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## **Meet the editor**

Dr. Chuan-Fa Chang started his research work in Glycobiology for his doctorate at Institute of Biological Chemistry, Academia Sinica. He developed new methodologies for carbohydrate organic synthesis and accomplished series of carbohydrate analogs by using exo-glycals as building blocks. He then joined Dr. Chi-Huey Wong's chemical biology lab as a postdoctoral

fellow and worked on discovering glycosidase inhibitors by high-throughput drug screening systems. In 2007, Dr. Chang moved to National Cheng Kung University and initiated his independent research career. He is also the member of Society of Glycobiology (since 2004) and participating investigator of Consortium for Functional Glycomics (CFG, since 2011). Recently, Dr. Chang is interested in investigating the roles of carbohydrates in infectious disease and the regulation of glycosylation in tumorigenesis and cancer metastasis.

Contents

**Preface IX**

**Section 1 Chemistry and Biochemistry 1**

Aurore Richel and Michel Paquot

Chapter 3 **Boron-Carbohydrate Interactions 37**

Chapter 2 **Conversion of Carbohydrates Under Microwave Heating 21**

Brighid Pappin, Milton J. Kiefel and Todd A. Houston

**Mass Spectrometry of Carbohydrates Derivatized**

**A Paradigm of C-Type Lectin Binding Inhibition 137**

**with Biotinamidocaproyl Hydrazide 55**

**Derivatives as Crystallization Modifiers 81**

Stephanie Bank and Petra Kapková

Chapter 6 **Glycosidases – A Mechanistic Overview 117**  Natércia F. Brás , Pedro A. Fernandes, Maria J. Ramos and Nuno M.F.S.A. Cerqueira

Chapter 8 **Broad Antiviral Activity of Carbohydrate-Binding**

Marijke Alen and Dominique Schols

**Agents Against Dengue Virus Infection 161**

Chapter 5 **Investigation of Carbohydrates and Their**

Josef Jampílek and Jiří Dohnal

**Section 2 Microbiology and Immunology 135**

Chapter 7 **DC-SIGN Antagonists –**

Marko Anderluh

Chapter 1 **Carbohydrate Microarray 3**  Chuan-Fa Chang

Chapter 4 **Liquid Chromatography –**

## Contents

### **Preface XIII**

Chapter 8 **Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection 161**  Marijke Alen and Dominique Schols

Contents VII

**Section 5 Biotechnology 439**

Chapter 20 **Carbohydrates from Biomass: Sources**

Chapter 21 **Starch and Microbial α-Amylases:**

Stefan Kraan

Chapter 23 **Biological Applications**

Luis Henrique Souza Guimarães

Ahmed El-Sayed and Noha Omar

Amira El-Fallal, Mohammed Abou Dobara,

**and Transformation by Microbial Enzymes 441**

Chapter 22 **Algal Polysaccharides, Novel Applications and Outlook 489**

**of Plants and Algae Lectins: An Overview 533** Edson Holanda Teixeira, Francisco Vassiliepe Sousa Arruda, Kyria Santiago do Nascimento, Victor Alves Carneiro,

Alexandre Holanda Sampaio and Benildo Sousa Cavada

Celso Shiniti Nagano, Bruno Rocha da Silva,

**From Concepts to Biotechnological Applications 459**

#### **Section 5 Biotechnology 439**

VI Contents

Chapter 9 **IL-13, Asthma and Glycosylation**

Chapter 11 **Digestion in Ruminants 245**  Barbara Niwińska

**Section 3 Carbohydeate Metabolism 259**

Chapter 12 **Resistant Dextrins as Prebiotic 261**

Chapter 14 **Carbohydrate Metabolism in Drosophila:**

and Juan Rafael Riesgo-Escovar

Anna Novials and Serafín Murillo

Chapter 17 **Environmental and Genetic Variation for**

Chapter 18 **Alterations in Root Morphology of Rootstock**

and Jorge Ernesto de Araújo Mariath

Chapter 19 **Plant Responses to Sugar Starvation 409** Iwona Morkunas, Sławomir Borek, Magda Formela and Lech Ratajczak

Monia El Bour

**Section 4 Animal and Plant 371**

Ali Ashraf Jafari

Chapter 16 **Adapting the Consumption of**

Chapter 15 **Starvation Conditions Effects on Carbohydrate Metabolism of Marine Bacteria 339**

**in Airway Epithelial Repair 187**

Chapter 10 **Metabolism of Carbochidrates in the Cell of Green Photosintesis Sulfur Bacteria 229**  M. B. Gorishniy and S. P. Gudz

> Katarzyna Śliżewska, Janusz Kapuśniak, Renata Barczyńska and Kamila Jochym

Chapter 13 **Food Structure and Carbohydrate Digestibility 289**  Suman Mishra, Allan Hardacre and John Monro

> **Reliance on the Disaccharide Trehalose 317** Alejandro Reyes-DelaTorre, María Teresa Peña-Rangel

**Carbohydrates for Diabetic Athletes 355**

**Water Soluble Carbohydrate Content in Cool Season Forage Grasses 373**

**Peach Trees Caused by Mycorrhizal Fungi 389** José Luis da Silva Nunes, Paulo Vitor Dutra de Souza, Gilmar Arduino Bettio Marodin, José Carlos Fachinello

Samuel J. Wadsworth, S. Jasemine Yang and Delbert R. Dorscheid

#### Chapter 23 **Biological Applications of Plants and Algae Lectins: An Overview 533**  Edson Holanda Teixeira, Francisco Vassiliepe Sousa Arruda, Kyria Santiago do Nascimento, Victor Alves Carneiro, Celso Shiniti Nagano, Bruno Rocha da Silva, Alexandre Holanda Sampaio and Benildo Sousa Cavada

Preface

in Glycobiology.

Carbohydrates consist of carbon, oxygen, hydrogen, and other important modification atoms. They are the major component of plants and comprise more than 40% of energy in human diets. In addition to be the basic energy source and biomaterial building blocks, carbohydrates are also involved in many physiological and pathological events. Because the carbon atom has four valence electrons, it can form chemical bonds with four different atoms. As a consequence, much of constitutional isomers and stereoisomers are found in carbohydrates. In addition, the cyclization of a linear carbohydrate to form five, six or seven membered hemiacetal/hemiketal ring also increases the complexity of glycan structures. Since the late nineteenth century, many chemists devoted their life to figure out these chemical and biochemical questions. The slow development of analytical methods and instruments has prevented the biological roles of carbohydrates from being investigated and characterized until the 1970s. Due to the difficulties of getting a lot of pure and homogeneous carbohydrates from natural sources, carbohydrate organic synthesis has become an essential and flourish subject

Because carbohydrates are involved in multiple disciplines, this book consists of 23 excellent chapters, classified into five catogories - Chemistry and Biochemistry, Microbiology and Immunology, Carbohydrate metabolism, Animal and Plant, and Biotechnology. In section one, organic reactions of carbohydrates and the analysis of carbohydrate derivatives are discussed. A high-throughputable carbohydrate microarray without any wash steps is also introduced. This reliable platform not only offers consistent results with literature - it can be applied for the determination of pathogen-carbohydrate interactions. The importance of glycans in infectious disease has been well studied, as well as the immune response and signal transduction pathway. Chapters in section 2 introduce the studies of DC-SIGN antagonists and IL-13 in a viral infection, and the biosynthesis of carbohydrates in a microorganism. The metabolism of carbohydrates in bacteria and drosophila is collected in section 3. This section also discusses the effects of starvation in carbohydrate metabolism in marine bacteria. Section 4 contains some non-popular topics about the digestion of carbohydrates in ruminants, the influences of environment and fungi in plant carbohydrates, and the responses of plants under sugar starvation. The observation, discovery and results of these chapters are specialized and unique. This book is also an

## Preface

Carbohydrates consist of carbon, oxygen, hydrogen, and other important modification atoms. They are the major component of plants and comprise more than 40% of energy in human diets. In addition to be the basic energy source and biomaterial building blocks, carbohydrates are also involved in many physiological and pathological events. Because the carbon atom has four valence electrons, it can form chemical bonds with four different atoms. As a consequence, much of constitutional isomers and stereoisomers are found in carbohydrates. In addition, the cyclization of a linear carbohydrate to form five, six or seven membered hemiacetal/hemiketal ring also increases the complexity of glycan structures. Since the late nineteenth century, many chemists devoted their life to figure out these chemical and biochemical questions. The slow development of analytical methods and instruments has prevented the biological roles of carbohydrates from being investigated and characterized until the 1970s. Due to the difficulties of getting a lot of pure and homogeneous carbohydrates from natural sources, carbohydrate organic synthesis has become an essential and flourish subject in Glycobiology.

Because carbohydrates are involved in multiple disciplines, this book consists of 23 excellent chapters, classified into five catogories - Chemistry and Biochemistry, Microbiology and Immunology, Carbohydrate metabolism, Animal and Plant, and Biotechnology. In section one, organic reactions of carbohydrates and the analysis of carbohydrate derivatives are discussed. A high-throughputable carbohydrate microarray without any wash steps is also introduced. This reliable platform not only offers consistent results with literature - it can be applied for the determination of pathogen-carbohydrate interactions. The importance of glycans in infectious disease has been well studied, as well as the immune response and signal transduction pathway. Chapters in section 2 introduce the studies of DC-SIGN antagonists and IL-13 in a viral infection, and the biosynthesis of carbohydrates in a microorganism. The metabolism of carbohydrates in bacteria and drosophila is collected in section 3. This section also discusses the effects of starvation in carbohydrate metabolism in marine bacteria. Section 4 contains some non-popular topics about the digestion of carbohydrates in ruminants, the influences of environment and fungi in plant carbohydrates, and the responses of plants under sugar starvation. The observation, discovery and results of these chapters are specialized and unique. This book is also an

#### XIV Preface

encouragement to people who work in the non-mainstream investigations. The final section is the technology and application of carbohydrates in microbes. They can be used as prebiotics, to form biomass, as well as healthy supplements.

I hope this comprehensive book can serve well as an uncomplicated introduction to different disciplines of carbohydrate investigators and glycobiologists. Consulting the literature cited in the chapters is suggested for readers who need further information. The corrections, improvements and suggestions from readers for all the chapters are welcome and appreciated.

I deeply appreciate the contributors to this book and the assistance from InTech in publishing this book.

**Chuan-Fa Chang** Department of Medical Laboratory, Science and Biotechnology, Medical College, National Cheng Kung University, Tainan city, Taiwan

**Section 1**

**Chemistry and Biochemistry**

**Chapter 1**

© 2012 Chang, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Chang, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Glycosylation adorns more than one half of the proteins in eukaryotic cells [1,2]. This post-translational modification plays an indispensible role in many important biological events, especially on cell surface [1,3]. Alterations in carbohydrate structures are known to correlate with the changes in protein stability and clearance, as well as various physiological functions including cell-cell adhesion, inflammation, tumor metastasis, and infection of bacteria and viruses [4-8]. Although glycosylation is essential for the formation and progression of various diseases, study of this subject is hampered by lack of effective tools available to date, in addition to structural heterogeneity and complexity of carbohydrates. A number of techniques have been developed to analyze the binding interactions between carbohydrates and proteins [2,9]. For instances, lectin blotting/binding assay has become a routine method to determine the glycan-protein interactions [10], but the relatively low sensitivity and the necessity of multiple wash steps/time-consuming have restricted the sensitivity and application. Surface plasmon resonance is another highly sensitive method which monitors the interactions in real time and in a quantitative manner [11-16]. However, sometimes the sensitivity is relatively low toward the use of low molecular-weight carbohydrates, though the problem can be overcome by labeling sugars with heavy metal ions [17]. In addition, fluorescence polarization and two-photon fluorescence correlation have been applied to study lectin-glycan interactions [18-21]. The most applicable technique is carbohydrate microarrays which immobilize oligosaccharides to a solid supports are developed and widely used to measure the carbohydrate binding properties of proteins, cells, or viruses [22-25]. For example, a high-content glycan microarray is developed by a robotic microarray printing technology in which amine-functionalized glycans are coupled to the succinimide esters on glass slides [26,27]. These microarrays have also been subjected for profiling the carbohydrate binding specificities of lectins, antibodies, and intact

**Carbohydrate Microarray**

Additional information is available at the end of the chapter

Chuan-Fa Chang

**1. Introduction**

viruses.

http://dx.doi.org/10.5772/51971

## **Chapter 1**

## **Carbohydrate Microarray**

## Chuan-Fa Chang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51971

## **1. Introduction**

Glycosylation adorns more than one half of the proteins in eukaryotic cells [1,2]. This post-translational modification plays an indispensible role in many important biological events, especially on cell surface [1,3]. Alterations in carbohydrate structures are known to correlate with the changes in protein stability and clearance, as well as various physiological functions including cell-cell adhesion, inflammation, tumor metastasis, and infection of bacteria and viruses [4-8]. Although glycosylation is essential for the formation and progression of various diseases, study of this subject is hampered by lack of effective tools available to date, in addition to structural heterogeneity and complexity of carbohydrates. A number of techniques have been developed to analyze the binding interactions between carbohydrates and proteins [2,9]. For instances, lectin blotting/binding assay has become a routine method to determine the glycan-protein interactions [10], but the relatively low sensitivity and the necessity of multiple wash steps/time-consuming have restricted the sensitivity and application. Surface plasmon resonance is another highly sensitive method which monitors the interactions in real time and in a quantitative manner [11-16]. However, sometimes the sensitivity is relatively low toward the use of low molecular-weight carbohydrates, though the problem can be overcome by labeling sugars with heavy metal ions [17]. In addition, fluorescence polarization and two-photon fluorescence correlation have been applied to study lectin-glycan interactions [18-21]. The most applicable technique is carbohydrate microarrays which immobilize oligosaccharides to a solid supports are developed and widely used to measure the carbohydrate binding properties of proteins, cells, or viruses [22-25]. For example, a high-content glycan microarray is developed by a robotic microarray printing technology in which amine-functionalized glycans are coupled to the succinimide esters on glass slides [26,27]. These microarrays have also been subjected for profiling the carbohydrate binding specificities of lectins, antibodies, and intact viruses.

