first coupling reaction, followed by cleavage from the solid support by treatment with NaI to furnish pure disaccharides in 85–91% yield. Reduction with Bu3SnH (AIBN) provided the desired 6-deoxydisaccharide. These impressive initial accomplishments using trichloroacetimidate glycosyl donors on the solid support underscore the potential this strategy holds for the

Solid Phase Oligosaccharide Synthesis

Synthesis of ␣ (1→2) mannosides by the trichloroacetimidate strategy.

17

Scheme 9

18

Seeberger

Scheme 10

Use of trichloroacetimidate donors in the preparation of a branched pentasaccharide.

synthesis of complex oligosaccharide structures containing a variety of glycosidic linkages. Before the assembly of larger constructs may be contemplated, the overall yield and recovery will have to be improved. Novel linkers that are completely stable throughout the synthesis as well as improved coupling protocols should remedy these shortcomings.

Solid Phase Oligosaccharide Synthesis

C.

19

Solid Support Synthesis of Oligosaccharides Using Thioglycosides

Next to anomeric trichloroacetimidates, thioglycosides are the glycosyl donors most widely used in the synthesis of complex oligosaccharides and glycoconjugats. Thioglycosides may be prepared in high yield and are exceptionally stable, allowing for prolonged storage even at room temperature. Reaction with thiophiles such as methyl triflate or dimethylthiomethylsulfonium triflate (DMTST) ensures efficient activation of thioglycosides for the formation of glycosidic linkages [41]. In solution phase, thioethyl glycosides have found application in the synthesis of very complex oligosaccharides and in the preparation of a combinatorial carbohydrate library [61]. Straightforward synthetic access, stability, and high-yielding, selective glycosylation reactions made thioglycoside donors ideal for application to the solid support synthesis of oligosaccharides. Nicolaou and coworkers utilized phenolic polystyrene equipped with a photolabile, readily available o-nitrobenzyl linker in the synthesis of heptasaccharide 72 from mycelial walls of Phytophthora megasperma (Scheme 11) [62]. This heptasaccharide consists entirely of ␤ (1→6) or (1→3) glucosidic linkages and had previously been prepared by using solution phase methods [63] and block synthesis on a soluble PEG polymer [13]. Silyl ethers and fluorenylmethyloxy carbonyl (Fmoc) protective groups were employed to provide temporary protection of the hydroxyl functionalities to be glycosylated in the coupling steps that followed. Removal of the silyl ether protecting group by fluoridolysis to yield 64 was followed by coupling of 4 equiv of the solution-bound thiodonor 65 in the presence of the activator DMTST. The coupling to form 66 proceeded in greater than 95% yield according to material recovered after cleavage from the resin. Consecutive deprotection and coupling steps resulted in assembly of the desired heptasaccharide 71 still attached to the polymeric support. Photolytic cleavage of the linker, treatment with sodium methoxide to remove the ester protecting groups, and hydrogenolysis to cleave the benzyl ethers yielded unprotected heptasaccharide 72. Alternatively, fully protected heptasaccharide was obtained by acetylation of the anomeric hydroxyl group support in 20% overall yield from 64 after photolytic cleavage from the solid support [62]. After having established a reliable and efficient reaction sequence for the synthesis of oligosaccharides Nicolaou et al. further improved upon their initial strategy [64]. A 4-benzyloxybenzoic acid spacer was incorporated between the photolabile linker and the anomeric position of the first glycoside to provide access to either unprotected oligosaccharides or oligosaccharide glycosyl donors (Scheme 12). Support-bound acceptor 73 was converted into trisaccharide 77 following the synthetic strategy established earlier (see above). Cleavage from the resin was effected photolytically to furnish the fully protected trisaccharide 80 in 63% yield from 73. Alternatively, the protected trisaccharide was cleaved from the polymer with concomitant activation by exposure to PhSSiMe3/ZnI2/nBu4NI, furnishing phenylthiotrisaccharide donor 79 (Scheme 12; pp. 21 and 22). Support-derived trisaccharide building block 79 was used in a block synthesis of dodecasaccharide 82 (Scheme 13; p. 23). Reaction of support-bound glycosyl acceptor 78 with 79 furnished hexasaccharide 81. Removal of a silyl protecting group and coupling with trisaccharide donor 79 was repeated twice, followed by photolytic cleavage of the linker to furnish dodecasaccharide 82 in 10% yield from 73 [64].

20

Synthesis of a heptasaccharide employing thioglycoside donors.

Seeberger

Scheme 11

Solid Phase Oligosaccharide Synthesis

Preparation of trisaccharide thioglycoside building blocks on a solid support.

21

Scheme 12

22

Continued.

Seeberger

Scheme 12

Solid Phase Oligosaccharide Synthesis

Scheme 13

Assembly of a dodecasaccharide by the building block strategy on the solid phase.

23

24

Seeberger

The two examples of solid support oligosaccharide synthesis employing thioglycosides demonstrate the power of this strategy in the preparation of large carbohydrate structures. For the first time, a very large oligosaccharide was constructed via a block synthesis strategy in which every block itself was prepared on a solid support. The synthesis of combinatorial carbohydrate libraries using this strategy should certainly be possible. D.

Synthesis of Oligosaccharides Using Pentenyl Glycosides

Fraser-Reid and coworkers have established n-pentenyl glycosides as a class of powerful and versatile glycosyl donors [65]. Activation by a variety of reagents [e.g., Niodosuccinimide (NIS), TESOTf] furnished various glycosidic linkages in high yield and excellent selectivity in solution phase synthesis. More recently, the application of n-pentenyl glycosides to the synthesis of oligosaccharides on a solid support has been explored [66]. Both donor- and acceptor-bound synthetic strategies were investigated initially. Attachment of a pentenyl glycoside through the C6 hydroxyl group to the solid support was achieved via a photolabile 3-amino-3-(2-nitrophenyl)propionyl linker. Glycosylation using NIS and TESOTf as activating agents did not result in clean formation of the desired disaccharide product but also yielded lactol by donor degradation. An acceptor-bound synthetic strategy was more successful. Model studies with a glucose acceptor that was anchored to the solid support via its anomeric position showed that ␣- and ␤-glucosidic and C2 amino glucosidic linkages could be furnished in average coupling yields of greater than 90%. Reaction development was aided by 13C gel phase NMR spectroscopy to analyze the formed products [69]. After these initial studies, the synthesis of a support-bound, fully deprotected trisaccharide was achieved (Scheme 14). Chiron’s polystyrene-grafted ‘‘crowns’’ were chosen as solid support material because they are more amendable to parallel synthesis than traditional polymer supports. A photolabile o-nitrobenzyl linker was selected to attach the first amino glucoside 83 through its anomeric position to the solid support. Removal of the C6 dinitrobenzyl protecting group was followed by coupling with pentenyl mannoside 84 and resulted in formation of the desired disaccharide 85. Removal of the C2 chloroacetyl protecting group and coupling of pentenyl galactose donor 86 furnished trisaccharide 87. Global deprotection followed by peracetylation and photolytic cleavage from the solid support provided trisaccharide 88, but no yield was reported [66]. E.

Bidirectional Glycosylation Strategy for the Solid Phase Synthesis of Oligosaccharides

The preceding sections discussed the strengths and weaknesses of the donor- and acceptor-bound synthetic strategies developed to date for the synthesis of complex oligosaccharides. Boons et al. recently described an approach that combines both strategies in a bidirectional solid phase oligosaccharide synthesis [67]. The use of orthogonal glycosylating agents during the synthesis is an absolute prerequisite for the success of this bidirectional strategy. Trichloroacetimidate glycosides may be activated and function as glycosyl donors in coupling reactions with glycosyl acceptors containing an anomeric thioglycoside. The C6 hydroxyl group of thioethyl glycoside 89 (Scheme 15) was connected to TentaGel resin via a succinate

Solid Phase Oligosaccharide Synthesis

Scheme 14

Application of n-pentenyl glycosides to solid phase synthesis: preparation of a trisaccharide.

25

26

Bidirectional strategy for the synthesis of oligosaccharide libraries.

Seeberger

Scheme 15

Solid Phase Oligosaccharide Synthesis

27

linker. First, the exposed C4 hydroxyl functionality of 89 served as glycosyl acceptor in the reaction with solution-based trichloroacetimidate donor 90 in the presence of TMSOTf as promoter. Without the need for further protecting group manipulations, support-bound disaccharide thioglycoside 91 was activated with NIS/TMSOTf [68] to serve as a glycosyl donor in the reaction with solution-based glycosyl acceptor 92. This strategy served to generate a combinatorial library of trisaccharides while minimizing the necessary protecting group manipulations. This bidirectional approach stands to combine the advantages of the acceptorbound approach, such as use of excess donor during the synthesis, with the opportunity to fashion glycoconjugates through the anomeric position formerly possible only in a donor-bound paradigm. Other combinations of orthogonal glycosylating agents and the applicability of this strategy to the synthesis of larger oligosaccharide structures will have to be addressed in future studies. V.

‘‘ON-RESIN’’ ANALYTICAL METHODS

Development of a reliable solid phase methodology for the synthesis of oligosaccharides and glycopeptides has been greatly hampered by the lack of convincing ‘‘on-resin’’ analytical techniques. In most of the syntheses outlined above, it was necessary to cleave the products or intermediates of multistep syntheses from the resin to allow for the use of classical spectroscopic means (e.g., solution state NMR and mass spectrometry). The cleavage method analysis is time-consuming and wasteful in the context of multistep syntheses. Polystyrene–PEG copolymers may be analyzed by gel phase NMR spectroscopy but are quite expensive and allow only for relatively low loading [69]. Two NMR-based approaches for the on-resin analysis of polystyrene resins have been developed to aid reaction development by monitoring of the solid support matrix. A.

High-Resolution Magic Angle Spinning NMR Spectroscopy

The effectiveness of the glycal assembly approach to solid support oligosaccharide synthesis was impressively documented by on-resin analysis. The ‘‘crude product’’ of the multistep synthesis of trisaccharide 32 (Scheme 16) was monitored by highresolution magic angle spinning NMR (HR-MAS) [70].

Scheme 16

Solid phase synthesis of a trisaccharide by the glycal assembly method.

28

Seeberger

HR-MAS experiments proved to be an ideal way of monitoring the solid support synthesis by obtaining 1H NMR, 13C NMR, and 1H-13C NMR spectra of high quality. The 1H NMR (Scheme 17) of the crude product of this synthesis showed that only one product was obtained. Spin-echo techniques were used to suppress NMR peaks derived from the polymeric matrix. Since its introduction, this technique has greatly facilitated the development of novel synthetic schemes of oligosaccharides and glycopeptides on a solid support. B.

Gated-Decoupling–

13

C NMR Spectroscopy After 13C Enrichment

Wong et al. monitored the solid support synthesis of the sialyl Lewisx tetrasaccharide using 13C-enriched linker and protecting groups [71]. The 13C-labeled linker was attached to the polymer support and functioned as an internal integration standard for the 13C-enriched protecting groups of the growing oligosaccharide. After each coupling reaction, the NMR signal of the protecting group at the growing end of the molecule was compared with the signal of the internal standard to quantify the coupling. This method does not require special NMR hardware such as an HR-MAS probe but can be carried out in standard 5 mm NMR tubes. On the other hand, this method yields no detailed structural information regarding the anomeric purity of the compound prepared. The chemical integrity of the glycosidic linkages formed must be determined after cleavage of the target molecule from the solid support.

VI.

CONCLUSIONS AND OUTLOOK

Over the last 5 years a number of very promising strategies for the solid support synthesis of oligosaccharides and glycoconjugates have been developed. Several of the glycosylating reagents successfully used in solution have yielded promising results under the soild phase paradigm. While a number of different solid support matrices and linkers have been explored, much work in this area remains to be done. The rapid developments in combinatorial chemistry on the solid support have created novel support matrices, which will find application to solid phase oligosaccharide synthesis. Innovative linkers will be required to allow for the use of highly flexible protecting group strategies in the synthesis of branched oligosaccharides. Given the progress in the development of methods for the on-resin analysis of oligosaccharides, reaction development and optimization will be drastically accelerated. Novel glycosylating agents and orthogonal glycosylating schemes will further impact solid support synthesis. Alternative glycosylation methods such as the use of glycosyltransferase-catalyzed reactions on the solid support have also made great strides. For the synthesis of diverse structures, including natural and nonnatural glycoconjugates, chemical synthesis will be of paramount importance. While much progress has been made, many challenges remain before a flexible, high-yielding and absolutely selective strategy for the synthesis of oligosaccharides on the solid support becomes available. Once these problems have been solved, the construction of an automated oligosaccharide synthesizer may become feasible. Detailed studies concerning the structure and function of oligosaccharides and glycoconjugates will be possible once rapid access to complex glycoconjugates is a reality.

Solid Phase Oligosaccharide Synthesis

Spectrum of 32 obtained by high-resolution magic angle spinning 1H NMR spectroscopy.

29

Scheme 17

30

Seeberger

REFERENCES 1.

2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30. 31. 32. 33.

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31

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71.

2 Dehydrative Glycosylation with 1-Hydroxy Donors David Gin University of Illinois at Urbana-Champaign, Urbana, Illinois

I.

INTRODUCTION

The preparation of complex carbohydrates has emerged as a major focus in synthetic organic chemistry, and this is no doubt a result of the growing awareness of the many important roles of this class of molecules in biology [1]. Perhaps the most important reaction in the chemical synthesis of carbohydrates is the formation of the glycosidic bond, for this is the primary means for the controlled assembly of complex oligosaccharides and glycoconjugates from monosaccharide precursors. Thus a variety of methods have been developed to effect the glycosylation process. Much of the effort has focused on the general coupling strategy outlined in Scheme 1 [2]. In this strategy, one begins with a carbohydrate coupling partner (1), which is subjected to initial derivatization whereby the anomeric substituent is transformed into a latent leaving group (LG). The resulting intermediate 2, or glycosyl donor, is typically isolated, and in a second step the anomeric leaving group is activated with an appropriate glycosylation promoter or catalyst. This process usually takes place in the presence of a nucleophilic glycosyl acceptor (Nu-H), which undergoes an effective displacement of the leaving group to form the anomeric bond in the product glycoside (3). Within the last century, a variety of leaving groups have been developed for the generation of various glycosyl donors that can undergo efficient coupling in the second step. Some of these are listed in Scheme 1, and all have proven to be useful to some extent in complex carbohydrate synthesis [2]. A far less developed strategy for glycosidic bond formation is a direct dehydrative coupling procedure in which one begins with a 1-hydroxy carbohydrate (1) 33

34

Scheme 1

Gin

Traditional glycosylation strategies [2].

as the glycosyl donor (Scheme 2). This approach offers a complementary if not more efficient strategy for glycosylation in that a distinct anomeric derivatization step to generate an isolable glycosyl donor 2 is obviated. As such, all the operations of anomeric derivatization, activation, and bond formation are combined into a one-pot procedure. Despite its potential advantages, however, this strategy has not been extensively employed in complex oligosaccharide synthesis. The establishment of a viable synthetic method calls for the overcoming of such inherent difficulties associated with this approach as the reversibility of the process and the propensity for hemiacetal self-coupling, in addition to the common glycosylation obstacles such as coupling efficiency and high anomeric stereoselectivity. This chapter summarizes recent advances in the development of nonenzymatic direct dehydrative glycosylations with 1-hydroxy glycosyl donors. The concept of direct dehydrative glycosylation is not a new one. One of the earliest glycosylation methods is the Fischer procedure [3], currently adopted for preparation of simple glycosides (Scheme 3). In this process an unprotected monosaccharide (4) is treated with an excess of an alkyl alcohol in the presence of an acid catalyst, resulting in the net loss of water and substitution at the anomeric position by the alcohol acceptor. Usually a desiccant is not present in this hemiacetalto-acetal exchange process; as a result, the equilibrium can favor the formation of the glycoside product 5 only through the use of a large excess of the alcohol acceptor (typically employed as the reaction solvent or cosolvent). In the original glycosylation procedure, HCl was used as the acid catalyst, with the coupling event usually proceeding at elevated temperatures. Over the years, a number of other Brønsted acid catalysts have been found to be effective in this process, including various inorganic and sulfonic acids [4,5] as well as acidic resins [6]. In addition, a host of Lewis acid catalysts have been employed [7], giving rise to substrate-specific variants of the Fischer protocol. In fact, with selected Lewis acid promoters such as FeCl3

Scheme 2

Direct dehydrative glycosylations.

Glycosylation with 1-Hydroxy Donors

Scheme 3

35

Fischer glycosylation [3]. R = methyl, ethyl, n-propyl, i-propyl, amyl, allyl,

benzyl, etc.

[8], it is possible to favor the formation of the kinetic furanoside product over the thermodynamically more stable pyranoside adducts [9]. While it is not the goal of this section of the chapter to present a comprehensive summary of Fischer glycosylation methods, it is worth emphasizing that this protocol and its variants have, over the last century, remained one of the most popular methods for the preparation of simple alkyl glycosides. Indeed, this venerable method, with its simplicity and versatility, has frequently been chosen as the starting point for the preparation of C1-protected monosaccharide building blocks for complex molecule syntheses. Despite the widespread use of the Fischer method, it has yet to be shown to be effective in the controlled assembly of complex oligosaccharides. Because of the acidic medium under which the acetal exchange process occurs, only simple alcohols devoid of acid-labile functionality are employed as glycosyl acceptors. Moreover, the necessity of a large excess of acceptor to favor equilibrium formation of glycoside 5 precludes the use of complex or valuable molecules as nucleophilic acceptors. Typically the preparation of oligosaccharides by modified Fischer protocols has been limited to the preparation of carbohydrate oligomers of varying size and complexity [10,11]. To establish a method for controlled glycosylation with 1-hydroxy carbohydrates, mild dehydrative coupling conditions are required that favor the cross-condensation of distinct hemiacetal donor and nucleophilic acceptor substrates. One approach toward this end is to employ a set of reagents that can rapidly and completely activate the C1-hydroxyl functionality in 1 to generate, in situ, a highly reactive intermediate that incorporates a transient leaving group at the anomeric position. The requirement for rapid activation of the anomeric hydroxyl is obviously necessary to minimize the extent of hemiacetal self-condensation, and the in situ formation of an extremely potent leaving group at C1 would allow for facile glycosidic bond construction with a nucleophilic acceptor without the need for isolation of an intermediate glycosyl donor. II.

COUPLING VIA GLYCOSYL HALIDES

Koto and coworkers have developed a dehydrative coupling procedure employing the in situ generation of a glycosyl bromide intermediate (6, Scheme 4) [12,13]. In this procedure, a stoichiometric quantity of TMS-Br is introduced into a reaction mixture containing equimolar quantities of the 1-hydroxy glycosyl donor 1, the alcohol acceptor (R⬘OH), CoBr2, and tetrabutylammonium bromide. The glycosyl bromide 6, generated in situ, then proceeds to glycosylate the alcohol acceptor. Glycosyl bromides are well-established donors in the Koenigs–Knorr procedure [14], wherein these intermediates are typically isolated and activated with a halophilic promoter to

36

Gin

Scheme 4 Dehydrative glycosylations via glycosyl bromides [12,13,15]. * Using alternate method: MeSO3H, CoBr2, Et4NClO4.

effect glycosylation. However, the method of Koto and coworkers is distinct in that the intermediate 6 is both generated and consumed in situ under the reaction conditions, resulting in a net dehydration via a one-pot coupling process. The authors tentatively attribute the feasibility of this one-pot procedure to (1) the favorable ˚ molecular sieves acting both as a HBr scavenger and as a desiccant, effects of the 4 A and (2) the functioning of CoBr2 both as an effective desiccant and as a glycosyl bromide activator in the coupling stage with R⬘OH. This procedure was used to glycosylate a number of alkyl alcohols with 2,3,4,6tetra-O-benzyl-D-glucose, resulting in very good to moderate yields, depending on the complexity of the alcohol acceptor (e.g., 8–12). In these couplings, only trace amounts of products arising from hemiacetal self-coupling were detected, and the presence of Bu4NBr favored the formation of the ␣ anomer of the product glycosides. It is worth noting that in an earlier related report, the reagent combination of [MeSO3H, CoBr2, Et4NClO4] was also employed by Koto to effect the desired dehydrative glycosylation [15]. Under these reaction conditions, it is presumed that HBr is generated, leading to the formation of 6; however, this protocol generally led to diminished yields (e.g., 8 and 10) compared with the above-mentioned TMS-Br ⭈CoBr2 procedure.

Glycosylation with 1-Hydroxy Donors

III.

37

COUPLING VIA GLYCOSYL SULFONATES

The in situ generation and coupling of glycosyl sulfonate esters of 1-hydroxy glycosyl donors has also been investigated in the context of direct dehydrative glycosylation. Early work by Leroux and Perlin highlighted the generation of glycosyl trifluoromethanesulfonates 13 as useful reactive intermediates for glycosylation in the presence of Bu4NBr and the acid scavenger s-collidine (Scheme 5) [16]. Their initial attempts at the glycosylation of the acceptor R⬘OH directly with triflate 13 in the absence of Bu4NBr were unsuccessful (i.e., 1 → 13 → 7), presumably as a consequence of the instability of the highly reactive triflate species. However, the introduction of Bu4NBr served to rapidly convert 13 into the more stable glycosyl bromide 6, which then proceeds to glycosylate the acceptor R⬘OH. With this protocol, isolation of the glycosyl bromide 6 is again unnecessary, allowing for an overall one-pot process. The authors also highlight this as a useful method for the generation of glycosyl bromides under nonacidic conditions.

Scheme 5 Dehydrative glycosylations via glycosyl triflates [16]. * Using alternate method: (Me2SO2)2O, s-collidine; R⬘OH.

38

Gin

With this procedure, a number of simple alcohols were glycosylated to generate carbohydrate products such as 14–17. With glycosyl donors incorporating a C2-acyl functionality such as an acetate ester, the corresponding orthoester adduct (e.g., 18) is generated, the obvious result of acceptor addition to the bicyclic oxygen-stabilized carbocation that arises through neighboring group participation. It should be noted that the use of methanesulfonic anhydride in place of triflic anhydride also led to similar glycoside adducts (e.g., 14 and 18). When (MeSO2)2O is used as the dehydrating reagent, however, the addition of Bu4NBr is not necessary because the intermediate glycosyl methanesulfonate, which can directly glycosylate the acceptor at ambient temperature, has increased stability. Although the use of triflic anhydride and s-collidine in the absence of bromide ion was ineffective for glycosylation, the use of triflic anhydride alone was found to be useful for the coupling of 1-hydroxy glycosyl donors. Pavia et al. [17] have shown that the treatment of 1-hydroxy donors with triflic anhydride in the absence of any acid scavenger led to the formation of (1,1⬘)-linked disaccharides 19, the products of self-coupling of the 1-hydroxy donor (Scheme 6). The presumed mechanism [18] of this transformation involves (1) activation of the hemiacetal with trace amounts of triflic acid for conversion to the glycosyl triflate 13 (or the corresponding oxocarbenium triflate), (2) the rapid coupling of the highly reactive intermediate 13 with unactivated 1, and (3) the action of triflic anhydride as a desiccant to favor formation of the (1,1⬘)-disaccharide 19. Although this is a relatively efficient method for the preparation of symmetrical trehalose analogs (e.g., 20 and 21), it is often the case that the self-condensation product of the hemiacetal donor is an unwanted side product of dehydrative glycosylation. However, if this coupling procedure is performed in the presence of an excess (2–4 equiv) of an alcohol glycosyl acceptor R⬘OH, heterocouplings do take place (e.g., 22 and 23). Pavia has used this protocol to prepare complex galactosyl-O-glycopeptides [19]. Koto has shown that glycosyl arylsulfonates can also be used as intermediates in couplings with 1-hydroxy glycosyl donors. The direct glucosylation of several simple alcohol acceptors with 2,3,4,6-tetra-O-benzyl-D-glucose can be accomplished with the ternary mixture of p-nitrobenzenesulfonyl chloride, silver triflate, and triethylamine as the acid scavenger (Scheme 7) [20]. Simple alcohols such as methyl alcohol, cyclohexylmethyl alcohol, and dihydrocholesterol are glycosylated efficiently via the glycosyl sulfonate 24, and the formation of trehalose by-products is minimized as a result of rapid anomeric sulfonylation. In addition, couplings performed in the presence of N,N-dimethylacetamide generally lead to higher proportions of the ␣ anomer (26) [21]. This procedure can be used to prepare glucuronides (27) [22], as well as other complex oligosaccharides such as the branched-chain oligosaccharides of the sarsasaponins (e.g., 28–30) [23]. Other dehydrative glycosylations employing glycosyl arylsulfonates include the formation of transient glycosyl tosylates from 1-hydroxy donors, reported by Szeja (Scheme 8) [24]. For obvious reasons, all the preceding dehydrative coupling methods call for care to exclude moisture from the reaction. However, the method of Szeja is distinct in that it effects a dehydrative coupling with 2,3,4,6-tetra-O-benzylD-glucose in an aqueous cosolvent under phase transfer conditions, presumably via in situ formation of the intermediate tosylate 31. Using a mixture of p-toluenesulfonyl chloride, an excess (4 equiv) of the alcohol acceptor, a solvent mixture of CH2Cl2 and 40% NaOH(aq), and BnEt3NCl as the phase transfer catalyst, simple alcohols

Glycosylation with 1-Hydroxy Donors

Scheme 6

39

Dehydrative glycosylations via glycosyl triflates [17–19].

such as benzyl alcohol, cholesterol, and even tertiary alcohols such at t-butyl alcohol can be glucosylated with good ␣-selectivity (e.g., 15, 32, 33). IV.

COUPLING VIA GLYCOSYL OXOPHOSPHONIUM INTERMEDIATES

The in situ generation of glycosyl oxophosphonium salts has been shown to be useful for one-pot couplings with 1-hydroxy glycosyl donors. The most common method employed for the generation of the carbohydrate oxophosphonium intermediates 35 occurs by way of the Mitsunobu protocol [25] or variants thereof (Scheme 9). The method typically employs the initial reaction of a dialkyl azodicarboxylate with a phosphine substrate in the presence of a weak acid to generate a quaternary phosphonium salt, 34. With this reagent, it is known that a variety of alcohols can be

40

Gin

Scheme 7 Dehydrative glycosylations via glycosyl arylsulfonates [20–23]. * Coupling performed in the presence of 2.5 equiv AcNMe2.

activated to form the corresponding oxophosphonium species, which can then be displaced (SN2) by a variety of weak-acid nucleophiles, including carboxylic acids, phenols, N-hydroxyimides, imides, and oximes. The process, being a one-pot dehydration, was therefore studied in the context of activation of the carbohydrate C1hemiacetal function. Early reports by Jurczak et al. [26] focused on the preparation of N-glycosylphthalimides from various furanoses and pyranoses employing diethyl azodicarboxylate, triphenylphosphine, and phthalimide as glycosyl acceptor; however, yields were typically low (5–43%). The use of N-hydroxyphthalimide as the glycosyl acceptor, on the other hand, led to a dramatic increase in coupling efficiency (e.g., 36) [27] with both pyranose and furanose donors. The utility of this particular glycosylation was demonstrated in the preparation of a key alkoxyamino glycosyl linker in Nicolaou’s synthesis of the oligosaccharide portion of calicheamicin ␥ [28].

Glycosylation with 1-Hydroxy Donors

Scheme 8

41

Dehydrative glycosylations via glycosyl arylsulfonates [24].

Simple alkyl alcohols are typically poor acceptor substrates for this type of coupling owing to the insufficient acidity of the acceptor component to aid in the formation of the phosphonium salt 34. To overcome this difficulty, Szarek and coworkers [29] introduced a mercuric halide salt to facilitate the formation of 34, thereby effecting dehydrative glycosylation of alcohol acceptors (e.g., 37) with hemiacetal donors. Carboxylic acids are generally good nucleophiles in the Mitsunobu reaction and have also been demonstrated to function as efficient glycosyl acceptors with this protocol. Smith has refined and used this method for glycosyl ester formation [30] as one of the key convergent steps (i.e., 38) in the synthesis of a (⫹)-phyllanthoside [31]. Likewise, phenolic nucleophiles have also been shown to be useful acceptors. Early reports involved efficient glycosylations to prepare simple phenyl glycosides [32– 34] followed by the preparation of more complex ones such as 39 [35], an intermediate in Ogawa’s total of synthesis of hygromycin A [36]. Roush’s preparation of the olivomycin fragment 40 [37], highlighting the utility of the method in complex molecule synthesis and its tolerance to a wide variety of sensitive functionalities, is of particular interest. Mukaiyama and coworkers have developed an alternate method for the generation of glycosyl oxophosphonium intermediates. This method is based on Hendrickson’s earlier work concerning the development of phosphonium anhydride dehydrating reagents, prepared by the reaction of triphenylphosphine oxide (2 equiv) and triflic anhydride (1 equiv) [38]. With the reagent combination of tributyl phosphine oxide (2 equiv) and triflic anhydride (1 equiv), Mukaiyama reported that the resulting diphosphonium salt 41 (Scheme 10) [39] efficiently converted the hemiacetal functionality of 1-hydroxy carbohydrates to the anomeric oxophosphonium species 42, an effective glycosylating agent. In the presence of i-Pr2NEt as an acid scavenger, glycosylation of a number of simple alcohols, O-TMS-protected alcohol acceptors, and even azide acceptors, can be effected with 2,3,5-tri-O-benzyl-D-furanose to afford the product glycosides (e.g., 43–45).

42

Scheme 9

Gin

Dehydrative glycosylations via glycosyl oxophosphonium intermediates [26– 37]. DEAD = diethyl azodicarboxylate; DIAD = diisopropyl azodicarboxylate.

Glycosylation with 1-Hydroxy Donors

Scheme 10

V.

43

Dehydrative glycosylations via glycosyl oxophosphonium intermediates [39].

COUPLING VIA GLYCOSYL OXOSULFONIUM INTERMEDIATES

We have developed a new method for dehydrative glycosylation involving the in situ generation of glycosyl oxosulfonium species [40]. The use of reactive dimethylsulfonium reagents for hydroxyl activation has been investigated in depth, primarily in the context of hydroxyl oxidation to form a carbonyl functionality [41]. Similar activation of a carbohydrate hemiacetal should therefore lead to the formation of an anomeric oxosulfonium species such as 47 (Scheme 11), which would likely function as an efficient glycosyl donor. The key challenge is the development of a reaction protocol in which sulfoxide displacement (or dissociation) at the anomeric center of 47 is favored over other possible reaction manifolds such as oxidation or elimination. In our procedure (Scheme 11), initial activation of diphenyl sulfoxide with triflic anhydride presumably generates diphenyl sulfide bis(triflate) 46. In situ activation of the hemiacetal hydroxyl function in 1 by 46 would afford the oxosulfonium triflate 47, which then undergoes coupling with the appropriate acceptor to afford the glycosylated product 3. By employing diphenyl sulfoxide, which does not incorporate protons adjacent to the sulfur center, ylide formation from 47 is precluded, and thus oxidation via intramolecular proton transfer (i.e., Moffatt–Swern oxidation) is avoided. Furthermore, triflic anhydride serves as an ideal reagent for sulfoxide activation in that (1) activation of dimethyl sulfoxide with triflic anhydride to induce nucleophilic attack at sulfur has been reported in a limited number of cases in the contexts of Swern-type oxidations [42], sulfilimine synthesis [43], alkene functionalization [44], and quinone methide generation [45]; and (2) the anionic by-product of activation, triflate, is a weak nucleophile and thus should not obstruct glycosidic bond formation with the desired acceptor.

44

Scheme 11

Gin

Dehydrative glycosylations via glycosyl oxosulfonium intermediates [40].

The glycosylation method just described was used to couple a variety of acceptors with 2,3,4,6-tetra-O-benzyl-D-glucopyranose. For example, phenol, ethanethiol, and 1,3,5-trimethoxybenzene underwent efficient glycosylation to yield the corresponding O-aryl, S-alkyl, and C-aryl glycosides in good yield (48–50). Moreover, the N-glycosylation of amide functionalities, which has been reported to occur with only the most reactive of nonenzymatic glycosylation procedures [46], was

Glycosylation with 1-Hydroxy Donors

45

found to proceed smoothly with N-(trimethylsilyl)trimethylacetamide to afford 51. In addition, the glycosylation of tertiary alcohols (e.g., 33) was shown to be equally efficient when this new method was used. To determine whether C2-neighboring group effects would influence the glycosylation stereochemistry in this procedure, 2,3,4,6-tetra-O-benzoyl-D-glucopyranose, a donor incorporating multiple electronwithdrawing protective groups, was also employed as a coupling partner. This donor was coupled with carbohydrate acceptors to form the corresponding (1→6)- and (1→4)-linked disaccharides 53 and 54 with complete ␤-selectivity. Furanose donors are also compatible with this method, as evidenced by the formation of 52, the product of dehydrative N-glycosylation of bis(O-TMS)-thymine. Finally, the free NH functionality of a carbamate is compatible under these reaction conditions, allowing access to 2-amino-2-deoxy-glycosides (e.g., 55) with this protocol. VI.

COUPLINGS VIA GLYCOSYL OXOTITANIUM AND THIO-TIN INTERMEDIATES

Glycosyl oxotitanium intermediates were found to be useful in the furanosylation of various glycosyl acceptors. As long ago as 1989, Mukaiyama and coworkers had shown that [1,2-benzenediolato(2-)-O,O⬘]-oxotitanium (56) is a useful Lewis acid catalyst for aldol [47] and Michael reactions of ketene silyl acetals [48]. Used in combination with triflic anhydride, this Lewis acid was also found to be an effective dehydrating agent (Scheme 12) [49]. Treatment of 56 with triflic anhydride presumably leads to the formation of the bis(titanium) salt 57, which activates a 1-hydroxy glycosyl donor to form the intermediate anomeric oxotitanium species 58. This intermediate was shown to be an effective glycosyl donor to various alcohol and trimethylsilyl ether nucleophiles (e.g., 59–61). Mukaiyama et al. also found the use of diphenyltin sulfide and triflic anhydride to be suitable for furanoside formation with C1-hydroxy furanose donors (Scheme 13) [50]. Following activation of the hemiacetal with this pair of reagents, a glycosyl acceptor is introduced, in the form of an alcohol or a TMS-ether, to yield the product glycoside. Excellent anomeric selectivity can be achieved with 2,3,5-tri-O-benzyl-Dribofuranose as the donor (e.g., 60, 63), with the selectivity depending on the presence or absence of LiClO4 as an additive. The authors presumed that in the absence of LiClO4, the reactive intermediate 64 (Scheme 14) is generated, whereby the ␣ face is sterically shielded by virtue of coordination of tin(IV) with the C2-oxygen of the donor, leading to near exclusive information of the ␤ adduct 65. Conversely, the presence of LiClO4 (15 equiv) may serve to convert 64 into the ion pair 66, in which the perchlorate anion is situated on the ␤ face, trans to the C2 substituent, favoring acceptor approach onto the ␣ face. It is worth noting that the intermediacy of the corresponding C1-O-TMS derivative of the 1-hydroxy donor has also been suggested in these coupling reactions when TMS–ether acceptors are employed [51]. VII.

COUPLINGS VIA GLYCOSYL ISOUREA INTERMEDIATES

Carbodiimide reagents have long been known to be effective dehydrating agents in coupling reactions [52]. Tsutsumi et al. have shown that simple carbodiimide reagents are also effective in the context of dehydrative couplings with 1-hydroxy glycosyl donors (Scheme 15) [53]. Reactions of alcohols with carbodiimides have been known

46

Gin

Scheme 12

Dehydrative glycosylations via glycosyl oxotitanium intermediates [49].

to occur in the presence of CuCl to form O-alkyl isoureas [54]. This transformation was applied to C1-glycosyl hemiacetals 1 with dicyclohexyl carbodiimide to generate the corresponding O-glycosyl isourea 68, which proceeds to glycosylate appropriate acceptors via expulsion of dicyclohexyl urea from the anomeric center. This one-pot procedure was found to be effective at elevated temperatures (80–85⬚C) with a variety of phenolic nucleophiles (e.g., 69–72) in addition to several thiophenolic nucleophiles (e.g., 73). The high ␤-selectivity of the coupling reactions presumably arises from SN2 displacement of the corresponding ␣-glycosyl isourea intermediate, which is generated at elevated temperatures in the activation stage. VIII.

COUPLING VIA LEWIS ACID CATALYSIS

Acid catalysis in glycosylations with 1-hydroxy glycosyl donors has traditionally employed the Fischer glycosylation procedure or variants thereof in which simple glycosyl acceptors are employed in vast excess. Within the last several years, a series

Glycosylation with 1-Hydroxy Donors

Scheme 13

47

Dehydrative glycosylations via glycosyl thio-tin intermediates [50,51].

of dehydrative glycosylations using Lewis acid catalysts in the presence of a desiccant has been reported, employing equimolar or only slight excesses of various nucleophilic acceptors (Scheme 16, Table 1). Inanaga and coworkers reported the use of the catalyst combination of Yb(OTf)3 and methoxyacetic acid (0.1 equiv of each) employing only 1.2 equiv of the glycosyl acceptor (Table 1, entries 1–3) [55]. Anomeric mixtures were obtained with pyranose donors, while the ribofuranose donor 77 afforded high ␤-selectivities.

Scheme 14

Perchlorate ion and anomeric selectivity [50].

48

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Scheme 15

Dehydrative glycosylations via glycosyl isoureas [53].

Scheme 16

Dehydrative glycosylations via Lewis acid catalysis: Selected coupling partners

[55–60].

Glycosylation with 1-Hydroxy Donors

Table 1

Entry

49

Dehydrative Glycosylations via Lewis Acid Catalysis Coupling partners

Catalyst(s)

Reagents

1

77 ⴙ 78

2

77 ⴙ 79

3

74 ⴙ 81

4

74 ⴙ 78

Yb(OTf )3, MeOCH2CO2H Yb(OTf )3, MeOCH2CO2H Yb(OTf )3, MeOCH2CO2H Zn(OTf )2

5

75 ⴙ 79

Zn(OTf )2

6

76 ⴙ 79

Zn(OTf )2

7

77 ⴙ 78

Sn(OTf )2, (TMS)2O

TMSCl (1.5 equiv), CH3CN (23⬚C) TMSCl (1.5 equiv), CH2Cl2 (23⬚C) TMSCl (1.5 equiv), CH2Cl2 (23⬚C) Drierite, MeNO2 (23⬚C)

8

77 ⴙ 79

Sn(OTf )2, (TMS)2O

Drierite, MeNO2 (23⬚C)

9

77 ⴙ 78

Sn(OTf )2, (TMS)2O

10

75 ⴙ 80

Sn(OTf )2TMSCl

11

77 ⴙ 78

[Ph3C]⫹ [B(C6F5)4]⫺

Drierite, LiClO4 (1.5 equiv), MeNO2 (23⬚C) Drierite, LiClO4 (3.5 equiv), PhH (23⬚C) Drierite, EtNO2 (0⬚C)

12

77 ⴙ 80

[Ph3C]⫹ [B(C6F5)4]⫺

Drierite, EtNO2 (0⬚C)

13

77 ⴙ 78

Ph2Sn — —S, AgClO4

PhH, 3A-MS (23⬚C)

14

77 ⴙ 79

Ph2Sn — —S, AgClO4

PhH, 3A-MS (23⬚C)

15

77 ⴙ 78

PhCH3, 3A-MS (23⬚C)

16

77 ⴙ79

Lawesson’s reagent, AgClO4 Lawesson’s reagent, AgClO4

4A-MS, CH2Cl2 (reflux) 4A-MS, CH2Cl2 (reflux) Cl(CH2)2Cl (53⬚C)

PhCH3, 3A-MS (23⬚C)

Yield (%) (␣ :␤ ) 92 (0:100) 98 (04:96) 85 (48:52) 76 (55:45) 61 (62:38) 43 (88:12) 97 (05:95) 97 (05:95) 96 (94:06) 86 (96:04) 90 (09:91) 92 (04:96) 89 (13:87) 90 (04:96) 79 (24:76) 90 (04:96)

Ref. 55 55 55 56 56 56 57 57 57 58 59 59 60 60 60 60

The authors found that simple thiols also are effective acceptors in this coupling ˚ molecular sieves, the glycoside yields were method, and, in the presence of 4 A uniformly high. The work of Susaki established that Zn(OTf)2 can be employed in dehydrative glycosylation [56]. The combination of Zn(OTf)2 and TMSCl was found to be useful for glycosidic bond formation with both glycosyl ester donors, as well as 1-hydroxy carbohydrate donors (entries 4–6). In these couplings, the ratio of donor to acceptor to TMSCl to Zn(OTf)2 is 1:2:1.5:0.3, with either acetonitrile or CH2Cl2 as the reaction solvent. Benzylated gluco-, galacto-, and mannopyranose donors generally afforded good yields of alkyl glycosides with anomeric selectivities that vary with

50

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the nature of the coupling substrates. It is worth noting that couplings were significantly less efficient when an excess (1.5 equiv) of Zn(OTf)2 was employed. In a variety of the Lewis acid catalysts also surveyed by Mukaiyama and coworkers, benzylated furanose and pyranose substrates were found to be effective donors [57,58]. With the catalyst combination of the Sn(OTf)2 (0.01 equiv) and TMS2O (0.1 equiv) in the presence of anhydrous calcium sulfate, 2,3,5-tri-O-benzylD-furanose 77 can be used to directly glycosylate the alcohols 78 and 79 with high ␤-stereoselectivity (Table 1, entries 7, 8). The couplings are presumed to proceed via the TMS ether derivatives of the coupling partners, which are generated in situ, and the high ␤-selectivity is likely a result of product formation under thermodynamic control. In the presence of LiClO4 (1.5 equiv) as an additive, however, a dramatic reversal of the anomeric selectivity is observed, likely owing to the formation of the oxocarbenium perchlorate 66 (Scheme 14) as the active glycosylating agent (entries 9, 10). Various other glycosyl acceptors in addition to those in Table 1 were shown to be compatible with the Sn(OTf)2 /TMS2O method, including Cnucleophiles such as 1,3,5-trimethoxybenzene and O-silyl ketene acetals. Although the Sn(OTf)2 /TMS2O Lewis acid system is highlighted in Table 1, Mukaiyama has also demonstrated that other Lewis acid catalyst systems can be used, such as those incorporating Yb(OTf)3, La(OTf)3, and SnCl2, leading to comparable coupling efficiencies and stereoselectivities. In addition to metallic Lewis acid catalysts, it has been demonstrated that trityl salts can be used for the efficient ribofuranosylation of alcohols [59]. With 77 as the donor and 3 mol% of [Ph3C]⫹[B(C6F5)4]⫺ catalyst in the presence of anhydrous CaSO4, high yields of various furanosides were obtained with only 1.3 equiv of the alcohol acceptors, affording high selectivity for the thermodynamic ␤ isomer (entries 11, 12). In these studies, other counterions for the trityl cation were investigated ⫺ ⫺ in Mukaiyama’s survey of catalysts, including BF⫺ 4 , ClO4 , and SbCl6 , but ⫹ ⫺ [Ph3C] [B(C6F5)4] was found to be the most efficient in terms of yield, anomeric selectivity, and ease of catalyst handling. Two-component dehydrative catalysts have also been reported by Mukaiyama and coworkers for ribofuranosylation of alcohols. One such system is based on an earlier report by these investigators of the Ph2Sn — —S/Tf2O reagent combination for dehydrative coupling. When a catalytic amount of Ph2Sn — —S in combination with AgClO4 (0.2 equiv each) [60] were employed in the coupling of 77 with alkyl alcohols (1.2 equiv), good yields were obtained favoring the ␤ anomer of the product furanosides (entries 13, 14). The presumed active catalytic agent in the reaction is the Sn(IV) species 82. In like manner, the two-component catalyst system of Lawesson’s reagent and AgClO4 (0.2 equiv each) was found to be effective for coupling transformations (entires 15, 16), where the phosphonium species 83 is believed to be the active catalyst.