© 2012 Chang, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Chang, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Carbohydrate microarray**

In our recent work, we have developed two novel carbohydrate microarrays: solution microarray [28] and membrane microarray [29]. Carbohydrate solution assay is a highthroughput, homogenous and sensitive method to characterize protein-carbohydrate interactions and glycostructures by in-solution proximity binding with photosensitizers (**Figure 1**). The technology, also called AlphaScreenTM, is first described by Ullman et al., and has been used to study interactions between biomolecules [30-34]. In these assays, a light signal is generated when a donor bead and an acceptor bead are brought into proximity. This method usually provides good sensitivity with *femto*-mole detection under optimized conditions, relying on the binding affinity between analytes. All the procedures are carried out in 384-welled microtiter plates, thus qualifying the protocol as high-throughput. Two particles of 200 nm are involved in this technology including streptavidin-coated particles (donor beads) and protein A-conjugated particles (acceptor beads). Biotinylated polyacrylamide (biotin-PAA)-based glycans that are immobilized on donor beads can be recognized by lectins or antibodies, and connected with acceptor beads through specific antibodies (**Figure 1**). A number of carbohydrate binding proteins, including eleven lectins and seven antibodies, are profiled for their carbohydrate binding specificity to validate the efficacy of this developed technology. This assay is performed in homogeneous solutions and does not require extra wash steps, preventing the loss of weak bindings that often occur in the repeating washes of glycan microarray. However, antigen/ligand excess effect may happen in the homogeneous solution assay if the concentrations of carbohydrate epitopes, proteins, or antibodies are too high. One mg of biotin-PAA-sugar can be applied for fifty thousand assays because minimal amount of materials are needed in this microarray system (a range of nano-gram is required per well). Although the detection limit of biotin-PAAsugar is good (2 ng per well), the linear range is too narrow for quantitative application.

Carbohydrate Microarray 5

Carbohydrate membrane microarray is fabricated by immobilization of the biotinconjugated PAA-based glycans on aldehyde-functionalized UltraBind via streptavidin. Streptavidin interacted strongly with biotin and formed covalent linkage with membranes after reductive amination, which prevented the loss of glycans from membrane during repeated wash steps. The use of PAA also avoided the nonspecific interactions that take place in other studies between some lectins and non-glycosylated proteins (e.g. HAS or BSA) [35]. The operation of this carbohydrate membrane microarray is similar to that of Western blotting and can be performed easily by anyone without prior intensive training.

**3.1. Carbohydrate binding profiles of lectins and antibodies (solution**

Fifty-four biotinylated polyacrylamide backboned glycans (biotin-PAA-glycans) (**Table 1**) are collected in total to examine fifteen carbohydrate-binding proteins, including eight lectins (Con A, DBA, GS-I, PNA, SBA, UEA-1, WFA and WGA), and six antibodies (anti-Lea, Leb, Lex, Ley, sialyl Lea and sialyl Lex). The resulting signals are indicated with bars as relative intensities (**Figures 2 and 3**). The natural carbohydrate ligands for these lectins are listed in **Table 2**. All of the lectins showed nearly the same carbohydrate binding preferences as those in literatures. For example, concanavalin A (Con A) bound preferentially to mannose (No. 3) and biantennary *N*-glycan (No. 53), and very weakly to 3- and 6-sulfated galactosides (No. 19, 23 and 25). DBA, a GalNAc-binding lectin, recognized GalNAcα1-3Gal-containing epitopes (No. 11 and 39). ECA interacted with LacNAc disaccharide, Galβ1-4(6-sulfo)GlcNAc, and Galβ1-4(α1-2Fuc)GlcNAc (No. 17, 24, 31 and 47), and weakly bound to Lec (Galβ1-3GlcNAc, No. 20). GS-I preferred interacting with Gal/GalNAc that contains α1-3 or 1-4 linkage (No. 11, 13, 14, 16, 40 and 42). MAA, in this study, recognized mainly to 3'-sulfated Galβ1-3GlcNAc, 3'-sulfated Galβ1-4GlcNAc and LacNAc and weakly to 3-sialylated galactosides (No. 26, 37 and 53). PNA interacted with Galβ1-3GalNAc (No. 15) and bound to some galactosides weakly (No. 12, 16, 20, 45 and 46). SBA preferentially interacted with α-linked galactosides (No. 16 and 42) and *N*acetylgalactosaminoside (No. 11). SNA, a well-known α2-6 sialoside-binding lectin, interacted strongly to 6'-sialyl lactose and sialylated diantennary *N*-glycan (No. 36 and 53). UEA-1 specifically bound with Fucα1-2Gal-containing glycans (No. 18, 31 and 49). Due to weak interaction with PAA, WFA is the only one lectin showing higher background signals than the others. It recognized nearly half of the glycans on the glycan library, such as GlcNAc- and NeuAcα2-3-Gal/NeuAcα2-6-Gal containing saccharides. WGA also bound to terminal Gal or GalNAc epitopes (GalNAcα1-3Gal, No. 11 and Galβ1- 4(6HSO3)GlcNAc, No. 24) according to some minor signals. Interestingly, WGA showed better interactions with chitotriose than with chitobiose and GlcNAc. In addition, the binding specificities of monoclonal anti-carbohydrate antibodies also revealed some interesting features. As shown in **Figure 3**, anti-Lea antibody bound tightly with Lex, but less with Leb and sialyl Lea. Anti-Leb antibody represented specificities for both Leb and

**3. Applications**

**microarray)**

**Figure 1.** In-solution proximity binding with photosensitizers which was developed to characterize the protein-carbohydrate interactions [28].

Carbohydrate membrane microarray is fabricated by immobilization of the biotinconjugated PAA-based glycans on aldehyde-functionalized UltraBind via streptavidin. Streptavidin interacted strongly with biotin and formed covalent linkage with membranes after reductive amination, which prevented the loss of glycans from membrane during repeated wash steps. The use of PAA also avoided the nonspecific interactions that take place in other studies between some lectins and non-glycosylated proteins (e.g. HAS or BSA) [35]. The operation of this carbohydrate membrane microarray is similar to that of Western blotting and can be performed easily by anyone without prior intensive training.

## **3. Applications**

4 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

In our recent work, we have developed two novel carbohydrate microarrays: solution microarray [28] and membrane microarray [29]. Carbohydrate solution assay is a highthroughput, homogenous and sensitive method to characterize protein-carbohydrate interactions and glycostructures by in-solution proximity binding with photosensitizers (**Figure 1**). The technology, also called AlphaScreenTM, is first described by Ullman et al., and has been used to study interactions between biomolecules [30-34]. In these assays, a light signal is generated when a donor bead and an acceptor bead are brought into proximity. This method usually provides good sensitivity with *femto*-mole detection under optimized conditions, relying on the binding affinity between analytes. All the procedures are carried out in 384-welled microtiter plates, thus qualifying the protocol as high-throughput. Two particles of 200 nm are involved in this technology including streptavidin-coated particles (donor beads) and protein A-conjugated particles (acceptor beads). Biotinylated polyacrylamide (biotin-PAA)-based glycans that are immobilized on donor beads can be recognized by lectins or antibodies, and connected with acceptor beads through specific antibodies (**Figure 1**). A number of carbohydrate binding proteins, including eleven lectins and seven antibodies, are profiled for their carbohydrate binding specificity to validate the efficacy of this developed technology. This assay is performed in homogeneous solutions and does not require extra wash steps, preventing the loss of weak bindings that often occur in the repeating washes of glycan microarray. However, antigen/ligand excess effect may happen in the homogeneous solution assay if the concentrations of carbohydrate epitopes, proteins, or antibodies are too high. One mg of biotin-PAA-sugar can be applied for fifty thousand assays because minimal amount of materials are needed in this microarray system (a range of nano-gram is required per well). Although the detection limit of biotin-PAAsugar is good (2 ng per well), the linear range is too narrow for quantitative application.

**Figure 1.** In-solution proximity binding with photosensitizers which was developed to characterize the

**2. Carbohydrate microarray**

protein-carbohydrate interactions [28].

## **3.1. Carbohydrate binding profiles of lectins and antibodies (solution microarray)**

Fifty-four biotinylated polyacrylamide backboned glycans (biotin-PAA-glycans) (**Table 1**) are collected in total to examine fifteen carbohydrate-binding proteins, including eight lectins (Con A, DBA, GS-I, PNA, SBA, UEA-1, WFA and WGA), and six antibodies (anti-Lea, Leb, Lex, Ley, sialyl Lea and sialyl Lex). The resulting signals are indicated with bars as relative intensities (**Figures 2 and 3**). The natural carbohydrate ligands for these lectins are listed in **Table 2**. All of the lectins showed nearly the same carbohydrate binding preferences as those in literatures. For example, concanavalin A (Con A) bound preferentially to mannose (No. 3) and biantennary *N*-glycan (No. 53), and very weakly to 3- and 6-sulfated galactosides (No. 19, 23 and 25). DBA, a GalNAc-binding lectin, recognized GalNAcα1-3Gal-containing epitopes (No. 11 and 39). ECA interacted with LacNAc disaccharide, Galβ1-4(6-sulfo)GlcNAc, and Galβ1-4(α1-2Fuc)GlcNAc (No. 17, 24, 31 and 47), and weakly bound to Lec (Galβ1-3GlcNAc, No. 20). GS-I preferred interacting with Gal/GalNAc that contains α1-3 or 1-4 linkage (No. 11, 13, 14, 16, 40 and 42). MAA, in this study, recognized mainly to 3'-sulfated Galβ1-3GlcNAc, 3'-sulfated Galβ1-4GlcNAc and LacNAc and weakly to 3-sialylated galactosides (No. 26, 37 and 53). PNA interacted with Galβ1-3GalNAc (No. 15) and bound to some galactosides weakly (No. 12, 16, 20, 45 and 46). SBA preferentially interacted with α-linked galactosides (No. 16 and 42) and *N*acetylgalactosaminoside (No. 11). SNA, a well-known α2-6 sialoside-binding lectin, interacted strongly to 6'-sialyl lactose and sialylated diantennary *N*-glycan (No. 36 and 53). UEA-1 specifically bound with Fucα1-2Gal-containing glycans (No. 18, 31 and 49). Due to weak interaction with PAA, WFA is the only one lectin showing higher background signals than the others. It recognized nearly half of the glycans on the glycan library, such as GlcNAc- and NeuAcα2-3-Gal/NeuAcα2-6-Gal containing saccharides. WGA also bound to terminal Gal or GalNAc epitopes (GalNAcα1-3Gal, No. 11 and Galβ1- 4(6HSO3)GlcNAc, No. 24) according to some minor signals. Interestingly, WGA showed better interactions with chitotriose than with chitobiose and GlcNAc. In addition, the binding specificities of monoclonal anti-carbohydrate antibodies also revealed some interesting features. As shown in **Figure 3**, anti-Lea antibody bound tightly with Lex, but less with Leb and sialyl Lea. Anti-Leb antibody represented specificities for both Leb and

Lea, but less for Lex and sialyl Lex. Anti-Ley antibody not only binding to Ley, but also recognized lactose, Lex, sialyl Lex and H type 2 structures. We also compared the binding patterns of lectins with the results reported by Blixt and coworkers at CFG in which 264 different glycans are studied by using the printed microarray (Ver. 2) (http://www.functionalglycomics.org/glycomics/publicdata/ primaryscreen.jsp). There are forty-seven glyco-epitopes are found to be identical in both analyses. Even the different principles and procedures of the two systems, the binding patterns of eight lectins are nearly the same, except for a few minor differences. For example, our characterized patterns of WFA and WGA show 90% similarity to the CFG data. Nevertheless, the interactions of SBA, WFA and WGA to β-GalNAc (No. 2) in the CFG's printed microarray are not observed in our system. Both of our method and the printed microarray indicate that MAA preferentially binds to sulfated glycans [36]. Because of the observed consistency shown by the two very different methods, we conclude the protein-glycan binding interactions are not affected by the PAA linker, the assay procedure (washing vs. non-washing) and the interacting microenvironment (2D for printed microarray vs. 3D for our solution microarray).

Carbohydrate Microarray 7

**Figure 2.** Carbohydrate binding specificities of eleven lectins characterized by glycan solution

**Figure 3.** Carbohydrate binding specificities of six antibodies characterized by glycan solution

microarray [28].

microarray [28].

**Table 1.** List of biotin-PAA-glycans (fifty-two) used in glycan solution microarray [28].

our solution microarray).

Lea, but less for Lex and sialyl Lex. Anti-Ley antibody not only binding to Ley, but also recognized lactose, Lex, sialyl Lex and H type 2 structures. We also compared the binding patterns of lectins with the results reported by Blixt and coworkers at CFG in which 264 different glycans are studied by using the printed microarray (Ver. 2) (http://www.functionalglycomics.org/glycomics/publicdata/ primaryscreen.jsp). There are forty-seven glyco-epitopes are found to be identical in both analyses. Even the different principles and procedures of the two systems, the binding patterns of eight lectins are nearly the same, except for a few minor differences. For example, our characterized patterns of WFA and WGA show 90% similarity to the CFG data. Nevertheless, the interactions of SBA, WFA and WGA to β-GalNAc (No. 2) in the CFG's printed microarray are not observed in our system. Both of our method and the printed microarray indicate that MAA preferentially binds to sulfated glycans [36]. Because of the observed consistency shown by the two very different methods, we conclude the protein-glycan binding interactions are not affected by the PAA linker, the assay procedure (washing vs. non-washing) and the interacting microenvironment (2D for printed microarray vs. 3D for

**Table 1.** List of biotin-PAA-glycans (fifty-two) used in glycan solution microarray [28].

**Figure 2.** Carbohydrate binding specificities of eleven lectins characterized by glycan solution microarray [28].

**Figure 3.** Carbohydrate binding specificities of six antibodies characterized by glycan solution microarray [28].

Carbohydrate Microarray 9

**Figure 4.** Fabrication, principle, and procedures of carbohydrate membrane microarray [29].

**Table 2.** Carbohydrate ligands of commercial available lectins.

## **3.2. Binding patterns of seventeen lectins and four antibodies (membrane microarray)**

The principle and procedures of carbohydrate membrane microarray are showed in **Figure 4**. The western blotting like procedures not only reduces the time and interference, but also increases the application of this platform. In order to look deep inside the carbohydrate binding preferences of proteins and microorganisms, the collections of biotin-PAA-glycans were to increased eighty-eight different structures (**Table 3**). The glycan binding specificities of sixteen lectins (six alkaline phosphatase (AP)-conjugated lectins, four FITC-conjugated lectins, six unconjugated lectins) and four Lewis blood-group antibodies are evaluated and showed in **Figures 5 and 6**. All the lectins recognized the glycans that are consistent with the literature. For instance, ECA preferentially interacted with LacNAc, lactose, GalNAc, and Gal terminal sugars; PNA specifically bound to the Galβ1-3GalNAc structure; SBA dominantly recognized α-linked GalNAc epitopes; 3-sulfate LacNAc is ligand for MAA [36]. Compare the patterns of unconjugated lectins with conjugated lectins (AP- or FITCattached) indicated that the glycan preferences of ECA and PNA are not interfered by conjugation. More binding signals are observed in the binding profiles of AP-conjugated MAA, SBA and WGA compared with unconjugated or FITC-attached ones. Additionally, the binding patterns of DBA, ECA, GS-I, MAA, SBA and VVA are highly consistent with those reported by Consortium for Functional Glycomics (CFG, printed microarray Ver. 2,). However, few inconsistencies are also observed in the study of MPA, PNA, UEA and WGA. Furthermore, the binding patterns of four Lewis blood group antibodies represented very high specificities (**Figure 6**).

**Table 2.** Carbohydrate ligands of commercial available lectins.

**microarray)**

high specificities (**Figure 6**).

**3.2. Binding patterns of seventeen lectins and four antibodies (membrane**

The principle and procedures of carbohydrate membrane microarray are showed in **Figure 4**. The western blotting like procedures not only reduces the time and interference, but also increases the application of this platform. In order to look deep inside the carbohydrate binding preferences of proteins and microorganisms, the collections of biotin-PAA-glycans were to increased eighty-eight different structures (**Table 3**). The glycan binding specificities of sixteen lectins (six alkaline phosphatase (AP)-conjugated lectins, four FITC-conjugated lectins, six unconjugated lectins) and four Lewis blood-group antibodies are evaluated and showed in **Figures 5 and 6**. All the lectins recognized the glycans that are consistent with the literature. For instance, ECA preferentially interacted with LacNAc, lactose, GalNAc, and Gal terminal sugars; PNA specifically bound to the Galβ1-3GalNAc structure; SBA dominantly recognized α-linked GalNAc epitopes; 3-sulfate LacNAc is ligand for MAA [36]. Compare the patterns of unconjugated lectins with conjugated lectins (AP- or FITCattached) indicated that the glycan preferences of ECA and PNA are not interfered by conjugation. More binding signals are observed in the binding profiles of AP-conjugated MAA, SBA and WGA compared with unconjugated or FITC-attached ones. Additionally, the binding patterns of DBA, ECA, GS-I, MAA, SBA and VVA are highly consistent with those reported by Consortium for Functional Glycomics (CFG, printed microarray Ver. 2,). However, few inconsistencies are also observed in the study of MPA, PNA, UEA and WGA. Furthermore, the binding patterns of four Lewis blood group antibodies represented very

**Figure 4.** Fabrication, principle, and procedures of carbohydrate membrane microarray [29].

Carbohydrate Microarray 11

**Figure 5.** Carbohydrate binding specificities of sixteen lectins (six alkaline phosphatase-conjugated lectins, four FITC-conjugated lectins, and six unconjugated lectins) characterized by carbohydrate

membrane microarray [29].

2-6 sialylated diantennary N-glycans :(NeuAc2-6Gal1-4GlcNAc1-2Man)21-3,6Man1-4GlcNAc1-4GlcNAc

**Table 3.** List of biotin-PAA-glycans (eighty-eight) used in carbohydrate membrane microarray [29].

 List of biotin-PAA-glycans (eighty-eight) used in carbohydrate membrane microarray [29].

(sulfate-TF) S-88 2-6 sialylated diantennary N-glycans-PAA-biotin

PAA-biotin

S-39 Gal1-2Gal-PAA-biotin S-84 Gal1-3GlcNAc1-3Gal1-4Glc-PAA-biotin S-40 6-HSO3-Gal1-4GlcNAc-PAA-biotin S-85 Gal1-4GlcNAc1-3Gal1-4Glc-PAA-biotin

S-41 Neu5Ac2-3Gal-PAA-biotin S-86 (NeuAc2-8)5-6-PAA-biotin

S-44 GlcNAc1-3GalNAc-PAA-biotin S-89 GalNAc--Ser-PAA-biotin

S-23 Gal1-3GalNAc-PAA-biotin S-68 Blood type A (tri)-PAA-biotin,

S-24 Gal1-3GalNAc-PAA-biotin S-69 Blood type B (tri)-PAA-biotin,

sp=(CH2)3NHCO(CH2)5NH-

sp=(CH2)3NHCO(CH2)5NH-

S-83 Gal1-3(Fuc1-2)Gal1-4GlcNAc-PAA-biotin

NHCOCH2NH-

**Figure 5.** Carbohydrate binding specificities of sixteen lectins (six alkaline phosphatase-conjugated lectins, four FITC-conjugated lectins, and six unconjugated lectins) characterized by carbohydrate membrane microarray [29].

Carbohydrate Microarray 13

(**Figure 7, Lane 6 to 9**). California/07/2009 and A/Taiwan/2024/2009 bound with 6-sulfate GlcNAc, 3'-sulfated Lea, 3'-sulfated Lex, all of the 2-3 sialylated glycans with the exception of NeuAcα2-3GalNAc, 6'-sialyl lactose, sialyl biantennary *N*-glycan, and α2-8 oligomers. A/Taiwan/942/2009 favored four sulfated glycans (3-sulafe Gal, 6-sulfate GlcNAc, 3'-sulfated Lea and 3'-sulfated Lex), most of the 2-3 sialylated glycans (except NeuAcα2-3Gal and NeuAcα2-3GalNAc) and three 2-6 sialylated glycans (NeuAcα2-6Gal, 6'-sialyl lactose and sialyl biantennary *N*-glycan). A/Taiwan/987/2009 recognized most of the PAA-sugar substrates tested, with the exception of the PAA backbone, muramyl dipeptide, αNeuAc, 3 sulafe Galβ1-4GlcNAc, 3-sulafe Galβ1-3GlcNAc and Galβ1-3(NeuAcα2-6)GalNAc. In addition, we found that pandemic (H1N1) viruses isolated in 2009 represented broader substrate specificities than the pandemic viruses isolated in 2010, especially to sulfated sugars. A/Taiwan/395/2010 and A/Taiwan/1477/2010 recognized NeuAcα2-3Gal, 3'sialyl lactose, NeuAcα2-3Galβ1-3GlcNAc, sialyl-Lea, NeuAcα2-6GalNAc, NeuAcα2-6Gal, 6'-sialyl lactose, and sialyl biantennary *N*-glycan (**Figure 7, Lane 11 to 12**). A/Taiwan/257/2010 accepted only six glycans including NeuAc (linked to *p*-aniline), 3'sialyl lactose, NeuAcα2- 6GalNAc, NeuAcα2-6Gal, 6'-sialyl lactose, and sialyl biantennary *N*-glycan (**Figure 7, Lane 9**).

I-1 Blank-PAA-biotin I-17 Neu5Gc2-6GalNAc-PAA-biotin I-2 -Neu5Ac-PAA-biotin I-18 Neu5Ac2-3GalNAc-PAA-biotin

PAA-biotin I-19 3'Sialyl-Lactose-PAA-biotin

I-7 -D-GlcNAc-6-sulfate-PAA-biotin I-23 Neu5Ac2-3Gal1-4GlcNAc-PAA-

I-9 3-HSO3-Gal1-4GlcNAc-PAA-biotin I-25 Neu5Ac2-3Gal1-3GalNAc-PAA-

I-10 3-HSO3-Gal1-3GlcNAc-PAA-biotin I-26 Gal1-3(Neu5Ac2-6)GalNAc-PAA-

(sulfate-TF) I-31 2-6 sialylated diantennary N-glycans

2-6 sialylated diantennary N-glycans :(NeuAc2-6Gal1-4GlcNAc1-2Man)21-3,6Man1-4GlcNAc1-4GlcNAc **Table 4.** List of biotin-PAA-glycans (thirty) fabricated on carbohydrate membrane microarray for

Biotin

Biotin

biotin

PAA-biotin

I-27 Neu5Ac2-3(Neu5Ac2-6)GalNAc-

I-4 MDP(muramyl dipeptide)-PAA-biotin I-20 6'Sialyl-Lactose-PAA-biotin I-5 -Neu5Gc-PAA-biotin I-21 3-HSO3-Lex-PAA-biotin I-6 -D-Gal-3-sulfate-PAA-biotin I-22 3-HSO3-Lea-PAA-biotin

I-8 Neu5Ac2-6GalNAc-PAA-biotin I-24 3'Sialyl-Lec

I-12 6-HSO3-Gal1-4GlcNAc-PAA-biotin I-28 Sialyl Lea-PAA-biotin I-13 Neu5Ac2-3Gal-PAA-biotin I-29 Sialyl Lex-PAA-biotin I-14 Gal1-4(6-HSO3)GlcNAcb-PAA-biotin I-30 (NeuAc2-8)5-6-PAA-biotin

I-3 -Neu5Ac-OCH2C6H4-p-NHCOOCH2-

I-11 Neu5Ac2-8Neu5Ac-sp\*\*-PAA-

I-15 3-HSO3-Gal1-3GalNAc-PAA-biotin

I-16 Neu5Ac2-6Gal-PAA-biotin I-32 H2O

carbohydrate binding surveillance of influenza clinical isolates [29] [50].

biotin, (Neu5Ac)2

**Figure 6.** Carbohydrate binding specificities of four antibodies characterized by carbohydrate membrane microarray [29].