Glycosylation with 1-Hydroxy Donors

IX.

51

CONCLUSION

The importance of the development of new glycosylation methods cannot be disputed. Although many new variants of the traditional glycosylation processes have been developed over the years, comparatively fewer methods have been developed for direct dehydrative coupling with 1-hydroxy donors. The methods summarized in this chapter address many of the inherent obstacles associated with this strategy. For example, with the appropriate dehydrating agent in the glycosylation, the formation of the undesired by-products of hemiacetal self-condensation can be minimized, even when only a slight excess of the glycosyl acceptor component is employed in the coupling. Further development of these and other dehydrative coupling strategies will certainly establish this to be an efficient approach for the controlled assembly of complex carbohydrates. REFERENCES 1.

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(a) Synthetic Oligosaccharides. Indispensable Probes for the Life Sciences, Kovac, P., ed. ACS Symposium Series 560; American Chemical Society: Washington, DC, 1994. (b) Dwek, R. A. Chem. Rev. 1996, 96, 683–720. (a) Preparative Carbohydrate Chemistry; Hanessian, S., Ed.; Marcel Dekker: New York, 1997, Chapters 12–22. (b) Boons, G.-J. Tetrahedron 1996, 52, 1095–1121. (c) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem. Int. Ed. Engl. 1996, 35, 1380–1419. (d) Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21–123. (e) Toshima, K.; Tatsuta, K. Chem. Rev. 1993, 93, 1503–1531. (f ) Sinay¨, P. Pure Appl. Chem. 1991, 63, 519–528. (a) Fischer, E. Chem. Ber. 1893, 26, 2400–2412. (b) Bochkov, A. F.; Zaikov, G. E. Chemistry of the O-Glycosidic Bond. Pergamon: Oxford, 1979. McCurry Jr., P. M.; Pickens, C. E. U.S. Patent 4,950,743, 1990. Wessel, H. P. J. Carbohydr. Chem. 1988, 7, 263–269. For example: Nepogod’ev, S. A.; Backinowsky, L. V.; Grzeszczyk, B.; Zamojski, A. Carbohydr. Res. 1994, 254, 43–60. For example: (a) Fischer, B.; Nudelman, A.; Ruse, M.; Herzig, J.; Gottlieb, H. E. J. Org. Chem. 1984, 49, 4988–4993. (b) Kuhn, M.; von Wartburg, A. Helv. Chim. Acta 1968, 51, 1631–1641. (c) Kochetkov, N. K.; Khorlin, A. J.; Bochkov, A. F. Tetrahedron Lett. 1964, 289–293. Lubineau, A.; Fischer, J.-C. Synth. Commun. 1991, 21, 815–818. Smirnyagin, V.; Bishop, C. T. Can. J. Chem. 1968, 46, 3085–3090. Defaye, J.; Gadelle, A. Carbohydr. Res. 1989, 186, 177–188. Brochette, S.; Descotes, G.; Bouchu, A.; Queneau, Y. J. Carbohydr. Chem. 1998, 17, 879–891. Koto, S.; Morishima, N.; Zen, S. Bull. Chem. Soc. Jpn. 1982, 55, 1543–1547. Koto, S.; Morishima, N.; Kusuhara, C.; Sekido, S.; Yoshida, T.; Zen, S. Bull. Chem. Soc. Jpn. 1982, 55, 2995–2999. Paulsen, H. Angew. Chem. Int. Ed. Engl. 1982, 21, 155–173. Koto, S.; Morishima, N.; Zen, S. Chem. Lett. 1976, 1109–1110. Leroux, J.; Perlin, A. S. Carbohydr. Res. 1978, 67, 163–178. Pavia, A. A.; Rocheville, J.-M.; Ung, S. N. Carbohydr. Res. 1980, 79, 79–89. Pavia, A. A.; Ung-Chhun, S. N. Can. J. Chem. 1981, 59, 482–489. Lacombe, J. M.; Pavia, A. A. J. Org. Chem. 1983, 48, 2557–2563. Koto, S.; Sato, T.; Morishima, N.; Zen, S. Bull. Chem. Soc. Jpn. 1908, 53, 1761–1762. Koto, S.; Morishima, N.; Owa, M.; Zen, S. Carbohydr. Res. 1984, 130, 73–83.

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22.

Koto, S.; Miura, T.; Hirooka, M.; Tomaru, A.; Iida, M.; Kanemitsu, M.; Takenaka, K.; Masuzawa, S.; Miyaji, S.; Kuroyanagi, N.; Yagishita, M.; Zen, S.; Yago, K.; Tomonaga, F. Bull. Chem. Soc. Jpn. 1996, 69, 3247–3259. Koto, S.; Morishima, N.; Uchino, M.; Fukuda, M.; Yamazaki, M.; Zen, S. Bull. Chem. Soc. Jpn. 1988, 61, 3943–3950. Szeja, W. Synthesis, 1988, 223–224. (a) Hughes, D. L. Org. Prep. Proced. Int. 1996, 28, 127–164. (b) Mitsunobu, O. Synthesis 1981, 1–28. Jurczak, J.; Grynkiewicz, G.; Zamojski, A. Carbohydr. Res. 1975, 39, 147–150. Grochowski, E.; Jurczak, J. Carbohydr. Res. 1976, 50, C15–C16. Nicolaou, K. C.; Groneberg, R. D. J. Am. Chem. Soc. 1990, 112, 4085–4086. Szarek, W. A.; Jarrell, H. C.; Jones, J. K. N. Carbohydr. Res. 1977, 57, C13–C16. Smith, A. B. III; Hale, K. J.; Rivero, R. A. Tetrahedron Lett. 1986, 27, 5813–5816. Smith, A. B. III; Rivero, R. A.; Hale, K. J.; Vaccaro, H. A. J. Am. Chem. Soc. 1991, 113, 2092–2112. Grynkiewicz, G. Carbohydr. Res. 1977, 53, C11–C12. Kometani, T.; Kondo, H.; Fujimori, Y. Synthesis, 1988, 1005–1007. Roush, W. R.; Lin, X.-F. J. Org. Chem. 1991, 56, 5740–5742. Chida, N.; Ohtsuka, M.; Ogawa, S. Chem. Lett. 1988, 969–972. Chida, N.; Ohtuka, M.; Nakazawa, K.; Ogawa, S. J. Org. Chem. 1991, 56, 2976–2983. Roush, W. R.; Lin, X.-F. J. Am. Chem. Soc. 1995, 117, 2236–2250. (a) Hendrickson, J. B.; Schwartzman, S. M. Tetrahedron Lett. 1975, 277–280. (b) Hendrickson, J. B.; Hussoin, S. Md. J. Org. Chem. 1989, 54, 1144–1149. Mukaiyama, T.; Suda, S. Chem. Lett. 1990, 1143–1146. Garcia, B. A.; Poole, J. L.; Gin, D. Y. J. Am. Chem. Soc. 1997, 119, 7597–7598. Tidwell, T. T. Synthesis 1990, 857–870. Hendrickson, J. B.; Schwartzman, S. M. Tetrahedron Lett. 1975, 273–276. Coburn, M. D.; Hayden, H. H.; Coon, C. L.; Mitchell, A. R. Synthesis 1986, 490–492. Nenajdenko, V. G.; Vertelezkij, P. V.; Gridnev, I. D.; Shevchenko, N. E.; Balenkova, E. S. Tetrahedron 1997, 53, 8173–8180. Corey, E. J.; Gin, D. Y.; Kania, R. S. J. Am. Chem. Soc. 1996, 118, 9202–9203. Kahne, D.; Walker, S.; Cheng, Y.; Van Engen, D. J. Am. Chem. Soc. 1989, 111, 6881– 6882. Hara, R.; Mukaiyama, T. Chem. Lett. 1989, 1909–1912. Mukaiyama, T.; Hara, R. Chem. Lett. 1989, 1171–1174. Suda, S.; Mukaiyama, T. Chem. Lett. 1991, 431–434. Mukaiyama, T.; Matsubara, K.; Suda, S. Chem. Lett. 1991, 981–984. Mukaiyama, T.; Matsubara, K. Chem. Lett. 1992, 1041–1044. Williams, A.; Ibrahim, I. T. Chem. Rev. 1981, 81, 589–636. Tsutsumi, H.; Ishido, Y. Carbohydr. Res. 1981, 88, 61–75. Schmidt, E.; Moosmu¨ller, F. Liebigs Ann. Chem. 1955, 597, 235–240. Inanaga, J.; Yokoyama, Y.; Hanamoto, T. J. Chem. Soc. Chem. Commun. 1993, 1090– 1091. Susaki, H. Chem. Pharm. Bull. 1994, 42, 1917–1918. Mukaiyama, T.; Matsubara, K.; Hora, M. Synthesis, 1994, 1368–1373. Uchiro, H.; Miyazaki, K.; Mukaiyama, T. Chem. Lett. 1997, 403–405. Uchiro, H.; Mukaiyama, T. Chem. Lett. 1996, 79–80. Shimomura, N.; Mukaiyama, T. Bull. Chem. Soc. Jpn. 1994, 67, 2532–2541.

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3 Chemistry of Glycosyl Triflates: Synthesis of ␤-Mannopyranosides David Crich University of Illinois at Chicago, Chicago, Illinois

I.

INTRODUCTION

Among the numerous and varied glycosyl donors known, the glycosyl sulfonates have been somewhat neglected in recent years, despite their considerable potential in stereoselective glycosylation having been demonstrated over 20 years ago by Schuerch and coworkers. This unfortunate circumstance perhaps arose because of the difficulties encountered by the early workers in the isolation of pure samples. Recent work from our laboratories in Chicago has revealed that glycosyl triflates may be very readily accessed from either glycosyl sulfoxides or thioglycosides and that these substances have excellent reactivity toward a broad spectrum of glycosyl acceptors even at ⫺78⬚C. The main part of this chapter is therefore concerned with the development of the chemistry of the glycosyl triflates and, especially, with their applications in the synthesis of ␤-mannopyranosides. However, there are useful lessons to be learned from the precedents alluded to above, and it is with this in mind that we begin with an overview of the earlier work. II.

GLYCOSYL SULFONATES

The first reported preparation of a glycosyl sulfonate, in 1929, involved the heating of acetobromoglucose with silver toluenesulfonate in diethyl ether; Helferich and Gootz noted that the product was white and crystalline but decomposed in a matter of hours in chloroform solution at room temperature [1]. Many years after this inauspicious start, Schuerch and his coworkers turned their attention to an exploration of the uses of glycosyl sulfonates as glycosyl donors. Thus, in a 1973 paper that presaged many of the later developments, Kronzer and Schuerch suggested that the 53

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Crich

treatment of 2,3,4,6-tetra-O-benzyl-␣-D-glucopyranosyl chloride (1) or bromide with silver triflate at ⫺78⬚C in dichloromethane or diethyl ether provided the anomeric triflate 2 (Scheme 1) [2]. They opined that, owing to the strongly electron-withdrawing nature of the triflate group, this substance probably had the ␣ configuration. It was also noted that subsequent couplings with methanol, conducted at ⫺78⬚C, were ␤-selective in dichloromethane but unselective in diethyl ether, and the difference was attributed to the superior shielding of the ␣ face provided by the tighter ion pair in dichloromethane [2]. The rapidity of the reactions, both the initial formation of the triflate and its subsequent reaction with methanol at ⫺78⬚C, were also noted at this time [2]. Prompted by the extreme reactivity of the triflates and the desire to work with isolable, characterized intermediates, Schuerch and Eby subsequently turned their attention to the use of toluenesulfonates and mesylates [3]. Thus, 2,3,4,6-tetra-Obenzyl-␣-D-glucopyranosyl chloride, or bromide, was allowed to react with silver toluenesulfonate in acetonitrile at room temperature. The authors were able to use standard vacuum line techniques to remove the silver halide by filtration and obtain an 1H NMR spectrum of the product in deuteriochloroform: the characteristics of the anomeric proton (␦ 6.1, 3J1,2 = 3.5 Hz) led to the attribution of the ␣ stereochemistry. In the case of the analogous 2,3,4-tri-O-benzyl-6-O-(N-phenylcarbamoyl) series, signals attributed to the ␤-toluenesulfonate (␦ 5.5, 3J1,2 = 8.0 Hz) accounted for approximately 15% of the reaction mixture [3]. In agreement with the earlier report of Helferich and Gootz, complete decomposition was noted after several hours at room temperature. Coupling reactions of the so-formed 2,3,4,6-tetra-O-benzyl-1-O-tosyl␣-D-glucopyranose, conducted with methyl 2,3,4,-tri-O-benzyl-␣-D-glucopyranoside in a range of solvents, gave moderate yields but little selectivity. Kinetic measurements, carried out polarimetrically, revealed no clear dependence of reaction rate on alcohol structure or concentration and suggested to the authors that the couplings were SN1 in character and involved a series of interchanging tight ion pairs [3]. Eby and Schuerch also noted that the anomeric toluenesulfonates formed from silver toluenesulfonate and peracylated (acetyl, benzoyl) glucosyl halides were considerably more stable, and less reactive, and upon exposure to methanol led to mixtures of ␣- and ␤-glucosides as well as to orthoesters. These latter results prompted them to suggest that the esterified toluenesulfonates would probably be of little use in glycosylation [3]. Schuerch and his coworkers also investigated the galactopyranosyl toluenesulfonates and triflates. As in the glucose series, these were formed from the corresponding bromides by metathesis reactions with the appropriate silver sulfonates [4,5]. With a series of differentially 6-O-protected 2,3,4-tri-O-benzylgalactopyranosyl

Scheme 1

Chemistry of Glycosyl Triflates

55

sulfonates, excellent yields and high ␣-selectivity were obtained upon coupling to a range of alcohols in various solvents. It was noted that the ␣-selectivity was minimized with the use of the triflate leaving group, albeit for reactions conducted at ⫺78⬚C, in contrast to the other members of the series when room temperature was used. In the case of the reaction of the 4,6-di-O-(N-phenylcarbamoyl)-2,3-di-O-benzylgalactopyranosyl triflate with methanol, high ␤-selectivity was exceptionally obtained. These results were again all interpreted in terms of more or less tight ion pairs [4,5]. Finally, Srivastava and Schuerch studied the formation of ␤-mannopyranosides and the related ␤-rhamnopyranosides, using a range of glycosyl sulfonates as donors [6,7]. In this most difficult series, the authors emphasized the increased anomeric effect resulting from the antiparallel dipoles of the C1—O and C2—O bonds and sought to maximize this contribution by installing a strongly electron-withdrawing, nonparticipating protecting group on O2. Thus, 3,4,6-tri-O-benzyl-2-O-mesyl-␣-Dmannopyranosyl chloride (4) was prepared and reacted with a range of silver sulfonates to give the corresponding ␣-mannosyl sulfonates. Subsequent exposure to methanol or cyclohexanol, usually in acetonitrile, then gave the mannopyranosides in high yield and excellent ␤-selectivity. Only one carbohydrate-based alcohol (6) was used as glycosyl acceptor, but this too gave excellent ␤-selectivity especially with the 2,2,2-trifluoroethanesulfonate 5 as donor (Scheme 2). In considering the mechanism of their mannosylation reaction, Srivastava and Schuerch suggested that the electron-withdrawing sulfonate ester at the 2-position served to render the reactive ion pair in the SN1 mechanism tighter on the ␣ face than was the case in the glucose and galactose series. In this manner the ␣ face was considered to be highly shielded toward approach of the nucleophile, with the displacement taking on the high stereoselectivity normally associated with SN2 processes [6,7]. Similar ␤-selectivities were obtained from a 3,4-di-O-benzyl-2-O-mesyl␣-L-rhamnopyranosyl tosylate [6,7]. Although the authors obtained excellent

Scheme 2

56

Crich

␤-selectivity and yields in their mannosylations and rhamnosylations, they repeatedly alluded to the high moisture sensitivity of the anomeric sulfonates, as in the earlier glucose and galactose series, and the need to work on vacuum lines. Presumably this, and the need to remove the somewhat unconventional 2-O-mesylate protecting group subsequent to coupling, prevented them from developing the full potential of the method and applying it in the synthesis of oligosaccharides. More or less contemporaneously with the work of Schuerch, Leroux and Perlin studied the sulfonylation of pyranoses. Thus, 2,3,4,6-tetra-O-benzyl-␣-D-glucopyranose was treated in cold dichloromethane with triflic anhydride in the presence of 2,4,6-collidine, leading to a purported anomeric triflate. However, the authors noted that subsequent addition of methanol or ethanol did not lead to the formation of the glycoside in acceptable yields [8,9]. Subsequently, it was discovered that conducting the triflation in the presence of tetrabutylammonium bromide resulted in the formation of the ␣-glucopyranosyl bromide and that addition of an alcohol then led cleanly to the glycoside. In these reactions Perlin and Leroux typically carried out the triflation/bromide displacement at ⫺70⬚C, noting that bromide formation was complete after 15–30 min; the reaction mixture was warmed to room temperature before addition of the alcohol. However, it was also noted that bromide formation could be conducted at room temperature without apparent detriment. These workers also investigated the use of 2,3,4,6-tetra-O-acetyl-␤-D-glucopyranose (8), with its potentially participating protecting groups, as substrate. In the presence of collidine but the absence of bromide ion, an orthoester (10) was isolated in 44% yield. When bromide ion was included in the reaction mixture, acetobromoglucose (11) was isolated in 50% yield and there was no indication of orthoester formation. It was suggested that both reactions proceeded by way of the ␤-triflate (9), which was trapped either by further pyranose or by bromide ion according to the conditions employed (Scheme 3) [8,9]. The reaction of methanesulfonic anhydride with 2,3,4,6-tetra-O-benzyl-␣-Dglucopyranose in dichloromethane in the presence of collidine, followed by addition of methanol, resulted in the formation of a 3:2 ␣/␤ mixture of the methyl glycosides, isolated in 87% yield. Since glycosylation was achieved without the need for addition of the quaternary ammonium bromide, unlike the case of triflic anhydride, it was concluded that the glucosyl mesylate was considerably more stable and allowed for the displacement reaction to take place. Moreover, the anomeric ratio suggested that the ␣ and ␤ anomers of this mesylate were in equilibrium, with displacement of the ␤ anomer occurring more rapidly. When 2,3,4,6-tetra-O-acetyl-␤-D-glucopyranose was treated with methanesulfonic anhydride, a crystalline product was obtained in 74% yield and assigned as the ␣-glucosyl mesylate. Indeed the characteristics of this substance were comparable to those of the compound obtained by Schuerch upon treatment of acetobromoglucose with silver methanesulfonate [8,9]. When methanesulfonyl chloride was allowed to react with 2,3,4,6-tetra-O-benzylglucopyranose and collidine in dichloromethane, the ␣-glucopyranosyl chloride was isolated regardless of whether the quaternary ammonium bromide was included. Addition of methanol to the reaction mixture resulted in the formation of an anomeric mixture of methyl glycosides. Similar results were obtained with toluenesulfonyl chloride, although it was noted that the initial sulfonylation was somewhat slower [8,9]. The use of tosyl chloride in the dehydrative coupling of alcohols with pyranoses was later revisited by Szeja and his coworkers, with the difference that aqueous

Chemistry of Glycosyl Triflates

57

Scheme 3

phase transfer conditions were used, and glycosyl toluenesulfonates were implied as intermediates [10,11]. Koto and coworkers investigated the coupling of tetra-O-benzyl-␣-D-glucopyranose and a range of acceptor alcohols with the aid of a mixture of 4-nitrobenzenesulfonyl chloride and silver triflate [12–15]. In the presence of triethylamine, the ␣-glycoside predominated, whereas the inclusion of N,N-dimethylacetamide resulted in the isolation of the ␤ anomer. It was suggested that the anomeric hydroxyl group was sulfonylated with the sulfonyl chloride to a glucopyranosyl 4nitrobenzenesulfonate and subsequently converted to the active glycosyl donor, the glucopyranosyl triflate, by the action of silver triflate. However, given the relative acidities of triflic and 4-nitromethanesulfonic acid and the related work of Szeja, carried out in the absence of silver triflate, the formation of a covalent glycosyl triflate in this work appears to be somewhat unlikely. The inversion of stereoselectivity on inclusion of the N,N-dimethylacetamide was explained by invoking the formation of an ␣-N,N-dimethylacetimidate ester [12,13]. Pavia et al. revisited the reactions of tetra-O-benzylglucopyranose (12) with trifluoromethanesulfonic anhydride, but in the absence of base. They discovered that the corresponding trehalose derivatives (13) were formed in good yield, predominantly as the ␣,␣ form (Scheme 4) [16]. Comparable results were obtained in the galacto-, manno-, and arabinopyranose series as with fructofuranose [16]. When trifluoromethanesulfonic anhydride was added to a mixture of a perbenzyl-protected glycopyranose and an acceptor alcohol, such as various serine, threonine and hydroxproline derivatives, coupling was achieved in good yield [17]. Pavia and coworkers carried out a careful study of the mechanism of these reactions using 19F-NMR spectroscopy. They concluded that they were observing simple acid-catalyzed dehydrative couplings in which water was removed from the

58

Crich

Scheme 4

equilibrium in the form of a salt with triflic acid (TfO⫺ H3O⫹), which is insoluble in dichloromethane. The authors specifically excluded the intermediacy of a covalently bound glucosyl triflate on the grounds that the reaction did not occur below 15⬚C, whereas Perlin’s reactions succeeded at ⫺70⬚C [18]. III.

MANNOSYL TRIFLATES FROM MANNOSYL SULFOXIDES AND THIOGLYCOSIDES: THE ␤-MANNOSYLATION REACTION

Several years ago in Chicago, we were engaged in developing a solution to the wellknown ␤-mannoside problem [19] involving the inversion of the much more readily accessible ␣-mannosides by a sequence of hydrogen atom abstraction, radical inversion, and diastereoselective quenching (Scheme 5) [20,21]. With the fundamental chemistry, which was developed using commercially available ␣-methyl mannopyranoside as substrate in hand, it became necessary to prepare a genuine ␣-disaccharide for inversion. We selected Kahne’s excellent sulfoxide method [22–24] for numerous reasons, which included the reported excellent yields for coupling to extremely hindered alcohols at low temperatures, the absence of any metal salt as promoter, and the implication that the coupling proceeded through quenching of the oxacarbenium ion, hence should provide the ␣-mannoside. Other than a brief footnote in their original communication noting that the anomeric stereoselectivity was a function of the stereochemistry at C2 of the sulfoxide donor [22], Kahne and his coworkers had not described the application of their method to the mannose series. Jarmila Brunckova thus prepared S-ethyl 4,6-benzylidene-␣-Dthiomannopyranoside by standard means and converted it to the 3-O-benzyl-2-Otert-butyldimethylsilyl derivative (14) by the aegis of dibutyltin oxide and benzyl bromide, then TBDMS triflate. Oxidation with mCPBA then gave the sulfoxide (15) required for coupling [21]. Brunckova noted the highly selective sulfoxidation process, which gave essentially a single diastereomer, in contrast to the unselective oxidations ␤-thioglycosides observed previously [23], but was unable to assign configuration at the time. In fact it was several years before Jan Mataka and Sanxing Sun were able to prepare crystalline derivatives suitable for X-ray analysis and so assign the configuration as SR [25]. Brunckova then mixed the sulfoxide with methyl

Scheme 5

Chemistry of Glycosyl Triflates

59

Scheme 6

2,3,4-tri-O-acetyl-␣-D-glucopyranoside (16) and 2,6-di-tert-butyl-4-methylpyridine (DTBMP), a hindered base, in diethyl ether at ⫺78⬚C and activated the sulfoxide by dropwise addition of triflic anhydride. In line with our expectations, a good yield of a 10:1 mixture of glycosides (17) favoring the ␣ anomer was obtained (Scheme 6) [21]. The TBDMS protecting group was removed and was replaced by the radical precursor, and the radical inversion procedure was conducted with moderate success, comparable to that seen with the ␣-methyl mannoside [21]. Sanxing Sun, a new student, sought to prepare more of the ␣-disaccharide (17) and subsequently to improve the radical inversion process. He repeated Brunckova’s preparation with the minor, but fortuitous, difference that the sulfoxide was activated with triflic anhydride before addition of the acceptor alcohol. To our amazement, an excellent yield was obtained of an anomeric mixture favoring the ␤-mannoside by a factor of roughly 10 (Scheme 7) [26,27]. Consideration of this unanticipated reversal of diastereoselectivity led us to propose a mechanism in which, under all conditions, the sulfoxide 15 (Scheme 8) undergoes rapid sulfonylation leading to a sulfonium ion 18. We then postulated that this sulfonium ion collapses to the oxacarbenium ion 19 which, when generated in

Scheme 7

60

Crich

Scheme 8

the presence of the donor a` la Brunckova, is trapped axially to give the anticipated ␣-mannoside (17␣). On the other hand, we suggested that, under the Sun conditions, the oxacarbenium ion is trapped by triflate anion to give an ␣-mannosyl triflate 20. Then, on subsequent addition of the acceptor, an SN2-like process occurs with formation of the ␤-mannoside (17␤) [27,28]. This mechanistic hypothesis also provided a reason for the poor ␤-selectivity observed with secondary alcohol acceptors, even under the Sun conditions, namely, the well-known retardation of SN2 reactions by steric hindrance, leading to the interference of a dissociative mechanism via a Curtin–Hammett type of kinetic scheme. We predicted therefore that reducing the size of the O2 protecting group on mannose would accelerate the SN2 process for any given alcohol and so lead to increased ␤-selectivity. We also predicted that a change in solvent from diethyl ether to dichloromethane would further shift any ion pair/covalent triflate equilibrium toward the covalent triflate and so similarly lead to enhanced ␤-selectivity [27,28]. Both hypotheses were readily tested and confirmed. Thus, the series of sulfoxides 15, 21 and 22, with decreasing bulk of the O2 protecting group, were prepared and coupled to the rhamnosyl acceptor 23. As seen from Table 1, selectivity increases both as the size of the protecting group decreases and as the solvent is changed from diethyl ether to dichloromethane. Ultimately, with the 2-O-benzyl donor in dichloromethane as solvent, the ␣ anomer of the product was not detectable [28]. These conditions were then applied to the ␤-mannosylation of a range of primary and secondary carbohydrate acceptors with considerable success (Table 2) [27,28]. Attention was next focused on the use of alternative, nonparticipating protecting groups for the mannosyl donor, and the allyl group was found to be satisfactory at both O2 and O3 (Scheme 9) [27]. Indeed, Zongmin Dai and Greg Barba subsequently used a 2-O-allyl protected donor to form the ␤-mannoside linkage in the Hyriopsis schlegelii trisaccharide and the caloporside disaccharide, respectively [29– 31]. Further work, however, revealed the 4,6-benzylidene group to be indispensable for high ␤-selectivity, inasmuch as a 2,3,4,6-tetra-O-benzyl protected mannosyl donor gave very poor selectivity (Scheme 10) [27,28].

Chemistry of Glycosyl Triflates

Table 1

61

Reaction of Glycosyl Donors with 23 in Ether and CH2Cl2 Product and yield (%)b

Donor

a

Protocola

␤-Mannoside

␣-Mannoside

␤:␣

Et2O

A

24␤, 49

24␣, 30

1.6 : 1

Et2O

A

25␤, 76

25␣, 15

5.1 : 1

Et2O

A

26␤, 74

26␣, 11

6.7 : 1

CH2Cl2

B

24␤, 82

24␣, 11

7.5 : 1

CH2Cl2

B

25␤, 92

25␣, 7

13.1 : 1

CH2Cl2

B

26␤, 90

26␣, 0

>25 : 1

Solvent

A, Protocol A: addition of ROH to premixed donor, Tf2O, and DTBMP in ether/benzene; Protocol B, addition of ROH to premixed donor, Tf2O, and DTBMP in CH2Cl2. b All except 26␤ and 26␣ isolated after treatment of the reacton mixture with TBAF.

Table 2

Coupling of Secondary Acceptors to 22 and 27 in CH2Cl2 Product and yield (%)

␤-Mannoside

␣-Mannoside

␤:␣

29␤, 93

29␣, 5

18.6 : 1

27

31␤, 90

31␣, 6

15.0 : 1

27

33␤, 94

33␣, 5

18.8 : 1

27

35␤, 31

35␣, 8

3.8 : 1a

27

37␤, 72

37␣, 13

5.5 : 1

27

39␤, 94

39␣, 3

31.3 : 1

33␤, 91

33␣, 7

13.0 : 1

Donor

a

Acceptor

The reaction mixture was allowed to come to room temperature and stirred there for 24 h before workup.

Chemistry of Glycosyl Triflates

63

Scheme 9

Scheme 10

With a series of successful couplings in hand, we returned to the question of mechanism and the hypothesis of glycosyl triflates as the key reaction intermediates. A donor 44, lacking any diastereotopic benzyl hydrogens, was prepared and its 1HNMR spectrum recorded in CD2Cl2 in the presence of DTBMP at ⫺78⬚C. Cold (⫺78⬚C) Tf2O was then added, and a rapidly recorded spectrum showed the sulfoxide to have been completely consumed and converted to a new carbohydrate species [32]. This new substance was characterized in the 1H-NMR spectrum by its anomeric proton, a broad singlet, which resonated at ␦ 6.20. In the 13C-NMR spectrum the anomeric carbon had a chemical shift ␦ of 104.6 and a 1JCH coupling of 184.5 Hz. These data indicated that a strongly electron-withdrawing group was covalently linked through oxygen to the ␣ position of the mannopyranose ring, providing strong support for the ␣-mannosyl triflate (45) hypothesis. Confirmatory evidence was obtained when treatment of the mannsoyl bromide 46 with AgOTf and DTBMP in CD2Cl2 gave indistinguishable spectra [32]. This experiment also provided strong support for Schuerch’s earlier hypothesis that glycosyl triflates were formed at low temperature upon treatment of glycosyl halides with AgOTf (see above). Addition of methanol at ⫺78⬚C to these NMR tube experiments resulted in the very rapid formation of mannosides (47) with high ␤-selectivity, in full agreement with the general mechanism proposed (Scheme 11) [32]. A tetra-O-methylmannosyl sulfoxide 48 was prepared as a surrogate for the unselective tetrabenzyl donor 42. Again, low-temperature 1H and 13C NMR experiments indicated clean formation of a covalently oxygen-linked intermediate and, again, the same spectra were obtained going out from the bromide and AgOTf [32]. A first clue to reasons underlying the differing selectivity of the tetramethyl (or benzyl) and 4,6-benzylidene series was obtained in the course of attempts to record a CH-gated, coupled 13C-NMR spectrum of 49. Substantial decomposition occurred

64

Crich

Scheme 11

over the acquisition time, unlike the case of the 4,6-benzylidene donor (44), which prevented us from obtaining the 1JCH coupling constant. This difference in stability was confirmed by a series of variable temperature experiments, which revealed the 4,6-benzylidene protected triflate (45) to decompose around ⫺10⬚C, whereas its tetramethyl congener (49) did so some 20 degrees lower (⫺30⬚C). These latter observations provide strong support for the notion that the ␣/␤-selectivity in these couplings is a function of the equilibrium between the covalently bound triflate and the ion pair. The less stable the triflate, as reflected in the lower decomposition temperature, the greater the population of the ion pair and the greater the likelihood that ␣-mannosylation will occur through a Curtin–Hammett type of kinetic scheme. The increased stability of the 4,6-benzylidene protected triflates may be rationalized in terms of Fraser-Reid’s concept of a torsionally disarming protecting group [33]. In effect, the sofa conformation of the oxacarbenium ion imposes a twist and torsional strain on the acetal ring, which increases the energy of the oxacarbenium ion with respect to that of the covalently bound triflate. This effect is not present in the perether protected systems.

The basic mechanistic hypothesis (Scheme 8) suggests that the ␤-mannosides are formed in an SN2-like manner from the ␣-triflate. The NMR experiments provide overwhelming support for the formation of these triflates and, in the 4,6-benzylidene series, for their stereochemistry. However, there remains the possibility, which we cannot rule out, that the covalently bound triflates simply serve as a reservoir for the storage of extremely reactive contact ion pairs. In this hypothesis, which echoes that of Schuerch (see above), the ␣-triflate dissociates to a contact ion pair in which the anion is intimately associated with the ␣ face of the oxacarbenium ion and so sterically prevents approach from that face. In this hypothesis, reduced selectivity, as

Chemistry of Glycosyl Triflates

65

Scheme 12

seen with the per-ether protected donors, arises from intervention of solvent and of solvent-separated ion pairs. This hypothesis therefore requires a change in the equilibrium between contact ion pairs and solvent-separated ion pairs in going from the 4,6-benzylidene series to the per-ether protected series. Again we fall back on the extra strain imposed on the oxacarbenium ion by the 4,6-benzylidene group, which will have the effect of shifting the CIP/covalent triflate equilibrium toward the covalently bound species, hence of reducing the likelihood of intervention of a solvent-separated ion pair. This variation of the original mechanism is presented in Scheme 12. Although we have provided compelling evidence for the formation of mannosyl triflates in our system, it is not implied that related triflates are formed in all sulfoxide glycosylation reactions. Indeed, Kahne and his coworkers subsequently investigated the mechanism of their reaction and found that in many cases the original sulfoxide is rearranged to isolable sulfenate esters, which also serve as glycosyl donors as shown, for example, in Scheme 13 [34]. The precise reasons for the different reactivity patterns are not clear and are the subject of ongoing research in our laboratory. In the course of our investigations into the mechanism of the reaction, we became aware of the extreme electrophilicity of the by-product benzenesulfenyl tri-

Scheme 13

66

Crich

Scheme 14

flate (53). As demonstrated by control experiments, this species is itself able to convert glycosyl sulfoxides into glycosyl triflates on a time scale comparable to that of the Tf2O activation (Scheme 14) [32]. Sanxing Sun was so struck by the electrophilicity of PhSOTf that he tested its ability to convert thioglycosides into triflates at ⫺78⬚C. His curiosity was immediately rewarded when low-temperature NMR experiments indeed showed this reagent to cleanly convert a mannosyl thioglycoside (54) into triflate (45) within the space of minutes at ⫺78⬚C (Scheme 15) [27,35]. Sun then went on and provided a series of examples of his new reaction, involving coupling to primary, secondary, and tertiary alcohols as set out in Table 3 [27,35]. The obvious advantage of this new method is that it eliminates the need to oxidize thioglycosides to sulfoxides. The disadvantage is that the reagent has to be freshly prepared in situ from AgOTf and benzenesulfenyl chloride which, itself, has only limited shelf life. In 1997 Gin and coworkers published an intriguing new method for the dehydrative coupling of pyranoses with alcohols in which the pyranose (61) is activated with a mixture of diphenyl sulfoxide and triflic anhydride before addition of the acceptor alcohol, leading to the formation of the coupled product (63) in excellent yield [36]. It was suggested that this chemistry, a development of that of Perlin and Pavia (see above), proceeds via the sulfonylation of the sulfoxide and attack of the pyranose on the so-formed sulfonium salt (62). In a NMR experiment, the mannosyl sulfonium salt (65) has been shown to be stable at low temperature in the absence of nucleophiles (Scheme 16). Indeed, it could even be generated by addition of diphenyl sulfoxide to the corresponding glycosyl triflate (49), which was generated from the fluoride (64) with trimethylsilyl triflate. Glycosyl triflates therefore appear not to be intermediates in this chemistry [37]. IV.

␤-THIOMANNOSIDES

Thiols are generally regarded as better nucleophiles than alcohols in substitution reactions. Accordingly, Hongmei Li investigated the use of thiols as nucleophiles in

Scheme 15

Chemistry of Glycosyl Triflates

Table 3

67

␤-Mannoside Formation from Thioglycosides with PhSOTf in CH2Cl2 Product and yield (%)

␤:␣

26, 95

>25 : 1

55

33, 95

<25 : 1

55

29, 95

23 : 1

55

31, 97

18 : 1

55

37, 85

5.1 : 1

55

58, 94

>25 : 1

Glycosyl donor

Glycosyl acceptor

(Table 3 continues on p. 68)

our ␤-mannosylation protocol, with the expectation that even higher ␤-selectivity would be observed. This proved to be the case with a range of primary, secondary, and tertiary thiols (Table 4) with no ␣-thiomannoside being detected in any case. The yields, although good, were nevertheless somewhat less than we had typically observed with alcohols [38]. We attribute this phenomenon to the partial capture of the soft nucleophiles by one or other of the several sulfur-based electrophilic byproducts present in the reaction mixture.

Table 3

Continued

Glycosyl donor 55

a

After treatment with TBAF.

Scheme 16 68

Glycosyl acceptor

Product and yield (%)

␤:␣

60, 90

>25 : 1

26,a 80

10 : 1

Chemistry of Glycosyl Triflates

Table 4 Acceptor

V.

69

Synthesis of ␤-Thiomannosides from Sulfoxide 27 Product

Yield (%)

69

74

70

77

72

54

Acceptor

Product

Yield (%)

74

71

76

63

␣-SELECTIVE MANNOSYLATION REACTIONS

In our continuing search for alternatives to the 4,6-benzylidene protecting group, and for 2,3-protecting groups that will confer enhanced selectivity even in the 4,6-benzylidene series, we have encountered several systems, which, contrary to our initial expectations, were highly ␣-selective. Zongmin Dai, recalling the very early work of Perlin in which the 2,3-O-carbonate was found to be highly ␤-selective when used in the mannosyl bromide/insoluble silver salt method [39], prepared the 2,3-O-carbonate (77) of S-phenyl 4,6-benzylidene-␣-D-mannothiopyranoside and investigated its reaction with 1,2;3,4-diacetone-D-galactose (78) by the benzenesulfenyl triflate method (Scheme 17). She found to her surprise that the only product isolated was the ␣-mannoside 79 [40]. Earlier Sanxing Sun had looked at the use of the 2,3;4,6diacetonide 80 as glycosyl donor in conjunction with methyl 2,3,4-tri-O-acetyl-␣-Dglucpyranoside as acceptor and had obtained a 1:1 ratio of the ␣- and ␤-mannosides [27]. Again, this result was surprising in view of the fact that Garegg had earlier successfully employed the 2,3;4,6-dicyclohexylidene protecting system in the synthesis of ␤-mannosides by the mannosyl bromide/insoluble silver salt method [41].

70

Crich

Scheme 17

We interpret both these results in terms of the general mechanism and the effect of the protecting groups on the covalent triflate/ion pair equilibrium. Thus, the fivemembered cyclic 2,3-O protecting groups lead to considerable flattening of the pyranose ring and so raise the ground state energy of the triflate. The barrier to formation of the flattened oxacarbenium is therefore reduced and the equilibrium shifted more in its favor. This effect is more pronounced in the carbonate than the acetonide, owing to the sp2 nature of the atoms in the bridge, and so leads to higher ␣-selectivity in that case. Zongmin Dai also investigated the use of a 2-O-TBDMS-3-O-benzoyl protected 4,6-O-benzylidene donor (81) but, again, excellent ␣-selectivity was observed [40]. This result must arise from neighboring group participation by the 3-O-benzoate. Models demonstrate that the twist–boat conformation (82) imposed by such participation on the pyranose ring is not unduly straining even though the system is 4,6benzylidene protected.

Weiling Cai prepared and investigated the Ley type [42,43] 3,4-bisacetal protected donor 83 in the expectation that the second ring would serve a similar rigidifying function as the 4,6-benzylidene group. This was not to be the case, and again excellent ␣-selectivity was observed in coupling to a number of acceptors (Table 5) [40].

Chemistry of Glycosyl Triflates

Table 5

71

␣-Mannoside Formation from Thioglycoside 83 with PhSOTf in CH2Cl2

Glycosyl acceptor

Product and yield (%)

␣:␤

84, 52

>95 : 5

85, 57

>95 : 5

86, 88

>95 : 5

87, 62

>95 : 5

88, 60

>95 : 5

72

Crich

With hindsight this result could have been predicted, inasmuch as Fraser-Reid’s earlier experimental and computational work on the formation of oxacarbenium ions from pentenyl glycosides showed this to be more facile with a six-membered ring bridging the 3- and 4-positions than in the absence of such a ring, and certainly much more facile than in the case of 4,6-benzylidene protected systems [33]. Thus, this system also fits the general mechanistic scheme, with the ␣-selectivity being again due to a shift in the covalent triflate/ion pair equilibrium. Furthermore, the reversal of selectivity seen across the series 4,6-benzylidene/tetrabenzyl/3,4-bisacetal is in full agreement with the increasing stability of the oxacarbenium ions, as predicted computationally by Fraser-Reid in the glucose series [33].