## **3.3. Surveillance of seasonal and pandemic H1N1 influenza A viruses (membrane microarray)**

Several methods are applied for exploring the carbohydrate-binding preference of influenza viruses or recombinant HA, including cell-based assays [37-39], immobilization of virus [40- 46] and glycan microarray technology [47-49]. In our study, 30 biotin-PAA-glycans (**Table 4**, most of them were sulfated and sialylated) were selected and fabricated on membrane and applied for the carbohydrate ligand surveillance of influenza clinical isolates. We subjected this influenza membrane microarray to characterize the glycan-binding features of five seasonal influenza A (H1N1) and seven A(H1N1)pdm09 clinical isolates. The binding patterns of all studied viruses are successfully profiled, with each virus exhibiting different and clear patterns, thereby enabling characterization (**Figure 7, Lane 1 to 12**). The majority of the seasonal H1N1 viruses bound strongly to 6'-sialyl lactose and sialyl biantennary *N*glycan (**Figure 7, Lane 1 to 5**). A/Taiwan/1156/2006, A/Taiwan/510/2008, and A/Taiwan/289/2009 isolates interacted with the two glycans, and with numerous α2-3 sialylated structures, including 3'-sialyl lactose, NeuAcα2-3(NeuAcα2-6)GalNAc, sialyl Lea, and sialyl Lex. In addition, A/Taiwan/1156/2006 isolate also recognized NeuAc (linked to *p*aniline), NeuGc, some of the sulfated glycans (3'-sulfated Lea, 3'-sulfated Lex, 6-sulfate LacNAc and 6'-sulfate LacNAc), NeuAcα2-6GalNAc, and a NeuAcα2-8 dimer. Contrary to seasonal H1N1 isolated in 2009, pandemic (H1N1) isolated in 2009 accepted more substrates (**Figure 7, Lane 6 to 9**). California/07/2009 and A/Taiwan/2024/2009 bound with 6-sulfate GlcNAc, 3'-sulfated Lea, 3'-sulfated Lex, all of the 2-3 sialylated glycans with the exception of NeuAcα2-3GalNAc, 6'-sialyl lactose, sialyl biantennary *N*-glycan, and α2-8 oligomers. A/Taiwan/942/2009 favored four sulfated glycans (3-sulafe Gal, 6-sulfate GlcNAc, 3'-sulfated Lea and 3'-sulfated Lex), most of the 2-3 sialylated glycans (except NeuAcα2-3Gal and NeuAcα2-3GalNAc) and three 2-6 sialylated glycans (NeuAcα2-6Gal, 6'-sialyl lactose and sialyl biantennary *N*-glycan). A/Taiwan/987/2009 recognized most of the PAA-sugar substrates tested, with the exception of the PAA backbone, muramyl dipeptide, αNeuAc, 3 sulafe Galβ1-4GlcNAc, 3-sulafe Galβ1-3GlcNAc and Galβ1-3(NeuAcα2-6)GalNAc. In addition, we found that pandemic (H1N1) viruses isolated in 2009 represented broader substrate specificities than the pandemic viruses isolated in 2010, especially to sulfated sugars. A/Taiwan/395/2010 and A/Taiwan/1477/2010 recognized NeuAcα2-3Gal, 3'sialyl lactose, NeuAcα2-3Galβ1-3GlcNAc, sialyl-Lea, NeuAcα2-6GalNAc, NeuAcα2-6Gal, 6'-sialyl lactose, and sialyl biantennary *N*-glycan (**Figure 7, Lane 11 to 12**). A/Taiwan/257/2010 accepted only six glycans including NeuAc (linked to *p*-aniline), 3'sialyl lactose, NeuAcα2- 6GalNAc, NeuAcα2-6Gal, 6'-sialyl lactose, and sialyl biantennary *N*-glycan (**Figure 7, Lane 9**).

12 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

**Figure 6.** Carbohydrate binding specificities of four antibodies characterized by carbohydrate

**3.3. Surveillance of seasonal and pandemic H1N1 influenza A viruses**

Several methods are applied for exploring the carbohydrate-binding preference of influenza viruses or recombinant HA, including cell-based assays [37-39], immobilization of virus [40- 46] and glycan microarray technology [47-49]. In our study, 30 biotin-PAA-glycans (**Table 4**, most of them were sulfated and sialylated) were selected and fabricated on membrane and applied for the carbohydrate ligand surveillance of influenza clinical isolates. We subjected this influenza membrane microarray to characterize the glycan-binding features of five seasonal influenza A (H1N1) and seven A(H1N1)pdm09 clinical isolates. The binding patterns of all studied viruses are successfully profiled, with each virus exhibiting different and clear patterns, thereby enabling characterization (**Figure 7, Lane 1 to 12**). The majority of the seasonal H1N1 viruses bound strongly to 6'-sialyl lactose and sialyl biantennary *N*glycan (**Figure 7, Lane 1 to 5**). A/Taiwan/1156/2006, A/Taiwan/510/2008, and A/Taiwan/289/2009 isolates interacted with the two glycans, and with numerous α2-3 sialylated structures, including 3'-sialyl lactose, NeuAcα2-3(NeuAcα2-6)GalNAc, sialyl Lea, and sialyl Lex. In addition, A/Taiwan/1156/2006 isolate also recognized NeuAc (linked to *p*aniline), NeuGc, some of the sulfated glycans (3'-sulfated Lea, 3'-sulfated Lex, 6-sulfate LacNAc and 6'-sulfate LacNAc), NeuAcα2-6GalNAc, and a NeuAcα2-8 dimer. Contrary to seasonal H1N1 isolated in 2009, pandemic (H1N1) isolated in 2009 accepted more substrates

membrane microarray [29].

**(membrane microarray)**

2-6 sialylated diantennary N-glycans :(NeuAc2-6Gal1-4GlcNAc1-2Man)21-3,6Man1-4GlcNAc1-4GlcNAc

**Table 4.** List of biotin-PAA-glycans (thirty) fabricated on carbohydrate membrane microarray for carbohydrate binding surveillance of influenza clinical isolates [29] [50].

Carbohydrate Microarray 15

**Figure 8.** Carbohydrate binding profiles of sixteen influenza B clinical isolates characterized by

The DNA sequences of hemagglutinin of the twelve influenza B clinical isolates were analyzed, translated into sequences of amino acids, and aligned with B/HongKong/8/73, Victoria (B/Victoria/02/87), and Yamagata (B/Yamagata/16/88) lineages using online software EBI ClustalW2. Surprisingly, the twelve influenza B clinical isolates could all be categorized into two groups depending on protein sequence alignment. Group 1 contained eight clinical isolates (B/Taiwan/314/2001, B/Taiwan/174/2002, B/Taiwan/288/2002, B/Taiwan/1729/2002, B/Taiwan/1994/2002, B/Taiwan/872/2004, B/Taiwan/913/2004 and B/Taiwan/1902/2004, **Figure 8, Table 5**) and Group 2 contained four viruses (B/Taiwan/262/2001, B/Taiwan/925/2001, B/Taiwan/966/2001, B/Taiwan/2284/2001). We found that all of the viruses in group 2 were isolated in 2001. In addition, there were twelve amino acid differences between the two groups. Most of the amino acids were located at the chain region and subunit interfaces, while four amino acids were located within the antigenic site (loop 160, amino acid number of B/HK/73) [51,52]. Additionally, four amino acids changed the charge property from group 1 to group 2 viruses (**Table 5**, No. 56, 116, 181 and 182, amino acid number of B/Yamagata/16/88). Different carbohydrate binding properties were also observed between some viruses of the two groups. Specifically, group 2 viruses interacted with three major sialylated glycans, while some of group 1 viruses bound not only strongly to the three glycans, but also weakly to sulfated and α2-3 sialylated glycans. Additionally, the number of charged amino acids was higher in group 2 than in group 1 viruses and

carbohydrate membrane microarray [50].

B/HongKong/8/73 (**Table 5**).

**Figure 7.** Carbohydrate binding profiles of twelve influenza A clinical isolates characterized by carbohydrate membrane microarray [50].

### **3.4. Surveillance of influenza B clinical isolates (membrane microarray)**

The influenza carbohydrate membrane microarray is subsequently subjected for the surveillance of twelve influenza B (IB) clinical isolates [50]. All of the viruses are collected in the Clinical Virology Laboratory of National Cheng Kung University Hospital between 2001 and 2004. The clinical isolates are amplified in MDCK cells and purified by a sucrose gradient. The binding patterns of the influenza B viruses are investigated and are all found to exhibit clear binding profiles (**Figure 8**). Three common glycans, including α-Neu5Ac (*p*-anilinyl linked), 6'sialyl-lactose, and α2-6 sialylated biantennary *N*-glycans are bound by all of the influenza B viruses. Furthermore, some additional bindings are also observed. For instance, B/Taiwan/314/2001 and B/Taiwan/1729/2002 also recognized sulfated sugars (3-sulafe Gal, 6-sulfate GlcNAc, Galβ1-4(6-HSO3)GlcNAc, and 3'-sulfated Lea) and two α2-6 sialosides (NeuAcα2-6GalNAc and NeuAcα2-6Gal). B/Taiwan/288/2002 and B/Taiwan/1902/2004 portrayed minor interactions with α2-3 (3'-sialyl lactose, NeuAcα2-3Galβ1-4GlcNAc, 3'-sialyl Lec and sialyl Lex) and α2-6 sialylated sugars (NeuAcα2-6GalNAc and NeuAcα2-6Gal). Although the signal is relatively low, B/Taiwan/872/2004 and B/Taiwan/913/2004 also interacted with sulfated sugars. Surprisingly, seven viruses showed weak bindings to α2-8 linked sialic acids, including B/Taiwan/314/2001, B/Taiwan/174/2002, B/Taiwan/288/2002, B/Taiwan/1729/2002, B/Taiwan/872/2004, and B/Taiwan/913/2004.

**Figure 7.** Carbohydrate binding profiles of twelve influenza A clinical isolates characterized by

**3.4. Surveillance of influenza B clinical isolates (membrane microarray)**

The influenza carbohydrate membrane microarray is subsequently subjected for the surveillance of twelve influenza B (IB) clinical isolates [50]. All of the viruses are collected in the Clinical Virology Laboratory of National Cheng Kung University Hospital between 2001 and 2004. The clinical isolates are amplified in MDCK cells and purified by a sucrose gradient. The binding patterns of the influenza B viruses are investigated and are all found to exhibit clear binding profiles (**Figure 8**). Three common glycans, including α-Neu5Ac (*p*-anilinyl linked), 6'sialyl-lactose, and α2-6 sialylated biantennary *N*-glycans are bound by all of the influenza B viruses. Furthermore, some additional bindings are also observed. For instance, B/Taiwan/314/2001 and B/Taiwan/1729/2002 also recognized sulfated sugars (3-sulafe Gal, 6-sulfate GlcNAc, Galβ1-4(6-HSO3)GlcNAc, and 3'-sulfated Lea) and two α2-6 sialosides (NeuAcα2-6GalNAc and NeuAcα2-6Gal). B/Taiwan/288/2002 and B/Taiwan/1902/2004 portrayed minor interactions with α2-3 (3'-sialyl lactose, NeuAcα2-3Galβ1-4GlcNAc, 3'-sialyl Lec and sialyl Lex) and α2-6 sialylated sugars (NeuAcα2-6GalNAc and NeuAcα2-6Gal). Although the signal is relatively low, B/Taiwan/872/2004 and B/Taiwan/913/2004 also interacted with sulfated sugars. Surprisingly, seven viruses showed weak bindings to α2-8 linked sialic acids, including B/Taiwan/314/2001, B/Taiwan/174/2002, B/Taiwan/288/2002, B/Taiwan/1729/2002,

carbohydrate membrane microarray [50].

B/Taiwan/872/2004, and B/Taiwan/913/2004.

**Figure 8.** Carbohydrate binding profiles of sixteen influenza B clinical isolates characterized by carbohydrate membrane microarray [50].

The DNA sequences of hemagglutinin of the twelve influenza B clinical isolates were analyzed, translated into sequences of amino acids, and aligned with B/HongKong/8/73, Victoria (B/Victoria/02/87), and Yamagata (B/Yamagata/16/88) lineages using online software EBI ClustalW2. Surprisingly, the twelve influenza B clinical isolates could all be categorized into two groups depending on protein sequence alignment. Group 1 contained eight clinical isolates (B/Taiwan/314/2001, B/Taiwan/174/2002, B/Taiwan/288/2002, B/Taiwan/1729/2002, B/Taiwan/1994/2002, B/Taiwan/872/2004, B/Taiwan/913/2004 and B/Taiwan/1902/2004, **Figure 8, Table 5**) and Group 2 contained four viruses (B/Taiwan/262/2001, B/Taiwan/925/2001, B/Taiwan/966/2001, B/Taiwan/2284/2001). We found that all of the viruses in group 2 were isolated in 2001. In addition, there were twelve amino acid differences between the two groups. Most of the amino acids were located at the chain region and subunit interfaces, while four amino acids were located within the antigenic site (loop 160, amino acid number of B/HK/73) [51,52]. Additionally, four amino acids changed the charge property from group 1 to group 2 viruses (**Table 5**, No. 56, 116, 181 and 182, amino acid number of B/Yamagata/16/88). Different carbohydrate binding properties were also observed between some viruses of the two groups. Specifically, group 2 viruses interacted with three major sialylated glycans, while some of group 1 viruses bound not only strongly to the three glycans, but also weakly to sulfated and α2-3 sialylated glycans. Additionally, the number of charged amino acids was higher in group 2 than in group 1 viruses and B/HongKong/8/73 (**Table 5**).

Carbohydrate Microarray 17

but the relative intensity of GlcNAc-binding lectins is not as good as those of Con A and SNA, which is the contribution of the interference by the terminal sialic acids [59]. All of the

results are similar to the analyses from lectin microarray or dot blot analysis [55-58].

**Figure 9.** Glycopattern profiling of six biotinylated proteins with ten lectins by glycan solution microarray. The six proteins include ovalbumin, porcine mucin, human serum albumin, human

In summary, carbohydrate-protein interactions are the key steps for many physiological and pathological evens. Hence, development of new carbohydrate microarrays is important for detecting these activities. Our studies have demonstrated a rapid, highly sensitive, and reliable method to characterize carbohydrate-protein interactions with minimized materials (in the range of ng per well). Lectins and antibodies are evaluated and most of the results are coherent to previous reports, including those of CFG. Solution microarray is the first homogeneous and washless carbohydrate microarray which offers a reliable alternative for characterizing sugar-binding features of lectins and proteins, as well as antibodies. In addition, membrane microarray is also easy handled with low cost (USD\$0.25 for one membrane microarray). The easy-handling feature avoids the necessity of repetitive training and accelerates the screening efficiency. Both of the microarrays represent a convenient and reliable way to examine the carbohydrate-binding features of various proteins, highthroughput drug screening, and the glycan binding surveillance of influenza viruses.

transferrin, fetuin and asialofetuin [28].

**4. Perspective**

**Author details**

Chuan-Fa Chang

*Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Taiwan*

**Table 5.** Critical amino acid differences between the two groups of influenza B clinical isolated viruses [50].

According to the binding preference for α2-6 and α2-3 sialylated oligosaccharides displayed by these clinical isolated influenza viruses, these clinical isolates might infect not only the surface of nasal mucosa, pharynx, larynx, trachea and bronchi (that are resided in the upper respiratory tract to express α2-6 sialosides predominantly), but also alveoli, bronchi (that are located in the lower respiratory tract), eyes, and tissues in the gastrointestinal tract (that contain both α2-3 and α2-6 sialosides) [53,54]. After we surveyed more clinical isolates, factors which affect the binding preference of IB could be dissected. We believe that application of the carbohydrate microarray for detailed analysis of sugar-binding structures will eventually build the connection between clinical symptoms and the genetic specification of influenza viruses.

## **3.5. Characterization the glycan structure of glycoproteins (solution microarray)**

The aforementioned lectins are further applied to characterize the glycan structures of six biotinylated proteins including ovalbumin, porcine mucin, human serum albumin, human transferrin, fetuin and asialofetuin. Distinctive glycopatterns are generated in accordance with the analysis of lectins (**Figure 9**). Porcine mucin is known to have *O*-linked fucosylated glycans with terminal GalNAc, GlcNAc and NeuAc residues. Our results showed positive signals in the tests with the ECA, DBA, UEA-1 and WGA lectins, indicating that the mucin contains the determinants of Galβ1-4GlcNAc, GalNAc, Fucα1-2Gal and GlcNAc/NeuAc, respectively. Manα1-3(Fucα1-6)GlcNAc and NeuAcα2-6Galβ1-4GlcNAc containing biantennary or triantennary *N*-glycans have been reported as the major glyco-structures of human transferrin [55-58]. Strong Con A and SNA signals are shown in our binding assay, but the relative intensity of GlcNAc-binding lectins is not as good as those of Con A and SNA, which is the contribution of the interference by the terminal sialic acids [59]. All of the results are similar to the analyses from lectin microarray or dot blot analysis [55-58].

**Figure 9.** Glycopattern profiling of six biotinylated proteins with ten lectins by glycan solution microarray. The six proteins include ovalbumin, porcine mucin, human serum albumin, human transferrin, fetuin and asialofetuin [28].

## **4. Perspective**

16 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

[50].

specification of influenza viruses.

**Table 5.** Critical amino acid differences between the two groups of influenza B clinical isolated viruses

According to the binding preference for α2-6 and α2-3 sialylated oligosaccharides displayed by these clinical isolated influenza viruses, these clinical isolates might infect not only the surface of nasal mucosa, pharynx, larynx, trachea and bronchi (that are resided in the upper respiratory tract to express α2-6 sialosides predominantly), but also alveoli, bronchi (that are located in the lower respiratory tract), eyes, and tissues in the gastrointestinal tract (that contain both α2-3 and α2-6 sialosides) [53,54]. After we surveyed more clinical isolates, factors which affect the binding preference of IB could be dissected. We believe that application of the carbohydrate microarray for detailed analysis of sugar-binding structures will eventually build the connection between clinical symptoms and the genetic

**3.5. Characterization the glycan structure of glycoproteins (solution microarray)**

The aforementioned lectins are further applied to characterize the glycan structures of six biotinylated proteins including ovalbumin, porcine mucin, human serum albumin, human transferrin, fetuin and asialofetuin. Distinctive glycopatterns are generated in accordance with the analysis of lectins (**Figure 9**). Porcine mucin is known to have *O*-linked fucosylated glycans with terminal GalNAc, GlcNAc and NeuAc residues. Our results showed positive signals in the tests with the ECA, DBA, UEA-1 and WGA lectins, indicating that the mucin contains the determinants of Galβ1-4GlcNAc, GalNAc, Fucα1-2Gal and GlcNAc/NeuAc, respectively. Manα1-3(Fucα1-6)GlcNAc and NeuAcα2-6Galβ1-4GlcNAc containing biantennary or triantennary *N*-glycans have been reported as the major glyco-structures of human transferrin [55-58]. Strong Con A and SNA signals are shown in our binding assay, In summary, carbohydrate-protein interactions are the key steps for many physiological and pathological evens. Hence, development of new carbohydrate microarrays is important for detecting these activities. Our studies have demonstrated a rapid, highly sensitive, and reliable method to characterize carbohydrate-protein interactions with minimized materials (in the range of ng per well). Lectins and antibodies are evaluated and most of the results are coherent to previous reports, including those of CFG. Solution microarray is the first homogeneous and washless carbohydrate microarray which offers a reliable alternative for characterizing sugar-binding features of lectins and proteins, as well as antibodies. In addition, membrane microarray is also easy handled with low cost (USD\$0.25 for one membrane microarray). The easy-handling feature avoids the necessity of repetitive training and accelerates the screening efficiency. Both of the microarrays represent a convenient and reliable way to examine the carbohydrate-binding features of various proteins, highthroughput drug screening, and the glycan binding surveillance of influenza viruses.

## **Author details**

Chuan-Fa Chang *Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Taiwan*

### **5.  References**

1375-1383

6140-6148

268-274

115, 149-152

1011-1017

*Biochem* 334, 36-47

	- [46] Gambaryan, A. S., Tuzikov, A. B., Pazynina, G. V., Webster, R. G., Matrosovich, M. N., and Bovin, N. V. (2004) *Virology* 326, 310-316

**Chapter 2**

© 2012 Richel and Paquot, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Richel and Paquot, licensee InTech. This is a paper distributed under the terms of the Creative Commons

Milder reaction conditions, associated to reduced run times (from several hours to a few minutes) and improved yields and selectivities, are the key advantages usually reported for

**Conversion of Carbohydrates**

**Under Microwave Heating**

Additional information is available at the end of the chapter

The non-energetic valorisation of renewable resources using efficient and eco-friendly methodologies is the central axis of the "green chemistry" concept. In particular, the chemical and chemo-enzymatical transformation of carbohydrates arising from the hydrolysis of nonedible vegetal feedstock (i.e., lignocellulosic biomass) is a widely explored thematic for the production of new high-added value materials, synthons, and platform chemicals (Bozell, 2010). The use of mono-, di- and polysaccharides for the production of new chemicals constitutes thus a subject of special relevance from both academic and industrial points of view (Lichtenthaler, 2004). Amongst the 12 principles defining this "green chemistry" concept, the development of new effective synthetic protocols, minimising wastes and energy-consumption while enhancing purity of the final product, is the corner stone (Anastas, 1998). In this regard, the use of microwaves as a non-conventional heating method has progressively gained attention due to commonly observed acceleration in reactions rates and improved (regio- and chemo-) selectivities and yields in synthetic organic transformations (Kappe, 2004; Caddick, 2009). The claimed cleaner reaction profiles of microwave-assisted processes have thus rapidly projected this kind of heating as a popular method in chemistry, which often replaces the "classical" heating ones. Numerous organic reactions are nowadays fully depicted in the peer-reviewed literature and books. They concern typical synthetic organic approaches (substitutions, alkylations, cycloadditions, esterifications, cyclisations, etc.), organometallic reactions, oxidations and reductions, or

Aurore Richel and Michel Paquot

polymerisation reactions (Bogdal, 2006).

**2. Scope of this contribution**

http://dx.doi.org/10.5772/50628

**1. Introduction**

**Chapter 2**

## **Conversion of Carbohydrates Under Microwave Heating**

Aurore Richel and Michel Paquot

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50628

## **1. Introduction**

20 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

and Bovin, N. V. (2004) *Virology* 326, 310-316

Wilson, I. A. (2006) *J Mol Biol* 355, 1143-1155

J., and Chang, C. F. (2012) *Fut Virol* 7, 13

*Microbiol* 47, 327-334

*Viruses* 2, 8

985-989

[46] Gambaryan, A. S., Tuzikov, A. B., Pazynina, G. V., Webster, R. G., Matrosovich, M. N.,

[47] Gall, A., Hoffmann, B., Harder, T., Grund, C., Hoper, D., and Beer, M. (2009) *J Clin*

[48] Stevens, J., Blixt, O., Paulson, J. C., and Wilson, I. A. (2006) *Nat Rev Microbiol* 4, 857-864 [49] Stevens, J., Blixt, O., Glaser, L., Taubenberger, J. K., Palese, P., Paulson, J. C., and

[50] Wang, Y. F., Lao, W. I., Kuo, Y. D., Guu, S. Y., Wang, H. C., Lin, C. H., Wang, J. R., Su, I.