VI.

4,6-BENZYLIDENE PROTECTED GLUCOSYL TRIFLATES

Weiling Cai prepared a 4,6-O-benzylidene-2,3-di-O-benzyl-␣-D-glucopyranosyl thioglycoside 89 and oxidized it to the sulfoxide 90 in the expectation that activation under the standard conditions for ␤-mannosylation would lead to the formation of ␤-glucosides. Low-temperature studies in CD2Cl2 showed that both the sulfoxide and the thioglycoside, upon activation with Tf2O or PhSOTf, respectively, provided the ␣-glucosyl triflate 91 (␦H1 = 6.3; ␦C1 = 100.6; 3JH1,H2 = 3.5 Hz) in high yield (Scheme 18). However, all couplings except that to methanol were highly ␣-selective (Table 6) [44]. It is unlikely that this reversal of selectivity can be explained by reduced stability of the glucosyl triflate: variable temperature NMR studies showed that decomposition of the triflate did not set in until above 0⬚C (i.e., above the decomposition temperature of the corresponding mannosyl triflate). We believe that this result is a function of the differing extent of the anomeric effect in the two series. In effect it is known that the anomeric effect is considerably stronger in mannopyranose than glucopyranose derivatives [45,46]. Thus we suggest that the ␣- and ␤-triflates are in dynamic equilibrium and that the reduced anomeric effect in the glucose series permits a population of the more reactive ␤-triflate sufficient for it to serve as a donor in an SN2-like fashion, with Curtin–Hammett kinetics, leading preferentially to the ␣-glucosides observed. A similar rationale was advanced decades ago by Lemieux

Scheme 18

Chemistry of Glycosyl Triflates

Table 6

73

␣-Glucoside Formation Product and yield (%)

␣:␤

92, 98

>95 : 5

89

93, 87

>95 : 5

89

94, 70

>95 : 5

89

96, 85

>95 : 5

98, 89

>95 : 5

99, 72

>1 : 7.5

Glucosyl donor

90

Glycosyl acceptor

MeOH

74

Crich

for the formation of ␣-glucosides from ␣-acetobromoglucose in the presence of added bromide ion [45,47]. VII.

SUMMARY

Glycosyl triflates may be prepared rapidly and cleanly at ⫺78⬚C in CH2Cl2 solution from anomeric sulfoxides or thioglycosides upon activation with triflic anhydride or benzenesulfenyl triflate, respectively. These triflates are extremely reactive glycosyl donors and, when applied in conjunction with the 4,6-benzylidene protecting group, provide a very facile entry into the ␤-mannopyranosides. ACKNOWLEDGMENTS I thank the numerous talented students and postdoctorals whose hard work and diligence has contributed, and continues to contribute, to the development of glycosyl triflate chemistry in my laboratory. The National Institutes of Health (GM 57335) are gratefully acknowledged for their support of this work. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27.

B Helferich, R Gootz. Ber Dtsch Chem Ges 62:2788–2792, 1929. FJ Kronzer, C Schuerch. Carbohydr Res 27:379–390, 1973. R Eby, C Schuerch. Carbohydr Res 34:79–90, 1974. V Marousek, TJ Lucas, PE Wheat, C Schuerch. Carbohydr Res 60:85–96, 1978. TJ Lucas, C Schuerch. Carbohydr Res 39:39–45, 1975. VK Srivastava, C Schuerch. Carbohydr Res 79:C13–C16, 1980. VK Srivastava, C Schuerch. J Org Chem 46:1121–1126, 1981. J Leroux, AS Perlin. Carbohydr Res 47:C8–C10, 1976. J Leroux, AS Perlin. Carbohydr Res 67:163–178, 1978. W Szeja, J Bogusiak. Synthesis 224–225, 1988. W Szeja. Synthesis 223–224, 1988. S Koto, N Morishima, S Zen. Carbohydr Res 130:73–83, 1984. N Morishima, S Koto, S Zen. Chem Lett 1039–1040, 1982. S Koto, S Inada, T Yoshida, M Toyama, S Zen. Can J Chem 59:255–259, 1981. S Koto, T Sato, N Morishima, S Zen. Bull Chem Soc Jpn 53:1761–1762, 1980. AA Pavia, JM Rocheville, SN Ung. Carbohydr Res 79:79–89, 1980. JM Lacombe, AA Pavia, JM Rocheville. Can J Chem 59:473–481, 1981. AA Pavia, SN Ung-Chhun. Can J Chem 59:482–489, 1981. F Barresi, O Hindsgaul. Synthesis of ␤-D-mannose containing oligosaccharides. In SH Khan, RA O’Neill, eds. Modern Methods in Carbohydrate Synthesis. Amsterdam: Harwood Academic Publishers, 1996, pp 251–276. J Brunckova, D Crich, Q Yao. Tetrahedron Lett 35:6619–6622, 1994. D Crich, S Sun, J Brunckova. J Org Chem 61:605–615, 1996. D Kahne, S Walker, Y Cheng, DV Engen. J Am Chem Soc 111:6881–6882, 1989. L Yan, D Kahne. J Am Chem Soc 118:9239–9248, 1996. C Thompson, M Ge, D Kahne. J Am Chem Soc 121:1237–1244, 1999. D Crich, J Mataka, S Sun, K-C Lam, AR Rheingold, DJ Wink. J Chem Soc Chem Commun 2763–2764, 1998. D Crich, S Sun. J Org Chem 61:4506–4507, 1996. D Crich, S Sun. Tetrahedron 54:8321–8348, 1998.

Chemistry of Glycosyl Triflates 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

47.

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D Crich, S Sun. J Org Chem 62:1198–1199, 1997. D Crich, Z Dai. Tetrahedron Lett 53:1681–1684, 1998. D Crich, Z Dai. Tetrahedron 55:1569–1580, 1999. D Crich, GR Barba. Tetrahedron Lett 39:9339–9342, 1998. D Crich, S Sun. J Am Chem Soc 119:11217–11223, 1997. CW Andrews, R Rodebaugh, B Fraser-Reid. J Org Chem 61:5280–5289, 1996. J Gildersleeve, RA Pascal, D Kahne. J Am Chem Soc 120:5961–5969, 1998. D Crich, S Sun. J Am Chem Soc 120:435–436, 1998. BA Garcia, JL Poole, DY Gin. J Am Chem Soc 119:7597–7598, 1997. BA Garcia, DY Gin. J Am Chem Soc 122:4269–4279, 2000. D Crich, H Li. J Org Chem 65:801–805, 2000. PAJ Gorin, AS Perlin. Can J Chem 39:2474–2485, 1961. D Crich, W Cai, Z Dai. J Org. Chem 65:1291–1297, 2000. PJ Garegg, T Iversen, R Johansson. Acta Chem Scand B34:505–508, 1980. A Hense, SV Ley, H Osborn, DR Owen, J-F Poisson, SL Warriner, KE Wesson. J Chem Soc Perkin Trans 1:2023–2031, 1997. J-L Montchamp, F Tian, ME Hart, JW Frost. J Org Chem 61:3897–3900, 1996. D Crich, W Cai. J Org Chem 64:4926–4930, 1999. RU Lemieux, AR Morgan. Can J Chem 43:2214–2221, 1965. RU Lemieux. Rearrangements and isomerizations in carbohydrate chemistry. In P De Mayo, ed. Molecular Rearrangements, Part 2. New York: Wiley Interscience, 1964, pp 709–769. RU Lemieux, KB Hendriks, RV Stick, K James. J Am Chem Soc 97:4056–4062, 1975.

4 C-Glycoside Synthesis: Recent Developments and Current Trends Maarten H. D. Postema and Daniel Calimente Wayne State University, Detroit, Michigan

I.

INTRODUCTION

The chemistry of C-glycoside compounds has been of considerable interest over the last 20 years. This class of compounds is defined as compounds in which the exocyclic oxygen atom of an O-glycoside has been replaced by a carbon atom (Fig. 1). C-Glycosides are not solely man-made, but are the creation of nature, as attested by the occurrence of many C-glycoside natural products. Unlike the corresponding Oglycosides, C-glycosides are unaffected by hydrolytic or enzymatic cleavage and therefore would seem to be ideally suited for use as stable sugar mimics. They possess a level of chemical stability comparable to that of cyclic ethers. Several reviews have appeared on the subject of C-glycoside preparation, as well as two monographs. Many methods for the preparation of both the ␣- and ␤-C-glycosides have been developed, and new approaches are still appearing in the literature. This chapter covers the literature from the end of 1994 to early 1999, with other inclusions as deemed appropriate. Following the organization of earlier work [1], we select the mode reactivity of the anomeric center to define the category. The chapter discusses free radical chemistry, C1 anions, Wittig chemistry–cyclization chemistry, transition metal mediated chemistry, sigmatropic chemistry, and approaches based on cationic chemistry. The chapter focuses on the most recent and novel developments in the field, but all the relevant references are included whenever possible. 77

78

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Figure 1

II.

FREE RADICAL APPROACHES

A.

Introduction

The use of free radical chemistry at the anomeric center to produce carbon–carbon bonds, especially in an intramolecular fashion, remains a popular method for Cglycoside synthesis. A review entitled ‘‘C-Glycosidation Technology with Free Radical Reactions’’ appeared in early 1998, and the reader is referred to that source [2] as well as to the more traditional ones for complete overviews on the subject. Again, the coverage here focuses on the most recent developments. B.

Intermolecular Approaches

1.

Anomeric Radicals

Wang has studied the trapping of anomeric pyranosyl radicals with t-butylisocyanide to give anomeric cyanides. The readily available acetylated gluco derivative 1 when exposed to t-butylisocyanide in the presence of AIBN and tris(trimethylsilylsilane), gave a 73% yield of the ␣ isomer 2. Other sugars were examined and gave products with yields in the range of 22–71%, (Scheme 1) [3]. Junker and Fessner examined the addition of anomeric radicals to vinyl phosphonic dialkyl esters to deliver Cglycosyl-phosphonates directly. Thus exposure of 3 to standard radical conditions in the presence of vinyl phosphonic dimethyl ester gave 4 in 31% yield. Several other sugars were examined and generally gave yields of similar magnitude with excellent ␣ :␤ ratios, (Scheme 1) [4].

Scheme 1

C-Glycoside Synthesis

79

Scheme 2

The use and application to C-glycoside preparation of a different radical initiator also have been reported [5]. Schwartz has reported that treatment of glycosyl halides with (Cp2TiCl2)2 leads to glycals, presumably via the anomeric organometallic intermediate 6. However, if the reaction is carried out in the presence of a Michael acceptor, such as MVK, then the initially formed radical can be trapped and undergo carbon–carbon bond formation to give the ␣-C-glycoside 9. Methyl acrylate and acrylonitrile were also examined as radical traps. The product is presumably formed as the titanium enolate 8, which should be useful for further transformations (Scheme 2) [6]. Witzack et al. also carried out the following intermolecular radical addition to the levoglucosenone 11. The addition was highly exo-face selective and product 12 was obtained in 26% yield along with the remainder of product being that of direct reduction of 10, (Scheme 3) [7]. Allylations have continued to serve as useful reactions, and Bertozzi et al., found that radical allylation of the 2-deoxy-2-phthalimido derivatives 13 and 14 gives good to excellent selectivity of the ␤-allylated isomers 15 and 16, (Scheme 4). This is contrasted to allylations of the 2-deoxy-2-acetyl derivative, which gives mainly the ␣ product, which is the normal stereochemical course for additions of these types. Presumably the steric bulk of the phthalimido group plays a role in directing the stereochemistry of the addition [8]. Praly et al. cleverly used radical allylation of sugars to obtain ␤-C-glycosides. Treatment of the 1-bromoglucopyranosyl chloride 18 with tri-n-butylallyltin under photolytic conditions gave the intermediate ␤-allyl compound 19. When 19 was further exposed to standard radical reducing conditions (Bu3SnH, h␯) the expected ␤-allyl C-glycoside 20 was formed in good yield. Alternatively, 19 could be treated

Scheme 3

80

Postema and Calimente

Scheme 4

Scheme 5

Scheme 6

with DBU in acetonitrile to afford the product of elimination 21 in good yield (Scheme 5). The same chemistry was carried out on the D-manno and D-galacto derivatives which gave the analogous compounds in similar yields [9]. Work by Magnusson dealt with the application of allylic sulfones as radical acceptors for allylic carbon–carbon bond formation at the radical center. Various acceptors were utilized, and the products are shown in Scheme 6 [10]. The Kolbe electrolysis of sugar acids to give radicals and their subsequent trapping with a radical source gave the chain-extended C-glycoside 27 as the major product (Scheme 7). Compounds such as 27 are useful as stable nonionic tensides and are expected to show liquid crystalline properties [11]. 2.

Radical Addition to Sugar Olefins

Vismara, Nicotra, and their colleagues used glycoexenitol, 30 and 32 as radical acceptors in the preparation of precursors for isosteres of glycolipids and glycoproteins. In this case, the formed radical is anomeric, and reduction by tin hydride from the axial direction should then give the ␤-C-glycoside. The additions proceeded cleanly to give only 31 and 33 as the major products of (␣ :␤ = 10:4) noting that the yields

C-Glycoside Synthesis

81

Scheme 7

Scheme 8

Scheme 9

are quantitative when based on the amount of recovered glycoexenitol. Gervay has added thiolacetic acid to 32 to obtain 34 in good yield, (Scheme 8) [12]. Motherwell published the full paper corresponding to his initial work on radical additions to difluoromethylene-linked C-glycosides. Scheme 9 shows an application of the atom transfer work developed by Curran with iodide 34 to give the bis-C,Cdialkylglycoside in fair yield [13]. C.

Intramolecular Approaches

Fraser-Reid has published a full account of his work on serial radical cyclizations with carbohydrates. The example shown (38 → 40, Scheme 10) illustrates the complexity of the frameworks that can be accessed by careful application of this methodology [14]. Work by Beau, Skrydstrup, and their colleagues has focused on the generation of anomeric radicals from anomeric sulfones by treatment with samarium iodide [15]. If a radical trap is present, the second electron transfer to form the C1 anion does not have time to occur to any appreciable extent, and the formed radical can then

82

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Scheme 10

be trapped. The trapping with a sugar acetylene to give the ␣-C-glycoside 44 is illustrated in Scheme 11. Hung and Wong used glycosyl phosphates as radical precursors en route to Cglycosides via intramolecular samarium diiodide mediated cyclization as shown in Scheme 11 [16]. Work by Czernecki et al. focused on intramolecular additions of 2deoxy-2-amino sugars. Exposure of the diallylated species 47 to tin hydride in the presence of triethylborane gave compound 48 in 74% yield as the sole product [17]. Lee et al. carried out intramolecular cyclizations to C-glycoside-like structures using aldehydes such as 49. Exposure of 49 to tributyltin hydride and AIBN gave an Ostannyl ketyl intermediate that underwent 5-exo cyclization to give 50 and 51 in a 46:54 ratio in 91% combined yield, (Scheme 11) [18].

Scheme 11

C-Glycoside Synthesis

83

Scheme 12

Although not radical in nature, this class of species is electron deficient and their inclusion here is warranted. Vasella has continued his work on glycosyl carbenes. The carbene generated from 52, when trapped with dimethyl fumarate of Nphenylmaleimide, gave 53 and 54, respectively (Scheme 12) [19]. III.

C1-ANION APPROACHES

A.

Introduction

The chemistry associated with the anomeric center is cationic by nature. One interesting approach to C-glycoside synthesis is to reverse the electronic character of this center from electrophilic to nucleophilic. Hence anions at the anomeric center (glycosyl anions) have now become fairly common in the synthesis of C-glycosides. Of course, one limitation of this method was the presence of heteroatoms at C2, since any discrete anion at C1 would surely cause an elimination reaction to occur and in turn produce the corresponding glycal (55 → 58, Scheme 13). This is in fact a very common way of preparing sugar glycals from sugars. As a consequence of this limitation, the chemistry of 2-deoxy C1 anions (56 → 59) has been extensively explored. An interesting development in this area came with the observation that if the hydroxyl group at O2 is unprotected, deprotonation followed by anion formation at C1 can occur to give species 57, which can go on to react with a suitable electrophile to deliver 60, after workup. It should be noted that a publication dealing with the preparation of anomeric stannanes from 2,3-dihydro-4H-pyran-4-ones has appeared [20]. A related, and important observation was that anomeric organosamarium species possessing a protected oxygen atom at C2 are stable enough to react via the anomeric carbon atom with suitable electrophiles to give access to C-glycosides (61 → 62, Scheme 13) [21]. B.

Dianion Approaches

1.

O2 Sugars

Work by the groups of Vasella and Kessler (Scheme 14) focused on the preparation of dianions from the corresponding 1,2-anhydro sugars. Thus treatment of 63 with Ph3SnLi gave 64 as the major product in 65% yield. Exposure to an excess of nBuLi resulted in formation of dianion 65, which was then quenched with a variety

84

Postema and Calimente

Scheme 13

of electrophiles to give the ␤ isomers (66–69 ) as the major compounds in all cases, attesting to the configurational stability of these anions at low temperature [22]. This strategy was then applied to the preparation of the anomeric ␤ and ␣ acids 72 and 76. The ␤ isomer 72 was prepared by application of the epoxide-opening strategy above, while the ␣ acid 76 was synthesized by using the procedure originally developed by Kessler and coworkers. Once prepared, the acids were coupled with the protected asparagine amino acid 78 to give the unnatural C-glycosylated amino acids 73 and 74 (Scheme 15) [23]. Glycosyl phenyl sulfoxides also serve as a good source of dianions. The ␣sulfoxide was metallated to the dianionic species, treated with a suitable aldehyde to give alcohol 81 and reduced product 82 (Scheme 16) [24]. 2.

2-Deoxyamino Sugars

The Kessler group then investigated the question of whether similar dianion chemistry could be carried out with the corresponding 2-deoxy-2-amino sugars. Accordingly, compound 83 was treated with thionyl chloride to give the intermediate 2-deoxy-2-aminoglucosyl chloride, which was then treated sequentially with n-butyllithium and lithium naphthalenide to give intermediate 84. Quenching with a variety of electrophiles gave modest to good yields of the 2-deoxy-2-amino-␣-C-gly-

Scheme 14

C-Glycoside Synthesis

85

Scheme 15

Scheme 16

Scheme 17

cosides 85 stereoselectively (Scheme 17) [25]. Similar chemistry was also carried out in the galacto series [26]. The corresponding ␤ dianion 84 could be accessed by first converting 83 to the ␣-pyranosyl chloride followed by SN2 displacement with tributylstannyllithium to give 84. Deprotonation and subsequent halogen metal exchange was followed by quenching with various electrophiles to provide the ␤-isomeric C-glycosides 87 [25]. The chemistry was then applied to the preparation of a C-glycoside-based analog of N-glucoaspargine 88 (Scheme 18) [27]. C.

Anomeric Organosamarium Species: O2 Sugars

Work in the area of O2 sugars was spurred by the following observations: upon treatment of 89 with samarium iodide (to obtain an anomeric free radical that would

86

Postema and Calimente

Scheme 18

then cyclize onto the pendant acetylene), three products were obtained. The first, 91, was the expected product of free radical cyclization in 25% yield; the product of elimination 58 (11%) and the product of simple deoxygenation 90 (60%) also were isolated (Scheme 19). This was very surprising, since the elimination product is produced from the organometallic anomeric samarium species that then undergoes a 1,2-elimination, but 90 is derived from protonation of the same intermediate organometallic species! This implies that the organometallic species is stable enough to be deprotonated to give 90 [28]. The workers then took advantage of this reactivity to generate anions at the anomeric center of 2-oxygenated sugars and have succeeded in showing that pyridyl sulfones make excellent precursors for such reactions. When O2 is protected at a tbutyldimethylsilyl ether, acceptable yields of ␤-C-glycosides are obtained from the corresponding ␤-pyridyl sulfone (92 → 93, Scheme 20). When the ␣-manno derivative 94 was used, a good yield of the corresponding ␣-C-glycoside 95 was obtained [15]. The authors attribute the discrepancy in yields to the ability of the manno intermediate to attain a reactive boat conformation that is more stable than the gluco derivative. With anomeric phosphates such as 96 as precursors, Wong has used a similar reaction to prepare ␣-C-manno pyranosides. Good yields of ␣-C-glycosides were obtained (Scheme 20). In the absence of the electrophile, the 1-deoxy sugars were the main products formed. The use of furanoside 98 gave good yields of Cglycoside products [16]. The results were obtained in the gluco series paralleled those of Beau and coworkers. Scheme 21 shows the reaction of the 2-deoxy-2-amino sugar 100 reacting with samarium iodide and cyclohexanone to give the desired C-glycoside 101 as the major compound [29]. When the azide was used in lieu of the protected amine function, only the product of elimination was formed. This indicates a possible complexation between the acetimido oxygen atom (Scheme 21, second equation) and the samarium atom, resulting in stabilization of the ␣-organosamarium species, since normally,

Scheme 19

C-Glycoside Synthesis

87

Scheme 20 anomerization to the more stable ␤-organosamarium species would be expected to take place. In this case the complexation holds the organosamarium intermediate in the ␣ configuration. It may also be possible that a chair flip of 102 occurs to give 103, which can then react via the equatorial direction to ultimately give the ␣-Cglycoside [30]. The same workers applied their methodology to the preparation of a C-glycoside analog of the Tn antigen. The Tn antigen epitope is expressed in over 70%

Scheme 21

88

Postema and Calimente

Scheme 22

of human epithelial cancers. This epitope is a major tumor-associated O-linked glycoprotein. The same antigen has also been identified as a partial structure of the HIV envelope glycoprotein gp120. The workers designed an analog in which the interglycosidic serine oxygen atom was replaced with a methylene group. The anomeric pyridyl sulfone 100 was coupled with aldehyde 104 to give 105 as a mixture of isomers. Radical deoxygenation was followed by standard manipulations to deliver the analog building block 106 (Scheme 22) [31]. Lindhart and coworkers also relied on a samarium-mediated condensation reaction to prepare ␣-C-glycosides related to N-acetylneuraminic acid (Neu5Ac). Neu5Ac is frequently found as the terminating residue of cell surface glycoproteins and glycolipids and is involved in a number of important biological events such as intracellular interactions, adhesion, and aggregation. A stable analog that would be hydrolytically inert was desired, and thus sulfone 109 was exposed to standard samarium iodide conditions (Scheme 23). However, it gave only the net product of reduction 110 [32]. When the same reaction was carried out with pyridyl sulfone 111 in the presence of the shown cyclic ketone, a 90% yield of 112 was obtained. Several other carbonyl compounds were also used as electrophiles with the corresponding pyridyl sulfone and gave good yields of coupled products. However, the reaction did not proceed with hindered carbonyl compounds [32]. Matsuda and coworkers have found that if such 2-keto-thioglycosides are treated with samarium iodide in THF followed by quenching with a carbonyl compound, good yields of C-glycosides are obtained, with the ␣ isomer (114) predominating in a ratio of 9:1 (Scheme 24) [33]. Kocienski et al. showed that the ␣-glycosyl copper(I) compound 115 reacts with the molybdenum complex 116 to give an excellent yield of the allylic ␣-Cglycoside 117 (Scheme 25). The corresponding ␤-glycosyl copper(I) reagent gave a

Scheme 23

C-Glycoside Synthesis

89

Scheme 24

Scheme 25 49% yield of the corresponding ␤-C-glycoside. When the mirror image of the cationic complex was used, the stereochemistry of the newly formed stereogenic center was found to be reversed [34]. IV.

APPROACHES BASED ON WITTIG CHEMISTRY

A.

Introduction

The reaction of sugar lactols with Wittig-type reagents and cyclization to C-glycosides is a traditional method for preparing C-glycosides. This section addresses the reactions of sugar lactones, the Wittig reaction of sugar lactols with unstabilized ylides and their subsequent cyclizations, and reactions of lactols with stabilized ylides and their conversion into C-glycosides. B.

Reaction of Sugar Lactones

Chapleur and coworkers have published a full account on their work dealing with the dichloroolefination of sugar lactones [35]. They found that sugar-based lactones give good yields of dichloroolefins when treated with a carbon tetrachloride-triphenylphosphine reagent combination. Some of the products along with their yields are boxed in Scheme 26. The same group has also found that reaction of certain chloroolefins such as 121 with mCPBA in dichloromethane gives compounds of the general structure 122 [36]. The reaction is thought to proceed via formation from dichloromethane of chlorine radicals that do not undergo addition to chloroolefins, since they are poor radical acceptors, but instead combine to give molecular chlorine that then undergo ionic addition to the double bond to give the observed products.

90

Scheme 26

Postema and Calimente

C-Glycoside Synthesis

91

Scheme 27

The same group also courageously discovered that the reaction of sugar lactones with the stabilized ylide 125 under high-temperature sealed-tube conditions gives good yields of the corresponding Wittig products (Scheme 27)! No mechanistic explanation is offered, but the sealed-tube conditions are crucial for success. No reaction occurs if the products are heated under reflux at similar temperatures under atmospheric pressure [37]. When the same reaction was carried out on sugar lactones that had a judiciously placed free hydroxyl, cyclized structures were obtained. For example, reaction of 126 with 125 gave a 1:1 mixture of 127 and 128. Compound 127 results from Michael addition of the free hydroxyl addition onto the ␣,␤-unsaturated ester function (Scheme 27) [38]. C.

Wittig Reaction with Unstabilized Ylides Followed by Cyclization

The Wittig reaction of a sugar lactol with an unstabilized ylide to give a hydroxy olefin poised to undergo electrophilic-induced cyclization is a common method for the preparation of C-glycosides. Martin et al. converted 129 to olefin 130 and followed this by inversion and introduction of the nitrogen substituent to give 131. Mercury-induced cyclization was followed by treatment with iodine to give compound 132. Presumably the benzyl carbamate participates to form a new C—O bond during the treatment of the organomercurial intermediate with iodine. Compound 132 could also be debenzylated and treated with base go give 133, the ␣ homoanalog of galactostatin. Alternatively, 132 was transformed into 134, and exposure to potassium carbonate then gave 135, the 1,N-anhydro derivative, a potential inhibitor of both ␣- and ␤-galactosidases, (Scheme 28) [39]. Work from the same group showed that olefin 137, available via Wittig reaction of 136, could be reductively aminated to give a mixture of amines 138 and 139 in a 5:2 ratio (Scheme 29). NIS promoted cyclization of 138, and 139 then gave 140 and 141, respectively, in good yield. Similar chemistry was carried out in the galacto series [40].

92

Postema and Calimente

Scheme 28

Nicotra et al. observed that 144 or 145, obtained by mercury-induced cyclization of 142 and 143, could not be converted to the desired iodo compound 146. The workers had to resort to introducing the 2-deoxynitrogen substituent after the phosphonate had been introduced as shown in Scheme 30. The ketone was converted to an oxime and with diborane gave the gluco isomer 149 in 64% diastereomeric excess [41]. Schmidt et al. also used electrophilic cyclization to gain access to C-glycosides, in this instance to produce 2-deoxy C-aryl glycosides. Known aldehyde 150 was olefinated and gave a mixture of isomeric olefins 151, which were converted to 152 and 153 and separated. Compounds 153a–c and 152c were exposed to NIS in acetonitrile and gave good yields of the ␤ isomers exclusively. Protecting group manipulation and removal of the iodine atom then gave the ␤-C-aryl glycosides 155a–c (Scheme 31) [42]. Work by Tius has shown that the preference for formation of the axial organomercurial is not due solely to coordination of the incoming mercurio species to the C2 oxygen substituent. Oxymercuration of 156, a compound in which there is no C2 substituent, gave a 60:40 ratio of 157 and 158, respectively. Interestingly enough, cyclization of the TBS derivative 159 gave 160 as the exclusive product (Scheme 32). Compound 160 was transformed into the known triacetate 162 by standard methods. The axial OTBS groups and equatorial methyl group are preferred because van der Waals repulsions that occur between the OTBS groups when they are equatorial are of greater energy than the 1,3-diaxial interactions that occur when the groups are disposed axially [43]. Hydroxy olefin 164 was epoxidized with mCPBA to give a mixture of Cglycosides 165 and 166 in the yields shown in Scheme 33. Compound 165 then served as a building block for C-nucleoside synthesis [44]. Two groups reported a novel cyclization reaction when a suitably protected sugar derivative was treated with triflic anhydride in the presence of pyridine. Thus, exposure of 136 to the aforementioned conditions gave an excellent yield of the vinyl C-furanoside 169. Several other sugars were examined, and all gave good yields of the corresponding furanosides. The reaction proceeds via triflation of the free hydroxyl group to give 167, and displacement with the C3 benzyloxy group provided 168, which is then debenzylated to deliver 169 [45,46]. Sharma and coworkers found that exposure of

C-Glycoside Synthesis

Scheme 29

93

94

Postema and Calimente

Scheme 30

alcohol olefins such as 170 to Wacker oxidation conditions gave a good yield of the corresponding keto-furanoside 171 and the C-glycoside 172 in a 4:1 ratio (Scheme 33) [47]. D.

Wittig Reaction with Stabilized Ylides Followed by Cyclization

1.

With Unprotected or Partially Protected Sugars

The situation with stabilized ylides is rather different from that with unstabilized ones because the formed product has the potential to undergo Michael addition to the corresponding C-glycoside. It is not clear what factors control the question of cyclization selectivity, but it is certain that reaction conditions and substrate structure both play an important role for the reaction in question. It has been shown that free sugars can be converted to the free olefins when exposed to stabilized ylides (173 → 174, Scheme 34) [48]. This reaction was later applied by others [49] to the preparation of open-chain sugar ␣,␤-unsaturated ester derivatives. As shown in the second equation of Scheme 34, compound 175 was then efficiently converted to 177, a compound that has been previously converted to KDO. Demailly and coworkers have shown that when Reformatsky ‘‘type’’ conditions are applied to this mode of bond formation, a preponderance of open-chain com-

Scheme 31

C-Glycoside Synthesis

Scheme 32

Scheme 33

Scheme 34

95

96

Postema and Calimente

Scheme 35

pounds such as 179 can be isolated. These results are contrasted to those obtained when the same conditions were applied to the protected sugar derivatives, in which chelation of the lone free hydroxyl with the ester carbonyl causes a template effect favoring stereoselective cyclization to the ␤-C-glycoside as shown by 181. In the unprotected case, the formed zinc bromide presumably ties up two vicinal hydroxyl groups, thereby disfavoring cyclization to the C-glycoside (Scheme 35) [50]. 2.

With Protected Sugars

Similar chemistry was applied to the protected glucosamine derivative 183, and the ␤-C-glycoside compound 184 was produced in 50% yield (Scheme 36) [51]. Murai and coworkers used the Wittig reaction on a protected aldehyde to give the Z-olefin in 78% yield along with 15% of the E-olefin after acid-catalyzed deprotection of the acetal. Exposure to base then gave the C-glycoside derivative 187, along with some dimer in 92% combined yield (Scheme 37) [52]. The thioamidephosphonate was reacted with the manno derivative 188 and gave a good yield of C-glycoside 189 (␣ :␤ ratio of 21:78). Compound 189 was then converted into the glycine-C-glycosyl compound 190 (Scheme 37) [53]. Davis and coworkers carried out a Wittig–cyclization sequence with the partially protected 2-deoxyamino sugar 191 using an amino acid based phosphonate. Reaction of 191 with the cesium enolate 192 gave a 53% yield of epimers 193 and 194 (1:1 ratio) in which epimerization of the 2-amino group had taken place. The manno isomer 194 was then epimerized to the more stable gluco 193 derivative by simple treatment with t-BuOLi in methanol (Scheme 38) [54].

Scheme 36

C-Glycoside Synthesis

97

Scheme 37

Scheme 38

V.

TRANSITION METAL MEDIATED APPROACHES TO C-GLYCOSIDES

A.

Introduction

Several years ago transition metal mediated reactions in the area of C-glycoside synthesis were primarily limited to palladium and to a lesser extent nickel and manganese. Over the last few years several other metals, including chromium, molybdenum, tungsten, cobalt, and rhodium, have been utilized in C-glycoside synthesis. This section discusses the chemistry of palladium, which is divided into Stille-type couplings and ␲-allyl complexes. This is followed by considerations of the chemistry of chromium and the above-listed metals. A review by Frappa and Sinou entitled ‘‘Transition Metal Catalysed Functionalization at the Anomeric Center of Carbohydrates’’ appeared in early 1997 [55]. B.

Use of Palladium

1.

Cross-Coupling Reactions

Falck et al. used the anomeric stannane 195 under palladium/copper-mediated coupling conditions with phenyl chlorothionoformate and ethyl chlorothiolformate to deliver 196 and 197, respectively. Coupling with benzoyl chloride proved sluggish and gave only low yields of the desired C-glycosides [56]. Work from the same group relied on a similar reaction as the key step in the short synthesis of the cytotoxic natural product (⫹)-goniofufurone (202). Compound 199 (available in one

98

Postema and Calimente

step from commercial D-glucurono-6,3-lactone) was converted to 200 in eight steps, and exposure to the proper conditions gave a 60–65% yield of product 201. Selective reduction and deprotection completed the sequence to provide natural 202 (Scheme 39) [57]. Snieckus has used his lithiation methodology in tandem with Suzuki–Miyama cross-couplings to produce C-glycoside-like structures. Vinyl iodide 204 was coupled with a variety of aryl boronic acids to give the corresponding aryl derivatives 205– 208 in excellent yield [58]. In work directed at synthesizing the core of the papulacandin natural products, Brimble and Chan carried out a Suzuki coupling of the boronic acid 211 with the iodo glycal 212 and obtained a 25% yield of the desired product 213, along with large amounts of the product of protiodeboronation. The use of the Stille reaction (both with and without copper as cocatalyst) for the coupling of 210 and 212 gave only 11% of the same desired product 213 (Scheme 40) [59]. Work from the same group addressing a Stille approach to C-glycoside derivatives of kalafungin has been published [60]. Lowary, Bock, and coworkers have prepared carbon-linked glycopeptides, such as 216, via Sonogashira coupling of glycosyl acetylene 214 and aromatic iodide 215 (Scheme 41) [61]. 2.

␲-Allyl Complexes

Sinou and coworkers published a full account of their work on the synthesis of 2,3unsaturated aryl C-glycosides via ␲-allyl-type methodology [62]. They showed that the reaction of aryl Grignard reagents with 217␣ under transition metal catalysis leads to either the ␣- or ␤-C-glycoside in good yield. When palladium is used as the metal, ␲-allyl complex formation is followed by intermolecular attack of the Grignard reagent from the bottom ␣ face, leading to 220, the product of net retention. A complementary approach involves the use of nickel catalysis to give the product of net inversion. In this case transmetallation of the ␲-allyl intermediate with the Grignard reagent gives 221 and reductive syn elimination then delivers 222. As far as catalyst choice is concerned, the catalysts PdCl2(dppf ) and NiCl2(dppe) gave the best results. Numerous Grignard reagents were also examined as part of this work (Scheme 42). Daves has continued his work directed at using palladium-mediated processes to achieve C-nucleoside synthesis. Coupling of iodide 223 with glycal 224 gave a good yield of adduct 225, which was subsequently desilylated and oxidized to furnish 226. The coupling of several other iodides and glycals was also examined [63]. A Spanish group recently employed ␲-allyl chemistry to prepare a potentially useful anti-HIV agent. Olefin 227 was treated with two of 1,3-diketones (R = Me, Ph) to give the diketo-2,3-unsaturated C-glycosides 228. Condensation with a variety of hydrazine derivatives then gave access to a small library of 2⬘,3⬘-unsaturated pyrazole C-glycosides 229a–j (Scheme 43) [64]. Exposure of 230 and 231 to diethyl allylmalonate in the presence of sodium hydride and tetrakis(triphenylphosphine)palladium in tetrahydrofuran gave C-glycosides 232 and 233, respectively, in good yield (Scheme 44). Metallo-ene cyclization of both 232 and 233 gave the same cis product of cyclization 234 in 83–89% yield [65]. Genet has used ␲-allyl chemistry to extend the chain of a preexisting C-glycoside. The example shown in Scheme 44 is illustrative. Exposure of 235 (available from the corresponding ␤-glycosyl aldehyde by condensation with vinyl magnesium

C-Glycoside Synthesis

Scheme 39

99

100

Postema and Calimente

Scheme 40

Scheme 41

Scheme 42

bromide followed by acylation) to Pd(dppe)2 and sodio malonate gave the chainextended C-glycoside derivative 236 in 71% yield. Several other examples were studied and gave comparable yields of C-glycoside products [66]. C.

Use of Cobalt

Luengo and Gleason utilized Murai’s cobalt chemistry to produce the equatorial Cglycoside 238 in 85% yield, followed by desilylation and conversion of 239 to 241. The final step, standard coupling, then gave 244 the potential GDP-fucose inhibitor (Scheme 45). Several other analogs were also prepared [67].

C-Glycoside Synthesis

Scheme 43

Scheme 44

Scheme 45

101

102

Postema and Calimente

Scheme 46

Photolysis of the anomeric cobaloxime 245 in the presence of maleic anhydride and PhSSPh gave 246 in 46% yield. The reaction proceeds via radical intermediates and the formed ␣-carbonyl radical is trapped with PhSSPh to give the formed adducts. Then syn-␤-H elimination led to 247 in quantitative yield (Scheme 46) [68]. D.

Use of Rhodium

McDonald et al. used a rhodium-catalyzed cyclotrimerization reaction to gain access to aryl C-glycosides (Scheme 47). Their work began with the conversion of lactone 248 to alkynyl glycal 249 by standard methods. Exposure of 249 or 251 to 254 in the presence of ClRh(Ph3P)3 in protic solvent furnished the product glycals 252 and 253, respectively, in the indicated yields [69]. Dialkyne 255 was also subjected to similar conditions and gave C-aryl spiroglycoside 257, a compound structurally related to the papulacandin natural products. E.

Chromium, Tungsten, and Molybdenum

Do¨tz and coworkers prepared several interesting and novel glycosylidene carbenes (259–261 ) by reaction of lithiated glycal 258 with the appropriate metal carbonyl derivative. The synthetic utility of the formed carbenes was demonstrated by reaction with 3-hexyne to give a mixture of complexed and uncomplexed adducts 262 and 263, respectively. The anomeric chromium carbene 264 was converted to 265 by exposure to ethoxy ethyne (Scheme 48) [70]. The same workers have carried out a similar chromium-mediated benzannulation (266 → 268), this time with the chromium on the aromatic fragment [71]. Other sugar-based carbenes have also been prepared [72]. Postema and coworkers have used ring-closing metathesis to prepare C1-glycals, compounds convertible to C-glycosides (Scheme 49). The sequence is both general and convergent. Ester formation (269 → 270) is followed by methylenation to give 271, and ring-closing metathesis is then carried out with the Schrock catalyst 272 to deliver the generic C1-glycal 273. The reaction tolerates alkyl and aryl groups at the anomeric center. Very hindered groups (R = t-Bu) were not good substrates because the methylenation step did not proceed in good yield. This was presumably due to the steric hindrance [73]. The chemistry has been extended to the preparation of a number 1,6-linked C-disaccharides. The conversion of 274 to 277 exemplifies the strength and versatility of this approach to C-glycoside synthesis. Compound 277 was converted to the 2-deoxy ␤-C-disaccharide and also to the ␤-C-disaccharide by standard methods (not shown). A small library of glycal C-disaccharides was prepared by use of this novel methodology [74].

C-Glycoside Synthesis

Scheme 47

103

104

Postema and Calimente

Scheme 48

Scheme 49

VI.

SIGMATROPIC APPROACHES

A.

Introduction

This section focuses on the use of concerted processes in the preparation of Cglycosides. The topics covered are cycloaddition reactions and Wittig rearrangements as approaches to C-glycoside synthesis. B.

Cycloaddition Approaches

1.

Diels–Alder

Several types of Diels–Alder reactions have been applied to the preparation of Cglycosides. Some of these include approaches to prepare the sugar ring or to append

C-Glycoside Synthesis

105

Scheme 50

a ring on an existing C-glycosyl derivative. Dipolar cycloadditions have also been utilized. (a) From Sugar Dienes or Glycals. Lubineau et al. reported an aqueous Diels– Alder approach to the trehalo derivative 281. Cycloaddition of 279 in water at 140⬚C gave a mixture of four isomers, of which the major compound, 280a, was separated, debenzoylated, and reduced to give 281. Compound 281 showed only modest activity (IC50 = 47 mM) as an inhibitor or porcine kidney trehalase (Km = 3 mM) (Scheme 50) [75]. In attempting to prepare sugar-based radical acceptors, Martin and Xie found that treatment of 282 with base to generate enone 283 gave instead the dimerized product 284; the result of a [4⫹2] cycloaddition reaction. Compound 283 was never isolated from the reaction mixture (Scheme 51) [76]. Quayle and coworkers used a Stille coupling approach to prepare a number of sugar-based dienes that then under-

Scheme 51

106

Postema and Calimente

Scheme 52

went a facile cycloaddition reaction with Cookson’s reagent 287 to give good yields of the cycloadducts. The reaction shown in Scheme 51 is illustrative [77]. Work in this area has also been carried out with noncyclic carbohydrates. Reaction of the nitro sugar 289 with cyclopentanedienone gave a mixture of 291 and 290 in a 1.9:1 ratio (Scheme 52) [78]. Dondoni used his masked formyl derivative in a hetero-Diels–Alder approach to carbon-linked disaccharides. Cycloaddition of 293 with ethyl vinyl ether gave a mixture of isomers; the major compound, 294, is shown in Scheme 52. The cycloadduct was then converted to 295 via standard methods [79]. (b) De Novo Ring Formation. Varelis and Johnson utilized a hetero-Diels–Alder reaction to prepare the pyran ring system 300. The olefin diastereomers were separated, and reduction followed by acetylation gave 298. Ireland ester rearrangement of 299 proceeded with syn selectivity to deliver the 1,6-dialkyl C-glycoside-like structure 300 (Scheme 53) [80]. Hanna and coworkers used a [2⫹2] cycloaddition approach for the preparation of precursor en route to forskolin. Dichloroketene cycloaddition with glycal 301 was followed by reductive dechlorination to give 302 in 80% overall yield. Several steps were required to convert 302 into the forskolin (304) Diels–Alder precursor 303 (Scheme 54) [81]. (c) Other Cycloadditions. Holzapfel and van der Merwe utilized a palladium-mediated [3⫹2] cycloaddition of nitroglycal 305 to deliver the C-glycosides 306 and 307 in a 1:2.4 ratio (Scheme 55) [82]. The 1,3-dipolar nitrone cycloaddition has also been employed for the preparation of pseudo-aza-C-disaccharides from glycals. Ex-

C-Glycoside Synthesis

Scheme 53

Scheme 54

107

Scheme 55

108

Postema and Calimente

posure of nitrone 308 to 3 equiv of glucal 309 in toluene at 100⬚C for 3 days gave a 61% yield of the adduct 310. The protecting groups were removed by standard methods to yield an intermediate polyol and cleavage of the N—O bond then delivered to the target compound 311 (48% over four steps; Scheme 55) [83]. 2.