[52] Wang, Q., Tian, X., Chen, X., and Ma, J. (2007) *Proc Natl Acad Sci U S A* 104, 16874-16879 [53] Kumlin, U., Olofsson, S., Dimock, K., Arnberg, N. (2008) *Influenza and Other Respiratory*

[54] Kogure, T., Suzuki, T., Takahashi, T., Miyamoto, D., Hidari, K. I., Guo, C. T., Ito, T.,

[56] Pilobello, K. T., Krishnamoorthy, L., Slawek, D., and Mahal, L. K. (2005) *Chembiochem* 6,

[57] Yang, S. N., Liu, C. A., Chung, M. Y., Huang, H. C., Yeh, G. C., Wong, C. S., Lin, W. W.,

[59] Angeloni, S., Ridet, J. L., Kusy, N., Gao, H., Crevoisier, F., Guinchard, S., Kochhar, S.,

[51] Wang, Q., Cheng, F., Lu, M., Tian, X., and Ma, J. (2008) *J Virol* 82, 3011-3020

Kawaoka, Y., and Suzuki, Y. (2006) *Glycoconj J* 23, 101-106 [55] Kolarich, D., and Altmann, F. (2000) *Anal Biochem* 285, 64-75

Yang, C. H., and Tao, P. L. (2006) *Hippocampus* 16, 521-530 [58] Karlsson, N. G., and Packer, N. H. (2002) *Anal Biochem* 305, 173-185

Sigrist, H., and Sprenger, N. (2005) *Glycobiology* 15, 31-41

The non-energetic valorisation of renewable resources using efficient and eco-friendly methodologies is the central axis of the "green chemistry" concept. In particular, the chemical and chemo-enzymatical transformation of carbohydrates arising from the hydrolysis of nonedible vegetal feedstock (i.e., lignocellulosic biomass) is a widely explored thematic for the production of new high-added value materials, synthons, and platform chemicals (Bozell, 2010). The use of mono-, di- and polysaccharides for the production of new chemicals constitutes thus a subject of special relevance from both academic and industrial points of view (Lichtenthaler, 2004). Amongst the 12 principles defining this "green chemistry" concept, the development of new effective synthetic protocols, minimising wastes and energy-consumption while enhancing purity of the final product, is the corner stone (Anastas, 1998). In this regard, the use of microwaves as a non-conventional heating method has progressively gained attention due to commonly observed acceleration in reactions rates and improved (regio- and chemo-) selectivities and yields in synthetic organic transformations (Kappe, 2004; Caddick, 2009). The claimed cleaner reaction profiles of microwave-assisted processes have thus rapidly projected this kind of heating as a popular method in chemistry, which often replaces the "classical" heating ones. Numerous organic reactions are nowadays fully depicted in the peer-reviewed literature and books. They concern typical synthetic organic approaches (substitutions, alkylations, cycloadditions, esterifications, cyclisations, etc.), organometallic reactions, oxidations and reductions, or polymerisation reactions (Bogdal, 2006).

## **2. Scope of this contribution**

Milder reaction conditions, associated to reduced run times (from several hours to a few minutes) and improved yields and selectivities, are the key advantages usually reported for

© 2012 Richel and Paquot, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Richel and Paquot, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

microwave-promoted reactions. Even if widely employed in common organic chemistry, microwaves find however fewer applications in carbohydrate chemistry (Corsaro, 2004; Cioffi, 2008; Richel, 2011a). This fact, usually associated to a high number of unwanted and uncontrolled typical sugars reactions such as carbonisation or browning processes, has been overcame by the design of novel sophisticated microwave reactors. Advantageously, microwaves provide nowadays, with remarkable atom efficiency and yields, novel carbohydrate-based chemicals that are not easily obtainable by any other "classical" means or only using painstaking multi-step and energy-consuming protocols (Richel, 2011b). This chapter describes thus the use of microwave processes to mediate key reactions in the field of carbohydrate chemistry. Some examples of benchmark reactions (glycosylations, hydroxyl groups' protection, etc.) under microwave conditions are displayed and highlight the benefits of this microwave approach in terms of yields, selectivities and environmental impact.

Conversion of Carbohydrates Under Microwave Heating 23

The ability of a given substance to convert electromagnetic energy into calorific heat, at specific frequency and temperature, is determined by the loss tangent factor (tan). This factor is described as the ratio between dielectric loss ('') and dielectric constant ('). Practically, substances with high tan values are required to ensure a convenient and fast microwave heating. By convention, substances are thus categorised into good (tan>0.5), medium (0.5>tan>0.1) and poor (tan<0.1) candidates for microwave (MW) applications. At the labscale, MW is conveniently applied to reactions performed in the liquid state in the presence of a solvent with a high tan. However, in order to provide a truly eco-friendly process, syntheses without solvent or in dry-media conditions are also described with reactants and/or catalysts impregnated on a solid microwave-absorbing support such as alumina or silica (Polshettiwar, 2008). In the field of carbohydrate chemistry, reactions under microwave (MW) assistance are usually achieved with saccharides in suspension or in solution in solvent and/or liquid reagents. Highly secured MW reactors, operating in batch or continuous flow conditions, are nowadays available for chemistry. They operate typically at 2.45 GHz and allow an accurate control of the temperature within the MW reaction vessel with an adjustable power, avoiding thus browning. Reactions can also be performed under "closed vessel

conditions", at temperatures exceeding the boiling point of the reaction medium.

**4. Chemical transformations of carbohydrates under microwave**

Saccharides, which contain several hydroxyl groups, are polar molecules particularly suited for MW applications. Typical MW-assisted transformations reported in the literature include either reactions involving hydroxyl groups for the production of novel entities (category 1) or dehydration reactions leading to the formation of furfural and related

**4.1. Microwave-assisted transformations involving hydroxyl groups (category 1)**

Most common reactions of the first category include protections/deprotections of hydroxyl groups, glycosylations, glycosamines formation and halogenations. These reactions are usually catalysed by chemical entities, even if enzymes are proved to offer convenient results for specific cases under microwave conditions. The several hydroxylated stereogenic centres in carbohydrates are difficult to chemically differentiate. Thus, the regioselective derivatisation of such compounds is a challenging task and requires multi-step protocols and purifications. Microwave heating has demonstrated to be a convenient solution, allowing an accurate control of the regioselectivity and the anomeric selectivity by an appropriate tuning of operating conditions. Some relevant examples are proposed herein.

Several methods and protocols have been reported for the MW-assisted regioselective protection of hydroxyls, especially in mono- and disaccharides (Corsaro, 2004). They include

**conditions**

platform molecules (category 2).

*4.1.1. Hydroxyls protections/deprotections*

## **3. Fundamental of microwave technology**

The pioneering works in the microwave-assisted chemistry are attributed to Gedye and Giguere using household microwave ovens (Gedye, 1986; Giguere, 1986). From a theoretical point of view, microwave heating originates from interactions between a given material (reagent, solvent or catalyst) and the electric component of the electromagnetic wave through a dipolar polarisation and/or ionic conduction mechanism. Polar substances like DMSO, ethylene glycol or ethanol are thus suited candidates for microwave applications by opposition to benzene, dioxane or carbon tetrachloride which do not possess permanent dipolar moment. It is recognised that accelerations of reactions rates under microwave conditions are to correlate with a thermal/kinetic effect. Compared to analogous heating under classical conditions (using an oil bath for instance), microwave ensures a fast, selective and homogeneous heating of the reaction vessel (Fig. 1). Convection currents and temperature gradients from vessel walls to the core of the reaction medium, commonly observed using classical heating options, are thus avoided under microwave conditions.

**Figure 1.** Difference between conventional heating (**a**) and microwave-assisted heating (**b**). In (**a**), heating is provided by thermal conduction. In (**b**), solvent and/or reagents absorb microwave heating and convert this energy into calorific components.

The ability of a given substance to convert electromagnetic energy into calorific heat, at specific frequency and temperature, is determined by the loss tangent factor (tan). This factor is described as the ratio between dielectric loss ('') and dielectric constant ('). Practically, substances with high tan values are required to ensure a convenient and fast microwave heating. By convention, substances are thus categorised into good (tan>0.5), medium (0.5>tan>0.1) and poor (tan<0.1) candidates for microwave (MW) applications. At the labscale, MW is conveniently applied to reactions performed in the liquid state in the presence of a solvent with a high tan. However, in order to provide a truly eco-friendly process, syntheses without solvent or in dry-media conditions are also described with reactants and/or catalysts impregnated on a solid microwave-absorbing support such as alumina or silica (Polshettiwar, 2008). In the field of carbohydrate chemistry, reactions under microwave (MW) assistance are usually achieved with saccharides in suspension or in solution in solvent and/or liquid reagents. Highly secured MW reactors, operating in batch or continuous flow conditions, are nowadays available for chemistry. They operate typically at 2.45 GHz and allow an accurate control of the temperature within the MW reaction vessel with an adjustable power, avoiding thus browning. Reactions can also be performed under "closed vessel conditions", at temperatures exceeding the boiling point of the reaction medium.

## **4. Chemical transformations of carbohydrates under microwave conditions**

Saccharides, which contain several hydroxyl groups, are polar molecules particularly suited for MW applications. Typical MW-assisted transformations reported in the literature include either reactions involving hydroxyl groups for the production of novel entities (category 1) or dehydration reactions leading to the formation of furfural and related platform molecules (category 2).

## **4.1. Microwave-assisted transformations involving hydroxyl groups (category 1)**

Most common reactions of the first category include protections/deprotections of hydroxyl groups, glycosylations, glycosamines formation and halogenations. These reactions are usually catalysed by chemical entities, even if enzymes are proved to offer convenient results for specific cases under microwave conditions. The several hydroxylated stereogenic centres in carbohydrates are difficult to chemically differentiate. Thus, the regioselective derivatisation of such compounds is a challenging task and requires multi-step protocols and purifications. Microwave heating has demonstrated to be a convenient solution, allowing an accurate control of the regioselectivity and the anomeric selectivity by an appropriate tuning of operating conditions. Some relevant examples are proposed herein.

#### *4.1.1. Hydroxyls protections/deprotections*

22 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

**3. Fundamental of microwave technology**

(a) (b)

and convert this energy into calorific components.

impact.

microwave-promoted reactions. Even if widely employed in common organic chemistry, microwaves find however fewer applications in carbohydrate chemistry (Corsaro, 2004; Cioffi, 2008; Richel, 2011a). This fact, usually associated to a high number of unwanted and uncontrolled typical sugars reactions such as carbonisation or browning processes, has been overcame by the design of novel sophisticated microwave reactors. Advantageously, microwaves provide nowadays, with remarkable atom efficiency and yields, novel carbohydrate-based chemicals that are not easily obtainable by any other "classical" means or only using painstaking multi-step and energy-consuming protocols (Richel, 2011b). This chapter describes thus the use of microwave processes to mediate key reactions in the field of carbohydrate chemistry. Some examples of benchmark reactions (glycosylations, hydroxyl groups' protection, etc.) under microwave conditions are displayed and highlight the benefits of this microwave approach in terms of yields, selectivities and environmental

The pioneering works in the microwave-assisted chemistry are attributed to Gedye and Giguere using household microwave ovens (Gedye, 1986; Giguere, 1986). From a theoretical point of view, microwave heating originates from interactions between a given material (reagent, solvent or catalyst) and the electric component of the electromagnetic wave through a dipolar polarisation and/or ionic conduction mechanism. Polar substances like DMSO, ethylene glycol or ethanol are thus suited candidates for microwave applications by opposition to benzene, dioxane or carbon tetrachloride which do not possess permanent dipolar moment. It is recognised that accelerations of reactions rates under microwave conditions are to correlate with a thermal/kinetic effect. Compared to analogous heating under classical conditions (using an oil bath for instance), microwave ensures a fast, selective and homogeneous heating of the reaction vessel (Fig. 1). Convection currents and temperature gradients from vessel walls to the core of the reaction medium, commonly observed using classical heating options, are thus avoided under microwave conditions.