Wittig Rearrangements

In 1997 Nakai and coworkers wrote a mini-review on the general area of subject of Wittig rearrangements [84]. Scheme 56 shows an application of the Wittig rearrangement (312 → 313) with a benzyl ether used as the starting material. Scheme 56 also illustrates the use of the propargylic substrates 314 and 317. The rearrangement proceeds with retention of configuration. This high selectivity is attributed to the tightly bound lithium-coordinated transition state, as in 315 [85]. Nakai has also employed the Wittig rearrangement in the preparation of the zaragozic acid core (319 → 320, Scheme 57). This work disclosed a violation of the principle of retention of configuration: when 321 was treated with butyllithium, a product with a net inversion of configuration at the anomeric center 322 was obtained. The authors suggest that steric hindrance of the groups on the ring caused radical recombination to occur from the less hindered bottom ␣ face (Scheme 57) [86]. VII. A.

CATIONIC APPROACHES Introduction

This section, the largest of the entire chapter in terms of number of citations, relies on the natural mode of reactivity at the anomeric center. Because of the large number of references, only an overview is presented here. Relevant references are included

Scheme 56

C-Glycoside Synthesis

109

Scheme 57

whenever possible. The section is divided into the formation of C-glycosides from reaction of glycals and 1,2-anhydro sugars, heteroatomic glycosyl derivatives, and sugar lactones with various nucleophiles. Section VII.E deals with the preparation of aryl C-glycosides. B.

Use of Glycals

1.

Reaction Under Lewis Acid Catalysis

The reaction of glycals with allylsilanes, enol ethers, or silyl acetylenes has received considerable attention over recent years. In 1996 Toshima et al. employed environmentally friendly montmorillonite K-10 (clay) to catalyze the addition of allylsilanes to sugar glycals for the purpose of obtaining allyl C-glycosides. Reaction of the galactal derivative 323 as shown in Scheme 58 gave a good yield of the ␣- and ␤allyl C-glycosides, with the 324 as the major isomer [87]. Work from the same group has also shown that the glycal does not have to be protected for the allylation to work, at least with conventional Lewis acid catalysts. This is exemplified by the conversion of 325 to 326 (Scheme 58) [88].

Scheme 58

110

Postema and Calimente

Scheme 59

Panek and Schaus continued their work with crotyl silanes and their addition to glycals (319 → 320) as shown in Scheme 59 [89]. Grieco et al. utilized [LiCo(B9C2H11)2] as a catalyst for the condensation of an allylic acetate with glycal 331 to give the C-glycosides 332. The use of this catalyst is interesting because it mimics the effect of highly polar media (5 M LiClO4 in Et2O) for reactions of these types [90]. Portella et al. showed that the acylsilanes and the glycal 309, in the presence of CF3SiMe3 and TBAF, react to give the C-glycosides 334 as a mixture of anomers [91]. Hayashi et al. cyanated (335 → 336) unprotected glycals in the presence of a catalytic amount of palladium(II) acetate (Scheme 59) [92]. Csuk et al. also examined some reactions of various glycals with a hindered silyl enol ether under Lewis acid catalyzed conditions (not shown) [93]. Isobe has continued his explorations of the reactivity of silyl acetylenes with glycals. The sugar-based acetylene 338 is condensed with glycal 337 to give the disaccharide 339 [94]. This area was reviewed in 1998 [95]. Steel et al. used actinium perchlorate to catalyze the dimerization of glucal. Three products were isolated in the yields shown. Separate treatment of 342 with boron trifuluoride etherate and triethylsilane then gave 343 in good yield (Scheme 60) [96]. 2.

With ‘‘Organometallic’’ Nucleophiles

Dorgan and Jackson [97] extended the work of Thorn and Gallagher [98], reaction of organozinc with glycals to give the corresponding C1 enitols (309 → 344), to the synthesis of C-linked glycosyl amino acids. This is exemplified by the conversion of 309 to 347 (Scheme 61). It has been reported that the glycal 348 undergoes cyclopropanation to give 349 or 350 as the major products, depending on the choice of reagent [99]. Treatment of 348 with ethyl diazoacetate in the presence of rhodium diacetate gave a 59% yield

C-Glycoside Synthesis

Scheme 60

111

112

Postema and Calimente

Scheme 61

of the ester cyclopropane 351 [100]. Hoberg and Claffey showed that treatment of the related glycal 352 under similar conditions gave 353 as the major product in 87% yield, with only trace amounts of the other three possible isomers (Scheme 62) [101]. The formed cyclopropanes have proved to be useful in several cases. For instance, 350 has been ring-expanded to the bicyclo olefin 354 by exposure to TMSOTf [102]. Compound 355 was also found to undergo ring opening to the dihalide 356, albeit in low yield [101]. In addition, 351 was converted to the Fuc-GDP analog 357, a potential transition state based inhibitor (Scheme 63) [103]. Van Boom and Sinay¨ have collaborated to show that the reaction of acetylides with 1,2-anhydro sugars under the influence of zinc chloride catalysis gives the ␣ isomer as the major product. Accordingly, reaction of 358 and 359 under the aforementioned conditions gave 360 in good yield. Standard protecting group manipulations then gave the C-disaccharide 361. The reaction is postulated to proceed via intermediate C [104]. Evans and coworkers have found that allylstannanes serve as good nucleophiles for oxirane opening of the 1,2-anhydro sugars under Lewis acid catalysis. Treatment of 358 with either 362 or 363 in the presence of tributyltin triflate gave good yields of the corresponding ␤-allyl-C-glycosides 364 and 365 (Scheme 64) [105].

Scheme 62

C-Glycoside Synthesis

113

Scheme 63

Scheme 64

Gervay et al. reported reduction of the diastereomeric epoxides, giving regioisomeric mixtures of alcohols (369 and 370) (Scheme 65) [106]. Related work (not shown) has been reported by Panza et al. [107]. C.

Use of Lactones with Organometallic-Based Nucleophiles

Dondoni and Scherrmann published a full account of their thiazole-based approach to formyl C-glycosides. Addition of the thiazololithio species to lactone 371 gave

114

Postema and Calimente

Scheme 65 the ␣-C-glycoside 372 as the major product. Reduction under standard conditions followed reduction–epimerization, and demasking of the protected aldehyde group then gave 373 (Scheme 66) [108]. Geneˆt et al. used a similar protocol to access ␤C-glycosyl aldehydes. Both the dithiane group (375 → 377) and an acetylene (378 → 377) served well as masked aldehyde equivalents (Scheme 66) [109]. Heterocyclic nucleophiles have also been utilized with pyranose sugars to prepare potential glucosidase inhibitors. In the case shown in Scheme 67, Lewis acid mediated silane reduction did not work owing to chelation of the Lewis acid with the imidazole group, and a slightly longer route was required to effect the reduction [110]. Epoxide alkyl ␤-C-glycosides, potential inhibitors of ␤-glucan hydrolases, have also been prepared from sugar lactones (374 → 377) [111]. The reaction of 2-deoxy-2-azido sugar lactones seems to parallel the reactivity of the corresponding 2-oxygenated lactones as shown for the conversion of 389 to 392 (Scheme 68) [112]. Martin and Saavedra utilized a conceptually related approach, the reduction of hemiacetals to give ␤-alkyl products, in the preparation of aza sugar analogs. Addition of (methoxymethoxy)methyllithium to lactone 374 gave 394, and this was followed by conversion to diketone 395. Reductive amination then furnished the protected C-aza sugar 396 (Scheme 68) [113]. Wightman et al. showed that in furanose sugars, reduction of C-glycoside lactols gives 1,2-anti products predominantly. It seems that silane reduction occurs preferentially from the same face as the vicinal oxygen substituent (Scheme 69) [114]. The reaction of enolates with lactones has also been carried out to give chainextended sugars. Three approaches have been published, two on samarium diiodide mediated chemistry [115,116] and one on classical Claisen-type chemistry [117]. Representative examples are shown in Scheme 70. D.

Use of Glycosyl Halides, S-Glycosides, Lactols, and O-Glycosides

1.

Glycosyl Halides

Glycosyl halides are popular electrophilic coupling partners for C-glycoside synthesis. The area of ␣-monohalo ethers in synthesis has been reviewed [118]. Heteroatoms at the 2-position have been well tolerated, as can be seen from the examples shown in Scheme 71. Alkylation of 407 with the potassium salt of diethylmalonate

C-Glycoside Synthesis

Scheme 66

115

116

Scheme 67

Scheme 68

Scheme 69

Postema and Calimente

C-Glycoside Synthesis

117

Scheme 70

Scheme 71

gave the ␤-C-glycoside 408 after decarboxylation [119]. Under the influence of silver ion, the displacement of the anomeric bromide in 410 with the tin acetylide shown, gave the the ␣ product 411 exclusively. Here, the stereochemical outcome is rationalized on the basis of attack of the formed oxonium from the ␣ face under the influence of the anomeric effect [120]. Similar chemistry has also been carried out

118

Postema and Calimente

Scheme 72

on the 2-oxygenated compounds. Aza sugars have also been found to be reactive toward electrophilic C-glycosidation upon conversion to the anomeric fluorides. Several nucleophiles were examined, and Scheme 71 shows the allylation and reaction with silyl enol ethers of 413 [121]. Veyrie`res et al. found that the allylsilane 417 reacts with a variety of pyranose derivatives to give good yields of allylated and functionalized C-glycosides (Scheme 72) [122]. 2-Thioglycosyl chlorides, derived from ionic addition of RSCl to glycals, have been found to react with Grignard reagents to give the ␤-gluco C-glycosides 426. The same chlorides also react with silyl enol ethers and allyltrimethylsilane to give the corresponding C-glycosides, 425 (Scheme 73) [123,124]. Intramolecular C-glycosidations have also been explored. For example, exposure of 427 to AgBF4 with strict exclusion of water gave a 74% yield of 428. The benzylic positions were oxidized to furnish 429, and ester cleavage then gave the free C-glycoside 430 (Scheme 74) [125]. Lo´pez and Go´mez found that treatment of pyranosyl chlorides with a suitable organolithium gave acceptable yields of the corresponding C1-glycals 432–434 (Scheme 75). No mechanistic explanation was offered. One possibility could be elimination of the benzyloxy group to generate the C1-chloro glycal, followed by addition of the organometallic to the double bond at C1 and subsequent elimination of chloride [126].

Scheme 73

C-Glycoside Synthesis

119

Scheme 74

Scheme 75

2.

Thioglycosides

Thioglycosides have received some attention over the last few years. The ethyl thioglycoside 435 has been found to be a good substrate for the selective formation of the ␣-glycosyl cyanide 436 (Scheme 76) [127]. Craig has continued his work on template directed C-glycosidations. Work directed at the C-glycosidation of 437, shown in Scheme 77 [128], is highlighted by the preparation of 2,3-dideoxy-D-manno-2-octulopyranosonic acid (444). Accordingly, thioglycoside 437 was exposed to AgOTf and gave the cis-fused C-glycoside 438 in 64% yield. Oxidative cleavage followed by reduction led to 442, which was desulfonylated to give the fragmentation product 443. Standard manipulations then afforded the target acid 444 [129]. 3.

Anomeric Acetates and Imidates

The formate group has been used at the anomeric center to install acetylenic moieties (445 → 446, Scheme 78) [130], while an acetate function served admirably as the activating function on a tetrahydropyran ring to install an allyl group in the synthesis of the F-ring 448 of the spongistatins. The TBS groups were first replaced with acetates to ensure ␤-C-glycosidation [131]. C-Glycosidation of furanosyl acetates is still a popular method for introduction of carbon-based groups at the anomeric center. Scheme 79 shows some recent examples. The entries highlight the use of a diethylthiocarbamoyl function as a partic-

Scheme 76

120

Postema and Calimente

Scheme 77

ipating group to control the facial selectivity of C-glycosidation [132]. This is significant, since the lack of a group at C2 hinders stereocontrol in the 2-deoxyribofuranosides [133]. It has also been found that scandium perchlorate serves as a good catalyst for the selective ␣-C-glycosidation of perbenzylated ribofuranosyl acetates [134]. Heteroatom-based siloxy dienes have received attention in recent years as an efficient method for building up ring frameworks. The subject has been reviewed, and only a few applications are illustrated here [135]. This is a powerful process, since in a relatively small number of steps several rings can be joined together. The first two equations [136,137] of Scheme 80 show how a single butenolide unit can be attached, while the last sequence illustrates an iterative methodology [138]. It should be noted that in the addition of trimethylsiloxy furan to the anomeric acetate,

Scheme 78

C-Glycoside Synthesis

121

Scheme 79

a mixture of four compounds is possible. Usually the erythro product 463 was obtained in a 6:4 ratio. The two examples in Scheme 81 illustrate the use of a cyclic enol ether reacting with anomeric acetate 467 to give 468 [139], as well as the reaction of an olefin with the same acetate to give the novel ring-fused product 469 [140]. The final example demonstrates the use of amino acid derived enol ether 470 reacting with a glycosyl imidate 469 under Lewis acid catalysis to give a low yield of the carbon-linked threonine isostere 471 (Scheme 82) [141].

Scheme 80

122

Postema and Calimente

Scheme 81

Scheme 82

4.

Lactols

There are two reports of the use of lactols as precursors to C-glycosides. Wang and Gross found that when triol 472 was treated with nitroethane under the outlined conditions, a 50% yield of the C-glycoside 473 was obtained as the major product [142]. D-Glucose (474) was found to undergo condensation with the barbituric acid derivative 475 to afford 476. Alkylation then gave the C-glycosyl derivative 477 (Scheme 83) [143]. 5.

Alkyl Glycosides

During their synthesis of the F-ring (480) of halichondrin B, Yonemitsu and coworkers relied on allylation of the methyl glycoside derivative 478 to deliver 479, which was converted to the target (Scheme 84) [144]. Johnson has carried out a

Scheme 83

C-Glycoside Synthesis

123

Scheme 84

highly selective allylation on the nojirimycin analog 481 to provide 482, which was then converted to the indolizine analog 438 (Scheme 84) [145]. Schmidt converted the TCP-protected glucosamine derivative to 484 via a cationic allylation reaction. Compound 485 was then converted to the amino acid analog 486. The ␤-selective allylation is noteworthy and is presumably due to the steric bulk of the phthalidebased amino protecting group, (Scheme 84) [146]. Ley et al. found that exposure 487 to stannic chloride furnished a good yield of the disaccharide derivative 488 as a 1:1 mixture of isomers (Scheme 84) [147]. Two groups have prepared C-glycoside analogs of the natural product (⫹)hydantocidin via Lewis acid mediated C-glycosidation of the acetal derivative 489 [148]. Sano and coworkers succeeded in converting 490 to the succinimide analog 493 (Scheme 85) [149]. A synthesis of the spirohydrouracil nucleoside analog of (⫹)-hydantocidin has also appeared [150]. E.

Aryl C-Glycosides

1.

Lewis Acid Monoarylation

Several different catalyst systems, anomeric activators, and applications of Lewis acid catalyzed reactions for aryl C-glycoside synthesis have been published over recent years and a few are presented in Scheme 86. Inazu et al. used the dimethylphosphinothioate groups [151], while Satoh et al. introduced the use of AgOTfa/ SnCl4 as a novel catalyst system [152]. Schmidt et al. continued the use of the trichloroacetimidate group, this time directed at the preparation of bis(C-glycosyl) flavanoid precursors [153]. Sato’s group has also used glycosyl fluorides to prepare precursors for C-glycosyl flavanoids (Scheme 86) [154]. Mallareddy and Rao prepared analogs of monocerin via intramolecular aryl C-glycosidation, (Scheme 86)

124

Scheme 85

Scheme 86

Postema and Calimente

C-Glycoside Synthesis

125

Scheme 87

[155]. Andrews and Larsen also carried out Lewis acid catalyzed glycosidations of naphthalene derivatives with anomeric acetates to gain access to the ring systems of the Angucycline antibiotics [156]. Suzuki has also published an account of his work on novel Lewis acid catalysts in organic synthesis [157]. Toshima et al. found that unprotected 2-deoxy sugars, as either reducing sugars or methyl glycosides, will undergo C-glycosidation to give aryl C-glycosides [158]. The reaction has been catalyzed by TMSOTf, and it has also been found that montmorillonite K-10, used in excess, also works with unprotected sugars (Scheme 87) [159]. Work by Park et al. deals with regioselective acylation of the 2⬘-hydroxyl group on a free aloesin glucopyranoside (not shown) [160]. The normal course for these O → C-glycoside rearrangement is to deliver the ␤ or equatorial product preferentially. Suzuki et al. found that if O4 and O3 are protected by extremely bulky groups, such as t-butyldiphenylsilyl, the ␣ anomer is the major product of the reaction. This result is contrasted to the case of the use of smaller groups, such as TBS groups, which produces ony the ␤ isomer (Scheme 88). So the stereochemistry of the glycosidation can be controlled by judicious choice of protecting groups [161]. 2.

Lewis Acid Mediated Diarylation

A few groups have carried out multiple arylation reactions to give bis-C-glycosyl compounds (Scheme 89). Schmidt et al. used their trichloroacetimidate methodology

Scheme 88

126

Scheme 89

Postema and Calimente

C-Glycoside Synthesis

127

Scheme 90

in a sequential process to deliver 522 [153]. Kumazawa et al. employed anomeric fluorides under boron trifluoride catalysis to give the corresponding bis-C-glycosyl flavanoid precursor 526 [162]. Satoh, using his SnCl4 /AgOTfa catalyst system, was able to introduce a second sugar unit onto an existing aryl C-glycoside (Scheme 90) [163]. VIII.

SUMMARY

This chapter provides an updated overview of recent developments in the synthesis of C-glycoside compounds. The stereoselectivity of the C-glycosidation step has improved over recent years, and milder conditions for C—C bond formation are still being developed. The use of unprotected C-glycosides is another important advance that has recently come to fruition [164]. Also the use of modern methods based on metals seems to be increasing in popularity. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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5 D-Arabinofuranosides

from Mycobacteria: Synthesis and Conformation Todd L. Lowary The Ohio State University, Columbus, Ohio

I.

INTRODUCTION

Unlike the majority of monosaccharides, the pentose arabinose exists in nature in all possible absolute and ring configurations, namely, D-arabinofuranose (D-Araf ), L-arabinofuranose (L-Araf ), L-arabinopyranose (L-Arap), and D-arabinopyranose (DArap). The pyranose forms are more rare and are found primarily either in protozoan parasites [1] or as constituents of plant saponins [2]. Oligo- and polysaccharides comprised of L-arabinofuranose are also widespread in the plant kingdom, where they are present as constituents of arabinoxylans [3], pectins [4], and hydroxyprolinerich glycoproteins (HPRGs) [5]. This chapter focuses on polysaccharides containing D-arabinofuranose and in particular the most prominent examples of these polymers, which are found as important components of the cell wall of members of the Actinomycetes family including the genera Mycobacteria, Corynebacteria, Nocardia, and Rhodococcus [6]. Although the majority of actinomycetes are benign to humans, among them are two important human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae [7]. Infection by these organisms causes, respectively, tuberculosis and leprosy. Mycobacterial infections have attracted renewed attention in recent years owing to their increasing incidence in the industrialized world as well as the emergence of drug-resistant strains of these organisms [8]. Additionally, AIDS patients and others with compromised immune systems are susceptible to opportunistic infections caused by ‘‘atypical’’ mycobacteria including M. avium and M. kansasii [9]. 133

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The total number of people infected with M. tuberculosis has been estimated at a third of the world’s population and almost 3 million deaths from tuberculosis occur each year, making it the single most lethal bacterial disease [10]. Treating mycobacterial infections is difficult and usually requires a 6–12 month course of antibiotic therapy [11]. The need for antibiotics over an extended period is due largely to the structure of the mycobacterial cell wall, which serves as a formidable barrier to the passage of antibiotics into the organism as well as into the immune system of the human host. Drug regimens that are in current use generally include one or two antibiotics that inhibit cell wall biosynthesis (e.g., ethambutol or isoniazid), in combination with others that have intracellular targets (e.g., rifampicin or streptomycin). When administered together, such combinations destroy the integrity of the cell wall, which in turn allows other antibiotics to pass to the cell more efficiently [12]. Even ignoring the issue of drug resistance, the arsenal of drugs that are active against mycobacteria is relatively small [8], and thus there is a pressing need for the identification of new antibiotics. When considered along with the emergence of drug-resistant mycobacterial strains, this need becomes even more urgent. In the search to identify new antibiotics, one area receiving particular attention is the identification of compounds that act by inhibiting mycobacterial cell wall biosynthesis. An increasing number of papers on this topic have appeared in the last few years [13–17]. However, a detailed understanding of the cell wall structure has really become available only in the last decade [6,13], and an in-depth understanding of its biosynthesis is unavailable [13]. Many more fundamental chemical and biochemical studies need to be completed before the rational design of such antibiotics can be realized. The mycobacterial cell wall is composed, to a very large degree, of polysaccharides and lipids [6]. The two major polysaccharides are an arabinogalactan (AG) and a lipoarabinomannan (LAM). The predominant lipid species are mycolic acids, branched long-chain lipids characteristic to the actinomycetes. What is particularly unique about both AG and LAM is that all the arabinose and galactose residues are present in the furanose form. Since oligosaccharides containing furanose residues are xenobiotic to mammalian biochemistry, the enzymes that assemble these polysaccharides are ideal targets for drug action. This chapter focuses on the arabinan portions of the AG and LAM. Sections II–IV outline the structure, biochemical importance, and biosynthesis of these arabinans. Section V discusses the chemical syntheses of oligosaccharides containing D-arabinofuranose residues. Such compounds are important tools in biochemical studies leading to a more detailed understanding of cell wall biosynthesis. Section VI focuses on work that we and others are doing to elucidate the solution conformation of oligo- and polysaccharides containing D-arabinofuranose residues, and Section VII offers some concluding remarks. This review covers work published up to the end of 1998. II.

ARABINAN STRUCTURE

Only recently has a detailed picture of the structure of the mycobacterial cell wall emerged [6,18,19]. Although the constituent species present have been known for many years [20,21], the assembly of these constituents into a coherent structure has required lengthy investigations and has relied on a number of techniques including,

D-Arabinofuranosides from

Mycobacteria

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in addition to methylation analysis, both NMR spectroscopy and fast-atom bombardment high-resolution mass spectrometry [6,18,19]. These studies were also greatly facilitated by the discovery [22] of hydrolytic enzymes capable of digesting both AG and LAM into small fragments, which are more amenable to these characterization methods. A.

Arabinogalactan

In common with gram-positive bacteria, mycobacteria have a layer of peptidoglycan immediately external to the cytoplasmic membrane. This peptidoglycan serves as the scaffold to which the AG is attached. Figure 1 is a schematic drawing of the overall structure of a single AG molecule [6]. A number of structural motifs are worthy of mention, and these are illustrated in Figure 2. Linkage of the AG to peptidoglycan is by way of a phosphodiester through a disaccharide comprised of rhamnose and N-acetylglucosamine (1). Attached to the 4-hydroxyl group of the rhamnose moiety is a linear chain of approximately 30 D-galactofuranose (D-Galf ) residues with alternating ␤-(1→5) and ␤-(1→6) linkages (2). Along the galactofuranose chain are branch points at which arabinan chains are attached. This arabinan contains in total approximately 70 D-arabinofuranose residues and consists primarily of linear ␣(1→5)-linked chains (3). However, there are periodic branch points at which another linear arabinan chain is attached via an ␣-(1→3) linkage (4). At the distal, nonreducing end of these chains is the branched hexasaccharide 5. In a single AG molecule, approximately two-thirds of these hexasaccharide motifs are esterified to mycolic acids at the four primary alcohols to provide 6. These lipids, which are unique to mycobacteria and other actinomycetes, are branched fatty acids containing 70–90 carbons as well as cyclopropane, ketone, hydroxyl, and methoxyl functionalities [23,24]. An example of one type of mycolic acid, a ketomycolate, 7, is shown in Figure 2. B.

Lipoarabinomannan

LAM, which has a less homogeneous structure than the AG, is attached noncovalently to the cytoplasmic membrane through the lipid portion of a phosphatidylinositol (PI) linker. A schematic drawing of LAM is presented in Figure 3 [6]. A mannan, comprised of ␣-(1→6) linked D-mannopyranose residues, is covalently bound via a glycosidic linkage to the inositol O6. Approximately half these mannose residues carry an ␣-(1→2) D-mannopyranosyl branch. An arabinan very similar to that found in the AG is attached at the termini of the mannan. In common with the AG arabinan, the major structural motif is an ␣-(1→5)-linked chain with periodic branch points, and hexasaccharide 5 at the nonreducing termini. However, there are fewer branch points, and thus the ratio of linear to branched arabinofuranose in LAM is greater than in AG. In addition, while the hexasaccharide motifs (5) found in the AG are esterified to mycolic acids, in LAM these moieties are found either unsubstituted or, in particularly virulent strains, capped with short mannopyranosyl oligosaccharides (8). When capped with the mannose residues, the polymer is referred to as ManLAM. Other structural features have been discovered more recently. For example, hexasaccharide 5 found in LAM of M. smegmatis is capped with inositol phosphates (9), thus providing a new type of LAM termed PI-GAM (phosphoinositol-glyceroarabino-mannan) [25,26].

136

Schematic representation of mycobacterial arabinogalactan (AG).

Lowary

Figure 1

D-Arabinofuranosides from

Figure 2

III.

Mycobacteria

137

Structural motifs present in mycobacterial AG and LAM.

ROLE OF THE AG AND LAM IN DISEASE PROGRESSION

The AG together with the mycolic acids comprise the major structural component of the mycobacterial cell wall; intercalated within this framework is LAM. These polymers play paramount roles in enabling these organisms to survive in the human host. For example, in contrast to many other microorganisms, mycobacteria can thrive in human macrophages instead of being killed by them [27]. This property is believed to be due, in large part, to the impenetrable nature of the cell wall envelope. In addition to protecting the organism from macrophage destruction, the cell wall is believed to substantially reduce the flow of antibiotics into the organism [12,28] and to be involved in the initial stages of the infection by the binding of cell wall oligosaccharides to host receptors [29]. A.

Arabinogalactan

The mycolic esters at the nonreducing ends of the AG present a formidable hydrophobic barrier that prevents the passage of antibiotics into the organism [28]. The currently accepted structural model of the mycolyl–AG complex was initially proposed by Minnikin in 1982 [24] and is illustrated in Figure 4. In this model the mycolic acid chains are packed side by side, perpendicular to the plane of the cy-

138

Schematic representation of mycobacterial lipoarabinomannan (LAM).

Lowary

Figure 3

D-Arabinofuranosides from

Figure 4

Mycobacteria

139

Schematic drawing of the mycolyl–AG complex.

toplasmic membrane. The tight packing of these chains results in very low fluidity of the cell wall, which in some cases is essentially crystalline [30,31]. The fluidity is dependent on the mycolic acids present, which in turn are species specific [31]. However, in all cases a major problem in treating mycobacterial infections is the passage of drugs through this lipid barrier into the host. This model was initially criticized by some, who suggested that the AG was too large to accommodate the tight packing of the lipid chains [28]. However, a more detailed look at the structure of the AG provides a solution to this problem. A distinguishing feature of mycobacterial AG is the almost exclusive presence of furanosyl (Araf or Galf ) residues. Furanoside rings are known to be much less conformationally well defined than their pyranoside counterparts [32]. It has been suggested that this inherent increased flexibility, in addition to the predominant coupling of these residues through linkages that involve primary hydroxyl groups, results in the polysaccharide being a scaffold of high flexibility. This in turn allows the polymer to adopt a conformation that facilitates the orientation of the mycolic acids into tightly packed parallel arrays. Additionally, this proposal provides a reasonable explanation for what, from an evolutionary standpoint, seems odd. Polysaccharides containing furanosyl residues are expected to be less thermodynamically stable than those possessing pyranose rings, and it therefore appears that these organisms have developed a system whereby glycan stability is traded for the protection afforded by the tightly packed mycolic acids. B.

Lipoarabinomannan

The major antigenic component of the mycobacterial cell wall is LAM. It should be pointed out that although not explicitly shown in Figure 4, LAM is interspersed throughout the mycolyl–AG framework, and this polymer is believed to be long enough that any groups found at the nonreducing terminus (e.g., the mannopyranosyl oligosaccharides) extend through the mycolic acid layer and are expressed on the outer surface of the cell wall [33]. LAM has been suggested to play critical roles in many immunomodulatory events occurring during progression of the disease. Among these are the inhibition of macrophage activation [34], the neutralization of potentially cytotoxic O2 free radicals [35], the induction of cytokines [36–38], the inhi-

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bition of protein kinase activities [39], and the induced expression of collegenases that destroy the extracellular matrix of the lung [40]. LAM in which the primary hydroxyl groups of 5 are capped with mannopyranosyl oligosaccharides (8, ManLAM) has been suggested to be involved in the infection process. It is believed that these terminal mannose residues initiate infection by adhering to human cells through their recognition by human mannose binding proteins [41–43]. In contrast to the capping of 5 by mannopyranosyl residues, mycobacterial species (e.g., M. smegmatis) in which this motif is capped with inositol phosphate residues (8) are avirulent. It has been proposed that the lack of pathogenicity of this species arises from the induction of tumor necrosis factor ␣ (TNF-␣) by the inositol phosphate moieties, which in turn results in the killing of the bacteria by macrophages [26]. Perhaps the most important recent discovery is that T cells recognize LAM via antigen presentation pathways that are independent of the major histocompatibility complex (MHC) [44,45]. It was shown that LAM is initially recognized by a mannose receptor and the protein CD14 before it is processed and then complexed with CD1b. This complex is then expressed on the surface of the antigen-presenting cell. Recognition by the T cell requires both the carbohydrate and lipid portion of LAM, and two distinct cell lines have been shown to differentiate between structurally different LAM fragments. Thus it appears that there is a significant amount of specificity in T-cell responses mediated by the LAM-complexed CD1 glycoproteins. IV.

ARABINAN BIOSYNTHESIS

Given the critical importance of AG and LAM to the survival of mycobacteria, there is increasing interest in developing new drugs for the treatment of these diseases, which act by inhibiting the enzymes involved in their biosynthesis. Inhibitors of the glycosyltransferases involved in the assembly of the furanosidic oligosaccharide portions of the glycoconjugates are particularly attractive targets. Glycans of this type are unknown in humans, and therefore blocking these biosynthetic pathways will have little deleterious effect on the host. However, for rational drug design to succeed, more details about the biosynthetic pathways leading to the formation of these polysaccharides are required. Although the overall features of this process are known [13,18,19], many of the details are not. The ␤-(1→2)- and ␣-(1→5)-linked arabinose residues are incorporated into the polymer from the activated polyprenyl sugar phosphate 10, which is in turn synthesized from glucose via 5-phosphoribose pyrophosphate (pRpp) [46–48]. Elongation of the polymer chain is believed to involve a family of arabinosyltransferases (AraT’s) that recognize both 10 and arabinofuranoside-based acceptors of differing structures (Fig. 5) [18,19,49,50]. In AG biosynthesis, the entire polysaccharide appears to be assembled as a polyprenol diphosphate intermediate, which is transferred to peptidoglycan prior to the addition of the mycolate esters [18]. In LAM biogenesis, the arabinan portion is believed to be synthesized as a polyprenol phosphate that is transferred to lipomannan [51]. Incorporation of arabinose into AG has been demonstrated through the use of synthetic, radiolabeled 10 and mycobacterial membrane extracts [50]. These reactions are believed to involve membrane-bound substrates and enzymes and to occur extracellularly. While to date none of these putative glycosyltransferases have been purified to homogeneity, the gene cluster that encodes for one or more of these AraT’s

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Figure 5

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141

A typical reaction catalyzed by mycobacterial arabinosyltransferases (AraT’s).

has been cloned and expressed [52–54]. In addition, an assay for their activity that uses mycobacterial membrane preparations as the enzyme source has been developed [49,50]. The assay measures the incorporation of radiolabeled arabinose from 10 into arabinan, and it has been used to screen both potential substrates and inhibitors [46,47,55,56]. Although the natural acceptor substrates for these enzymes are lipid-bound intermediates, these AraT’s also recognize small arabinofuranosyl oligosaccharides. A few of the oligoarabinosides that have been shown to be substrates for these enzymes are illustrated in Figure 6 [49,55]. Arabinofuranose disaccharides appear to be the minimum epitopes. Furthermore, investigation of the AraT recognition of the disaccharide ␣-D-Araf-(1→5)-␣-D-Araf-R (13–15 ) and the trisaccharide ␣-D-Araf(1→5)-␣-D-Araf-(1→5)-␣-D-Araf-R (16, 17) revealed that glycosides with R = octyl (14, 17) are better substrates and those with R = methyl (13, 16). Surprisingly, the dodecyl glycoside 15 was a poorer substrate than 13. In the investigation of 14 and 17, the products of the enzymatic reactions were isolated and their structures elucidated [49]. In both cases, mixtures of two products, corresponding to addition of an Araf residue to either the 2- or the 5-position of the terminal residue in the oligosaccharide acceptor, were obtained. From this study it was concluded that 10 was the source of the ␤-(1→2)-, and ␣-(1→5)-linked Araf residues. Less is known about the formation of the ␣-(1→3) branch points than about the ␤-(1→2) and ␣-(1→5) linkages. Only ␤-(1→2) and ␣-(1→5) linkages are formed by the incubation of small oligosaccharide substrates with 10 in the presence of a mycobacterial membrane preparations [49]. However, additional work [57] demonstrated that when radioactive 10 is incubated with endogenous acceptors and a mycobacterial membrane preparation, a polymer essentially identical to native AG can be obtained. Additionally the radioactivity in the polymer was equally distributed

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Figure 6 Arabinofuranosyl oligosaccharides tested as substrates for mycobacterial arabinosyltransferases.

throughout, which suggests that 10 is the major, and possibly sole, source of Araf residues. The lack of ␣-(1→3) linkages formed in the initial study [49] could be attributed to a number of factors: (1) the instability or absence of ␣-(1→3) AraT activity in the membrane preparation, (2) the possibility that this enzyme recognizes oligosaccharide substrates larger than those investigated, or (3) the possibility that another activated donor (e.g., a sugar nucleotide) is used by this AraT. Although the presence of UDP-Araf in mycobacteria has been reported [58], the incorporation of arabinose from this source into arabinan has not been demonstrated. The range of donor substrates that are tolerated by these enzymes has also been explored. A series of analogs was prepared, each one differing from 10 only in the identity of the lipid chain, and the set was screened for biological activity [56]. It was discovered that conjugates of C50 and C55 prenols were substrates, while those compounds with shorter (
Action of Ethambutol

From the discussion above is it clear that both AG and LAM play paramount roles in the infection and survival of mycobacteria in the host. Drugs that act by inhibiting the biosynthesis of these polysaccharides are expected to show antimycobacterial action. One of the commonly used antituberculosis agents, ethambutol [(S,S⬘)-2,2⬘(ethylenediimino)di-1-butanol, 22] (Fig. 7), has been used for the treatment of tuberculosis since 1961, when it was first reported to have antimycobacterial activity

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Figure 7

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Structure of the antituberculosis drug ethambutol.

[59]. This drug is active only against mycobacteria [8], and while it was thought for many years to be involved in inhibiting cell wall biosynthesis [60,61], its precise target was not elucidated until recently. The structure of the drug is relatively simple and bears little resemblance to any cell wall component, which have complicated the identification of its precise biochemical target. It is now known that the drug acts as an AraT inhibitor and disrupts the biosynthesis of the arabinan portions of both AG and LAM [43,47,62–64].

V.

SYNTHESIS OF D-ARABINOFURANOSIDES

In comparison with the synthesis of oligopyranosides, the preparation of oligosaccharides containing furanose residues has received relatively little attention. However, this will likely change as the biological importance of glycans containing these residues becomes more widely appreciated. The methodologies used to date for assembling these molecules have closely paralleled the methods used for the assembly of pyranosidic linkages. Nevertheless, these interesting structures also open up the possibility for new methodologies to be developed, and these will undoubtedly emerge. In this section are outlined the chemical synthesis of D-arabinofuranosides that have been reported. As explained in Section IV, although the normal acceptor substrates for mycobacterial arabinosyltransferases are high molecular weight glycolipids, some (and probably most, if not all) of these enzymes will recognize small oligosaccharide fragments of these large glycans [49,55]. In this regard, these enzymes appear to be similar to mammalian glycosyltransferases [65]. Therefore, these small oligosaccharides are invaluable tools not only for mapping out substrate specificities of these enzymes but also as templates for the design of potentially inhibitory analogs. A.

Route to Arabinofuranose Donors

At equilibrium in solution, most sugars exist predominantly in the pyranose forms. For example, in water, D-arabinose is found in approximately a 95:5 pyranose-tofuranose ratio [66]. Therefore, the initial step in any synthesis of an oligofuranoside is the conversion of this mixture of pyranoses to a furanosyl derivative that can be incorporated into oligosaccharides. This can be done either directly or indirectly as outlined below.

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Fisher Glycosylation

Furanose glycosides of simple alcohols are conveniently obtained by subjecting a reducing sugar to a controlled Fisher glycosylation reaction. The furanosides are the kinetic products of this reaction, and excellent yields of the product (as a mixture of anomers) are usually obtained if the proper conditions are chosen [67]. The alcohol generally used in this reaction is methanol, which serves as both a reactant and the solvent. The methyl glycosides thus obtained must then be protected and converted to a suitable glycosylation agent. It is also possible to synthesize glycosides of other alcohols by this method; however, since the alcohol is usually employed as the solvent, the choice is somewhat limited. Therefore, from a practical point of view, this method is most useful for the preparation of glycosides of relatively volatile alcohols (e.g., methanol, ethanol, and allyl alcohol). This method works well for the preparation of methyl D-arabinofuranosides, and an excellent preparation is available [68] that can be used to prepare multigram quantities of the product (Fig. 8). Treatment of D-arabinose (23) with acidic methanol at room temperature provides a 1:1 ␣ : ␤ mixture of methyl arabinofuranosides (24). Benzoylation provides 25, again as a mixture of anomers; however, the ␣ isomer can be selectively crystallized from ethanol. With either 24 or 25 in hand, it is possible to prepare, in 1–3 steps, the glycosyl donors shown in Figure 9. These include glycosyl acetates 26 [69] and 27 [70,71], glycosyl halides 28 [68,72], 29 [49], 30 [73], 31 [73], and thioglycosides 32–34 [55,74–76]. 2.

Cyclization of Acyclic Dithioacetals and Related Derivatives

An old [67,77] but recently rediscovered [78] method for synthesizing furanose glycosides involves the cyclization of acyclic sugar S,O-acetals (e.g., 35) in the presence of mercuric salts (Fig. 10). This method is highly selective, producing furanosides in the absence of pyranosides, and is especially useful for the synthesis of galactofuranosides (e.g., 36) of both simple and carbohydrate alcohols [67,78]. This approach has not yet been used for the synthesis of D-arabinofuranosides; however, two related syntheses have been reported (Fig. 11). Work by Zinner and coworkers [79] demonstrated that the cyclization of the 5-O-benzoyl derivative of D-arabinose diethyl dithioacetal (37) in mercuric chloride afforded modest yields of thioglycoside 38. More recently, the isopropylidene derivative 39 has been prepared in good yield from 37 by treatment with mercuric chloride in acetone [80]. Conversion of 37 to any of the derivatives shown in Figure 9 can be achieved in a few steps. 3.

Ozonolysis of Glycals

The most recently reported route to the D-arabinofuranose ring nucleus involves an oxidative cleavage of protected glucal derivatives as illustrated in Figure 12 [74].

Figure 8

Fisher glycosylation route 2,3,5-tri-O-benzoyl-␣-D-arabinofuranoside (25).

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Figure 9

Mycobacteria

Synthesis of commonly employed D-arabinofuranosyl donors.

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Figure 10

Synthesis of ␤-galactofuranosides from acylic S,O-sugar acetals.

Ozonolysis of 3,4,6-tri-O-acetyl D-glucal (40), followed by reductive workup with dimethyl sulfide, affords the formyl aldehyde 41. The formate ester can be selectively cleaved in the presence of the acetates by refluxing in acidic methanol yielding the reducing sugar 42. Attempts to use 42 directly in glycosylation reactions via in situ activation of the anomeric hydroxyl group [81,82] failed, but this derivative could be converted in two steps and excellent yield to thioglycoside 33 [74]. Protected D-glucal derivatives are readily available, and consequently this methodology is an attractive alternative to more conventional routes to arabinofuranosides. In particular, the conversion of fully 13C-labeled glucose to labeled arabinose oligosaccharides by this route has been demonstrated [79]. For the synthesis of 13C-labeled oligoarabinosides, this method is preferable to a strategy involving Fisher glycosylation of labeled D-arabinose because the cost of the starting material (13C-labeled D-glucose) is fourfold cheaper, while the overall yields of the transformations are similar. B.