**Figure 1.** Difference between conventional heating (**a**) and microwave-assisted heating (**b**). In (**a**), heating is provided by thermal conduction. In (**b**), solvent and/or reagents absorb microwave heating

Microwaves

Several methods and protocols have been reported for the MW-assisted regioselective protection of hydroxyls, especially in mono- and disaccharides (Corsaro, 2004). They include

mainly acetal formation with aldehydes and acylation with acetic anhydride or an acyl chloride. All these reactions are promoted using (homogeneous or heterogeneous) acids or bases (Söderberg, 2001) or enzymes (Chen, 2001) in variable solvents, reagents concentrations and operating conditions (temperature and microwave exposure time). The total or regioselective protection (and subsequent deprotection) of specific hydroxyl groups is necessary for the synthesis of building blocks suited for the production of drugs, biologically active materials, and novel high-added value materials. Particularly, acetylation reactions are often the prerequisite step in the synthesis of such complex carbohydrates (Fig. 2). The conventional per-*O*-acetylation of saccharides is mostly achieved using anhydride acetic as both reagent and solvent. Although this step is high-yielding, long reaction times (several hours) are required and noxious pyridine is mandatory to promote the reaction. Microwave dielectric heating has thus appeared as a more eco-friendly option. Indeed, replacement of pyridine by catalytic amounts of non-toxic sodium acetate or Lewis acid such as zinc chloride is found practically quantitative after only a few minutes of MW heating (Limousin, 1997). With ZnCl2, an equimolar mixture of and pentaacetates is obtained under MW conditions, whilst a ratio of 7/3 is recovered under oil bath conventional heating (Fig. 2a). Microwave heating affords also convenient results for the complete acetylation of totally *O*unprotected mono- and disaccharides in 90 sec at 720 Watt under closed vessel conditions using indium(III) chloride catalyst. Reactions performed in acetonitrile with stoichiometric amounts of anhydride acetic are quantitative and affords predominantly the peracetylated form (Das, 2005). Partial acetylation of carbohydrates is usually achieved with acetyl chloride and pyridine. With 1,2:5,6-di-*O*-isopropylidene--D-glucofuranose as the starting material, Söderberg demonstrates that the synergic combination of microwaves and polystyrenesupported base catalysts offers a convenient strategy to obtain in 85% yield the expected mono-acetylated analogous without formation of side-products (Fig. 2b). This MW approach provides an eco-friendly and time-saving option as the catalyst can be recovered by filtration at the end of the process, thus minimising wastes and purification (Söderberg, 2001).

Conversion of Carbohydrates Under Microwave Heating 25

the 1,6-*O*-diacetate monosaccharide is formed under MW exposure, the 6-*O*-monoacetate adduct is preferentially formed under classical oil bath heating conditions. Reactions go to completion when conducted using acetic acid as a catalyst in neat anhydride acetic for 3 x 25 min under MW. For per-*O*-TMS-galactoside, a 52% yield in diacetylated adduct is recovered using 2 equivalents of acetic acid. When the galactoside is stirred for 2 days with 2 equiv of acid, the reaction shows a 50% completion with yields after column chromatography

**Figure 3.** MW-assisted selective acetylation of per-*O*-TMS-galactoside using protecting group exchange

 AcOH Ac2O, pyridine

r.t., 48 h

MW

Regioselective benzoylation and pivaloylation of carbohydrates is another major item for free hydroxyls protection. Typically performed using benzoyl chloride and pivaloyl chloride, these reactions are only viable when using a base catalyst. Due to the steric bulk of the pivaloyl groups, the protection of hydroxyls through formation of pivaloyl esters is very slow under conventional heating method and requires several hours. Substantial reduction in reaction times (10-20 min) is however encountered under MW conditions. Pivaloylation of 1,2:5,6-di-*O*isopropylidene--D-glucofuranose, catalysed by pyridine, provides thus the corresponding 3- *O*-protected target in 68% yield after 10 min at 160°C in a microwave reactor (Fig. 4a). However, presence of a side-product is recovered after MW exposure as a result of 5,6- to 3,6 acetal migration prior to acylation. The formation of this unwanted structure is repressed when using supported polystyrene-bases, i.e. *N,N*-(diisopropyl)aminoethylpolystyrene (PS-DIEA) and *N*-(methylpolystyrene)-4-(methylamino)pyridine (PS-DMAP) , as alternatives to pyridine. 79 and 88% yields are thus detected after 15-20 min at 160-180°C in the microwave cavity, whilst only 88% yield is obtained after 300 min at room temperature (Söderberg, 2001).

The regioselective benzoylation of carbohydrates affords convenient results using dibutyltin oxide mediators under microwave exposure (Herradón, 1995). Intermediate formation of dibutylstannylene acetal allows hydroxyl regioselective benzoylation after less than 10 min of microwave exposure with a 500 Watt output (Fig. 4b). This MW methodology is more advantageous than its "classical" analogous involving heating, for several hours, at reflux of a toluene suspension of saccharides and dibutyltin oxide with azeotropic removal of water. Tuning of operation conditions, including both the solvent and the MW power output, modulates the selectivity of dibutylstannylene acetal-mediated benzoylation of

representative carbohydrates, such as -D-mannopyranoside (Fig. 5).

purification reaching 35% of 6-*O*-monoacetate (Witschi, 2010).

strategy.

**Figure 2.** Peracetylation (a) and acetylation (b) of selected carbohydrates under MW conditions.

More recently, Witschi et al. describe a novel procedure for the selective acetylation of hydroxyls groups. It relies on a protecting group exchange strategy under microwave assistance starting from per-*O*-trimethylsilylated pyranosides (Fig. 3). Interestingly, whilst the 1,6-*O*-diacetate monosaccharide is formed under MW exposure, the 6-*O*-monoacetate adduct is preferentially formed under classical oil bath heating conditions. Reactions go to completion when conducted using acetic acid as a catalyst in neat anhydride acetic for 3 x 25 min under MW. For per-*O*-TMS-galactoside, a 52% yield in diacetylated adduct is recovered using 2 equivalents of acetic acid. When the galactoside is stirred for 2 days with 2 equiv of acid, the reaction shows a 50% completion with yields after column chromatography purification reaching 35% of 6-*O*-monoacetate (Witschi, 2010).

24 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

mainly acetal formation with aldehydes and acylation with acetic anhydride or an acyl chloride. All these reactions are promoted using (homogeneous or heterogeneous) acids or bases (Söderberg, 2001) or enzymes (Chen, 2001) in variable solvents, reagents concentrations and operating conditions (temperature and microwave exposure time). The total or regioselective protection (and subsequent deprotection) of specific hydroxyl groups is necessary for the synthesis of building blocks suited for the production of drugs, biologically active materials, and novel high-added value materials. Particularly, acetylation reactions are often the prerequisite step in the synthesis of such complex carbohydrates (Fig. 2). The conventional per-*O*-acetylation of saccharides is mostly achieved using anhydride acetic as both reagent and solvent. Although this step is high-yielding, long reaction times (several hours) are required and noxious pyridine is mandatory to promote the reaction. Microwave dielectric heating has thus appeared as a more eco-friendly option. Indeed, replacement of pyridine by catalytic amounts of non-toxic sodium acetate or Lewis acid such as zinc chloride is found practically quantitative after only a few minutes of MW heating (Limousin, 1997). With ZnCl2, an equimolar mixture of and pentaacetates is obtained under MW conditions, whilst a ratio of 7/3 is recovered under oil bath conventional heating (Fig. 2a). Microwave heating affords also convenient results for the complete acetylation of totally *O*unprotected mono- and disaccharides in 90 sec at 720 Watt under closed vessel conditions using indium(III) chloride catalyst. Reactions performed in acetonitrile with stoichiometric amounts of anhydride acetic are quantitative and affords predominantly the peracetylated form (Das, 2005). Partial acetylation of carbohydrates is usually achieved with acetyl chloride and pyridine. With 1,2:5,6-di-*O*-isopropylidene--D-glucofuranose as the starting material, Söderberg demonstrates that the synergic combination of microwaves and polystyrenesupported base catalysts offers a convenient strategy to obtain in 85% yield the expected mono-acetylated analogous without formation of side-products (Fig. 2b). This MW approach provides an eco-friendly and time-saving option as the catalyst can be recovered by filtration

at the end of the process, thus minimising wastes and purification (Söderberg, 2001).

6.5 min, 125°C : 98% yield

15 min, 180°C : 85% yield (81% under classical heating after several hours)

(83% under classical heating) Ac2O, ZnCl2

Ac = acetyl

(a)

(b)

**Figure 2.** Peracetylation (a) and acetylation (b) of selected carbohydrates under MW conditions.

AcCl, Base

MW

MW

More recently, Witschi et al. describe a novel procedure for the selective acetylation of hydroxyls groups. It relies on a protecting group exchange strategy under microwave assistance starting from per-*O*-trimethylsilylated pyranosides (Fig. 3). Interestingly, whilst

**Figure 3.** MW-assisted selective acetylation of per-*O*-TMS-galactoside using protecting group exchange strategy.

Regioselective benzoylation and pivaloylation of carbohydrates is another major item for free hydroxyls protection. Typically performed using benzoyl chloride and pivaloyl chloride, these reactions are only viable when using a base catalyst. Due to the steric bulk of the pivaloyl groups, the protection of hydroxyls through formation of pivaloyl esters is very slow under conventional heating method and requires several hours. Substantial reduction in reaction times (10-20 min) is however encountered under MW conditions. Pivaloylation of 1,2:5,6-di-*O*isopropylidene--D-glucofuranose, catalysed by pyridine, provides thus the corresponding 3- *O*-protected target in 68% yield after 10 min at 160°C in a microwave reactor (Fig. 4a). However, presence of a side-product is recovered after MW exposure as a result of 5,6- to 3,6 acetal migration prior to acylation. The formation of this unwanted structure is repressed when using supported polystyrene-bases, i.e. *N,N*-(diisopropyl)aminoethylpolystyrene (PS-DIEA) and *N*-(methylpolystyrene)-4-(methylamino)pyridine (PS-DMAP) , as alternatives to pyridine. 79 and 88% yields are thus detected after 15-20 min at 160-180°C in the microwave cavity, whilst only 88% yield is obtained after 300 min at room temperature (Söderberg, 2001).

The regioselective benzoylation of carbohydrates affords convenient results using dibutyltin oxide mediators under microwave exposure (Herradón, 1995). Intermediate formation of dibutylstannylene acetal allows hydroxyl regioselective benzoylation after less than 10 min of microwave exposure with a 500 Watt output (Fig. 4b). This MW methodology is more advantageous than its "classical" analogous involving heating, for several hours, at reflux of a toluene suspension of saccharides and dibutyltin oxide with azeotropic removal of water. Tuning of operation conditions, including both the solvent and the MW power output, modulates the selectivity of dibutylstannylene acetal-mediated benzoylation of representative carbohydrates, such as -D-mannopyranoside (Fig. 5).

Conversion of Carbohydrates Under Microwave Heating 27

+

classical heating conditions, 83% yield of a mixture of geranial and neral sucrose acetals (*E* and *Z* isomers of citral acetals) after 2 min at 100°C. When applying microwave as the heating source, yield drops to 42% after an identical runtime in open vessel conditions. Cleavage of the glycosidic linkage is denoted and leads also to the formation of unwanted

For deprotection processes, microwave heating is decisive and powerful, as a drastic diminution of reaction times is observed without affecting the initial anomeric configuration (Corsaro, 2004). An efficient and green protocol concerns the use of neutral alumina to ensure the regioselective depivaloylation of a set of carbohydrate derivatives (Fig 8a). Whilst the depivaloylation does not occur using a conventional heating source, the regioselective deprotection of primary hydroxyl functions is observed after a few minutes à 75°C under MW assistance (Ley, 1993). The fast, efficient and clean deacetalation of several carbohydrate 4,6‐di‐*O*‐benzylidene acetals is also reported using silica supported reagents under microwave irradiation in solvent-free conditions (Fig. 8b). Yields are superior to 80% after 7 min in a domestic oven at 500 Watt, without anomerisation effect (Couri, 2005).

Al2O3

MW

SiO2 AcOH, H2O

75°C, 6-30 min MW

500 W, 7 min

89%

90%

(*E, Z*) 32-42%

R = 17-26%

**Figure 8.** MW-assisted deprotection initiatives: deacylation (a) and deacetalation (b).

*O*-glycosylation consists in the specific derivatisation of the anomeric hydroxyl position in carbohydrate structures. This reaction is one of the most emblematic in carbohydrate

*4.1.2. O-glycosylations*

side-products (17-26% yield after 2 to 10 min of MW exposure) (Salanski, 1998).