Glycosylation Reactions with Arabinofuranose Donors

The glycosyl donors illustrated in Figure 9 have been the ones most commonly employed in the preparation of oligosaccharides containing D-arabinofuranosyl residues. The vast majority of glycosidic bonds in mycobacterial arabinan are ␣ link-

Figure 11

Cyclization of arabinose dithioacetals to give furanose derivatives.

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Figure 12

Synthesis of protected arabinofuranose reducing sugars by ozonolysis of acetylated D-glucal.

ages, and thus the relationship between the groups at C1 and C2 are trans. Consequently, the synthesis of these linkages can be achieved through the use of glycosyl donors possessing acyl protecting groups on O2. As would be expected, neighboring group participation resulting in the formation of an acyloxonium ion provides, in most cases, good to excellent yields of the 1,2-trans-glycoside. Donors 26–29 , 32, and 33 have been used effectively for these glycosylation reactions; however, with some donors the stereocontrol is poorer than in others. For example, it has been demonstrated that while thioglycoside 33 provides good yields of the desired product, peracetate 27 gave significant amounts of the 1,2-cis product [74]. Similar observations have been made with other arabinofuranosyl glycosyl donors [71,83]. In addition to 1,2-trans-D-arabinofuranosyl linkages, mycobacterial arabinan also contains ␤-D-arabinofuranosyl residues (1,2-cis-glycosides). These linkages are directly analogous to ␤-D-mannopyranosides, which are among the most difficult of all glycosidic linkages to prepare [84]. However, in contrast to the ␤-D-mannoside case, no significant effort has been directed toward the stereocontrolled synthesis of ␤-D-arabinofuranosides. A minimum prerequisite to the synthesis of any such linkage are donors possessing a nonparticipating group at O2. For this purpose, the 2-Obenzyl derivatives 30, 31, and 34, have been used [73,85,86] to produce, in most cases, mixtures from which the desired compounds could be isolated. Characterizing the stereochemical course of these glycosylation reactions is easily done by means of standard one-dimensional 13C and 1H NMR experiments [83]. The anomeric carbon of the ␣ anomers resonate between 107 and 110 ppm; the ␤ anomers between 97 and 104 ppm. Furthermore, in the ␣ anomers 3JH1,H2 is small (0–2 Hz), whereas in the ␤ anomers the magnitude of this coupling is larger (3–5 Hz). It should also be mentioned that although 1JC1,H1 values are unambiguous determinants of anomeric stereochemistry in pyranosides [87], they cannot be used to make these assignments in arabinofuranosides as their magnitudes have been shown to be insensitive to anomeric configuration [83]. 1.

Syntheses of 1,2-trans-Linked Oligosaccharides

The disaccharide ␣-D-Araf-(1→5)-␣-D-Araf comprises the core structure of the linear sections of the arabinan. A number of syntheses of this glycan nucleus, as glycosides of a variety of alcohols have been reported. The first synthesis was of the allyl glycoside 48, which was prepared as illustrated in Figure 13 [72]. Orthoester 43 was prepared from the bromide 29 and then rearranged to give the allyl glycoside 44. A series of protection and deprotection steps afforded the trityl ether 45, which was subsequently glycosylated with orthoester 46 (also prepared from 29). Deprotection afforded the target, 48. In the same paper, this route was used to prepare the benzyl glycoside starting from a benzyl orthoester analogous to 43.

148

Synthesis of disaccharide 48: All, allyl; Tr, triphenylmethyl.

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Figure 13

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149

As outlined in Figure 14, a more recent synthesis [55,74] of this disaccharide as the methyl glycoside (13, Fig. 6) made use of thioglycoside donor 33. This synthesis began with the preparation of alcohol 49 from 24␣, through a sequence of silylation, benzoylation, and desilylation. Glycosylation promoted by N-iodosuccinimide and silver triflate afforded the disaccharide 50, which could be deprotected with sodium methoxide to provide 13. A number of other derivatives of this disaccharide have been synthesized, namely, the 2⬘-chloroethyl, octyl (14), dodecyl (15), and hexyl glycosides [49,71]. These syntheses have employed either acetate 27 or chloride 28 as the glycosylating agent. The trisaccharide ␣-D-Araf-(1→5)-␣-D-Araf-(1→5)-␣-D-Araf is also a core fragment of the linear arabinan, and two synthetic derivatives have been reported. The chloride 28 has been used to prepare the S-octyl glycoside analog (17), as well as the methyl glycoside (16) [49]. Another route to 16 is illustrated in Figure 15 [55]. Monosaccharide 33 was converted to the differentially protected thioglycoside 51 in three steps. Glycosylation of 49 with 51 provided the protected disaccharide 52, which upon treatment with tetrabutylammonium fluoride was transformed to alcohol 53. The trisaccharide was then obtained by glycosylation of 53 with 33 and then deprotection. Four other oligosaccharides (18–21 , Fig. 6) corresponding to fragments of either the linear or the branched hexasaccharide moieties of the arabinan have been synthesized as methyl glycosides [55,74]. These were obtained in a manner similar to the disaccharide and trisaccharide via glycosylation of methyl glycoside acceptors with thioglycoside donors.

Figure 14

Synthesis of disaccharide 13: TBDPS, t-butyldiphenylsilyl; NIS, N-iodosuccinimide; AgOTf, silver trifluoromethanesulfonate.

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Figure 15 Synthesis of trisaccharide 16: TBDPS, t-butyldiphenylsilyl; NIS, N-iodosuccinimide; AgOTf, silver trifluoromethanesulfonate.

2.

Syntheses of 1,2-cis-Linked Oligosaccharides

The stereocontrolled synthesis of ␤-arabinofuranosides has received little attention since the reports, over 30 years ago, that solvolyses of 2-O-benzylated ␣-D-arabinofuranosyl halides (e.g., 30 or 31) with methanol gave mixtures of glycosides in which the ␤ isomer predominates [86,87]. It is of tangential interest to note that kinetic studies were done, and it was proposed that this reaction proceeds through an ion pair SN1, not SN2 mechanism. More recently, this method has been used to synthesize octyl ␤-D-arabinofuranosides from 30 and 31 [73]. As outlined in Figure 16, reaction of these labile chlorides in the presence of octanol without a promoter gave modest yields of ␤glycosides 54 and 55. The reaction of 30 under these conditions gave only the desired product; however, in the case of 31, traces of the ␣ isomer were also detected. Although the yields are modest, the ease by which the starting material can be prepared (two steps from commercially available 2,3,5-tri-O-benzyl arabinofuranose, 56) makes this an attractive route to these products. Unfortunately, while this method can be used for the synthesis of glycosides of simple primary alcohols, attempts to glycosylate secondary carbohydrate alcohols by this method failed, and thus the method appears to be of limited utility for the synthesis of oligosaccharides. On the other hand, reaction of the pyridyl thioglycoside 34 with alcohol 57 has been shown [76] to provide good yields of the ␤-linked disaccharide 58 (Fig. 17). No mention was made of the formation of any ␣-glycoside product in this

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Figure 16

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Synthesis of ␤-D-arabinofuranosides by alcoholysis of glycosyl chlorides.

glycosylation reaction. This disaccharide product was then used as a synthon for the preparation of a pentasaccharide fragment (62) of the hexasaccharide motif found at the nonreducing terminus of AG and LAM. Coupling of 58 with alcohol 59 followed by deprotection of the silyl group afforded trisaccharide 60. Glycosylation of 60, again with 58, provided the protected pentasaccharide 61, which was subsequently hydrogenated to afford the product. In this synthesis the first glycosylation proceeded stereoselectively to afford the ␣-linked product, but in the second case, a separable 3:2 ␣ : ␤ mixture was produced. Unquestionably, the stereocontrolled synthesis of ␤-D-arabinofuranosides is an area ripe for investigation. Indeed, no general methods for their preparation currently exist. Although new strategies will likely appear, it is probable that many of the methodologies used for ␤-mannoside synthesis can be applied to this problem. Indeed, recent work [88,89] has shown that Ogawa’s [90,91] intermolecular aglycone delivery method for ␤-mannoside synthesis can be used for the stereoselective synthesis of ␤-D-fructofuranosides (e.g., 65) (Fig. 18). Given the very close structural similarities between D-arabinofuranose and D-fructofuranose, it is likely that the approach can be used, but this has yet to be demonstrated.

VI.

CONFORMATIONAL STUDIES

A detailed appreciation of the biological function of a molecule requires that its conformation be understood. Therefore, in recent years a great deal of effort has been directed toward elucidating the solution conformation of oligosaccharides [92]. The work, however, has been heavily focused on oligopyranosides. At the same time, a wealth of information detailing the conformation of furanose rings in nucleotides and nucleic acids has accumulated [93]. However, except for sucrose [94,95], only recently have conformational investigations of oligosaccharides containing furanose residues been reported [96,97]. Outlined in this section are some recent investigations that have explored the solution conformation of methyl ␣-D-arabinofuranoside and oligosaccharides containing ␣-D-arabinofuranosyl residues.

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Figure 17

Synthesis of 62: IDCP, iodine dicollidine perchlorate; TBDPS, t-butyldiphen-

ylsilyl.

A.

Background

In oligosaccharides containing pyranosyl residues, most, if not all, of the constituent rings are rigid, existing in one of two possible chair conformations. Thus, the major degrees of freedom available to an oligopyranoside are rotation about glycosidic bonds, which can be described by two torsion angles (⌽, ⌿), and rotation about the exocyclic C—C bond (torsion angle ␻), as indicated in Figure 19 [98]. Three possible staggered conformations of ␻ are possible (gg, gt, and tg, Fig. 20) and usually a mixture is present in solution [99]. NMR studies focused on elucidating oligosaccharide structure have relied heavily on the measurement, of 3JH,H to determine ring conformations and rotamer populations of exocyclic hydroxymethyl groups of the

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Figure 18

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Synthesis of ␤-D-fructofuranosides by intramolecular aglycone delivery.

constituent monosaccharide residues [92,99]. The value of ⌽ is fixed by the exoanomeric effect, which place one of the lone pairs of the glycosidic oxygen antiperiplanar to the C1-ring oxygen bond, and the value of ⌿ is determined largely by steric constraints. The ⌽, ⌿ angles for a glycosidic linkage cannot be determined by means of 3JH,H values. Therefore, to orient the individual residues together in three-dimensional space, most investigations have employed either interresidue nuclear Overhauser effect (NOE) constraints [92] or 3JC,H values [92,100–104] measured across glycosidic linkages. In oligofuranosides, the problem is more complex. In addition to rotation about glycosidic and exocyclic C—C bonds, the rings themselves are flexible. Similar to substituted cyclopentanes, furanose moieties exist in either envelope (E) or twist (T) conformations [105]. At the monosaccharide level, often two of these conformations are of roughly equal energy and are thus present in equilibrium. These puckered conformers generally interconvert through pseudorotation, a process whereby, through a series of small molecular reorganizations, two conformations can equilibrate. An alternate pathway, inversion through the planar ring form, is disfavored because in the planar form, all substituents on the ring are eclipsed, which results in a significant energy penalty due to torsional strain [106]. A particular ring conformer can be described by two parameters, the puckering amplitude (␶m) and the pseudorotational phase angle (P), which can be illustrated by the pseudorotational wheel shown in Figure 21 for a D-aldofuranose [106]. The radius of the circle is ␶m and P, which defines the part of the ring that is most puckered,

Figure 19 furanosides.

Glycosidic, hydroxymethyl group, and ring torsion angles in pyranosides and

154

Figure 20

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Staggered rotamers about the C5–C6 bond in a pyranose ring (C4–C5 in

furanoses).

is indicated along with the associated envelope or twist conformer. A consequence of ring pucker is that in addition to the ⌽, ⌿, and ␻ torsion angles, furanoses possess a ring torsion angle, ␹ (Fig. 19). In solution, furanose rings typically exist as an equilibrating mixture of conformers, one in the northern hemisphere and another in the southern, respectively termed the north (N) and south (S) conformers. Given two low-energy conformations, for example 3T2 and 2T3, equilibration can occur through one of two pathways, either via the east or west. In the east pathway, usually favored for D-pentofuranoses, equilibration proceeds through the following series of conformers: 3T2 ↔ 3E ↔ 3T4 ↔ E4 ↔ ⬚E ↔ ⬚T1 ↔ E1 ↔ 2T1 ↔ E2 ↔ 2T3 [106]. In the case of furanose rings, where a mixture of conformers is equilibrating rapidly on the NMR time scale, conformational analysis becomes complicated because averaging of coupling constants is observed. To facilitate analysis, a commer-

Figure 21 Pseudorotational wheel for a D-aldofuranosyl ring: T, twist; E, envelope; superscript numbers indicate the atoms lying above the plane, while subscript numbers are atoms displaced below the plane.

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155

cially available least-squares minimization program (PSEUROT 6.2) can be used, given the observed intracyclic ring 3JH,H and the ␶m for each conformer, to calculate the N/S ratio and to provide P values for both conformers from which the ring forms (e.g., 3T2) can be determined [107]. Similarly, equations have been derived for calculating populations of rotamers about the exocyclic C—C bond from coupling constant data [99,108,109]. B.

NMR Studies

Mindful of the discussion above, it is clear that when one is probing the conformation of an oligofuranoside, the critical first issue to be addressed is that of the conformational equilibrium of the constituent monosaccharide rings. Once this equilibrium is understood, the three-dimensional orientation of the sugar residues relative to each other can be assessed to provide an overall picture of the shape of the molecule. In recent investigations [110], the equilibrium populations of ring conformers in methyl ␣-D-arabinofuranoside (24␣, Fig. 8) and a series of related di-, tri-, and tetrasaccharides (13, 16, 18–21 , Fig. 6) have been studied. These investigations have led to a better understanding of D-arabinofuranose ring conformation and have allowed the determination of the effect of ring substitution on conformer equilibria. High-field (600 and 800 MHz) NMR spectroscopy has been used for these studies. The oligosaccharides of interest contain only ␣-D-arabinofuranose residues, and consequently the potential for spectral overlap is high. However, in all the compounds studied to date, the spectral resolution at these field strengths has been good enough that standard one- and two-dimensional experiments (e.g., 1H-1H COSY, 1H-1H TOCSY, HMQC) can be used not only in assigning all the 1H and 13C resonances, but also in the measurement of coupling constants. Once the couplings have been obtained, PSEUROT analysis has provided conformer identities and their populations. As illustrated in Figure 22, this analysis has identified that the ring in 24␣ adopts a roughly equimolar distribution of the ⬚T4 (N) and 2T3 (S) conformations. In both these conformers the anomeric methoxy group is oriented in a pseudoaxial orientation, as would be expected on the basis of the endo anomeric effect. Furthermore, in both conformers the hydroxymethyl group lies in a pseudoequatorial fashion, as would be favored on the basis of steric interactions. In the N conformer, both the secondary OH groups adopt sterically favored pseudoequatorial orientations; however, in the S conformer, these groups are pseudoaxial. Although conformers possessing this orientation of substituents might not be expected to be energy minima on the basis of sterics, gauche effects [111] between these hydroxyl oxygens stabilize this conformer. Similar effects have been observed in the furanose rings of nucleic acids [112]. Comparison of the two solution conformers with that of the crystal structure [113] of octyl ␣-D-arabinofuranoside (66) and 24a [114] reveals that the rings in these crystals adopt a conformation (E4) very similar to the solution N conformer. Extension of these investigations to the oligosaccharides showed that both the conformations present and their populations can, in some cases, be affected by glycosylation. Figure 23 illustrates the N and S conformers present for each ring in disaccharides 13 and 18. In the ␣-(1→5)-linked disaccharide (13), each ring adopts the same two conformations as the monosaccharide, and in essentially the same populations. However, in the ␣-(1→3)-linked disaccharide (18), while the nonre-

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Figure 22

Solution N and S conformers for 24␣ as determined by PSEUROT analysis.

ducing end residue behaves like the monosaccharide, the reducing end residue is profoundly affected by glycosylation. In the 3-O-substituted ring, the S conformer remains at 2T3 but the identity of the N conformer changes, slightly, to ⬚E. In addition and more strikingly, the N conformer is favored with an equilibrium population of 86%. Clearly, glycosylation of a D-arabinofuranoside at O3 has a substantial influence upon the conformers available to the ring. Steric arguments can be used to rationalize these results, and similar trends are observed (Fig. 24) when these studies are carried out on the larger oligosaccharides (16, 19–21 ). From the data available to date, some general conclusions cn be drawn. 1.

Glycosylation, and likely any form of substitution, of a primary hydroxyl group in an arabinofuranose ring does not significantly alter the conformers present or their populations.

Figure 23 ulations.

Ring conformers present in disaccharides 13 and 18 and their relative pop-

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Figure 24

Ring conformers present in oligosaccharides 16 and 19–21 and their relative populations. 157

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2.

3.

4.

Similarly, replacement of a methyl group at the anomeric center with a monosaccharide residue does not alter the equilibrium. Hence, all terminal Araf residues behave like the monosaccharide. Glycosylation of O3 on an arabinofuranose ring does alter the conformational equilibria of the ring significantly. The identity of the N conformation changes from ⬚T4 to ⬚E, and this conformation is favored at equilibrium. In cases in which a ring is glycosylated at both O3 and O5, as in 19 and 21, favoring of the N conformation becomes even more pronounced.

The populations of rotamers about the C4—C5 bond group are relatively constant and are insensitive to ring conformation. Rotamer populations about this bond have been calculated using 3J4,5R and 3J4,5S values, and in all the oligosaccharides, the gg orientation is most heavily populated (47–53%). The gt rotamer is also present in significant amounts (34–40%), while the tg rotamer is the least populated (12– 15%). In the gg and gt orientations a gauche effect [111] between the ring oxygen and O5 stabilizes these rotamers over the tg conformational isomer, in which O5 is trans to the ring oxygen. C.

Computational Work

The initial computational studies on D-arabinofuranosides were molecular mechanics calculations of methyl ␤-D-arabinofuranoside done by Pe´rez and coworkers [115]. Through a process by which the MM3 force field was used to optimize 360 unique conformations, two energy minima, the 3T2 (N) and 2T3 (S) conformations, were identified. The N conformer is about 1.3 kcal/mol more stable than the S, and the favored conformation about the C4—C5 bond was the gt orientation, which was favored over the gg rotamer by less than 2 kcal/mol. Additionally, the most favorable pathway for pseudorotation was shown to be through the east. More recently, gas phase ab initio (HF/6-31G*, HF/6-31⫹G**, MP2/631⫹G**, MP3/6-31⫹G**) and density functional theory (B3LYP/6-31G*, B3LYP/ 6-31⫹G**) calculations have been carried out on methyl ␣-D-arabinofuranose [116]. A protocol developed by Serianni and coworkers [117,118], was used to construct the 10 possible envelope forms by fixing four atoms in a plane; then, upon minimization, the other parameters (bond lengths, bond angles, etc.) were optimized. These studies identified 3E and 2E as the N and S minima, respectively, with the latter being the global minima (Fig. 25). B3LYP/6-31⫹G** calculations indicated that in the gas phase, the N conformer was 1.5 kcal/mol less stable than the S conformer. Additionally, comparison of the relative energies of the conformers showed, as expected, that pseudorotation through the east was preferred. The highest energy conformer along this pathway is ⬚E (3.2 kcal/mol less stable than the global minima), whereas pseudorotation through the west would require proceeding through the E conformer, which is 7.0 kcal/mol less stable than the global minima. Inter⬚ estingly, these studies also showed that the planar species is approximately equivalent in energy to the E conformer, and thus inversion through the planar ring form is a ⬚ viable alternative to pseudorotation through the west. However, neither pathway is expected to be important because the east pseudorotation pathway is more favorable. In 9 of the 10 envelopes, the favored rotamer about the C4—C5 bond is the gg orientation; the gt rotamer is favored in the other. In the gg orientation O5 can

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Mycobacteria

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N (3E) and S (2E) conformers for 24␣ as determined by gas phase density fashion theory (B3LYP/6-31G*) calculations.

Figure 25

form an intramolecular H bond with OH2 and this capability appears to be of major importance in stabilization of the conformations in the gas phase. Indeed, in the two most stable ring conformers, 2E and E1, an intramolecular H bond is formed between these two groups (OH2 ⭈ ⭈ ⭈ O5) in addition to another such interaction between OH3 and O1 (OH3 ⭈ ⭈ ⭈ O1). These H bonds undoubtedly stabilize the conformers to a significant degree. This observation is consistent with other recent gas phase calculations on sugars, which revealed that the formation of intramolecular H bonds is critically important in the stabilization of these molecules [119]. VII.

CONCLUDING REMARKS

There is now a thorough understanding of the structure of mycobacterial arabinan, and biosynthetic studies are underway that will lead to the elucidation of the pathways by which it is assembled. These investigations will depend critically on the access to synthetic oligosaccharides and oligosaccharide analogs that can be used as potential substrates or inhibitors of these enzymes. Consequently, the synthesis of arabinofuranosides and more generally oligofuranosides will prove to be an interesting and important research area in the future. Additionally, if inhibitors of the enzymes that biosynthesize these glycans are to be identified, a much better understanding of the conformational preferences of oligofuranosides will be necessary. In this area too, although there have been some fundamental investigations, a great deal more must be accomplished before even a rudimentary understanding of the conformation of these molecules is available. REFERENCES 1. 2. 3.

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6 Chemical Synthesis of Bioactive Steroidal Saponins Biao Yu and Yongzheng Hui Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

I.

INTRODUCTION

Saponins, glycosides of steroids or triterpenes, exist widely in terrestrial plants and usually in considerable amounts [1]. They are constituents of many significant foods, forage crops, and herbal medicinal plants. The foodstuffs containing steroidal saponins include such familiar items as oats, leeks, onions, garlic, chives, asparagus, sarsaparillas, and yams. Saponins are of special importance in traditional Chinese herbs, and the list of those containing saponins as active components is long [2]. The herb genera commonly used in China that contain steroidal saponins as active principles include Dioscorea, Paris, Polygonatum, Ophiopogon, Liriope, Asparagus, Yucca, Allium, Smilax, Solanum, and Ruscus. Saponin extracts from these herbs have been used in China to treat various human diseases with surprisingly good precision. However, the precise mechanisms of the actions of these saponins remain ambiguous by modern standards. One reason is that each saponin extract from a herbal plant normally contains several dozens saponin components. These components have such similar structural patterns, and some components exist in such tiny amounts, that separation of each component is, if not impossible, an extremely formidable task. The structural diversity of saponins lies mainly in their ‘‘glycoforms’’ (a term used to describe a set of sugars attached to the same peptide chain in animals) [3]. And many investigations have shown that the ‘‘glycoform’’ of a saponin is important to its bioactivity [1]. Chemical synthesis should provide a realistic way to determine the availability of homogeneous saponins, thus affording new opportunities for understanding and applying this important group of natural products. However, only sporadic studies 163

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on the chemical synthesis of saponins have been conducted and reported [4]. Two recent examples are noted. Randolph and Danishefsky used their ‘‘glycal assembly’’ process to synthesize a tigogenyl tetrasaccharide from Digitalis purpurea [5], and Yamada and Nishizawa synthesized a sweet saponin (osladine) from Polypodium vulgare [6]. We set out to synthesize saponins in recent years as one of numerous research projects in China focusing on traditional Chinese herbs. This chapter presents our study of the chemical synthesis of bioactive steroidal saponins. II.

TWO BASIC STRATEGIES

Structurally, steroidal saponins have two quite distinct parts: a steroid part and a sugar part. The chemical synthesis of steroids was one of the hottest fields during 1950s to 1970s after the discovery of steroidal hormones and contraceptives [7]. Almost all the famous synthetic chemists of that period did something related to steroids. And then a big steroid industry was established. Some steroids of the steroidal saponins are important industrial materials (e.g., diosgenin, tigogenin, and hecogenin, etc.); others, which can be readily prepared from them and their industrial degradation products, include pregnenolone, dehydroisoandrosterone, and progesterone (Scheme 1). It is also practical to isolate considerable amounts of some steroids from the corresponding plants after removal of the ‘‘glycoforms.’’ The chemical synthesis of oligosaccharides has been a major focus since the 1980s [8] as a result of our increased understanding of the importance of these sugars in many life processes [3]. Although with low efficiency and sometimes with tremendous difficulties, almost all the sugar linkages known in nature have been synthesized. Since the chemical syntheses of oligosaccharides and steroids have been studied intensively, the key to synthesizing a saponin turns out to be the construction of the glycosidic bond between the oligosaccharide and the steroid. Accordingly, there are two basic strategies, outlined in Scheme 2: one is to fabricate an oligosaccharide donor and then attach it to the aglycone (strategy 1); the other is to begin by con-

Scheme 1

Some important industrial steroids.

Synthesis of Bioactive Steroidal Saponins

Scheme 2

165

Two basic strategies for the synthesis of saponins.

necting the first monosaccharide to the aglycone, then manipulate the protecting groups of the sugar moiety, and extend the sugar chain sequentially (strategy 2). Strategy 1 looks straightforward; however, it is risky, considering the unavailability of generally applicable glycosylation protocols [8]. In employing strategy 2, the formation of the glycosidic bond between the sugar and the steroid can be sought to be stereospecific and in high yields at the monosaccharide level, but the subsequent protecting group manipulation becomes lengthy and complicated. From another point of view, a series of saponins with identical oligosacchride chains but different steroids can be readily prepared by means of strategy 1, by connecting the oligosaccharide donor to various steroids; if strategy 2 is used, a series of saponins with the same steroid but different oligosaccharide chains similar in pattern can be readily prepared by employing a number of monosaccharide donors in each glycosylation steps. The systematic synthesis of both series of saponins would facilitate the evaluation of the respective biological roles of the sugar part and the steroid part of saponins. A.

Strategy 1: Synthesis of OSW-1 (1)

Saponin OSW-1 (1) is the major component of a small group of cholestane saponins isolated by Sashida et al. from the bulbs of Ornithogalum saudersia. This molecule, while showing little toxicity to normal cells in vitro, exhibited remarkable activities against a broad spectrum of malignant tumor cells, with IC50 between 0.1 and 0.7 nM, which is about 10–100 times more potent than such clinical anticancer agents as mitomycin C, adriamycin, cisplatin, camptothecin, and taxol [9]. According to strategy 1, OSW-1 (1) was broken down into steroid 2 and disasccharide donor 3 (Scheme 3). Glycosyl trichloroacetimidate with a neighboring participation group (3) is believed to be one of the most reliable donors [8b]. Since

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Scheme 3

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Retrosynthesis of OSW-1 (1).

either acidic or basic conditions might cause migration or cleavage of the acetyl (Ac) and 4-methoxybenzoyl (MBz) groups and the degradation of the whole molecule of OSW-1, protecting groups [tert-butyldimethylsilyl (TBS) and triethylsilyl (TES) for hydroxyls and ethylene glycol ketal for the keto] were selected to allow complete removal under neutral or near neutral conditions. Steroid 2 was prepared from dehydroisoandrosterone in a total of 12 steps and 10% yield (Scheme 4) [10]. The stereochemistries involved in the transformations were completely controlled by the steroid substrates themselves (4 → 5, 5 → 6, 8 → 9, 9 → 2). The 3-OH was first protected by a tert-butyldiphenylsilyl (TBDPS) group because the TBS protection was found to be susceptible in the ene reaction (4 → 5). Then, because it was found in a model reaction that the conditions for removal of the 3-OTBDPS group resulted in considerable migration and cleavage of the Ac and MBz groups in the sugar moiety of the target molecule (1), the 3OTBDPS was transferred into TBS protection (7 → 8). The disaccharide donor 3 was prepared as shown in Scheme 5. Coupling of the arabinopyranoside diol 11 with xylopyranosyl imidate donor 10 gave 12, the 3OH (equatorial hydroxyl) glycosylated product preferentially, in 70% yield. Since

Synthesis of Bioactive Steroidal Saponins

167

Scheme 4

Preparation of the aglycone of OSW-1 (2). (a) Ph3PEtBr, tert-BuOK, 78%; TBDPSCl, 100%. (b) (CH2O)n, BF3OEt2, 75%. (c) Dess–Martin periodinane, 86%; 3-methylbutylmagnesium bromide, 96%. (d) PDC, 83%; HOCH2CH2OH, 96%. (e) TBAF, 95%; TBSCl, 96%. (f) OsO4, 41%. (g) ClCOCOCl, DMSO, 78%; NaBH4, CeCl3, 93%.

Scheme 5 Preparation of the disaccharide donor 3. (a) BF3OEt2. (b) TESOTf, 70% (two steps). (c) H2, Pd/C, 50%. (d) CCl3CN, DBU, 65%.

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anomeric benzyl group was very difficult to remove (13 → 14), forcing conditions (50 atm H2, Pd/C, 50⬚C, 3 days, 50%) were used. Glycosylation of steroid 2 with disaccharide imidate donor 3 under the promotion of trimethylsilyl trifluoromethanesulfonate (TMSOTf) provided the desired glycoside 15 stereospecifically and in good yield (69%). Finally, all the protecting groups (one TBS, three TES, and one ethylene glycol ketal) were removed cleanly in one step by employing Pd(MeCN)2Cl2 as a catalyst to furnish the target OSW-1 (1) (Scheme 6) [11]. When strategy 1 was used, the first total synthesis of OSW-1 (1) was accomplished from dehydroisoandrosterone, L-arabinose, and D-xylose in a total of 27 steps, with the longest linear sequence being 14 steps, and in 6% yield [11]. The present route, at least after some optimization, would provide a practical entry to various OSW-1 analogs, especially those with the same sugar moiety as OSW-1, which can be readily prepared by coupling disaccharide donor 3 to various steroids. B.

Strategy 1: Synthesis of Maidong Saponin C (16)

Maidong, one of the most commonly used Chinese herbs, is held to nourish ‘‘yin’’ (tonic effect; ‘‘yin’’ is one of the basic philosophical concepts in traditional Chinese medicine). Saponins are believed to be the active principles in Maidong plants, which are found in several species, including some in the Ophiopogon and Liriope genera. Thus far more than 60 steroidal saponins have been isolated from these species [2c]. Yu et al. isolated Maidong saponin C (16) from the tuber of Liriope muscari [12]. Pharmacological studies showed that 16 possessed strong anti-inflammatory activity [13]. According to strategy 1, Maidong saponin C (16) was broken down into trisaccharide donor 17 and steroid 18 (Scheme 7). Again, a trichloroacetimidate donor (17) was used; without a neighboring participation group, an SN2 reaction is expected to achieve the formation of a ␤-glycosidic bond [8b]. Steroid 18 was conceivably derived from diosgenin by introducing a 1␤-OH. The 3-OH of 18 was believed to be more active than the 1␤-OH, and therefore protected as a TBS ether. Steroid 18 was prepared from diosgenin in 6 steps and 14% yield (Scheme 8). The configuration of the 1␣-OH (22) could not be inverted by using SN2-type reactions, probably owing to the stereohindrance of this position. Therefore an oxidation–reduction sequence was used to epimerize the 1␣-OH (22 → 18). The trans-

Scheme 6

Assembly of OSW-1 (1). (a) TMSOTf, 69%. (b) Pd(MeCN)2Cl2, 79%.

Synthesis of Bioactive Steroidal Saponins

Scheme 7

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Retrosynthesis of Maidong saponin C (16).

formation of 20 to diol 21 under Li/NH3 reduction was found to be capricious; the ⌬6 product or other products sometimes dominated. Schmidt glycosylation [8b] was used twice to prepare the trisaccharide donor 17 (Scheme 9). Attempts to use xylopyranosyl imidate donor 28 in the regioselective glycosylation of diol 26, which has an equatorial 3-OH and an axial 4-OH as diol 11, were not successful. The deprotection of the anomeric allyl group of trisaccharide 29 was found to be problematic. When reactions were catalyzed by palladium spe-

Scheme 8 Preparation of aglycone 18. (a) DDQ, 69%. (b) H2O2, 68%. (c) Li/NH3, 53%. (d) TBSCl, 90%. (e) PDC, 92%. (f) NaBH4, 70% (for 18).

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Scheme 9 Preparation of the trisaccharide donor 17. (a) BF3OEt2, 73%; 50% HOAc, 93%. (b) CH3C(OEt)3, 87%. (c) BF3OEt2, 90%. (d) I(CF2)6CL, Na2S2O4, NaHCO3; Zn, NH4Cl, 50%. (e) Cl3CCN, DBU, 83%.

cies, a mixture of three inseparable products, including the expected deallylation product and two Wacker oxidation products, resulted. We recently developed a new procedure for the deprotection of allyl group: the allyl group was converted into a ␤-iodide-␥-perfluoroalkane derivative using the perfluoroalkylation reaction and then reductive eliminated [14]. This procedure worked well on trisaccharide 29, giving 30 in good yield (50%). With donor 17 and steroid 18 at hand, we sought to effect the final glycosylation to assemble the target molecule 16. However, at this time we were not as lucky as we were in the OSW-1 synthesis. The final glycosylation was found to be difficult and capricious. Under very carefully controlled conditions, the corresponding glycosides were produced in moderate yields and in a mixture of their anomers (␣ : ␤ = 1:4.6). Deprotection of the ␤ anomer (31) gave the target molecule 16 (Scheme 10) [15].

Scheme 10 Assembly of Maidong saponin C (16). (a) TMSOTf (0.2 equiv). (b) TMSOTf (2.0 equiv); NaOMe, 85% (for two steps).

Synthesis of Bioactive Steroidal Saponins

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It is obvious that the present synthetic approach to Maidong saponin C (16) is of no practical use because the final glycosylation is so inefficient. From this example, we sensed the disadvantage in applying strategy 1 to the saponin synthesis, especially when the final glycosylation would involve either an oligosaccharide in the absence of a neighboring group (containing 1→2 linkage) or a steroid with a stereohindered hydroxyl group. C.

Strategy 2: Synthesis of Two Series of Diosgenyl Saponins

Diosgenyl saponins are the most abundant existing steroidal saponins [1,16]. A typical structural pattern of the ‘‘glycoforms’’ in this family is one with a ␤-D-glucopyranoside as the first sugar attched to diosgenin, which in turn has an ␣-L-rhamnopyranose substituted at 2-OH and another sugar or sugar chain at either the third (series 1) or the fourth (series 2) hydroxyl of the glucopyranose moiety (Scheme 11). Many members of these two series of diosgenyl saponins have been found in a number of traditional Chinese herbs, such as those in species of Paris, Ophiopagon, Dioscorea, and Allium. Some members, which have been isolated in sufficient amounts to permit investigators to conduct bioactivity evaluations, have demonstrated various interesting antitumor, cardiovascular, and antifungal activities. According to strategy 2, the glucopyranose residue is coupled to diosgenin first. At this stage, a large array of glucopyranosyl donors and the glycosylation conditions are readily screened. Some results are listed in Scheme 12. The glycosylation of a steroid was known to be a rather special glycosylation reaction [17]. Two side reactions—namely, acetyl group transfer and orthoester formation—took place easily and under some conditions dominated. It was found that acyl group transfer could be avoided completely by substituting a benzoyl group for the acetyl group as neighboring group; orthoester formation depended on the promoter used. Finally, we disclosed that the combined use of benzoyl-protected trichloroacetimidate as donor and TMSOTf as promoter produced the normal glycoside quantitatively (entry 6 of Scheme 12). Moreover, this reaction was conveniently conducted at a 40 g scale without any loss in yield [18]. Diosgenyl disaccharides 44 and 45 with only 3⬘-OH and 4⬘-OH free, respectively, were two key intermediates in the synthesis of the two series of diosgenyl saponins. Their preparations are shown in Scheme 13. Coupling of the benzoylprotected glucopyranosyl imidate 34 with diosgenin, followed by removal of the benzoyl protection, afforded the diosgenyl ␤-D-glucopyranoside (trillin, 37) quantitatively and on a large scale. Attempts to regioselectively glycosylate diol (38) with rhamnopyranosyl imidate 41 were not successful. Therefore, a bulky protecting group

Scheme 11

Two series of diosgenyl saponins.

172

Scheme 12

Yu and Hui

Glycosylation of diosgenin with various glucopyranosyl donors.

[TBS or trimethylacetyl (Piv)] was used to monoprotect one of the two hydroxyls of 38. The Piv group was selectively protected at the 3-OH, producing 40 in 64% yield, with the corresponding 2-OPiv product in 9% yield and 2,3-di-OPiv product in 5% yield. The regioselectivity of TBS in this case was moderate, producing 39 in 52% yield with the corresponding 2-OTBS product in 35% yield [19,20]. Glycosylation of diosgenyl disaccharide 44 with monosaccharide trichloroacetimidates (24, 28, 41, 46) produced the corresponding fully protected diosgenyl trisaccharides (47–50 ) in excellent yields (84–98%). Cleavage of the benzylidene group under 80% HOAc followed by removal of the acetyl groups under NaOMe provided four series 1 diosgenyl saponins (51–54 ) (Scheme 14) [19]. Saponin 51 (gracillin), which has been isolated from many medicinal plants, including species of Paris, Dioscoea, and Costacea, was shown to be cardioactive and hemostatic [21,22]. Saponin 52 has been isolated from Ophiopogon, 53 from Taccacheancer, and 54 from Paris [1,16]. Glycosylation of diosgenyl disaccharide 45 with monosaccharide trichloroacetimidates (41, 46) and thiodisaccharide donors (55–57 ) produced the corresponding fully protected diosgenyl trisaccharides (58, 59) and tetrasaccharides (60–62 ) in good to excellent yields (65–93%). Removal of the acyl groups under NaOH provided the corresponding five series 2 diosgenyl saponins (63–67 ) (Scheme 15) [20]. Saponin 63 (dioscin), which exists widely in plants, including many species intensively used in traditional Chinese herbal medicine, itself exhibits antitumor activity (GI50 = 0.40 ␮g/mL against P-388) and antifungal activity [23]. Saponin 64 (polyphyllin D)

Synthesis of Bioactive Steroidal Saponins

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Scheme 13

Preparation of the key intermediates 44 and 45. (a) PhCH(OMe)2, 89%. (b) TBSCl, 52%. (c) PivCl, 64%. (d) BF3OEt2, 100%. (e) TBAF, 80%. (f) TsOH, 80%; BzCl, 75%.

Scheme 14 Synthesis of series 1 saponins (51–54 ). (a) BF3OEt2, 84–98%. (b) 80% AcOH; NaOMe, 75–91%.

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Scheme 15

Synthesis of series 2 saponins (63–67 ). (a) BF3OEt2 (for 41, 46); NIS/AgOTf (for 55–57 ), 65–93%. (b) NaOH, 75–99% (for 67, 80% HOAc was used to remove the benzylidene).

has been isolated from species of Paris and other genera and shows very promising cardioactive and antitumor properties [21,22]. Saponins 65 and 66 are components of Paris polyphylla [16]. Saponin 67 (balanitin 7) is one of the cytostatic components isolated from the East African medicinal plant Balanites aegyptica, with the ED50 of 0.22 ␮g/mL against P-388 tumor cells [24]. When strategy 2 was used, as long as the steroid has been connected to a sugar moiety, normal chemistry for the oligosaccharide synthesis could be applied to extend the sugar chain. As shown in Scheme 16, a facile ‘‘one-pot’’ glycosylation protocol [25] was efficiently used: treatment of the thioglycoside acceptor 68 with imidate donors (41 and 34, respectively) and TMSOTf, followed by the addition of diosgenyl disaccharide acceptor 45 and N-iodosuccinimide (NIS), generated the corresponding diosgenyl tetrasaccharides 69 and 70 in excellent yields (96 and 98%, respectively). Deprotection of 69 and 70 produced saponins 65 and 66, respectively [26]. III.

SUMMARY

Saponins are distributed widely in plants, including many significant foodstuffs and medicinal plants. In traditional Chinese herbal medicine, saponin components are of special importance; many of them exhibit various interesting bioactivities. However, research on saponins has been limited to the isolation of these compounds and the determination of their structure. Pharmacological evaluation of saponins has been mainly retained at the folkloric level. The difficulty in isolation of saponins from

Synthesis of Bioactive Steroidal Saponins

Scheme 16

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‘‘One-pot’’ glycosylation in the synthesis of saponins.

plants, where they exist as complicated ‘‘glycoforms,’’ is one reason, and another is the long neglect of their importance, due to insufficient understanding and application of saponins by modern standards [27]. The increased interest in traditional herbal medicine and in ‘‘carbohydrate drugs’’ [28] should bring broad attention to this important group of natural glycosides. And chemical synthesis, as shown in the foregoing examples, can make homogeneous saponins readily available. ACKNOWLEDGMENT Appreciation is expressed to our excellent coworkers whose names are included in the references. Financial support was provided by the Ministry of Science and Technology of China. B. Y. is also grateful to a NSFC grant (No. 29925203). REFERENCES 1.

2.

3.

(a) K Hosttettmann, A Marston. Saponin. Cambridge: Cambridge University Press, 1995. (b) GR Wall, K Yamasaki, eds. Advances in Experimental Medicine and Biology, vol 404: Saponins Used in Traditional and Modern Medicine. New York: Plenum Press, 1996. (c) GR Wall, K Yamasaki, eds. Advances in Experimental Medicine and Biology, vol 405: Saponins Used in Food and Agriculture. New York: Plenum Press, 1996. (a) C Huang. The Pharmacology of Chinese Herbs. Boca Raton, FL: CRC Press, 1993. (b) LG Guo, J Ying, eds. Contemporary Research and Clinical Application on Chinese Herbs (in Chinese). Xueyuan Press, 1994. (c) GJ Xu, LS Xu, eds. Species Systemization and Quality Evaluation of Commonly Used Traditional Chinese Herbs (in Chinese), vol 1–2. Fujian Science Press, 1995, 1997. (d) ZC Lou, B Xu, eds. Species Systemization and Quality Evaluation of Commonly Used Traditional Chinese Herbs (in Chinese), vols 1–3. Beijing: Beijing Medical University and Union Medical University Press, 1995, 1996. (a) RA Dwek. Chem Rev 96:683–720, 1996. (b) A Varki. Glycobiology 3:97–127, 1993.

176 4.

5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

24. 25. 26. 27. 28.

Yu and Hui (a) T Ikeda, T Kajimoto, J Kinjo, K Nakayama, T Nohara. Tetrahedron Lett 30:3513– 3516, 1998. (b) LN Atopkina, NI Uvarova, GB Elyakov. Carbohydr Res 303:449–451, 1997. (c) S Saito, Y Sasaki, K Kuroda, Y Hayashi, S Sumita, Y Nagamura, K Nishida, I Ishiguro. Chem Pharm Bull 41:539–543, 1993. JT Randolph, SJ Danishefsky. J Am Chem Soc 117:5693–5700, 1995. H Yamada, M Nishizawa. J Org Chem 60:386–397, 1995. B Asbell. The Pill: A Biography of the Drug That Changed the World. New York: Random House, 1995. (a) H Paulson. Angew Chem Chem Int Ed Engl 21:155–224, 1982. (b) RR Schmidt. Angew Chem Chem Int Ed Engl 25:212–235, 1986. (c) K Toshima, K Tatsuta. Chem Rev 93:1053–1531, 1993. (d) GJ Boons. Contem Org Synth 173–200, 1995. Y Mimaki, M Kuroda, A Kameyama, Y Sashida, T Hirano, K Oka, R Maekawa, T Wada, K Sugita, JA Beutler. Bioorg Med Chem Lett 7:633–636, 1997. C Guo, PL Fuchs. Tetrahedron Lett 39:1099–1102, 1998. SJ Deng, B Yu, Y Lou, YZ Hui. J Org Chem 64:202–208, 1999. BY Yu, Y Hirai, J Shoji, GJ Xu. Chem Pharm Bull 38:1931–1935, 1990. Q Xu, R Wang, BY Yu. J China Pharm Univ 24:98–101, 1993. (a) B Yu, JB Zhang, SF Lu, YZ Hui. Synlett 29–30, 1998. (b) B Yu, B Li, JB Zhang, YZ Hui. Tetrahedron Lett 39:4871–4874, 1998. (a) MZ Liu, B Yu, YZ Hui. Tetrahedron Lett 39:415–418, 1998. (b) MZ Liu, B Yu, XY Wu, YZ Hui, KP Fung. Carbohydr Res 329:745–754, 2000. SB Mahato, AN Ganguly, NP Sahu. Phytochemistry 21:959–978, 1982. (a) G Wulff, G Rohle. Angew Chem Int Ed Engl 13:157–216, 1974. (b) FJ Urban, BS Moore, R Breitenbach. Tetrahedron Lett 31:4421–4424, 1990. SJ Deng, B Yu, JM Xie, YZ Hui. J Org Chem 64:7265–7266, 1999. (a) C Li, B Yu, MZ Liu, YZ Hui. Carbohydr Res 306:189, 1998. (b) C Li, B Yu, YZ Hui. J Carbohydr Chem 18:1107–1120, 1999. (a) SJ Deng, B Yu, YZ Hui. Tetrahedron Lett 39:6511, 1998. (b) SJ Deng, B Yu, YZ Hui, H Yu, XW Han. Carbohydr Res 317:53–62, 1999. J Zhou. Pure Appl Chem 61:457–460, 1989. T Namba, XL Huang, YZ Shu, SL Huang, M Hattori, N Kakiuchi, Q Wang, GJ Xu. Planta Med 55:501–505, 1989. (a) T Nakamura, C Komori, YY Lee, F Hashimoto, S Yahara, T Nohara, A Ejima. Biol Pharm Bull 19:564–566, 1996. (b) CD Hufford, S Liu, AM Clark. J Nat Prod 51:94– 98, 1988. GR Pettit, DL Doubek, DL Herald, A Numata, C Takahashi, R Fujiki, T Miyamoto. J Nat Prod 54:1491–1502, 1991. (a) H Yamada, T Harada, T Takahashi. J Am Chem Soc 116:7919–7920, 1994. (b) H Yamada, T Harada, H Miyazaki, T Takahashi. Tetrahedron Lett 35:3979–3982, 1994. (a) B Yu, H Yu, YZ Hui, XW Han. Tetrahedron Lett 40:8591–8594, 1999. (b) H Yu, B Yu, XY Wu, YZ Hui, XW Han. J Chem Soc Perkin Trans 1 1445–1453, 2000. AM Rouhi. Chem Eng News 73 (Sept 11):28–35, 1995. JC McAuliffe, O Hindsgaul. Chemistry & Industry (London) 5:170–175, 1997.

7 Recent Developments in Technology for Glycosylation with Sialic Acid Randall L. Halcomb and Mark D. Chappell University of Colorado, Boulder, Colorado

I.

INTRODUCTION

Sialic acids are a class of nine-carbon monosaccharides found at the termini of oligosaccharides in many mammalian cellular systems [1]. This class is represented by the prototypical congener N-acetylneuraminic acid (NeuAc, 1, Scheme 1). These unique sugars are ubiquitous, and they are present as components of both glycolipids and glycoproteins. Some examples of sialic acid containing oligosaccharides are shown in Scheme 2. Sialic acids are found in variety of glycosidic linkages, some more common of which are ␣-2,3- or ␣-2,6-linkages to galactose residues. Additionally, they frequently exist as ␣-2,8-linked oligomers or polymers. The lack of efficient technology to accomplish glycosylations with sialic acid is one of the long-standing deficiencies in carbohydrate chemistry [2]. Owing to the central role of sialic acids in carbohydrate recognition events, the development of high-yielding and operable methods to synthesize sialic acid glycosides has been the subject of considerable research. The development of such methods allows the construction of complex sialic acid containing glycoconjugates for the investigation of their roles in biochemical and cellular processes. Most classical methods for synthesizing sialic acid glycosides are based on the reaction of an activated sialic acid such as 2 with an oligosaccharide glycosyl acceptor bearing a hydroxyl group nucleophile (Scheme 3) [2]. The leaving group is typically a halogen, such as a chloride or bromide, and the activator is typically a heavy metal salt [3,4]. A regioselective union of the two reacting partners of course relies on appropriate protecting group patterns for both the glycosyl donor and acceptor components. Many of the early methods are generally plagued with side reactions, low yields, and poor stereoselectivity. There are several reasons for these 177

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Scheme 1

shortcomings. The electron-withdrawing carboxylate at the anomeric carbon disfavors the formation of the oxocarbenium ion 6 (7) en route to glycoside bond formation (Scheme 4). This oxocarbenium ion intermediate is also somewhat sterically hindered, so attack of hydroxyl nucleophiles can be slow. Therefore an elimination to provide 9 can be a significant competing reaction pathway. Additionally, since the reacting carbon is insulated from the nearest stereogenic center by either O6 or C3, there is very little steric biasing of one face of this oxocarbenium ion over the other. As a consequence, the stereoselectivity commonly observed is less than desirable. Complicating the issue further, the anomeric effect causes ␣-glycosides to be thermodynamically less stable than the corresponding ␤ anomers. Heating a sialic acid ester in the presence of a primary alcohol and an acid catalyst results in exclusive formation of the ␤-glycoside (Scheme 5) [5]. As a result of these factors, glycosylation reactions with sialyl donors often provide low yields of the desired sialic acid ␣-glycosides. Thus the synthesis challenge of efficient glycosylation with sialic acid is a daunting one indeed. More recently, this long-standing problem has been revisited in a number of laboratories. This renewed focus was influenced significantly by the recent explosion of discoveries concerning the roles of oligosaccharides in biochemical processes of all types. Reviewed in this chapter are some selected recent advances in both chemical and enzymatic glycosylation with sialic acid. II.

IMPROVED VARIATIONS OF THE CLASSICAL METHODS

A.

Optimized Leaving Groups and Activation Methods

␣-Glycosides of sialic acid are classically prepared by the Koenigs–Knorr [3] or Helferich [4] method, each of which involves the activation of a glycosyl halide by a metal salt and subsequent displacement by an alcohol (Scheme 6). Typically a metal salt such as silver(I) (Koenigs–Knorr) or mercury(II) (Helferich) complexes to a ␤-glycosyl halide 5 to form an activated intermediate 12. This intermediate can then be directly displaced to give the product 8 or dissociate to form an ion pair 6. It has been hypothesized that an ion pair may block the top face of the molecule and force bottom face attack to also provide the desired ␣-glycoside 8. solvent polarity plays an important role in determining the mechanistic course of the reaction. One hypothesis is that more polar solvents favor the ion pair 6 because they can solvate the charged species, while the SN2 pathway is preferred in nonpolar solvents. Many variations of the metal promoters have been examined to probe the effect of the activator on the Koenigs–Knorr glycosylation yield and stereoselectivity. The

Glycosylation with Sialic Acid

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Scheme 2

180

Scheme 3

Scheme 4

Scheme 5

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Glycosylation with Sialic Acid

Scheme 6

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promoters display a wide range of activity depending on the metal and counterion present. The order of reactivity among promoters with glycosyl halides was experimentally determined to be AgOTf > Ag2CO3 > silver salicylate > HgBr2 > Hg(CN)2 [6]. Generally, the more reactive silver promoters furnish higher stereoselectivities than the mercury salts, but give lower overall yields. In the majority of cases examined, the compound of choice was the most active promoter, AgOTf, or the least reactive, Hg(CN)2. Glycosyl halides are the typical sialyl donors in these processes. Sialyl chlorides are generally less reactive than bromides, but they are not as susceptible to decomposition and are sufficiently active under silver ion promotion to make them the most common halogenated glycosyl donor [2]. Sialyl fluorides are useful for glycosylation reactions but are usually activated under Lewis acidic conditions. An advantage of using fluoride glycosyl donors is their inherent preference to form ␤glycosides. Under Koenigs–Knorr conditions, ␤-sialyl chloride 13 leads to predominantly the ␣-glycoside 15 in a glycosylation with primary alcohol 14 [7a]. However, when the ␤-fluoride 16 is activated with BF3 ⭈ OEt2, the ␤-glycoside 17 is afforded in a 5:1 ␤ :␣ mixture of anomers (Scheme 7). Methods derived from the Koenigs–Knorr approach are useful for glycosylation reactions involving reactive alcohols, such as primary alcohols. Unfortunately,

Scheme 7

Glycosylation with Sialic Acid

183

when more hindered secondary alcohols are employed, the greater steric bulk of the nucleophile leads to lower stereoselectivities and yields. This is an important factor because many naturally occurring sialosides contain linkages to secondary alcohols. Sialyl bromides can be employed as more reactive donors in these instances. As a representative example, Paulsen and Von Reessen used a sialyl bromide to apply the Koenigs–Knorr method to the synthesis of an ␣-2,3-linked disaccharide (Scheme 8) [7b]. Glycosylation of the galactose acceptor 19 with sialyl bromide 18 led to the desired disaccharide 20, although with poor stereoselectivity (ca. 1:1 ␣ :␤). The low yields of Koenigs–Knorr glycosylations with secondary alcohols are partly due to elimination of the sialyl halide and formation of the 2,3-dehydro derivative 21. Many of the methods of activation using Koenigs–Knorr conditions require heterogeneous promoters and long reaction times at room temperature. In addition, the expense and toxicity of silver and mercury makes these methods impractical for large-scale syntheses. Consequently much research has focused on the development of glycosyl donors that could be activated at low temperature, under mild conditions, and without the need for heavy metal promoters. Sialic acid thioglycosides, although initially synthesized for use as biological probes, have been found to be excellent glycosyl donors. Hasegawa and coworkers utilized thioglycosides in the synthesis of several ␣-2,3- and ␣-2,6-linked disaccharides. The methylsulfide donor 22 was activated by using the thiophile dimethyl(methylthio)sulfonium triflate (DMTST) at ⫺15⬚C in the presence of either acceptor 23 or 25 to provide compounds 24 and 27, respectively, as exclusively the ␣-glycosides (Scheme 9) [8]. High stereoselectivities were observed for these glycosylations when the reactions were performed in acetonitrile, even though the thioglycoside starting material was a 1:1 mixture of anomers. This finding suggests that the stereoselectivity was not derived from the donor configuration as in Koenigs– Knorr glycosylations, but must depend on the reaction conditions. In an attempt to determine the source of the stereocontrol, Sina¨y and coworkers examined glycosylations of the xanthate donor 27 in polar and nonpolar solvent systems (Scheme 10) [9,10]. Glycosylation of 28 in acetonitrile under DMTST activation afforded the ␣-2,3-linked disaccharide 29 in 26% yield as a 7:1 ␣ :␤ mixture of diastereomers. However, the same reaction in CH2Cl2 gave exclusively the ␤glycoside 30 in 25% yield (Scheme 10). The major by-product in both reactions was the 2,3-dehydro NeuAc derivative. Sina¨y proposed that the glycosylation in CH2Cl2 probably involved an oxocarbenium ion 31 that underwent axial attack to form the ␤ product (Scheme 11). However in acetonitrile, formation of a ␤-nitrilium intermediate 32, which undergoes SN2 attack to afford the ␣-glycoside, may be the stereocontrolling element. The effect of acetonitrile has been studied in glucose and galactose systems, where the presence of axial nitrilium ions was supported by trapping and spectroscopic studies, both performed at low temperatures [10]. The Whitesides group achieved a more efficient synthesis of ␣-sialosides from sialyl xanthates by modifying the reaction conditions of Sina¨y’s system [11]. Previously, Lo¨nn and coworkers had demonstrated that lower temperatures and the use of milder promoter, methylsulfenyl triflate, increased the product yield and reaction stereoselectivity [12]. Whitesides and coworkers introduced an even milder promoter, phenylsulfenyl triflate (PST), which itself was prepared in situ from silver triflate and phenylsulfenyl chloride [11]. When sialylations of xanthate donor 27 were pro-

184

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Scheme 8

Glycosylation with Sialic Acid

Scheme 9

Scheme 10

185

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Scheme 11

moted by PST at ⫺70⬚C in a 2:1 ratio of CH3CN to CH2Cl2, isolated yields of 60– 80% and stereoselectivities of 19:1 ␣ :␤ were observed. A representative example is shown in Scheme 12. Interestingly, when the reaction was conducted under high dilution (0.01 M in donor) the stereoselectivity improved to better than 99:1 ␣ :␤, albeit at the expense of overall yield (52%). Regarding the reaction mechanism, Whitesides and coworkers also postulated that the presence of a ␤-nitrilium ion was the source of stereocontrol. In their proposed mechanism, the xanthate 27 initially reacts with the PST promoter to form 35 (Scheme 13). Subsequent loss of 36 leads to the oxocarbenim intermediate 31. Attack by acetonitrile is presumed to occur from the less hindered ␣ face and leads to the presumably thermodynamically more favorable equatorial nitrilium ion 37 because of the reverse anomeric effect [13,14]. However the equatorial nitrilium ion is believed to be in equilibrium with the more reactive ␤ species 32. Acetonitrile is then displaced from this species by the glycosyl acceptor in an SN2-like manner for form the ␣-glycosidic linkage in compound 38 The aforementioned experimental data do support this mechanism to some extent. First of all, the stereoselectivity is independent of the anomeric configuration of the starting material. A 1:1 mixture of xanthate anomers reacts to give predominantly the ␣-glycoside in acetonitrile, and the reaction by-product 36 was isolated and fully characterized. These data suggest that the xanthate is activated by the thiophilic promoter and glycosylation does not proceed through an SN2-type mechanism. Finally, Whitesides proposes that the gain in stereoselectivity that was observed by performing the reaction dilute in acetonitrile corresponds to an increase in the [MeCN]/[acceptor] ratio. The authors believe that a competition exists between acetonitrile and the acceptor alcohol for the oxocarbenium ion intermediate. When the concentration of the glycosyl acceptor is high enough, the alcohol reacts directly with oxocarbenium ion 32 to form the ␤-glycoside. However, an increase in the amount of acetonitrile favors nitrilium ion formation over direct glycosylation, and since nitrilium ion formation is the stereocontrolling element, higher ␣ :␤ ratios are observed. Takahashi and coworkers examined a novel approach to stereoselective sialylations by incorporating an auxiliary into the ester [15]. An electron-donating group with an appropriate linker to the carboxylate carbon was anticipated to stabilize an oxocarbenium ion through long-range participation. Activation of compound 39, where X is an electron-donating group, would produce oxocarbenium ion 40. This intermediate could be stabilized by X to provide an equilibrating mixture of ␤ and

Glycosylation with Sialic Acid

Scheme 12

187

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Scheme 13

␣ ‘‘onium’’ isomers, 41 and 42, respectively (Scheme 14). The authors predicted that the ␤-onium species 41 would preferentially undergo attack to afford the ␣glycoside 44. Preliminary experiments revealed that a sialyl donor containing a methyl sulfide and a 2-carbon spacer within the ester was the optimum substrate for these studies. This sialyl donor 48 was synthesized as an approximately 1:1 mixture of anomers and was activated with N-iodosuccinimide and trifluoromethanesulfonic acid. Glycosylations of compound 48 were compared with the methyl ester control 45 in various solvents to determine the extent of the enhancement of stereoselectivity (Scheme 15). Interestingly, glycosylations of 2-[2-(2-azidoethoxy)ethoxy]ethanol with donor 45 in acetonitrile or dichloromethane gave the same product ratios (entries 1 and 2), suggesting that for this system acetonitrile does not provide an increase in stereoselectivity. However, the tethered donor 48 predominantly favored formation of the ␣-glycosides (entrites 3, 4, and 5), with the solvent dimethoxyethane (DME) providing the highest ␣ :␤ ratios. Next, the sialyl donor 48 was utilized in the synthesis of ␣-2,6- and ␣-2,3-linked disaccharides under the optimal reaction conditions (entires 6 and 7). The selectivities observed were similar to those in the earlier

Glycosylation with Sialic Acid

189

Scheme 14

examples, although increased formation of products derived from elimination caused the reaction yields to drop significantly. A useful and powerful method that has been developed independently by Wong [16] and by Schmidt [17] and their colleagues utilizes sialyl phosphites as glycosyl donors. Phosphites are synthesized from the corresponding anomeric alcohols by reacting the latter with a phosphoramidite. This greatly shortens the reaction sequence needed to obtain the sialyl donors because many transformations that ordinarily would be needed to install a nonoxygen atom at the anomeric carbon are precluded, and several phosphoramidite reagents are readily available. An additional and significant advantage of these donors is that they are activated by many commonly used Lewis acids such as TMSOTf. Lewis acid activation is now a standard for glycoside synthesis, thus making these methods compatible with the most commonly used protecting group patterns and assembly strategies. Furthermore, sialyl phosphites are activated at low temperatures, and as a result generally provide products in high stereoselectivity. Some typical examples of the use of phosphites for

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Scheme 15

sialylation are the Wong lab’s synthesis 53 [18], an intermediate en route to the sialyl Lexis X antigen (Scheme 16) and the Schmidt lab’s synthesis of 56 (Scheme 17) [17a]. B.

Protection Strategies to Optimize Yields and Regiocontrol

The course and outcome of glycosylation reactions with sialic acid can be greatly influenced in some instances by seemingly insignificant modifications of the protecting groups on the glycosyl donor or acceptor. For example, simply employing a benzyl ester rather than the more commonly used methyl ester to protect the carboxylic acid of NeuAc, in combination with a sialyl chloride as donor using Koenigs–Knorr-type conditions, has profound consequences on the stereoselectivity of the glycosylation [19]. This fact was recorded by Ratcliff and coworkers, and has been widely utilized [19]. A representative example is shown in Scheme 18. The Boons laboratory has observed a dependence of the yield of glycosylation on the nature of the protecting group of the sialic acid C5 nitrogen [20]. When the nitrogen was engaged in an N,N-diacetylimide 60, yields significantly increased relative to the simple acetamide, the latter bearing only one acetyl group. This observation was utilized to efficiently construct NeuAc-␣-2,8-NeuAc linkages such as that in compound 62 (Scheme 19). The glycosylation of O8 of sialic acid by a sialyl

Glycosylation with Sialic Acid

Scheme 16

191

192

Scheme 17

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Scheme 18

Glycosylation with Sialic Acid

193

Scheme 19

donor is notoriously difficult to accomplish, and the donor 60 was nicely used to construct this linkage. A similar observation has been made by Hossain and Magnusson [21].

III.

USING DIRECTING GROUPS FOR STEREOCONTROL

Although several improved glycosyl donors have been developed to accomplish ‘‘direct’’ sialylations of acceptors, elimination reactions of the sialyl oxocarbenium ion intermediate often are significant competing nonproductive reaction pathways. Additionally, control of the stereochemistry at the NeuAc anomeric center is difficult and nontrivial. To alleviate these problems, several laboratories have investigated the use of stereodirecting auxiliaries at C3 of NeuAc. The postulate is that this group Y in 63 (Scheme 20) could, after activation, assist in stabilizing the positive charge of the oxocarbenium intermediate 64. In doing so, an intermediate such as 65 would be formed. Not only would this suppress elimination reactions, but one could imagine that the trajectory of attack of the glycosyl acceptor would be guided anti to the three-membered onium ion. As a result, the anomeric configuration might be assembled in a more predictable and ␣-selective fashion. Goto and coworkers, who were early contributors, synthesized compounds of this nature from the 2,3-dehydro sialic acid 21 [22]. The Goto group initially investigated hydroxyl groups at C3 as directors and used the reliable Koenigs–Knorr method for glycosylation (Scheme 21) [22]. The prototypical donor was the bromide 67. As a means of examining the versatility of this sialyl donor, the ␣-2,9 and ␣-2,8 NeuAc dimers were chosen as the synthetic targets. Treatment of 67 with silver triflate and a selectively protected sialic acid glycal 68 afforded 42% of the ␣-2,9 dimer 69 along with a 21% of the ␤ anomer. The secondary alcohol 70 was also glycosylated with 67 to give 26% of disaccharide 71. Although the yield of the glycosylation was rather modest, the stereoselectivity was good (3:1 ␣ :␤). The Goto lab later found that bromides at C3 could also be used as directing groups [23]. This prompted a great deal of research into more convenient C3 substituents that would provide higher stereocontrol.

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Scheme 20

Ogawa and coworkers introduced C3 phenylselenyl and phenylthio substituents as stereocontrolling auxiliaries [24,25]. These directing groups had already been developed in the context of stereoselective syntheses of 2-deoxyglycosides by several groups [26]. Phenylthio and phenylselenyl neighboring groups were anticipated to provide better charge stabilization than a C3 hydroxyl substituent because of the increased polarizability of sulfur and selenium [26]. Initial glycosylation attempts with the phenylselenyl substituent proceeded with good stereoselectivity, although yields were low owing to elimination of a cationic selenium species to provide the corresponding 2,3-dehydro NeuAc derivative as the major product of the reaction [24]. The less polarizable phenylsulfide auxiliary was found to circumvent his problem [25]. Mercury-promoted glycosylation of the acceptor 73 with the donor 72 afforded the GM3 precursor 74 in good yield with no trace of the ␤ isomer (Scheme 22). An additional benefit of using a sulfur auxiliary over a hydroxyl was its relative ease of removal in one step by a radical reduction. Treatment of 74 with Bu3SnH and AIBN provided the trisaccharide 75 in 75% yield. The phenylsulfide auxiliary was also used to synthesize an ␣-2,8 NeuAc dimer [25b]. The Ogawa group immobilized these types of sialyl donor on solid support resins and demonstrated their ability to act as donors in solid phase glycosylations [25c]. Thioglycoside donors combined with C3 phenylthio directing groups have been effectively employed by the Magnusson laboratory [27]. A disadvantage of auxiliary-directed glycosylation is that additional steps are required for the synthesis of the sialyl donor. To address this problem, the Whitesides group developed an expeditious route to a sialyl donor in two steps from the NeuAc glycal 21 (Scheme 23) [28]. Treatment of 21 with 2,4-dimethylbenzenesulfenyl chloride afforded the crystalline intermediate 76 in 85% yield. This compound was quan-

Glycosylation with Sialic Acid

Scheme 21

195

196

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Scheme 22

Glycosylation with Sialic Acid

197

Scheme 23

titatively converted to the thioglycoside 77 by treatment with sodium methanethiolate. Sialylations of 77 were achieved in good yield and stereoselectivity by using phenylsulfenyl triflate (PST) as a promoter and di-tert-butylpyridine (DTBP) as a proton scavenger. The most impressive application of this donor was for the synthesis of the ␣-2,3-linked disaccharide 78, which proceeded in 78% yield with no trace of the ␤ isomer (Scheme 24). It was also demonstrated that the auxiliary could be efficiently removed by reduction with Bu3SnH and AIBN in 94% yield. In 1998 the Schmidt group looked to improve the efficiency and simplicity of sialyl glycosylations through the development of a phosphite donor containing a directing auxiliary at C3 [29]. The primary objective was to develop an auxiliary that could be more easily introduced and removed and would more effectively direct the stereochemistry while minimizing side reactions. It was postulated that a better auxiliary might be one that could form a cyclic five-membered onium intermediate rather than a three-membered one. Compound 79 was found to meet the requirements. Acid-catalyzed activation of sialyl phosphite 79 should lead to oxocarbenium ion 80 without the need for stoichiometric promoters (Scheme 25). The intermediate oxocarbenium ion should readily undergo attack by the thionobenzoate auxiliary to form the intermediate 81. Incorporation of the thionobenzoate auxiliary, which provides anchimeric assistance through a larger, more favorable five-membered ring, should provide higher stereocontrol than the three-membered episulfonium ions. Glycosylation should then proceed from the ␣ face to provide the desired sialoside 82. Following this reaction, the thionobenzoate auxiliary should be easily removed by treatment with Bu3SnH/AIBN. Many of the earlier syntheses of NeuAc derivatives containing auxiliaries were long and tedious because it was so difficult to obtain the correct stereochemistry at

198

Scheme 24

Scheme 25

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Glycosylation with Sialic Acid

199

Scheme 26

C3. However, Schmidt and coworkers discovered that the reaction of glycal 21 with OsO4 led to stereoselective dihydroxylation from the ␤ face, providing diol 83 as the only product (Scheme 26). Subsequent regioselective introduction of the thiobenzoyl auxiliary was accomplished by treatment with N,N-dimethyl-␣-chlorobenzimidium chloride followed by dihydrogen sulfide. Compound 84 was obtained as the product of this sequence in 92% yield. Finally, the alcohol was converted to the glycosyl phosphite 79 in a straightforward manner. The utility of this donor was demonstrated in the synthesis of an ␣-2,3-disaccharide and an ␣-2,8-linked NeuAc dimer. Glycosylation of the protected lactose derivative 73 with donor 79 afforded an 88% yield of the protected GM3 analog 85, with no trace of the ␤-glycoside (Scheme 27). Likewise, glycosylation of glycal 86 provided an unprecedented 83% yield of the ␣-2,8 NeuAc dimer 87, once again with no trace of the undesired ␤ diastereomer. In both cases the auxiliary was removed with Bu3SnH/AIBN in good yield. IV.

USING SIALYLTRANSFERASES FOR THE SYNTHESIS OF SIALOSIDES

Enzyme-mediated glycosylations are powerful methods for the synthesis of complex carbohydrates, including those that contain sialic acid [30,31]. In mammalian systems, Leloir pathway glycosyltransferases are responsible for the biosynthesis of most glycoconjugates [32]. Sialyltransferases are a subset of the glycosyltransferases that transfer the NeuAc component of cytidinyl-5⬘-monophospho-␤-N-acetylneuraminic acid (88, CMP-NeuAc) to acceptor hydroxyl groups with inversion of configuration at the anomeric center (Scheme 28) [30,31]. Transferase-mediated sialylations are not burdened by some of the pitfalls of chemical glycosylations, such as un-

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Scheme 27

Glycosylation with Sialic Acid

201

Scheme 28

wanted reaction pathways and the need for tedious protecting group schemes or stereocontrolling auxiliaries. In addition, sialyltransferases have been shown to tolerate several acceptor modifications [33], although less is known about their donor specificity because the requisite CMP-NeuAc sugar donors are so difficult to synthesize. Kinetic isotope effect studies with a particular transferase, namely, rat liver ␣2,6-sialyltransferase, have shown that there is significant charge buildup at the anomeric center in the transition state [34]. This suggests that the glycosylation proceeds through an SN1-like mechanism. CMP-NeuAc appears to be the common donor for all the sialyltransferases. However, each enzyme varies in regioselectivity and to some degree in acceptor specificity. Under the premise that an enzyme exists for the formation of each natural sialoside, there should be at least nine sialyltransferases responsible for the synthesis of the following disaccharides: NeuAc-␣-2,6-Gal, NeuAc-␣-2,3-Gal, NeuAc-␣-2,6-GalNac, NeuAc-␣-2,4-Gal, NeuAc-␣-2,4-GlcNac, NeuAc-␣-2,6-GlcNAc, NeuAc-␣-2,6-Man, NeuAc-␣-2,8-NeuAc, and NeuAc-␣-2,9NeuAc. However, only a small subset of the known and postulated sialyltransferases has been isolated and used for synthesis purposes. The sialyltransferases are membrane-bound proteins located in the endoplasmic reticulum (ER) and in the Golgi apparatus. Information about their sequence homology is limited, but they do appear to share a common topography [35]. A catalytic domain resides at the C-terminus followed by an N-terminal segment that anchors the enzyme into the ER or Golgi membrane. Soluble, catalytically active sialyltransferases that lack the anchor segment have been isolated from milk, serum, and other body fluids, suggesting that this N-terminal anchor is not necessary for the enzyme to retain catalytic activity. However, the ability to obtain from natural sources quantities of most sialyltransferases that would be needed for synthesis applications is hampered by low tissue concentrations and difficult purifications. The genes for members of some of the most common classes of sialyltransferases have been cloned and expressed [33a]. Expressing and isolating membranebound enzymes in high catalytic activity can be difficult; however, Paulson and coworkers replaced the anchor segment with a cleavable peptide in the expression of Gal-␤-1,4-GlcNAc ␣-2,6-sialyltransferase [35a,b]. This allowed the enzymes to be secreted from the cell into the medium, thus simplifying the process of isolation. This technology has been used to express several sialyltransferases in quantities

202

Halcomb and Chappell

Scheme 29

suitable for synthesis. Gaining sialyltransferase accessibility is becoming a less serious problem as these enzymes become commercially available. A.

Synthesis of CMP-NeuAc and Related Derivatives

The primary method for the synthesis of CMP-NeuAc 88 is through the use of CMPNeuAc synthetase, an enzyme that catalyzes the condensation of cytidine triphosphate (CTP) with sialic acid to produce CMP-NeuAc (Scheme 29) [30]. CMP-NeuAc synthetase has been cloned from microbial sources and has been isolated from mammalian tissues [36,37]. The substrate specificity of each synthetase has been studied to some degree [36,37]. The mammalian version accepts C9 and some C8 modifications of NeuAc, as well as variations at the C5 position, such as replacement of the acetamide with OH (KDN) or hydroxylation of the acetamide (NeuAc). The microbial CMP-NeuAc synthetase has a high activity for C9-modified sialic acids, but does not tolerate alterations at the C5 position. Unfortunately, the microbial version is the more readily available enzyme, thus limiting the variety of analogs that can be prepared in this manner. Scheme 30 gives a representative sample of some important C9-derivatized CMP-NeuAc analogs [37]. Modifications at the C5 position are somewhat limited by the specificity of CMP-NeuAc synthetase for an amide at this position. Nevertheless, several analogs were prepared that incorporate sterically and electronically diverse substituents at C5 (Scheme 31) [37].

Scheme 30

Glycosylation with Sialic Acid

203

Scheme 31

Wong and coworkers first attempted the nonenzymatic synthesis of CMPNeuAc by employing phosphoramidite chemistry in a key step involving the ligation of a sialyl phosphoramidite and a selectively protected cytidine analog (Scheme 32) [38]. Treatment of NeuAc derivative 99 with 2-cyanoethyl chlorophosphoramidite resulted in the formation of the ␤-sialyl phosphoramidite 100 in 89% yield. The sialyl donor was then coupled to the protected cytidine 101 under promotion by 1H tetrazole to afford the intermediate phosphite, which was immediately oxidized with tert-butyl hydroperoxide to provide the protected CMP-NeuAc 102. Deprotection of this intermediate was not reported; however, the Wong group demonstrated that the CMP-NeuAc core structure could be successfully synthesized utilizing phosphoramidite chemistry.

Scheme 32

204

Halcomb and Chappell

The Schmidt group utilized a sialyl phosphite in a very different synthesis strategy (Scheme 33) [39]. Upon treatment of sialyl donor 54 with cytidine phosphoric acid 103, a phosphite–phosphate exchange reaction occurred to give compound 104 exclusively as the ␤ isomer. Deacylation by treatment with sodium methoxide followed by ester saponification through the addition of water provided CMP-NeuAc 88. This method circumvented the need for an oxidation step or phosphorus deprotection. This method was also applied to the synthesis of another naturally occurring CMP-NeuAc derivative 105 [40]. Hata and coworkers took a different approach to the ligation by utilizing cytidine phosphoramidite 107 and the tertiary anomeric alcohol of 106 as coupling partners (Scheme 34) [41]. The coupling reaction successfully provided a phosphite intermediate, which was subsequently oxidized with tert-butyl hydroperoxide to provide the trialkyl phosphate 108. Attempts to purify the phosphotriester failed, but the production of 108 was supported by 31P NMR spectroscopy of the crude reaction mixture. The protecting groups were then removed under mild conditions by treatment with tetrakis(triphenylphosphine)palladium(0) in the presence of the allyl scavenger n-butylammonium bicarbonate. Following purification by size exclusion chromatography, CMP-NeuAc 88 was obtained in acceptable overall yield (25% for three steps). In a related synthesis of CMP-NeuAc, Kajihara and coworkers also used a cytidine phosphoramidite in the coupling reaction, although with different protecting group patterns on the coupling partners (Scheme 35) [42]. The reaction of 99 with phosphoramidite 109 provided the phosphite 110 as a mixture of phosphorus diastereomers. Phosphite oxidation and subsequent treatment with methoxide led to decomposition. As an alternative, the cyanoethyl protecting group was first removed with DBU; then complete deprotection by treatment with sodium methoxide and sodium hydroxide afforded CMP-NeuAc 88 in good yield. This stragety was also applied to the synthesis of CMP-NeuAc-␣-2,8-NeuAc 111 [43]. Halcomb and Chappell developed a route to CMP-NeuAc 88 that promises to be general for the synthesis of virtually any derivative thereof [44,45]. The route (Scheme 36) utilizes a condensation of sialic acid derivative 99 with the phosphoramidite 112 to afford the phosphite 113 in 62% yield. Oxidation of the phosphite provided the phosphotriester 114 [46], which was taken directly to the next transformation without purification (owing to its instability to chromatography). Deallylation of the phosphate gave compound 115 (61% for two steps), which was stable to silica gel chromatography. Compound 115 was deacylated with methoxide, and its methyl ester was subsequently saponified with NaOH to provide CMP-NeuAc 88. The derivatives shown in Scheme 37 were synthesized according to this protocol and were investigated as substrates for sialyltransferases (see below). The synthesis of a CMP-NeuAc derivative that was bound to a solid support through the 9-position of the sialic acid has been reported by the Kajihara group [47]. This derivative is quite useful in that it can be utilized to immobilize glycoproteins onto a solid support by transferring the sialic acid to the terminus of the carbohydrate chain of the glycoprotein. B.

Synthesis of Wild-Type and Mutant Sialosides

The Brossmer and Paulson groups have studied the sialyltransferase donor specificity with a series of C5 and C9 conjugates, all of which were prepared through the CMP-

Glycosylation with Sialic Acid

205

Scheme 33

206

Halcomb and Chappell

Scheme 34

NeuAc synthetase route [37]. The relative rates of these C9-modified CMP-NeuAc derivatives shown in Scheme 30 were compared against the natural donor CMPNeuAc in sialyltransferase assays that utilized enzymes from different sources with their appropriate natural acceptors. The rat liver ␣-2,6-sialyltransferase tolerated a wide range of functional groups without significant decreases in the relative rates. Synthetically useful relative rates were observed for most of the CMP-NeuAc analogs with porcine sialyltransferase and rat liver ␣-2,3-sialyltransferase. The exception was the 9-amino analog, which was a poor substrate for both enzymes. Overall, these assays demonstrated that a wide variety of modifications at the C9 position are tolerated by these sialyltransferases. The relative rates of sialylation for the C5-modified CMP-NeuAc derivatives shown in Scheme 31 were also determined [37]. All the C5-modified sialyl donors were accepted by the transferase enzymes, but the variants containing free amines were poor substrates for all the sialyltransferases examined. Generally, higher relative

Glycosylation with Sialic Acid

Scheme 35

207

208

Halcomb and Chappell

Scheme 36

Glycosylation with Sialic Acid

Scheme 37

209

210

Halcomb and Chappell

rates were observed for both ␣-2,6-sialyltransferases over the porcine ␣-2,3-sialyltransferase, although all the rates are high enough to be synthetically useful. This study has shown that numerous C5-modified CMP-NeuAc sialyl donors can be successfully utilized in the synthesis of modified glycoproteins. The sialyltransferases investigated were found to accept many variations at C5 and C9 of the sialic acid moiety. Halcomb and Chappell confirmed this fact with studies of sialyltransferases from different sources [45]. The C9- and C5-modified substrates in Scheme 37 were good substrates. However no activity was detected when compounds 111 and 117 were assayed. In addition to these examples, other CMP-NeuAc analogs that have been prepared and successfully transferred include C9 fluorescent compounds, C9 thioacetyl, C5 thioacetyl, and C4 dexoy [37]. A particularly useful modification introduced by the Wong lab incorporates a mercury atom at C9 (Scheme 38) [48]. Transfer of this sialic acid to glycoproteins could greatly aid in X-ray crystallographic analysis of these biomolecules. The field of solid phase synthesis has also benefited from enzyme technology. Several reports have described the use of sialyltransferases in the solid-supported synthesis of oligosaccharides that bear sialic acid [49]. Additionally, metabolic pathways have been harnessed by Bertozzi and coworkers to synthesize glycoconjugates on cell surfaces that bear modified sialic acids [50]. C.

Cofactor Regeneration

Enzymatic methods are efficient for the assembly of complex oligosaccharides on a small scale, although the high cost of sugar nucleotides and problems with product inhibition detract from this approach in large-scale reactions. The synthesis of CMPNeuAc by CMP-NeuAc synthetase requires stoichiometric amounts of expensive reagents, such as CTP, while the CMP by-product of the sialyltransferase reaction is an enzyme inhibitor. Ichikawa and Wong used the concept of in situ cofactor regeneration to alleviate these problems and increase the efficiency of sialylation (Scheme 39) [51]. The cascade begins with stoichiometric amounts of phosphoenolpyruvate (PEP), ␤-allyl-N-acetyl lactosamine 120, NeuAc 1, and catalytic quantities of ATP and CMP. Initially, CMP is converted to CDP by nucleoside monophosphate kinase (NMK) in the presence of ATP. The CDP produced reacts with PEP under pyruvate kinase (PK) catalysis to form CTP. Next, CMP-NeuAc synthetase catalyzes the in situ formation of the sialyl donor from NeuAc and CTP. The pyrophosphate byproduct is decomposed to inorganic phosphate by inorganic pyrophosphatase (PPase). Subsequently, the ␣-2,6-sialyltransferase accomplishes the sialyation of the lactosamine acceptor 120 and produces the transferase inhibitor CMP as a by-product. The CMP concentrations are kept low by conversion to CDP, and in so doing the problem of product inhibition is minimized. The cycle afforded 21% of the sialylated trisaccharide 121, which is remarkable considering the complexity of the system and number of synthetic steps that can be avoided. V.

USING SIALIDASES FOR THE SYNTHESIS OF SIALOSIDES

A.

Sialylhydrolases

Sialidases are another class of enzymes that have been used in the synthesis of sialylated oligosaccharides. In addition to hydrolysis activity, sialidases are capable

Glycosylation with Sialic Acid

Scheme 38

211

212

Halcomb and Chappell

Scheme 39

Glycosylation with Sialic Acid

213

of sialoside transfer to alcohol donors. Using the 4-nitrophenylglycoside 122 as a sialyl donor, sialylidases can catalyze a transsialylation to an alcohol acceptor to generate the product 89, or hydrolyze to NeuAc 1 (Scheme 40). To complicate matters further, the product 89 may also undergo sialidase-catalyzed hydrolysis to form NeuAc. So if any product is to be isolated, the rate of sialyl transfer must be much faster than either hydrolysis rate. Thiem and Sauerbrei examined this concept to determine whether various sialidases could be used synthetically [52]. The rate of condensation, or reverse hydrolysis, was found to be negligible. However, the product hydrolysis rate was competitive with the rate of transsialylation to an alcohol acceptor. In an attempt to minimize product loss, reactions were stopped after 65–75% of the starting material had been consumed. Interestingly, a mixture of ␣-2,3 and ␣-2,6 regioisomers was obtained for reactions with an immobilized Vibrio cholerae sialidase (Scheme 41). In all cases, the ␣-2,6 isomer predominated, probably because of a combination of faster hydrolysis of the ␣-2,3 products (123 → 1 ⫹ 125) and lesser steric hinderance of the primary alcohol. Variations in the donor/acceptor ratio (1:7 optimized) had an effect on both reaction yield and regioselectivity, although most of the examples afforded only 2–3:1 ␣-2,6/␣-2,3 product ratios in 14–20% overall yield. Ajisaka et al. examined different sialidase sources and found that Newcastle disease virus (NDV) sialidase afforded predominantly the ␣-2,3 regioisomers, while Arthrobacter ureafaciens and Clostridium perfringens sialidases, in addition to the Vibrio cholerae sialidase examined by Thiem, favored the ␣-2,6-linked products [53]. Unfortunately, the reaction yields did not improve for the new enzymes, varying from 0.8% to 3.6% isolated yield. In the case of NDV sialidase, the high selectivity for ␣-2,3-sialosides stemmed from a large ␣-2,6/␣-2,3 hydrolysis ratio. Hydrolysis of the ␣-2,6 products was found to be 28 times faster than the ␣-2,3 isomers. Inter-

Scheme 40

214

Halcomb and Chappell

Scheme 41

estingly, the ␣-2,6 preference of the other three enzymes was not correlated to product hydrolysis rates. B.

trans-Sialidases

The Trypanosoma cruzi trans-sialidase catalyzes the reversible transfer of NeuAc from a NeuAc-␣-2,3-Gal-␤-OR1 sequence to an acceptor bearing the Gal-␤-OR2 motif (Scheme 42) [54]. The enzyme is a particularly useful sialidase because it has very little hydrolytic activity and tends to almost exclusively catalyze transsialylation to a galactose. However a major drawback to this method is that to drive the glycosylation to completion, there is a need for large quantities of complex ␣-2,3-linked sialyl donors, which are generally difficult to obtain from natural sources. Other natural donors with ␣-2,6- or ␣-2,8-linked sialic acids have been examined but were discovered to be poor sialyl donors for ␣-2,3-sialylations catalyzed by T. cruzi transsialidase [55]. Simple aryl ␣-sialosides, such as the 4-nitrophenyl glycoside 122 and methylumbelliferone glycoside 130 (Scheme 43), have been found to be excellent

Glycosylation with Sialic Acid

215

Scheme 42

substrates because of the irreversibility of the sialyl transfer, and these have become the most utilized sialyl donors for trans-sialidase-catalyzed glycosylations. The donor specificity of T. cruzi trans-sialidase was examined with various side chain modified NeuAc glycosides. Vandekerckhove and coworkers found that methoxy or deoxy modifications to C9 of a NeuAc-␣-2,3-Gal-␤-OR donor did not affect trans-sialidase activity, although the same modifications at C4, C7, or C8 completely prevented transfer of the sialic acid. In addition, the C4- and C8-modified compounds were found to be mild inhibitors of the enzyme [56]. Lee and Lee extended these studies to more drastically modified NeuAc aryl glycosides [57]. The triol side chain of the methylumbelliferone-␣-ketoside 130 was cleaved with periodate to afford the C7 aldehyde, which underwent reductive amination with different amines to provide several novel sialyl donors. Three of these derivatized sialic acids (those of 132, 134, and 136) were successfully transferred to a lactose acceptor on analytical scales (50 nmol), albeit in much lower yield than NeuAc from aryl glycoside 130 (Scheme 44). NeuAc analogs containing longer alkyl chain and terminal amine substituents were found not to be substrates for the trans-sialidase. Nonetheless, these studies helped to define the substrate requirements for trans-sialidase acceptance, and the latter work provides a novel method for the synthesis of chromophore labeled sialylated oligosaccharides. Ito and Paulson designed a cofactor regeneration system that overcomes several limitations of trans-sialidase-catalyzed sialylations (Scheme 45) [58]. The trans-sialidase is used in conjunction with ␣-2,3-sialyltransferase to effectively broaden the

Scheme 43

216

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Scheme 44

Glycosylation with Sialic Acid

Scheme 45

217

218

Halcomb and Chappell

substrate specificity of the sialyltransferase. The ␤-trimethylsilyl galactoside 139 is known to be a poor substrate for ␣-2,3-sialyltransferase; however, it is readily accepted by T. cruzi trans-sialidase. The sialylated oligosaccharide 138 was synthesized by the CMP-NeuAc regeneration system developed by Ichikawa and Wong [51]. Then 139 underwent a transsialylation with 138 to provide the desired GM4 precursor 141. The regeneration of 138 from the by-product 140 by sialyltransferase drives the equilibrium toward the product 141. This example demonstrates that trans-sialidases have some synthetic utility on their own but are much more general tools for oligosaccharide synthesis when coupled with sialyltransferases.

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8 The Chemistry and Biology of Multivalent Saccharide Displays David A. Mann and Laura L. Kiessling University of Wisconsin–Madison, Madison, Wisconsin

I.

INTRODUCTION

Recognition of the importance of carbohydrate binding proteins in regulating biological systems is fueling efforts to understand and manipulate these processes. Lectin–ligand interactions have been found to play key roles in a wide variety of life processes: bacterial and viral attachment, immune system function, transplant rejection, fertilization, cell targeting, and the inflammatory response [1–7]. Despite these significant functions for protein–carbohydrate interactions, observed dissociation constants are typically on the order of 10⫺4 –10⫺3 M for a monovalent carbohydrate adhesion to a single lectin binding site [3,8–10]. The low affinity of these interactions raises a critical question: How do natural systems exploit weak binding events? An attractive idea is that protein–carbohydrate interactions garner the requisite strength and specificity required to mediate important biological processes through multivalent contacts. Thus, multiple simultaneous interactions can occur between an array of associated lectin binding sites and a multivalent presentation of saccharide ligand epitopes [8,11–15]. This chapter describes recent efforts to investigate and manipulate multivalent binding events involving protein–carbohydrate interactions. We focus on how synthetic ligands have served as critical tools for dissecting, illuminating, and controlling these complex recognition processes and their functional consequences. There are several excellent reviews that discuss other aspects of multivalent protein–carbohydrate interactions [9,15–17]. II.

STRUCTURAL FEATURES AND ENERGETICS

A number of specific components contribute to the complex energetics of lectin– ligand interactions. The observed affinities (functional affinities, see Section II.B.1) 221

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for these processes arise from the features of the monovalent and multivalent binding interactions. As a prelude to an examination of specific multidentate interactions, this section describes the features of monovalent protein–saccharide complexation and the factors that contribute to the high functional affinities observed for many multivalent interactions. A.

Monovalent Interactions

The structural features of monovalent lectin–ligand complexes have been elucidated through X-ray crystallographic analysis [1,18–20]. These complexes have helped facilitate an understanding of the energetics of monovalent carbohydrate binding to proteins; they have facilitated structure-based free energy calculations, as well as providing a basis for interpretation of titration microcalorimetry experiments [10,21– 23]. Although proteins can bind carbohydrates tightly, lectin binding sites are often solvent-exposed indentations rather than deep binding pockets. In these shallow binding sites, contacts are made with a limited portion of the carbohydrate ligand (Figs. 1 and 2). Monovalent interactions appear to rely on hydrogen bonding, metal chelation, water displacement and reorganization, hydrophobic contacts, and Coulombic interactions. We will use specific protein–lectin interactions to illustrate these energetic contributions. 1.

Hydrogen Bonding

The density and diversity of stereochemical orientations available to saccharide hydroxyl groups suggests that hydrogen bonding interactions might play a role in their recognition by proteins. Although the energetic contributions of specific hydrogen bonding interactions are difficult to dissect, the directionality of these interactions is important for recognition and discrimination of saccharide ligands. For example, in the structure of the lectin concanavalin A (ConA) complexed with ␣-methyl mannopyranoside, hydrogen bonding interactions occur between the protein and the 3-, 4-, and 6-position hydroxyl groups of the saccharide [19]. There is no direct contact with the 2-position hydroxyl group, and ConA interacts with saccharides of both the gluco and manno configurations. The hydrogen bonding interactions, however, allow the lectin to discriminate between galactose and mannose (or glucose) derivatives. The amino acid side chains as well as the peptide backbone can contribute to recognition (Fig. 2). The side chain amide hydrogen of asparagine 14 can form a hydrogen bond with the 4-position hydroxyl group, whereas the backbone amide hydrogens of leucine 99 and tyrosine 100 are within reach of the ring and 6-position hydroxyl groups, respectively. This structure also illustrates another common feature of lectin–saccharide complexes: many side chains form bidentate hydrogen bonds. For example, the aspartic acid 208 carboxylate oxygens can interact with both the 4- and 6-position hydroxyl groups of mannose. In addition to direct contacts between the lectin and the saccharide, water-mediated hydrogen bonds commonly occur [22,24]. 2.

Metal Chelation

Many lectins depend on divalent metal cations, especially Ca2⫹, for carbohydrate binding. Two roles have been observed for metal cations. First, to preorganize side chains for saccharide binding, and second, to directly ligate saccharide hydroxyl

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Figure 1

The main chain of concanavalin A as determined by X-ray crystal structure analysis. The homotetramer and monomeric subunits are shown with bound ligand.

groups. Examples of the former can be found in the legume lectin structures, as observed in ConA, where Ca2⫹ and Mn2⫹ assist in organizing local structure and also in orienting protein functional groups for saccharide interaction (Fig. 2) [19]. Both modes of metal ion function are important in C-type lectin recognition. Direct coordination of a metal to both the complexed lectin and the target saccharide ligand is observed in C-type lectin structures [4,5] such as that of rat serum mannose binding protein A (MBP-A). In the model derived from X-ray crystallographic analysis, the

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Figure 2

Concanavalin A binding site showing metal chelation and hydrogen bond

networks.

saccharide hydroxyl groups act as ligands for the protein-bound calcium ion (Fig. 3). In addition, Ca2⫹ binding organizes protein side chain conformation to facilitate saccharide complexation [25]. The addition to these metal-cation-dependent lectins of chelating agents, such as EDTA, abrogates ligand binding, a result that emphasizes the importance of divalent metal ions in lectin activity. 3.

Hydrophobic Effects

Lectin binding sites, like the saccharides that bind to them, are amphiphilic. In addition to the hydrogen bonds that are important for orienting saccharides, the binding sites contain many hydrophobic groups that can contribute to saccharide binding affinity. The structure of MBP-A derived from X-ray crystallography illustrates the importance of hydrophobic binding regions complementary to those on the ligand (Fig. 4) [1]. For example, isoleucine 207 is located proximal to the 6-position methylene group of mannose, which should result in favorable van der Waals contacts. In many lectin–saccharide complexes, aromatic residues have been found to pack against one or more faces of the saccharide [26]. In the structure of MBP-A, the side chain of histidine 189 is near the 4-position C—H and 6-position O—H bonds, suggesting that interactions between the aromatic residue and nonpolar areas of the saccharide contribute to mannose binding in this system. 4.

Coulombic Interactions

Coulombic interactions between carbohydrate binding proteins and their ligands are pervasive. Carbohydrates often bear anionic charges, which are installed through attachment of sialic acid residues or by phosphorylation or sulfation of uncharged

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Figure 3

The mannose binding protein A (MBP-A) binding site, featuring calcium coordination to protein side chains as well as the 3- and 4-position hydroxyl groups of mannose.

Figure 4

The mannose binding protein A (MPB-A) binding site featuring van der Waals contacts with the mannose ligand.

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saccharides [27]. For proteins that bind sulfated polysaccharides such as heparin, salt bridges have been observed between positively charged lysine and arginine side chains and the anionic sulfate and carboxylate groups of the saccharide [28]. In contrast, some lectins bind anionic ligands without formation of salt bridges. For example, in the structure of sialic acid bound to influenza virus hemagglutinin, the carboxylate group of sialic acid hydrogen bonds to water and to a backbone amide proton [18]. Structural studies of the protein–saccharide complex in a related example, an MBP-A variant that binds the tetrasaccharide sialyl Lewis acid x (sLex) [29], also revealed that interactions between the protein side chains and the anionic carboxylate of sialic acid occurred indirectly rather than through a direct salt bridge. 5.

The Role of Water

The ability of water to act as both donor and acceptor of hydrogen bonds makes it an excellent mediator in protein–carbohydrate complexes. Results of numerous Xray crystallographic studies of protein–carbohydrate complexes attest to the pervasiveness of water-mediated hydrogen bonds in protein–carbohydrate interactions. Lectin-bound water molecules are proposed to act as an extension of the protein surface itself [1]. This idea is supported by the fact that water molecules occupy conserved sites in the structures of related lectins [24]. In addition, water molecules can mediate similar hydrogen bonding interactions in the complexes of different proteins with a specific carbohydrate ligand or in complexes of the same lectin with different saccharide ligands [1]. The energetic contribution of water to carbohydrate binding is controversial. From crystallographic and thermodynamic data and molecular modeling calculations, Lemieux [21,22] and Toone [10] have speculated that changes in hydration are a driving force for carbohydrate binding. They postulate that because carbohydrates and their binding sites are amphiphilic, an advantageous reorganization of water occurs upon binding. To gather experimental support of the importance of hydration in binding, Lemieux’s research group has generated oligosaccharide derivatives with polar groups attached to positions outside the binding site. These modifications, which should not disrupt critical binding interactions but do alter the saccharide solvation shell, affect ligand binding [22,30]. Most of the changes in saccharide solvation were detrimental to binding, a result that underscores the significant and impalpable role of water in carbohydrate–protein complexation. B.

Modes of Multivalent Ligand Function

Saccharides constitute one class of biologically important molecules whose function depends on multivalent presentation. In physiological settings, saccharide epitopes are not usually encountered in isolation, but rather in multivalent arrays. For example, high ligand density glycoproteins can serve as scaffolds for the presentation of saccharides. Alternatively, saccharides can be displayed in multivalent arrays through glycolipid clustering. Moreover, many saccharide binding proteins are multivalent because they form oligomeric quaternary structures or are present in multiple copies on a cell surface. To understand carbohydrate function, therefore, we need insight into how multivalent saccharide presentation affects recognition. Natural systems rely on assembling multiple weak monovalent binding events

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into multivalent arrays, and this strategy can lead to enhancements in the strength and specificity of the subsequent interaction. The importance of ligand multivalency, which is also referred to as the ‘‘cluster effect’’ [3], is widely recognized, but additional investigations are required to understand the increases in activity that are observed. Several types of interaction that can augment the observed activities of multivalent ligands are discussed in the following sections. Because of the difficulties associated with dissecting the mechanisms that contribute to the activity of a multivalent ligand, we use the term ‘‘functional affinity’’ to refer to the measured activity of a particular ligand. Functional affinity is a descriptor that has been used in studies of antibody–antigen interactions, which are also multivalent. Functional affinity is useful for the purposes of comparing monovalent and multivalent ligand activities because it describes a measurable quantity: the apparent association constant (or inhibition constant). The conditions under which an assay is conducted, however, must also be carefully considered. Many multivalent binding events, both in vivo and in vitro, do not occur under equilibrium conditions. Thus, the functional affinities of multivalent ligands can vary between assays, even when those of their monovalent counterparts do not (see Section III). 1.

Functional Affinity

As noted, monovalent binding energies are often weak, displaying dissociation constants in the millimolar range. The relative affinity preference of a lectin for two structurally related monovalent saccharide ligands can be slight. Presenting lectin binding sites in an array that can simultaneously recognize multiple clustered carbohydrate epitopes facilitates multivalent binding, a process that enhances not only the strength of the resulting interaction, but also the specificity. For example, a fourfold affinity preference for monovalent mannose over glucose binding to ConA is magnified to a 160-fold preference for a multivalent display of mannose over an analogous glucose polymer [31]. This enhancement in affinity and avidity, functional affinity, is thought to be the result of the combination of numerous multivalent binding modes. Characterizing the molecular features of these binding modes is a critical step in understanding lectin–ligand interactions. 2.

Antibody–Antigen Binding: A Context for Exploring Multivalent Saccharide Recognition

Multivalent interactions involving antibody binding to multivalent antigens have been studied extensively. Antibodies themselves can be multivalent, existing as the bivalent immunoglobulin D, E, and G types, the tetravalent IgE, or the immunoglubulin M type, displaying 10 associated binding sites. Models for multivalent antibody binding were developed by Crothers and Metzger in the 1970s [32–34]. These early studies emphasized that for multidentate ligands to simultaneously occupy multiple binding sites, they must present recognition epitopes that are both properly spaced and sufficiently flexible. As expected, these variables are also important in multivalent carbohydrate binding. Still, an understanding of how much increase in activity is to be gained by such presentation and how the structures of multivalent ligands should be optimized for maximal potencies remains a mystery. The following sections describe some of the molecular features and general mechanisms that lead to enhanced activities.

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The Chelate Effect

The simultaneous placement of saccharide residues from a multivalent ligand into multiple binding sites on an oligomeric lectin can lead to high functional affinities if the ligand epitopes are properly oriented to interact favorably with appropriate sites on the receptor. This situation, in which multiple receptor sites are occupied by a multidentate ligand, is an example of the chelate effect. The term ‘‘chelate effect’’ arises from the observation that ligands possessing more than one donor group are more effective at coordinating electrophilic metal centers than those that possess a single donor. Thus, the chelate effect describes multiple interactions, occurring after the first contact is made, as effectively intramolecular (Fig. 5A) [35]. The observed free energy of interaction (⌬Gobs) for an oligomeric receptor R1 —R2, . . . , —Rn with an oligomeric ligand, L1 —L2, . . . , —Ln will be related to the sum of the free energies of binding for each individual receptor. Thus, the binding is often estimated as follows: ⌬Gobs = ⌬G(R1 —L1) ⫹ ⌬G(R2 —L2) ⫹ ⭈ ⭈ ⭈ ⫹ ⌬G(Rn —Ln) This model predicts that the formation of additional contacts should give exponential affinity increases because only the first binding event exacts a translational entropy penalty. However, the entropic costs related to conformational flexibility are likely to be much higher for a multivalent protein–carbohydrate interaction. Page and Jencks recognized the potential utility of the chelate effect for devising highly effective inhibitors of target enzymes [35,36]. This idea was extended to devising multidentate ligands as potent inhibitors for multivalent receptors. For example, a synthetic nonnatural trimer of the glycopeptide antibiotic vancomycin binds with high functional affinity to the corresponding trimer of d-Ala-d-Ala, its dipeptide ligand. The affinity of this complex, stabilized by the chelate effect, is similar to that of the avidin–biotin interaction [37]. However, large distances (>40 ˚ ) separate the saccharide binding sites in many oligomeric lectins; therefore, long A spacers are required to link saccharide epitopes for multivalent binding. Thus, many attempts to design effective multivalent saccharide derivatives have been thwarted by the increases in conformational entropy inherent in the long linkers required for multivalent binding. The conformational entropy term is affected by the structural features of the multivalent ligand, such as the rigidity of the scaffold displaying the binding determinants and the relative orientation of each recognition element [15]. The functional affinity of multivalent ligands will reflect both the advantages of translational entropy gains and the disadvantages of conformational entropy penalties. 4.

Rebinding Leads to Decreased Off-Rates

The display of a receptor or ligand at high density can result in its partner exhibiting increased functional affinity, even if multipoint binding does not occur. The increase in apparent affinity can be rationalized by modeling the binding event as a two-step process. In the first phase, receptor and ligand form a transitory encounter complex. The second stage involves the transitory species proceeding to generate a stable complex or dissociating as a result of nonproductive binding (Fig. 5B). In the case of a multivalent partner, the probability of rebinding increases and that of dissociation decreases. Because the off-rates are slower for such an interaction, the observed

Multivalent Saccharide Displays

Figure 5

Modes of multivalent binding. 229

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functional affinities are higher. For example, ligand affinities for proteins clustered on a membrane or immobilized on a surface can appear to be significantly higher than that of the corresponding species in solution [38,39]. Like the chelate effect, this mode of statistical enhancement benefits from a high effective concentration of the binding partner, although the magnitude of enhanced activity is likely to be smaller than that for systems that exploit chelation. An advantage of such a statistical effect, however, is that optimal spacing of the ligand epitope is not crucial. 5.

Receptor Clustering

For a multivalent ligand, an alternative to occupying multiple saccharide binding sites within an oligomeric receptor is to bind multiple receptors at the cell surface (Fig. 5C). This mode of interaction is similar to the chelate effect, although the entropic costs are different. Moreover, the resulting effects of complexation can be quite distinct. For example, once the initial translational penalty for ligand binding has been paid, additional interactions will result in some restriction of receptor mobility. Nevertheless, this cost is moderated because receptors in a fluid membrane diffuse in two dimensions rather than three. An advantage over the chelate effect is that the ligand scaffold need not precisely orient epitopes for binding as proper spacing can be adopted by receptor movement in the membrane. Consequently, there will be more orientations in which multiple receptor–ligand complexes can form. Finally, the interaction of multivalent ligands with membrane-bound receptors can lead to their clustering. Bringing receptors together via liganded clustering may have useful functional consequences [40,41]. 6.

Subsite Binding

Some lectins have demonstrated the presence of secondary binding sites that aid in ligand discrimination [1,42–46]. In binding to these lectins, ligand substituents may either interact with a second independent saccharide binding site or with regions adjacent to the primary binding site (Fig. 5D). Secondary saccharide binding sites can impose ligand selectivity that results in specificity for complexation of one oligosaccharide (or one multivalent ligand) over another [43]. Similarly, an extended building site can engage in further interactions with substituents on the ligand, such as an appended carbohydrate residue or a portion of the scaffold upon which the primary saccharide ligand is displayed [47–49]. Although the physiological relevance of such sites remains unknown, multivalent ligands that can target subsites and extended sites will be more effective and selective inhibitors. 7.

Steric Interference

Steric stabilization can result in increases in the activities of multivalent ligands designed to inhibit the interaction of receptors and ligands on opposing surfaces, as in the case of cell adhesion. Whitesides and coworkers have suggested that the binding of high molecular weight multivalent ligands to one surface (e.g., a virus) can preclude the attachment of a competing surface (e.g., a cell) by sterically blocking access to neighboring binding sites (Fig. 5E) [15,50]. This mechanism may be especially important for ligands that target cell surface receptors displayed at high densities (or high local densities). Moreover, a large multivalent ligand that can bind to one receptor may also block access to other receptors located at interstitial sites. For multivalent ligands operating by this mechanism, the nature of the scaffold em-

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ployed will play a role in steric repulsion in addition to providing conformation flexibility or rigidity. 8.

The Polyelectrolyte Effect

Many naturally occurring multivalent saccharide ligands are anionic. Placement of specific anionic groups can be critical for saccharide recognition (see Section II.A.4), but the charge density resulting from a multivalent display of such groups can influence their binding activities. In analogy to protein–DNA interactions, protein binding to highly anionic saccharide derivatives (such as heparan sulfate) can benefit from the polyelectrolyte effect. Charged functional groups on both lectin and ligand mobilize counterions when solvated. Ligand binding releases these ions into the bulk solution, a process that is entropically favorable (Fig. 5F). Additional energetic gains will result from multiple successive attachments [51]. III.

ANALYZING MULTIVALENT BINDING EVENTS

Publications focusing on assessing multivalent interactions often highlight the difficulties associated with assessing binding kinetics and thermodynamics for such processes [11,15,39]. Determining the true equilibrium constant for a multivalent binding event is complicated because, as described above, multiple types of binding interaction may contribute to the interaction under investigation. Thus, an inability to dissect the contributions of monovalent binding, multiple site binding, subsite binding, rebinding of multiple epitopes within one or more binding site, and other effects such as steric stabilization renders the analyses of these systems difficult. The evaluation of protein–carbohydrate interactions is complicated by the tendency of these substances to participate in multivalent binding and the low affinities of their monovalent interactions. These features dictate that the assay used to measure ligand activities be chosen carefully. Assays of several different types may be required to properly evaluate ligand potency. Moreover, generating data relevant to ligand function under physiological conditions can be a difficult and onerous task. Because throughput can be limited in assays that closely mimic physiological conditions, the best assay for screening potential inhibitors might necessarily be artificial, and also may not identify the best ligand (see Sections VI.4 and VII.B.2). Some of the more effective methods that have been employed to evaluate inhibitors of protein–saccharide interactions are described in the subsequent sections. A.

Fluorescence Anisotropy

Fluorescence anisotropy is a technique that can be used to monitor low-affinity monovalent lectin–ligand interactions [52–54]. An advantage of this technique is that it can be used directly to determine ligand binding constants and also to confirm that ligands are competing for a specific saccharide binding site. The basis of the method is the detection of changes in the fluorescence anisotropy of a ligand-bound fluorophore. When a protein binds the labeled ligand, there is an increase in the rotational correlation time of the fluorophore because the resulting complex is large and tumbles more slowly than the free ligand. An increase in fluorescence anisotropy results (Fig. 6), and these changes can be analyzed to afford a binding constant. Moreover, binding affinities of unlabeled ligands can also be obtained accurately and

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Figure 6 Schematic depiction of a fluorescence anisotropy assay. Fluorescently tagged molecules are excited by plane-polarized light; only molecules in the proper orientation are excited. Emitted light is detected in the original plane and in the perpendicular plane. The quantity of fluorescence observed in the two orientations is determined by the rate of tumbling, which depends on particle size and relates to binding.

efficiently by monitoring their ability to compete with a fluorescently labeled control ligand. The disadvantage of this method is that high concentrations of protein are needed to accurately determine the weak association constants typically observed for protein–carbohydrate interactions. Even when the protein can be readily produced or isolated, the high concentrations required can result in protein aggregation and/or precipitation. Overall, the method has many advantages, but its applicability depends on the system. B.

Turbidimetric Analysis

Turbidimetric analysis is useful for the study of multivalent interactions that result in the formation of large protein aggregates. The method is typically employed to evaluate the ability of multivalent saccharide derivatives to promote lectin crosslinking (Fig. 7). To conduct the assay, multivalent ligand and lectin are mixed and allowed to form cross-linked complexes; the formation of large protein aggregates is monitored by changes in the optical density of the solution [55–57]. The abilities of various multivalent ligands to cross-link lectin is correlated with their relative affinities. As a measure of monovalent ligand function, inhibitory potencies of these species can be obtained by evaluating their abilities to inhibit lectin cross-linking by a specific multivalent ligand. This analysis is complicated, however, by the possibility

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Figure 7 Multivalent ligand-induced lectin cross-linking. Multivalent ligands, which are able to simultaneously span two lectin molecules, can form cross-linked aggregates. These aggregates are detectable by spectroscopic means in a turbidimetric assay.

that the monovalent ligands could initially contribute to optical density increases. In principle, they could occupy binding sites on the lectin that do not directly result in cross-linking, thereby releasing epitopes on the multivalent ligand that then attract more lectin to the aggregate. The complexities of comparing monovalent and multivalent ligands by turbidimetric analysis are not the only disadvantages of this method. One potential problem is that it does not effectively report on the activities of multivalent ligands that act by simultaneously binding to multiple sites within a lectin oligomer. In addition, the relative activities of a multivalent ligand for two related lectins cannot be determined because the inherent solubilities of the lectins and their cross-linked complexes will differ. Importantly, because precipitation of the lectin–ligand complex occurs, the process is generally under kinetic control; consequently, the relative potencies of ligands may not correlate with their intrinsic affinities for the lectin. As with most assays of multivalent ligand function, the species being detected is not the initial protein–ligand complexation event. Still this assay is widely used because it can be conveniently and rapidly performed. C.

Enzyme-Linked Immunosorbant Assays

A wide range of variations on the enzyme-linked immunosorbant assay (ELISA) have been used to evaluate monovalent and multivalent inhibitors owing to the convenience and utility of these methods (Fig. 8) [58,59]. Generally, one binding partner is immobilized on the bottom of a microtiter plate, and a binding counterpart, labeled with a reporter, is allowed to adhere to the immobilized partner. Monovalent and multivalent compounds can be tested for their ability to inhibit the binding interaction. The reporter group can be a radiolabel, fluorophore, biotin, conjugated antibody, peroxidase, or other enzyme conjugate. In the case of a protein–saccharide interaction, either the lectin or the ligand can be immobilized. Inhibitory potencies obtained from ELISA and related assays provide an estimate of the functional affinity of various ligands, although confounding parameters can complicate the analysis. The immobilized partner is often displayed in a random mixture of orientations, some of which may not be optimal for binding. All soluble ligands will encounter these various receptor orientations; therefore a comparison of ligand activity generally affords the proper qualitative relative ranking. One problem with the variety of orientations observed is that they may not reflect those found in natural settings. Consequently, the artificial ligand spacing inherent in the ELISA can introduce a bias to

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Figure 8 An example of an ELISA. Monomeric lectin is immobilized in the bottom of a microtiter well. Labeled multivalent ligand binds to the lectin. The assay can also be set up to observe lectin binding to immobilized ligand.

the assay. ELISAs, however, are simple to perform and can be used for rapid screening of multiple inhibitors. D.

Cell Agglutination

The activities of both monovalent and multivalent ligands can be assessed in a cell agglutination assay if the targeted receptor has the ability to agglutinate cells. This assay is generally applied to evaluate the potencies of ligands for a multivalent lectin that recognizes saccharide epitopes on red blood cell surfaces, thereby causing hemagglutination (Fig. 9) [60]. The lectin and isolated cells are combined in a microtiter plate with conical wells. In the absence of lectin, or in the presence of an inhibitor, the cells eventually pellet by gravity in the bottom of the well. In contrast, when the cells are cross-linked they form a large aggregate network that covers the well. Inhibition of agglutination is detected by visual inspection. The human component of the analysis renders this assay subject to potential bias, and it also makes it difficult to rapidly screen ligand libraries. Nevertheless, hemagglutination is a convenient method of identifying either monovalent or multivalent inhibitors of lectin–ligand binding. E.

Surface Plasmon Resonance

Surface plasmon resonance (SPR) is an analytical method that can be used to evaluate protein–ligand interactions. It has only recently been applied to probe lectin–ligand interactions [11,61–66]. In SPR, the amount of material bound to a surface is quantified by monitoring the absorption of an evanescent wave. One binding partner is attached to the surface by covalent or noncovalent interactions, and its counterpart

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Figure 9 Hemagglutination of red blood cells. Red blood cells are combined with a lectin that can recognize cell surface carbohydrate ligands. In the presence of inhibitors, the cells pellet, forming a visually identifiable spot in the well. Because cross-linked cells cannot pellet; the well maintains a homogeneous red tint.

is allowed to flow over the surface (Fig. 10). Receptor–ligand association is monitored to generate an apparent on-rate, and dissociation to afford the apparent offrate. From these values, an equilibrium constant can be obtained. To overcome the complications associated with monitoring ligand function in a heterogeneous system, a competition assay can be employed in which inhibitors are evaluated for their ability to block the target binding interaction. Thus, multiple ligands can be screened rapidly with a single binding surface [67–69]. One advantage of SPR is that only small amounts of material are required for the analysis. In addition, the availability of automated sample injection and monitoring facilitates the rapid screening of large numbers of compounds. Since confounding interactions can complicate the analysis, the design and implementation of an optimal binding surface is critical for the generation of meaningful data. IV.

PLANT LECTIN–SACCHARIDE INTERACTIONS

The carbohydrate binding properties of plant lectins have been widely studied and exploited. Because many plant lectins can be readily isolated, numerous studies of

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Figure 10 Surface plasmon resonance analysis. A surface displaying ligand is examined under conditions of flow. Injection of lectin results in an increase in signal observed in the association phase of the curve. At equilibrium the surface achieves maximal binding. Measurement of the dissociation can yield off-rates. Competing ligands in solution can also be assayed.

these receptors have been conducted. The results serve as a foundation for understanding protein–carbohydrate interactions. Plant lectins can be classified by their source, and these groups include the legume, cereal, Amaryllidaceae and related families, and Moraceae and Euphorbiaceae lectins [5]. The lectins in all these classes assemble into multimeric proteins and can adopt a range of different quaternary structures including di- and tetrameric arrangements. The multimers formed afford multivalent displays presenting saccharide binding sites in a variety of spacings and orientations with documented multiplicities of 2 to as many as 12 binding sites, as in the case of the snowdrop lectin [70]. X-ray crystallographic studies have revealed the spatial relationships in many of these multiple binding site arrangements [1,5,42,71,72]. With the diversity of multivalent binding site displays and the large amount of structural and biophysical data available on them, highly accessible plant lectins serve as excellent receptors for the study of multivalent protein–carbohydrate interactions. A.

Concanavalin A

One of the most widely studied lectins to date is the glucose/mannose binding legume lectin from Concanavalia ensiformis (ConA) [73]. ConA is a tetramer at neutral pH, and X-ray crystallographic analyses revealed the tetrahedral arrangement of the protomers (Fig. 1) [19]. Each subunit contains a saccharide binding site; therefore, ConA can bind up to four saccharide epitopes simultaneously. Significant insight into the

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molecular basis for ConA–saccharide interactions has been obtained through X-ray crystallographic analyses of ConA and its saccharide complexes (Figs. 1 and 2) [19]. The binding site is organized by side chain interactions with two divalent metal cations, Mn2⫹ and Ca2⫹ (Fig. 2), and the minimum saccharide determinants that bind ConA are glucose and mannose residues [73]. The lectin exhibits a three- to fourfold binding preference for mannose over glucose and does not bind fucose or galactose appreciably [74–76]. Hydrogen bonding contacts from the protein to the 3-, 4-, and 6-position hydroxyl groups of the carbohydrate dictate ConA saccharide binding specificity. ConA binds relatively weakly to monosaccharides, but it interacts with higher affinities with oligosaccharides and glycoproteins [77]. For example, ConA binds the trimmanoside methyl 3,6-di-O-(␣-D-mannopyranosyl)-␣-D-mannopyranoside with approximately 100-fold higher affinity than methyl-␣-D-mannopyranoside [20,23,78,79]. The well-characterized molecular features of ConA–saccharide interactions and the protein’s ability to participate in multivalent recognition events render ConA an excellent subject for the study of multivalent recognition. 1.

Synthetic Multivalent Ligands Are Effective Inhibitors of Concanavalin A

A number of natural multidentate ligands, including oligosaccharides and glycoproteins [73,77,80,81], serve as highly effective ligands for tetrameric ConA. While these ligands provide useful information about ConA binding, the structures are so difficult to vary systematically that it is difficult to analyze binding data from such substrates. Synthetic ligands have been used, therefore, to address fundamental issues with regard to multivalent recognition by ConA. Major ligand classes that have been employed include low molecular weight derivatives, dendrimeric ligands, and linear polymers. Some of the most potent inhibitors of ConA are multidentate ligands synthesized by the ring-opening metathesis polymerization (ROMP). ROMP has emerged as an attractive approach to the synthesis of functional polymers [82]. The advent of defined, functionalized group tolerant, metal alkylidine initiators has offered new opportunities for the creation of tailored materials with significant biological properties. A generalized mechanism for the polymerization involves an initiation through coordination of the metal alkylidine with a strained cyclic alkene monomer (Fig. 11). A [2 ⫹ 2] cycloaddition between the initiator (2) and a monomer (1) unit generates a metallocyclobutane intermediate (3) that undergoes a ring opening to relieve strain, thereby generating a new metal alkylidene. This intermediate metallocarbene (4) can react with another monomer, and the sequence of events can continue until the monomer is consumed. The reaction can be terminated by the addition of an electron-rich enol ether (5), which results in the production of an unreactive metal carbene (7) and a terminated polymer chain (6). Because ROMP can be a living process and because the rate of initiation exceeds that of propagation, oligomers of controlled length and narrow molecular mass distribution can be generated. In addition, both the catalyst and the electron-rich enol ether afford an opportunity to attach unique end labels to either polymer terminus. Thus, ROMP offers advantages for the production of biologically active polymers [83]. To explore interactions of multivalent ligands with ConA, polymers with pendant mannose or glucose residues have been generated by ROMP. The saccharide epitopes were attached to norbornene monomers through C-glycoside linker units

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General scheme for ring-opening metathesis polymerization (ROMP). Monomer 1 and catalyst 2 continue to react until the monomer has been consumed yielding polymer 4, which can be end-capped with functionalized allyl ether 5.

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and polymers of high molecular mass were produced. These materials were highly effective inhibitors of ConA-facilitated red blood cell agglutination: they were up to 105-fold more potent than ␣-methyl glucopyranoside [31]. Other nonnatural saccharide-substituted polymers have also been found to be effective inhibitors of ConA [84,85]. In addition, dendrimers [86–88] and low molecular weight ligands [44,89– 93] show levels of activity that exceed those of mannose, although they do not approach those of the ROMP-generated polymers. Together, these results highlight the range of inhibitory activities that can be manifested by synthetic multidentate ligands. 2.

Specificity in Multivalent Recognition: Concanavalin A Discriminates Between Related Multidentate Ligands

The specificity of ligand binding for a multivalent protein–carbohydrate interaction was explored by using ConA. ConA binds both glucose and mannose residues, but it binds monovalent C-mannoside 9 with a slightly (0.2 kcal/mol) more favorable free energy than the corresponding C-glucoside 8 (Fig. 12) [54]. Whether the small difference observed in the monovalent binding event would be manifested with the corresponding multivalent ligands has been explored. By examining the ability of glucose- and mannose-substituted polymers to inhibit ConA-mediated agglutination, the inhibitory potencies of mannose-modified polymer 11 could be compared with the corresponding glucose counterpart 10 in a hemagglutinaton assay (Fig. 12). Not surprisingly, no difference in potency was detected between the glucose and mannose monovalent C-glycosides 8 and 9. The activity difference between the glucose- and mannose-substituted polymers, however, is greater than 100-fold [31]. Consequently, multivalent interaction yields higher specificity as well as affinity. An enhancement of specificity through multivalent saccharide display is also observed in the binding of cluster glycosides of N-acetylgalactosamine to the mammalian hepatic Gal/ GalNAc specific receptor with subnanomolar binding constants (see Section V.C) [94]. When evaluated for binding to the lectin from Bauhinia purpurea, resin-bound saccharides also elicited a dramatic increase in observed functional affinity [38]. Enhanced specificity through polyvalent binding to multivalent carbohydrates has also been observed in interactions with cell surface L-selectin (see below: Sections VII.B.2–VII.B.3) [95]. Enhancement of specificity through multivalent recognition is likely to be an important feature of saccharide recognition events in nature. 3.

The Chelate Effect: Multivalent Ligands That Can Span Two Saccharide Binding Sites Within the ConA Tetramer Are the Most Potent Inhibitors

Multipoint binding leads to an amplification in specificity, perhaps at least partly accounting for the high selectivity of multidentate ligand binding by ConA. In multipoint binding, multiple saccharide epitopes would coincidentally contact ConA, and if this model is important, ligands that could simultaneously occupy more than one saccharide binding site within the ConA tetramer would be expected to be more potent inhibitors. The large distance between the binding grooves, predicted to be ˚ from X-ray crystallographic analysis, is easily spanned by linear approximately 65 A polymers. Although few polymerization strategies offer control over polymer size, ROMP can be used to generate polymers of defined length.

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Figure 12 Kiessling’s C-gluco- (8) and mannopyranosides (9) demonstrate increased functional affinity and specificity when displayed as bivalent mannose (13) and glucose (12) ligands.

Kanai et al. set out to prepare and determine the ConA inhibitory potencies of series of mannose-substituted oligomers differing in length (Fig. 13) [84]. Studies by the Grubbs group suggested that ruthenium carbene complexes can carry out living polymerization with the initiation step approximately eightfold faster than propagation [82]. Therefore, by varying the ratio of monomer 14 to initiator 15 in the reaction mixture, multidentate mannose ligands 16a–c of controlled length could be assembled. The effect of polymer length on inhibitory potency was assessed in a ConA-mediated cell agglutination assay. Interestingly, the relative potency of the

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Figure 13

ROMP polymers of defined length. Varying the ratio of monomer (14) to catalyst (15) affords control over the average polymer 16 length (n).

multivalent displays (on a saccharide residue basis) initially increased significantly with increasing polymer length, but eventually reached a plateau. Molecular modeling studies suggest that when polymer 16 is composed of about 25 repeat units, it ˚ required to simultaneously place saccharide residues in two bindcan span the 65 A ing sites within the ConA tetramer (Fig. 14). As predicted, the potency of the oligomers beyond that length did not increase. An independent assay, in which surface plasmon resonance (SPR) was used to monitor inhibition of ConA binding to a glycolipid surface, reproduced the trends [68]. Specifically, a maximal functional affinity increase was observed for polymer 16b, which consisted of 25 monomer units. ConA structural data provides a context for interpreting mechanisms of multivalent ligand function; consequently, these studies represent strong evidence that lectins can use the chelate effect to increase the strength of a protein–carbohydrate interaction. Such mechanisms are likely also important in vivo.

Figure 14

(A) Multivalent polymer binding within one binding site. (B) Polymers of sufficient length can span two binding sites simultaneously. (C) Longer polymers yield no increase in potency.

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High Local Ligand Concentration Contributes to ConA Binding

Despite the importance of the chelate effect in the increased functional affinities of ConA for the multidentate ligands described above, the chelate effect is not the only contributing mechanism. If chelation was the only important process, oligomers composed of more residues than are required to span the binding sites should display decreased potencies when evaluated on a saccharide residue basis because noninteracting residues would detract from the observed affinity. This was not the case, however, inasmuch as potency did not decrease. Therefore, the potencies of the longer polymers are greater than the chelation model predicts (Fig. 14). We suspect that the high local concentration of saccharide residues available with these polymers decreases the rate of dissociation of the multivalent ligands, making rebinding more favorable. This statistical enhancement mode of multivalent binding also contributes to the increase in the observed affinity of ligands unable to span two sites (i.e., <25 residues in length). Moreover, the large number of mannose residues available for binding with these ligands suggests that a number of different lectin–ligand complexes can form. Finally, ConA clustering and steric stabilization (see Section VI.C) may also contribute to the efficacies of the longer polymers (Fig. 5). 5.

Spacing and Orientation of the Saccharide Epitopes Is Important for ConA Interaction

Low molecular weight divalent and trivalent ligands have also been generated to probe ConA ligand recognition. As had been shown with the hepatic lectin (see Section V.C) [96,97] and influenza virus hemagglutinin (see Sections VI.E–VI.F) [98], low molecular weight ligands can achieve high potencies, displaying inhibitory potencies 100- to 1000-fold higher than those of the corresponding monovalent saccharides. To investigate the effect of epitope spacing and orientation in ConA binding, Roy and coworkers constructed a series of low molecular weight bivalent ligands in which the saccharide epitopes were appended through linkers differing in length and composition (Fig. 15) [89,90]. The ability of these bivalent molecules to inhibit ConA binding to immobilized mannan (a yeast glycoprotein) was tested in an enzyme-linked lectin assay (ELLA) (Fig. 8). The structure of the linking group had subtle effects on the ConA inhibitory properties of the bivalent molecules. The bivalent ligand with a six-carbon linker, 17c, found to be superior in this study, was 5.3 times more effective per saccharide at inhibition than ␣-p-nitrophenyl-mannopyranoside [89]. Roy and coworkers also examined the effects of higher order valency in a related series of related ligands (Fig. 16) [57]. Bi-, tri-, and tetravalent ligands, 18, 19, and 20, were 138, 210, and 231 times more active than ␣-Memannose, respectively, when analyzed on a molar scale. However this translates to 69-, 70-, and 57.8-fold increases in potency when determination is made on a persaccharide-residue basis. On a multivalent display, additional ligands that do not contribute productively to binding can detract from the observed potency [86]. This has also been observed in the case of polyacrylamide displays targeting influenza virus, where maximal activity is observed at a low ligand density (see Sections VI.A–VI.B) [99–101]. The abilities of small bi-, tri-, and tetravalent molecules to inhibit ConA likely arise from their propensities to cross-link lectin multimers. Molecular modeling of Roy’s ligands suggests that the spacers linking the saccharide recognition epitopes

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Figure 15

Roy’s bivalent mannose ligands directed against ConA. To investigate spacing effects, the ligands are designed with variable length linkers.

˚ required to simultaneously occupy two binding are not long enough to span the 65 A sites within a single ConA tetramer (D. Mann, unpublished results, 1999). Another potential mode of action for these ligands is suggested by Brewer’s finding that many oligosaccharides and glycopeptides can form cross-linked lectin complexes [102]. A turbidimetric assay monitoring the formation of cross-linked ConA precipitates supports this model for Roy’s ligands (Fig. 7). Thus, the best small-molecule inhibitors of ConA may be those that can cross-link the lectin. Dendrimers that exhibit enhanced inhibitory activity toward ConA also appear to function by this mechanism [87,88]. Regardless of their mode of action, data generated with the bivalent ligands highlight the necessity for proper spacing between ligands to elicit optimal multivalent interactions. 6.

Multivalent Saccharide Display on Beads Alters the Ligand Preference

The role of ligand presentation in affinity is also highlighted by the fact that functional affinity trends observed with monovalent ligands do not necessarily mirror the effects observed when these ligands are assembled into multivalent displays. Seeking to identify highly active monovalent ligands, Kahne and colleagues developed a combinatorial synthesis yielding a library of disaccharide ligands on TentaGel beads [38]. A mixed population of the beads was assayed for activity against peroxidaselabeled Bauhinia purpurea lectin (also a hemagglutinin). Beads bearing the two most active ligands identified from the library, compounds 21a and 22a, were shown to have significantly higher functional affinities than beads bearing the known Bauhinia purpurea ligand Gal␤1,3GalNAc, compound 23a (Fig. 17). However, when the monovalent analogs were tested in competition experiments inhibiting Bauhinia purpurea lectin binding to Gal␤1,3GalNAc-coated beads (23a), known ligand 23b was more active than the two best compounds from the library, 21b and 22b. One interpretation of these results is that ligand presentation on the beads altered the relative potencies of the ligands; however, other factors, such as saccharide residue density

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Figure 16

Roy’s bi-, tri-, and tetravalent ligands 18, 19, 20 all demonstrated similar potencies on a per-saccharide basis

for ConA.

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Figure 17

When presented on beads, Kahne’s carbohydrate-based ligands had relative activities different from those for corresponding monomers in solution.

and hydrophobicity of the saccharides and beads themselves, might influence the activities of the immobilized library members. 7.

Bivalent Ligands Distinguish Between Related Proteins ConA and the Lectin from Dioclea grandiflora

Many lectins can bind the same carbohydrate epitopes, but small molecules that can selectively target one lectin over another are desirable. To explore this issue, several groups have compared the specificity of carbohydrate binding to ConA with that of the lectin from Dioclea grandiflora [44,76,103]. Examination of the fine saccharide binding specificity of Dioclea grandiflora lectin and ConA reveals a great deal of similarity [103,104]. The two proteins have amino acid sequences that are more than 75% identical, and both bind glucose and mannose residues, exhibiting similar preferences for most naturally occurring saccharides. When assayed in fluorescence anisotropy competition experiments, synthetic bivalent C-glycoside derivatives were able to discriminate between ConA and the D. grandiflora lectin, even though monovalent C-allyl mannose and glucose analogs behaved similarly. Specifically, bivalent mannose and glucose, compounds, 13 and 12, bound selectively to ConA but not to the D. grandiflora lectin (Fig. 12) [44]. Turbidimetric analysis suggested that ConA cross-linking was not occurring, although soluble cross-linked complexes could be ˚ between primary present [102]. The divalent ligands are unable to span the 65 A binding sites on the tetramer, but the molecular basis for this discrimination may lie in the subtle structural differences between ConA and D. grandiflora lectin in regions proximal to the saccharide binding site [79,105,106]. These results indicate that changes in carbohydrate presentation, such as switching from a monovalent to a bivalent ligand, can have dramatic effects on the specificity of protein–carbohydrate recognition. Multivalent ligands can thus be used to distinguish between highly related lectins. V.

HEPATIC LECTIN

The mammalian hepatic lectin (also referred to as the asialoglycoprotein receptor) is found on the surface of liver cells. The protein directs the clearance from the serum

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of glycoproteins that possess terminal galactose or N-acetylgalactosamine residues. Upon recognition by the hepatic lectin, asialoglycoproteins are endocytosed and degraded [107]. Ligands for the protein can be generated from tri- and tetra-antennary N-linked carbohydrate epitopes by release of terminal sialic acid residues to expose galactose residues or by chemical synthesis [97,108–110]. The physiological function of the receptor and the identity of its relevant ligands are matters still under investigation. We will focus on efforts to utilize the hepatic lectin mediated recognition and endocytosis to target various therapeutic agents to the liver. A.

Multivalent Hepatic Lectin Is a Heteromultimer

The hepatic lectin is displayed on hepatocytes as a heteromultimer; in the human and the rabbit, the protein is composed of two subunits, and in mice and rats it consists of three subunits [107,111,112]. Hepatic lectin is a member of the C-type lectin family, a group of mammalian lectins requiring calcium for ligand binding [113]. The subunits act in concert to recognize branched saccharide ligand epitopes, which accounts for the saccharide epitope selectivity described above [108,114–116]. The relationship between multivalent ligand binding and endocytosis is unclear because both monovalent and multivalent peptide-based ligands can lead to an increase in the rate of receptor internalization [114]. It is clear, however, that characterization of the recognition and binding properties of the hepatic lectin is critical for understanding its function and devising strategies for selectively targeting liver tissues. B.

Low Molecular Weight Trimeric Galactose Displays Can Be Potent Ligands for the Hepatic Lectin on the Cell Surface

Multivalent displays of galactose and N-acetylgalactosamine epitopes have been synthesized to identify the features important for glycoconjugate recognition and endocytosis by the hepatic lectin [117,118]. Initial studies, performed with galactosesubstituted neoglycoproteins and polyacrylamide gels, demonstrated that the receptor binds more efficiently to multivalent ligands [119]. Since many natural saccharides contain branched or multiantennary structures, several groups postulated that highaffinity recognition of carbohydrates in biological systems requires the presentation of clustered saccharide residues [108,118]. To test this idea and to elucidate structure–function relationships, Lee’s group and others have synthesized a number of nonnatural low molecular weight multivalent compounds [97,108,109,120]. The compounds were evaluated for their ability to inhibit the binding to hepatocytes of radiolabeled asiolomucoid, a ligand for the hepatic lectin. A synthetic trivalent compound, YEE(ah-GalNAc)3 (24, Fig. 18) in which GalNAc (N-acetylgalactosamine) residues were appended to a glutamic acid template (YEE), was found to be 10,000fold more active than monovalent GalNAc in the assay. Other trivalent derivatives were less effective. For example, displays based on the aminotris(hydroxymethyl) methane template were only 70-fold more active than their monovalent analogs at inhibiting asialoorosomucoid binding to isolated hepatocytes. The modest potency increases suggest that the spacing of the saccharide residues presented on this scaf˚ ) is not complementary to that of the carbohydrate binding sites on the fold (ca. 9 A cell surface. This result is consistent with investigations suggesting that the best ˚ [3]. inhibitors display galactose residues separated at distances between 15–30 A These studies established that low molecular weight synthetic multivalent ligands

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could serve as highly potent ligands. Moreover, they emphasize the importance of template design in generating effective multidentate ligands. C.

Low Molecular Weight Trimeric Galactose Arrays More Effectively Inhibit Cell-Surface-Bound than Soluble Hepatic Lectin

The large increases in functional affinities of multivalent Gal- and GalNAc-substituted ligands (such as 24) for the hepatocyte cell surface are not observed in assays involving inhibition of the isolated hepatic lectin. Specifically, the best multivalent inhibitors of soluble lectin activity are approximately 100-fold more active than their monovalent counterparts; in a similar comparison of potencies using cell-surfacebound lectin, however, the multivalent displays exhibit a 10,000-fold enhancement [96,108]. Two potential rationales for the differences are as follows: the subunit organization of the lectin is different on the cell surface, and/or the multivalent ligands bridge more than one lectin on the cell surface. Interestingly, multivalent ligands directed against influenza virus hemagglutinin display similar binding properties; they show greater potency in cell surface binding assays than when evaluated against isolated trimeric lectin (see Section VI.F). Together, these results suggest that small multivalent ligands may give rise to high functional affinities because they can bridge or cluster lectins at the cell surface (Fig. 5). D.

Ligand Spacing Allows Selective Targeting of the Hepatic Lectin over a Related Receptor

As described above, ligands with the proper saccharide epitope spacing and flexibility are more effective at binding the hepatic lectin [108,121,122]. Altering the scaffold for ligand display can also give rise to selectivity for one protein over another, as discussed in the case of ligands that discriminate between ConA and the lectin from Dioclea grandiflora. For the hepatic lectin to function in cell-selective targeting, the carrier ligands must discriminate between this receptor and the related macrophage Gal/GalNAc-specific lectin. Moreover, galactose receptors are also found on hepatocarcinoma cells [123]; demonstrating the potential for cell selectivity would provide support for the concept of designing agents that target hepatocarcinoma cells over hepatocytes. To identify selective ligands, Ozaki et al. compared the binding of a series of multivalent GalNAc presenting cluster ligands for their abilities to inhibit asialomucoid binding to different cell lines. This study revealed that YEE(ahGalNAc)3, 24, can selectively target cell lines producing recombinant hepatic lectin. Specifically, a 20-fold increase in activity for trivalent derivative 24 over monovalent Gal was observed with the cell lines displaying the macrophage Gal/GalNAc-specific lectin, while an increase of approximately 106-fold was obtained with cells displaying the hepatic lectin [94,124]. These studies suggest that although these cell surface lectins possess similar amino acid sequences, their quaternary structures differ. Significantly, ligands can be generated that exploit these differences in lectin, quaternary structure and/or cell surface organization. E.

Applications of Ligands for the Hepatic Lectin: Exploiting Hepatic Lectin for Selective Targeting

The hepatic lectin is a key model system for the development of receptor-mediated drug delivery [125–131]. The observations that the hepatic lectin can endocytose

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ligands is restricted to selected cell types and can be inhibited by potent smallmolecule ligands prompted efforts to exploit this receptor for cell-specific targeting. Several research groups have explored the scope of molecules that can be targeted and endocytosed through a hepatic lectin binding ligand to develop novel therapeutic strategies. For example, oligonucleotide–saccharide ligand conjugates have been generated to deliver genes or antisense oligonucleotides to hepatocytes [132–137]. A similar approach has been employed to direct drugs to the liver [138,139]. In addition, approaches that take advantage of the degradative events that result from hepatic lectin mediated endocytosis have been investigated as a means of targeting and clearing unwanted proteins and associated molecules from circulation [134,140,141]. The examples discussed below focus on the use of low molecular weight multidentate ligands to achieve selective targeting of associated conjugates to hepatocytes through the hepatic lectin. 1.

Using Synthetic Saccharide Determinants for Gene Delivery via Hepatic Lectin

Wu and coworkers conducted pioneering studies in which they used polylysine– asialoglycoprotein conjugates to deliver genes to hepatocytes through the hepatic lectin [132,138,142–145]. The nucleic acid component binds the polylysine portion through Coulombic interactions, and the glycoprotein moiety interacts with the hepatic lectin for delivery of the conjugate. The feasibility of using this receptor to achieve selective uptake of nucleic acids in vivo was demonstrated, and this result launched efforts to optimize this approach. Since obtaining homogeneous galactosesubstituted glycoproteins is difficult, other ligands for the hepatic lectin are under investigation as potential targeting agents. These include glycoprotein and glycopeptide conjugates [146,147] and liposomes [141,148–151]. Although most applications of the targeting strategy have employed macromolecular targeting agents, defined small multivalent displays have valuable properties as drug delivery agents. They are homogeneous, their structures can be more easily optimized, and they are not immunogenic. Nonnatural multivalent galactose derivatives can also function to target genes to cells displaying the hepatic lectin. This approach was illustrated by the studies of Plank et al., in which a tetrameric galactose derivative was linked to unsubstituted polylysine [152]. This conjugate was able to selectively target cell lines displaying the hepatic lectin. Another study assessed in vivo as well as in vitro delivery of the luciferase (Luc) gene. The high-affinity ligand YEE(ah-GlcNAc)3, 24, was used to target hepatocytes [137]. In a strategy that is similar to Wu’s design, Merwin and colleagues covalently linked the YEE(ah-GalNAc)3 cluster ligand to polylysine by conjugation through human serum albumin (Fig. 18). The Luc gene then associated with the conjugate through Coulombic interactions, 28 (Fig. 19). Expression of the Luc gene was observed both in vitro and in vivo. These results indicate the promise of these targeting strategies to improve the design of gene–saccharide conjugates that may result in therapeutically useful delivery agents. 2.

Selective Uptake by the Hepatic Lectin of an Antisense Oligonucleotide–Saccharide Epitope Conjugate

In analogy to the results obtained for targeting genes, it is expected that the endocytotic abilities of the hepatic lectin could also delivery oligonucleotides for antisense

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Figure 18 Covalently linked oligonucleotides could be delivered to hepatocytes via attachment to the GlcNAc cluster ligand YEE(ah-GalNAc)3 24.

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Figure 19

Construct 28 was used for luciferase gene delivery to hepatocytes by noncovalent association of the gene to GalNAc cluster 24.

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applications. Building on their most tissue-selective trivalent galactosamine cluster, Lee and coworkers covalently attached a radiolabeled oligonucleotide methylphosphonate analog to YEE(ah-GalNAc)3 via the heterobifunctional linkage SMCC (25) to generate (27), which was devised to examine cell targeting specificity (Fig. 18) [135]. These investigations revealed that the oligonucleotide analogs could be selectively delivered to hepatocytes. After a 24 h incubation, the conjugate associated preferentially with a cell line that displayed the hepatic lectin, HepG2, over those that did not. The observed selectivity in uptake was 260-fold over leukemic HL60 and 49-fold over fibrosarcoma HT1080 cells. The fate of the conjugate and its location within the cell were not investigated in this initial study. The results demonstrate selective targeting, but whether this strategy can be used to deliver an antisense agent to the proper cellular location remains to be determined. 3.

Cholesterol-Lowering Conjugates Designed to Function Through the Hepatic Lectin

In contrast to the approaches described above in which molecules designed to function within the liver are delivered through their interaction with the hepatic lectin, a strategy to use the hepatic lectin to eliminate unwanted species from serum has also been investigated. Specifically, Biessen et al. attempted to take advantage of hepatic lectin mediated endocytosis and subsequent ligand proteolysis to lower serum cholesterol levels [134]. It was anticipated that targeting the proteins responsible for serum cholesterol transport, high-density lipoprotein (HDL) and low-density lipoprotein (LDL), to hepatocytes via the hepatic lectin would lead to their degradation, thereby lowering serum cholesterol. Bifunctional ligand 29 was designed, in which cholesterol was linked to a trivalent galactose derivative (Fig. 20). When added to serum, compound 29 formed complexes with HDL and LDL. Intravenous injection of 29 into rats resulted in a decrease in detected serum cholesterol levels by 44%. This approach may prove useful for patients who do not respond to the commonly

Figure 20 LDL in rats.

The cholesterol-linked galactoside cluster 29 was used to lower serum HDL and

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employed treatment, which involves administering inhibitors of cholesterol biosynthesis. Moreover, this strategy may be generally useful to recruit serum proteins to specific sites in vivo. VI.

TARGETING INFLUENZA VIRUS

Many pathogens use protein–carbohydrate interactions to seek out host cells; influenza virus is one example [15,153]. Because influenza virus infection can cause serious illness and even death, the development of strategies to inhibit it is clearly significant. The virus infects its host, using the sialic acid binding lectin hemagglutinin (HA) to bind sialic acid residues on the cell surface. HA exists as a homotrimer that is densely displayed on the viral surface [15,18]. HA binds terminal sialic acid residues found on target cell glycoproteins and is involved in membrane fusion of the virus with the target cell [154,155]. Although structures of HA complexed with different sialic acid derivatives have been determined by X-ray crystallographic analysis [18,156], progress in identifying high-affinity, selective, monovalent inhibitors has been slow [52]. As an alternative to generating high-affinity monovalent inhibitors, researchers have investigated the ability of multivalent ligand arrays to inhibit influenza virus HA. A variety of different ligand classes have been investigated, ranging from low molecular weight ligands [157–161] to dendrimers [9,162–164] to liposomes and high molecular weight polymers [99–101,162,165–173]. The data amassed in the course of these studies illuminate the structural features important in multivalent ligand design and the mechanisms underlying multivalent ligand function. A.

Sialic Acid Substituted Polymers Are Highly Effective Inhibitors

The medical relevance of HA inhibitors has prompted several efforts to generate, characterize, and understand the recognition properties of sialic acid substituted polymers. In a multivalent display of epitopes convalently linked to a scaffold, receptor binding determinants are presented at a high effective concentration and often give rise to high functional affinities. Thus, ligand off-rates decrease because binding is favored by the multiplicity of recognition elements available in the vicinity of the binding site, a statistical binding enhancement. Linear polymeric displays also present recognition units in a variety of distances and orientations. This feature increases the probability that a multivalent ligand will interact favorably with a cell surface receptor through the chelate effect. Large polymeric displays can also benefit from steric stabilization. Moreover, with this range of variation, a multivalent ligand may simultaneously take advantage of several different multivalent binding modes. The following examples illustrate the functional properties of linear arrays. Several groups have generated sialic acid substituted acrylamide polymers and investigated the abilities of these materials to inhibit influenza virus HA [101,162, 166,170,174]. In these investigations, the mole fraction of N-acetylneuraminic acid (NeuAc) residues displayed by the different acrylamide polymers has been varied. The potency of the materials generated depends on the degree of substitution on the polymers. Maximal polymer activity was observed when approximately 10–20% of the units were substituted with NeuAc. Lower amounts of substitution resulted in decreased functional affinities, presumably because these materials were less likely

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to bind through the chelate effect and/or related entropically favorable mechanisms. When the mole fraction of NeuAc was higher than 0.2, the potency of the resulting materials typically decreased. This drop in activity may be due to the addition of binding epitopes that do not contribute to productive binding interactions. Moreover, the high charge density of a sialic acid bearing polymer may impede binding, in addition to saccharide substituents that could sterically impede binding. Thus, multivalent, high molecular weight polymers are extremely effective HA inhibitors, and their inhibitory activities are dependent on the degree of saccharide substitution. B.

Method of Polymer Synthesis Affects Activity

Typically, saccharide-substituted copolymers are synthesized through the reaction of a carbohydrate-substituted monomer along with an unsubstituted derivative; many variations have been described [16,175–181]. Alternatively, acrylamide polymers have been generated by postsynthetic modification, in which an electrophilic acrylic acid derivative is polymerized and saccharides are coupled to the resulting template [101,182,183]. The Whitesides group directly compared the activities of materials with identical compositions assembled by these different synthetic methods. First, they generated sialic acid substituted polymers by copolymerization. Reactions were conducted with a sialic acid functionalized acrylamide monomer and different acrylamide monomers displaying other functional groups such as amides, alcohols, glycerols, glucose residues, carboxylic acids, and amines (Fig. 21) [99,100,168–170]. Sialylated acrylamide monomer 30 was reacted with functionalized monomer 31 to generate a series of copolymers with varied steric bulk and backbone flexibility. The most active polymers in a viral hemagglutination assay were those generated by reaction of 30 with simple acrylamide 31 (R = NH2). They found that the materials exhibiting maximal potencies were those generated from the reaction of 0.2 mole fraction sialic acid monomer 30 and acrylamide. The optimized polymer was 6600fold more effective than monomeric sialic acid at inhibiting agglutination [168]. For comparison, polymers with activated esters, such as 36 were also made to explore the effect of postsynthetic modification on polymer activity (Fig. 22) [99]. When two polymers of the same composition with different synthetic histories were compared, the material produced by the postsynthetic modification route was 75-fold more potent. Because monomers differ in their rates of polymerization, the postsynthetic modification strategy can give rise to materials with broader distributions of epitope substitution patterns. A statistically random distribution of binding epitopes may be advantageous as the resulting materials have the capacity to interact with their receptors by a variety of mechanisms. C.

Polymers Act by Chelation and Steric Stabilization

To explore structure–function relationships and optimize their activities, NeuAcsubstituted acrylamide polymers were prepared with a variety of appended functional groups (Fig. 22) [99]. These studies generated polymers with very high inhibitory potencies toward HA. For example, an acrylamide copolymer substituted with 10% benzylamine and 20% NeuAc residues had a KI of 0.6 nM [100]. Both the chelate effect and steric stabilization were mechanisms suggested to contribute to the potent activity of this and related materials (Fig. 5). The NeuAc-substituted acrylamide

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Figure 21

Whitesides’s copolymerization of SA acrylamide 30 and various copolymerants 31 yielded mixed copolymers of altered carbohydrate density.

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Figure 22

Two routes to polyfunctional acrylamide polymers. (A) Formation of acrylamide polymers by reacting functionalized monomers 33, 34, and acrylamide. (B) Postpolymerization coupling of activated polyacrylate ester 36.

polymers inhibited the adhesion of polyclonal antivirus antibodies that do not bind HA, thus supporting the hypothesized role of steric stabilization [169]. Additional support was provided by the observation that the addition of monovalent neuraminidase inhibitors to a solution of virus-bound polymer increased the potency of the polymer [50]. Neuraminidase binds sialic acid residues but does not contribute measurably to agglutination; consequently, the addition of neuraminidase inhibitors could promote the release of polymer side chains involved in nonproductive neuraminidase binding. The result would be an increase in the effective volume of the polymer, which should increase the observed activity of the polyvalent ligand through steric stabilization.

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Hemagglutination Inhibition Data Fails to Correlate with Viral Infectivity Data

Liposomes are another class of multivalent ligands that have been used to inhibit HA [167,173,184]. One advantage of this form of multivalent ligand presentation is that glycolipids can diffuse laterally to adopt proper spacing for optimal binding. Therefore, the need to precisely engineer the distance required for maximal binding by the multivalent ligand is circumvented. In addition, the ligand density can be easily varied by adjusting the proportion of nonglycosylated lipid included in the liposomal preparation. To assess the ability of multivalent liposomal displays to inhibit viral hemagglutination, Whitesides et al. synthesized the sphingosine-inspired, sialic acid functionalized glycolipid 38 (Fig. 23A) [167]. The introduction of 38 into liposomes containing phosphatidylcholine and cholesterol created assemblies that were approximately 104-fold more effective than monovalent inhibitors, on a per-saccharideresidue basis, at inhibiting agglutination. The high inhibitory potency exhibited by this molecular assembly is likely the result of a combination of the chelate effect and steric inhibition as well as other mechanisms. The size of the liposomes may interfere with red blood cell binding because when bound to the virus, these assemblies occupy a large surface area. As observed with the acrylamide glycopolymers, liposomes too may garner added potency by obscuring free neighboring HA sites from interacting with cells. The relative inhibitory potencies of multivalent liposomal displays in a viral infectivity assay did not correspond to those determined in a hemagglutination assay. Bednarski and coworkers designed NeuAc displays composed of glycolipids possessing a dialkyne group within the lipid portion (Fig. 23B) [173]. The liposomal array could be covalently cross-linked by irradiation at 254 nm, which limits saccharide epitope reorganization within the mosaic. Glycolipid 39 was introduced into liposomes along with the corresponding lipid 40 at varying densities. Testing these cross-linked multivalent displays for their abilities to inhibit influenza virus in a hemagglutination assay and a viral plaque forming assay revealed different activity trends for the two assays. Liposomal preparations consisting of 10% glycolipid proved the most potent inhibitors of agglutination, exhibiting a 30,000-fold increase in activity over monovalent analogs. Liposomes containing 1% glycolipid did show some activity, albeit about 10-fold less than the 10% preparation, and they were unable to inhibit agglutination completely. Significantly, the preparation containing 1% glycolipid proved to be the most active at inhibiting viral attachment in the plaque assay. The two assays measure viral attachment to different cell types, red blood cells and MDCK cells, and they monitor two different functions, hemagglutination inhibition and cell lysis. The difference in potencies observed could be explained by differences in ligand presentation on the cells being studied, which affects both virus–cell recognition and the functional consequences of this process. E.

The Affinity of Bivalent Ligands Depends on Linker Length and Flexibility

Because HA is a protein bound to viral membrane, multivalent ligands can interact with a single trimer or by spanning neighboring trimers. Molecules that can place multiple sialic acid residues in binding sites within a trimer might be expected to

258 (A) Whitesides’s sphingosine-inspired glycolipid 38. (B) Bednarski’s cross-linkable liposomal constructs 39 and

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Figure 23 40.

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have more potent activities. Bivalent compounds designed to bind HA in this mode have been synthesized. Several compounds in which the NeuAc residues were connected by various linker units were assembled to explore the role of linker length and flexibility in HA binding [98,160]. Sabesan and coworkers investigated oligosaccharide templates to generate bivalent sialic acid presenting ligands that could occupy multiple sites within a single HA trimer [160]. Heptasaccharides 41–45 , differing only in the linkage pattern to the core galactosyl residue, were assembled using a chemoenzymatic synthesis (Fig. 24). Nuclear Overhauser effect measurements made by 1H NMR spectroscopy and Monte Carlo simulations were used to determine the distances separating the NeuAc residues within the bivalent ligands. Compounds 44 and 45 had the shortest NeuAc ˚ , and the NeuAc residues were spaced 17–19 separation of approximately 9–10 A ˚ A apart in the other analogs. The heptasaccharides were assayed for their ability to inhibit hemagglutination by influenza virus [60]. Modest increases in activity were observed; the most effective compounds in the series, the 3,6-linked (44) and 4,6galactose-linked (45) heptasaccharides, were 10- and 8-fold more potent than monovalent analogs, respectively, when analyzed per sialic acid residue. Given the distances separating the saccharide epitopes, it is unlikely that these compounds bind to different primary sialic acid binding sites within an HA trimer. They may, however, occupy both a primary and a secondary site on HA [18,185]. The importance in multivalent binding of distance spanned and relative ligand geometries observed

Figure 24 Sabesan’s heptasaccharide influenza virus hemagglutinin (HA) inhibitors present bivalent sialic acid linked through disubstituted galactose core.

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suggests that proper ligand spacing is important, a result echoed in other lectin– ligand interactions. F.

Bivalent Ligands Function by Binding Neighboring HA Trimers on the Viral Surface

Glick et al. used nonnatural linkers to synthesize a series of bivalent sialosides [98]. Three series of bivalent compounds were generated in which different linker groups were used to connect two NeuAc residues (Fig. 25). The linkers employed were based on ethylene glycol (50), glycine (52), and piperazine (51) and were used to ˚ , possessing varying degrees of assemble bivalent compounds spanning 27 to 65 A conformational flexibility. The binding of these molecules to free HA trimer was measured by an 1H NMR shift assay [186]. None of the compounds had activities greater than that of ␣-methyl NeuAc when tested against free HA trimer. Potency was also measured by inhibition of virus-mediated hemagglutination. The most effective bivalent ligand was derived from the glycine series and possessed a linker with four amino acid residues between each NeuA group and the aromatic ring, 53 (n = 4). This derivative was approximately 400-fold more effective than NeuAc at inhibiting cell agglutination when analyzed on a per-saccharide-residue basis. The best polyethylene-based inhibitor, 50 (n = 2, m = 1) was only eight-fold more potent than NeuAc. These inhibitors ˚ span between the sialic acid moieties. Interestingly, the piperapproximate a 57 A azine series did not yield any active inhibitors even though compound 44 (n = 2, m ˚ span. Conformational flexibility, which is influenced by = 1) also modeled a 57 A the structure of the linker, appears to have an important effect on multivalent HA binding. The distances required for optimal activities in this series are appropriate for occupying multiple binding sites within an HA trimer or for bridging multiple binding sites between HA trimers. Given that different results obtained when the inhibitor activity was compared against HA trimer in solution versus that against the intact virus, the active ligands do not bind to multiple sites within a single HA trimer. Rather, they appear to be capable of spanning trimers on the surface of the virus and/or crosslinking viral particles. This result is similar to that obtained with multivalent ligands for the hepatic lectin (see Section V.C). VII.

TARGETING THE SELECTINS

The selectins are a family of C-type lectins that play key roles in leukocyte recruitment into extravascular tissues during the inflammatory response [187–193]. Leukocytes routinely patrol the vascular endothelial cell walls scanning for sites of tissue damage or infection. When action is required, signals are sent that elicit cellular tethering, rolling, firm adhesion, and subsequent transmigration [194,195]. Selectins E-, L-, and P-mediate the initial leukocyte interaction with the endothelium and facilitate leukocyte rolling. L-selectin is found on leukocytes and recognizes glycoprotein ligands on endothelial cells; E- and P-selectin are found on endothelial cells and recognize glycoproteins on leukocytes. All three selectins weakly bind the tetrasaccharide determinant sialyl Lewis acid x (sLex), 54 (Fig. 26). Unchecked leukocyte extravasation is detrimental to the surrounding tissues and can result in inflammatory

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Figure 25 Bivalent sialic acid dimers target HA by varying linker length. (A) Route to polyethylene glycol and piperazine linked compounds. (B) Route incorporating glycine spacers.

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Figure 26

The sialyl Lewis acid x (sLex) determinant 54 is recognized by the selectins.

disorders. The possibility of modulating the inflammatory response at the initial stages has fueled efforts to generate inhibitors targeting this family of lectins. Several reviews highlight efforts to develop selectin inhibitors [17,196,197]. Our focus is on new applications of multivalent displays to target the selectins and inhibit their functions. A.

Multivalent Selectin Ligands

Although soluble forms of the selectins appear to be monomeric in solution, the selectins are transmembrane proteins and their state of clustering on the cell surface in vivo is unknown. Many of the physiological selectin ligands identified to date are glycoproteins that possess multiple copies of their saccharide epitopes [191,192,198,199]. For example, GlyCAM-1, the best-characterized glycoprotein ligand for L-lectin, presents multiple copies of sialylated and sulfated carbohydrate epitopes [200–203]. In addition, L-selectin is clustered on microvilli, suggesting that this family member is poised to interact through multipoint attachment [204,205]. A variety of multivalent ligand mimics that target the selectins have been synthesized to dissect the structural requirements for selectin binding and create high-affinity inhibitors. These structures range from large macromolecular structures to low molecular weight multivalent ligands [175,206–225]. 1.

Low Molecular Weight Multivalent Ligands Show Some Affinity Enhancement for the Selectins

Low molecular weight multivalent ligands have been designed to target the selectins, with most efforts focusing on E-selectin. Overall, these bi- or trisubstituted derivatives are slightly more effective than their monovalent counterparts in static binding assays, with typical affinity enhancements of three- to five-fold. For example, Defrees et al. used a chemoenzymatic approach to synthesize bivalent sialyl Lewis acid x derivatives in which the saccharides were linked by either a carbohydrate or other acyclic spacer [207]. The most effective inhibitor of E-selectin in a static cell adhesion assay (inhibition of HL-60 cells binding to immobilized E-selectin) was that in which two sLex epitopes were linked through the 3- and 6-positions of galactose, 55 (Fig. 27). This ligand was approximately five-fold more potent than monovalent sLex. Ligands in which the sLex groups were tethered with more flexible polyethylene

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Bivalent sLex ligands generated by Wong. The selectin epitopes were linked through the 3- and 6-positions of galactose (55) and via a polyethylene glycol chain (56).

Figure 27

glycol spacers exhibited similar activities in a related assay; the best ligand, 56, was approximately five-fold more active than monovalent sLex (Fig. 27) [226]. Other bivalent derivatives with flexible linking groups afforded similar results [221]. Trivalent sLex derivatives reported by Kretzschmar and coworkers afforded similar enhancements in affinity (Fig. 28). Molecules displaying sLex epitopes on glycopeptide backbones exhibited higher activities than did those possessing more flexible scaf-

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Kretzchmar’s trivalent sLex conjugate.

folding. For example, trivalent glycopeptides were up to 10-fold more active than their monovalent counterparts [209,216,223]. Bivalent ligands displaying nonnatural residues, mannose biphenyl glycosides, were devised by Kogan et al. [225] to mimic di-sLex, an oligosaccharide that appears to bind more tightly to E-selectin than sLex. Compounds in a series with variable spacer residues connecting the mannose moieties were tested against all three selectins in a static cell binding assay. The most active ligand, 58, demonstrated increased potencies (ca. sixfold for E- and L-selectin) consistent with binding within a single lectin domain (Figure 29) [225]. Trivalent ligands in this series were no more potent than their bivalent counterparts. The modest inhibitory potency increases observed with the small multivalent ligands explored to date are likely due to secondary interactions near the binding site.

Figure 29

Kogan’s biphenyl-based sLex mimic 58, made bivalent through an alkyl linker.

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Attempts to target L-selectin with large multivalent oligosaccharides have resulted in the identification of ligands with higher functional affinities. Renkonen and colleagues have prepared bi- and tetravalent sLex ligands in which the selectin epitope is linked to N-acetyllactosamine and polylactosamine through the 3- and 6positions of galactose (Fig. 30) [215,217]. Whereas bivalent ligand 59 demonstrated modest inhibitory potency increases, approximately twofold, against L-selectin-mediated adhesion of lymphocytes to endothelial cells, the tetravalent sLex analog, 60, had increased potency, of approximately 50- to 100-fold [217]. The tetravalent sLex derivative, composed of 22 saccharide residues, may be able to span the distance required to bind to multiple L-selectin receptors on the cell surface. Alternatively, the tetravalent ligand may be able to occupy distal binding sites within a single Lselectin molecule. Nonfucosylated derivatives did not inhibit binding, suggesting that inhibition is dependent on the presence of the sLex epitope. 2.

Multivalent Ligands Are Effective Selectin Inhibitors

Although low molecular weight displays have not exhibited high functional affinities for the selectins, larger multivalent ligands are more effective inhibitors. For exam-

Renkonen’s bi- and tetravalent sLex displays 59 and 60, made using chemoenzymatic synthesis.

Figure 30

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ple, bovine serum albumin (BSA) conjugates bearing 16 sLex/BSA surpassed monovalent sLex at inhibiting HL-60 cells. They bound to immobilized E-selectin with reported IC50 values of 1 ␮M (16 ␮M saccharide residue concentration) versus 1 mM for sLex [208]. The BSA conjugate also inhibited neutrophil rolling on human umbilical vein endothelial cells (HUVECs) more effectively than sLEx (an IC50 value of 16 ␮M vs. ⬃0.3 mM). Similarly, a polyacrylamide substituted with 3⬘-sulfoLewisx epitopes inhibited the binding of an L-selectin–IgG chimera to immobilized 3⬘-sulfo-Lewisx with IC50 values around 1 ␮M (vs. >1 mM for sLex) [211,212,224]. ROMP generated polymers bearing the simple sulfated saccharide derivatives 3sulfo- and 3,6-disulfogalactose were effective inhibitors of E-, P-, and L-selectin in a cell binding assay. Significantly, the 3,6-disulfogalactose-substituted derivative specifically targeted P-selectin. The IC50 of the polymer was 84 ␮M, as determined on a per-saccharide-residue basis, while the IC50 of sLex was 3.4 mM [227]. Large liposomal arrays are also highly effective inhibitors of the selectins. For instance, multivalent liposomes bearing sLex inhibited binding of HL-60 cells to immobilized E-selectin with potencies 750-fold higher than monovalent analogs [228]. Other liposomal sLex displays with enhanced affinities also have been reported [210,223,229]. These higher order multivalent displays can act by several mechanisms including multisite binding and statistical and steric effects. Factors governing specificity and affinity for the lectins discussed earlier are also relevant in these examples. B.

Inhibitory Potencies of Multivalent Ligands for the Selectins Are Influenced by Assay Method

It is difficult to develop an appropriate assay for all potential selectin ligands because multivalent ligands function by means of a very broad range of mechanisms. An added complication is that the selectins function under conditions of shear flow. Conducting high-throughput assays under conditions of flow is complex, and therefore the activities of potential ligands are generally evaluated under static conditions. A widely employed method involves monitoring the abilities of molecules to inhibit cell binding to an immobilized selectin (Fig. 8). This assay is convenient and can lead to the identification of potential lead structures; however, the cell surface presentation of the selectins may be quite important for their function [205,230–233]. Moreover, the static assay conditions may not reproduce ligand activities that might be observable under flow conditions [95]. 1.

The Presentation of the Receptor Is Important for Assessing Multivalent Ligand Activity

The importance of selectin presentation was illustrated in experiments conducted by Stahn et al., who compared the E-selectin inhibitory activity of BBA2, an anti-Eselectin monoclonal antibody, to that of sLex-decorated liposomes in two different static assays [223]. When the agents were evaluated for inhibition of HepG2 cell binding to E-selectin immobilized in a microtiter well, the antibody and the liposomes had similar activities. The antibody, however, was approximately 100-fold more active than the liposomal preparation at inhibiting binding of HepG2 cells to activated HUVEC cells expressing E-selectin. These results demonstrate the need to examine multiple assays and to critically evaluate the data obtained.

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Static Assays Afford Results Different from Those Carried Out Under Dynamic Conditions

Many biological processes take place under dynamic rather than static conditions. For example, the ability of the selectins to mediate the dynamic process of leukocyte rolling is a critical aspect of their function; yet most selectin inhibitors have been evaluated in static assays. A study evaluating multivalent L-selectin inhibitors revealed dramatic differences in their activities in static cell free versus dynamic cell rolling assays. To explore the recognition of multivalent ligands by L-selectin, the ring-opening metathesis polymerization method (ROMP) was used to generate materials presenting Lex epitopes differing in sulfation pattern [95]. These compounds were designed to mimic the glycoprotein mucins that bind L-selectin, such as GlyCAM-1, by displaying multiple copies of sulfated saccharide epitopes on an extended polypeptide backbone. Oligomers bearing 3⬘,6⬘-disulfo Lewis x(Glc) (61) and 3⬘,6-disulfo-Lewis x(Glc) (62) were more potent than their monovalent counterparts, 63 and 64, in assays to determine their ability to inhibit an L-selectin–IgG fusion protein binding to immobilized heparin (Fig. 31). However, their abilities to inhibit the rolling of L-selectin-transfected cells on GlyCAM-1 were very different. Only the 3⬘,6disulfo-Lewis x(Glc) polymer 62 (Fig. 31) was able to prevent rolling, and it did so with high efficacy (IC50 = 1 ␮M; 15 ␮M based on saccharide residue concentration). These results suggest that fluid flow may play an important role in the specificity of selectin–ligand interactions. Intriguingly, multivalent ligand 62 may bind cell surface L-selectin through multipoint attachment. Thus, multivalent synthetic ligands along with their natural counterparts may possess the ability to cluster L-selectin on the cell surface (Fig. 5C). 3.

Synthetic, Multivalent Ligands Designed to Induce L-Selectin Shedding

The release of a cell surface protein, often termed shedding, can serve to regulate its density on the cell surface and also affects the concentration of the soluble form in circulation [234]. L-selectin shedding is believed to occur during the inflammatory response, and this process may be involved in regulating cell rolling rates [187]. We hypothesized that multivalent ligand binding may cluster L-selectin and thereby trigger shedding. To test this idea, the ability of the 3⬘,6⬘-disulfo-Lewis x polymer 62 to cause L-selectin release from the cell surface was evaluated [41]. The amount of L-selectin on the cell surface of neutrophils after treatment with ligand was assessed by flow cytometry using the anti-L-selectin antibody, DREG56. Although the monovalent compound 64 had no effect on L-selectin levels, exposure of neutrophils to polymeric compound 62 led to a reduction in the amount of cell surface L-selectin. The ability of multivalent ligand 62 to induce L-selectin shedding suggests that natural ligands might function similarly. The demonstration that synthetic ligands can be designed to promote the shedding of a cell surface receptor has implications for understanding the mechanisms cells use to control protein display and for manipulating the concentration of cell surface receptors. The ability to trigger this response by exposure to a synthetic multivalent ligand also suggests a new therapeutic strategy for targeting the inflammatory response.

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Figure 31 Kiessling’s disulfated Lex constructs 63 and 64 elicited different physiological effects when appended on multivalent displays to afford 61 and 62.

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SUMMARY

Multivalent protein–carbohydrate interactions are prevalent in biological recognition processes. Despite their important roles, an understanding of these interactions is only beginning to emerge. This understanding is facilitated by new developments in the chemical synthesis of multivalent ligands and the use of those ligands to explore and manipulate multivalent recognition processes. Applications for such ligands are widespread, ranging from the investigation of cellular recognition to agents for tissue-selective drug delivery to molecules that can be used to evoke cellular responses. With a growing appreciation of the importance of multivalent interactions, these systems offer intriguing opportunities and challenges for chemistry and biology.

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