[H+]

RCH(OMe)2

MW, 100°C 2-30 min

**Figure 7.** Protection of sucrose through acetal formation.

(a)

(b)

Toluene, maximum MW power output Toluene, minimum MW power output Acetonitrile, minimum MW power output Yield: 35% 6% 0% 25% 33% 41% 10% 0% 0%

**Figure 5.** MW-promoted benzoylation of -D-mannopyranoside with a tin catalyst.

MW, 9 min uncontrolled T°

TBDPS = *tert*-Butyldiphenylsilyl

For carbohydrates not stable in acid conditions, such as 4,6-*O*-benzylidene--D-glucopyranose, benzoylation can be achieved via transesterification with methyl benzoate using a week and non nucleophilic base promoter like potassium carbonate and a phase transfer agent (Limousin, 1998). MW permits again a tuning of the regioselectivity. Indeed, after 15 min in a domestic microwave oven, a 82% yield of di-benzoylated adduct is recovered, whilst classical oil bath heating affords preferentially the monobenzoylated structures (Fig. 6).

**Figure 6.** Competition between mono- and dibenzoylation with base catalyst.

Due to the sensitivity of some carbohydrate derivatives at elevated temperatures, microwave methodology suffers however from several limitations, notably for hydroxyls protection processes through acetal formation. As an illustration, the mono-acetalation of *O*unprotected sucrose in the presence of *p*-toluenesulfonic acid as the catalyst is reported by Salanski and Queneau (Fig. 7). Using citral dimethylacetal, transacetalation provides, under classical heating conditions, 83% yield of a mixture of geranial and neral sucrose acetals (*E* and *Z* isomers of citral acetals) after 2 min at 100°C. When applying microwave as the heating source, yield drops to 42% after an identical runtime in open vessel conditions. Cleavage of the glycosidic linkage is denoted and leads also to the formation of unwanted side-products (17-26% yield after 2 to 10 min of MW exposure) (Salanski, 1998).

**Figure 7.** Protection of sucrose through acetal formation.

26 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

**Figure 4.** MW-assisted pivaloylation (a) and benzoylation (b).

TBDPS = *tert*-Butyldiphenylsilyl

Bu2SnO, PhCOCl Et3N, solvent

MW, 9 min uncontrolled T°

(b)

Toluene, maximum MW power output Toluene, minimum MW power output Acetonitrile, minimum MW power output

(a)

**Figure 5.** MW-promoted benzoylation of -D-mannopyranoside with a tin catalyst.

Bz = benzoyl (C6H5-CO)-

PivCl, Base

MW

oil bath heating affords preferentially the monobenzoylated structures (Fig. 6).

**Figure 6.** Competition between mono- and dibenzoylation with base catalyst.

BzOCH3, K2CO3 Bu4NBr, MW

For carbohydrates not stable in acid conditions, such as 4,6-*O*-benzylidene--D-glucopyranose, benzoylation can be achieved via transesterification with methyl benzoate using a week and non nucleophilic base promoter like potassium carbonate and a phase transfer agent (Limousin, 1998). MW permits again a tuning of the regioselectivity. Indeed, after 15 min in a domestic microwave oven, a 82% yield of di-benzoylated adduct is recovered, whilst classical

Yield: 35% 6% 0%

Bu2SnO toluene

MW

Piv = pivaloyl (side-product)

25% 33% 41%

MicrowaveR1 = R2 = Bz Classical heating R1= Bz, R2 = H

R1= H, R2= Bz

10% 0% 0%

Due to the sensitivity of some carbohydrate derivatives at elevated temperatures, microwave methodology suffers however from several limitations, notably for hydroxyls protection processes through acetal formation. As an illustration, the mono-acetalation of *O*unprotected sucrose in the presence of *p*-toluenesulfonic acid as the catalyst is reported by Salanski and Queneau (Fig. 7). Using citral dimethylacetal, transacetalation provides, under For deprotection processes, microwave heating is decisive and powerful, as a drastic diminution of reaction times is observed without affecting the initial anomeric configuration (Corsaro, 2004). An efficient and green protocol concerns the use of neutral alumina to ensure the regioselective depivaloylation of a set of carbohydrate derivatives (Fig 8a). Whilst the depivaloylation does not occur using a conventional heating source, the regioselective deprotection of primary hydroxyl functions is observed after a few minutes à 75°C under MW assistance (Ley, 1993). The fast, efficient and clean deacetalation of several carbohydrate 4,6‐di‐*O*‐benzylidene acetals is also reported using silica supported reagents under microwave irradiation in solvent-free conditions (Fig. 8b). Yields are superior to 80% after 7 min in a domestic oven at 500 Watt, without anomerisation effect (Couri, 2005).

**Figure 8.** MW-assisted deprotection initiatives: deacylation (a) and deacetalation (b).

#### *4.1.2. O-glycosylations*

*O*-glycosylation consists in the specific derivatisation of the anomeric hydroxyl position in carbohydrate structures. This reaction is one of the most emblematic in carbohydrate

chemistry and is extensively reviewed (Demchenko, 2008). Several parameters are found to influence the (stereoselective) formation of glycosyl linkages such as solvent, catalyst, additives, nature of both leaving groups and surrounding protecting groups. In particular, the formation of alkyl- and aryl-glycosides has find special attention in both academic and industrial worlds, due to the importance of these molecules as non-toxic and biodegradable surfactants, liquid crystals, and pharmaceutical agents (Razafindralambo 2011 & 2012). Under microwave conditions, the introduction of alkyl substituents on the OH1 position is the most extensively developed, notably for the large-scale production of surface-active agents, and relies on the reaction of an alkyl alcohol with unprotected saccharide (Fischertype glycosylation) or with completely *O*-protected carbohydrate compounds (Helferichtype glycosylation). These glycosylations are catalysed either by chemical promoters or by enzymes. Alkylation of D-glucose by fatty alcohol using acid catalysts is the oldest reaction reported under MW conditions (Fig. 9). Fatty alcohol advantageously plays both the role of solvent and reagent, even if glucose is insoluble in most alcohols. After 10 min of MW exposure with a 200 Watt maximum output, a 15% yield in glycosylated adduct is obtained using homogeneous Brönsted (or Lewis) acids or heterogeneous promoters (zeolites, ion exchange resins, montmorillonites). No control of the temperature is provided using this domestic MW equipment. Such a low yield is to correlate with unwanted glycoside decomposition under acidic conditions during the run and to browning reactions (Limousin, 1997). This result is not completely satisfactory but remains quite noteworthy compared to other classical protocols. The versatility of this MW Fischer glycosylation is now published for various carbohydrates, including mostly easily available monosaccharides like Dmannose, D-galactose, *N-*acetyl-D-glucosamine and *N*-acetyl-D-galactosamine. Glycosylation of representative long chain alcohols is efficient with acidic ion-exchange resins between 90 and 120°C. Typically, the reaction goes to completion after less than 2 min of MW heating, compared with 4h at reflux, and affords preferentially the thermodynamic adduct. The anomeric distribution seems quite identical under MW and conventional oil bath heating conditions (Bornaghi, 2005). A valuable scaling-up of this Fischer-type glycosylation is proposed by Nüchter up to the kg-scale with an efficient improvement of economic efficiency, using an appropriated commercial MW batch reactor (Nüchter, 2001). An accurate control of the temperature inside the reactor cavity provides a good quality result. Practically, monosaccharide (glucose, mannose or galactose) reacts with a 3-30-fold excess of alcohol (methanol, ethanol, butanol or octanol) in the presence of sulphuric acid catalyst. Nearly quantitative yields are recovered after 20-60 min of MW heating at 60-140°C, as a function of the nature of the alcohol. Further assays, using continuous flow methods, afford quantitative yields with a MW chamber heated at 110-140°C. More recent work highlights the use of Brönsted acids immobilised on silica as convenient solid catalysts with advantages in manufacturing scale synthesis (Fig. 9). The Fischer-type glycosylation of Dglucose with *n*-decanol in solvent-less conditions leads to the expected adduct in 82% yield as an equimolar mixture of and anomers (Richel, 2011b). This protocol offers workup, economic and environmental advantages as catalyst can be recovered by filtration and reused.

Conversion of Carbohydrates Under Microwave Heating 29

**Figure 9.** Fischer-type glycosylation of D-glucose with *n*-decanol under MW conditions for the

MW [H+] *n*-decanol

An alternative three-step protocol (complete acylation-glycosylation-deprotection) is proposed under MW. Due to the increased thermal stability of acylated compounds, MW Helferich-type reactions are expected to be more effective (Fig. 10). Indeed, addition of decanol on per-*O*acetylated glucose offers a 74% yield (mainly the -pyranose form) after a running time of 3 min at 113°C using zinc chloride as the catalyst. Extended MW exposure times have a detrimental effect over the yield (25% after 300 min) due to decomposition of decyl glycoside. No rationalisation between the furanose-pyranose (and anomeric) distribution and processing conditions is found at this stage (Limousin, 1997). The first elements of answer are proposed by Kovensky and Ferlin in 2008. A microwave temperature of 115°C is found as a good compromise to obtain high glycosylation rates and low decomposition products contents. Both yields and anomeric distributions are dependent on the boiling/melting points and the polarity of the selected alkyl alcohol. Polar alcohols with low boiling/melting points lead to the formation of the corresponding thermodynamic adduct, while long chain alcohols provide preferentially the kinetic anomer in less than 5 min. For each alcohol, an optimum reaction time, beyond which the formed alkyl glycoside is deteriorated, is estimated (Ferlin, 2008).

**Figure 10.** Helferich-type glycosylation of per-*O*-acetylated glucose with alkyl alcohols. Influence of the

mixture

*n*-octanol 1 min 72% = 56:44

*n*-hexadecanol 5 min 71% = 17:83

Yield > 70%

CnH2n+1

5 min 61% = 82:18

7 min 35% = 88:12

2. Ac2O, Pyridine

1. CnH2n+1OH ZnCl2 115°C 1 -20 min

n = 6 to 16

**Figure 11.** MW-assisted trans-glycosylation of several carbohydrates acceptors and propane-1,2-diol.

MW major

production of a potent surface-active agent.

chain length on both yields andratio.

R = Ph R = 4-*O*-Gluc

**Figure 9.** Fischer-type glycosylation of D-glucose with *n*-decanol under MW conditions for the production of a potent surface-active agent.

reused.

chemistry and is extensively reviewed (Demchenko, 2008). Several parameters are found to influence the (stereoselective) formation of glycosyl linkages such as solvent, catalyst, additives, nature of both leaving groups and surrounding protecting groups. In particular, the formation of alkyl- and aryl-glycosides has find special attention in both academic and industrial worlds, due to the importance of these molecules as non-toxic and biodegradable surfactants, liquid crystals, and pharmaceutical agents (Razafindralambo 2011 & 2012). Under microwave conditions, the introduction of alkyl substituents on the OH1 position is the most extensively developed, notably for the large-scale production of surface-active agents, and relies on the reaction of an alkyl alcohol with unprotected saccharide (Fischertype glycosylation) or with completely *O*-protected carbohydrate compounds (Helferichtype glycosylation). These glycosylations are catalysed either by chemical promoters or by enzymes. Alkylation of D-glucose by fatty alcohol using acid catalysts is the oldest reaction reported under MW conditions (Fig. 9). Fatty alcohol advantageously plays both the role of solvent and reagent, even if glucose is insoluble in most alcohols. After 10 min of MW exposure with a 200 Watt maximum output, a 15% yield in glycosylated adduct is obtained using homogeneous Brönsted (or Lewis) acids or heterogeneous promoters (zeolites, ion exchange resins, montmorillonites). No control of the temperature is provided using this domestic MW equipment. Such a low yield is to correlate with unwanted glycoside decomposition under acidic conditions during the run and to browning reactions (Limousin, 1997). This result is not completely satisfactory but remains quite noteworthy compared to other classical protocols. The versatility of this MW Fischer glycosylation is now published for various carbohydrates, including mostly easily available monosaccharides like Dmannose, D-galactose, *N-*acetyl-D-glucosamine and *N*-acetyl-D-galactosamine. Glycosylation of representative long chain alcohols is efficient with acidic ion-exchange resins between 90 and 120°C. Typically, the reaction goes to completion after less than 2 min of MW heating, compared with 4h at reflux, and affords preferentially the thermodynamic adduct. The anomeric distribution seems quite identical under MW and conventional oil bath heating conditions (Bornaghi, 2005). A valuable scaling-up of this Fischer-type glycosylation is proposed by Nüchter up to the kg-scale with an efficient improvement of economic efficiency, using an appropriated commercial MW batch reactor (Nüchter, 2001). An accurate control of the temperature inside the reactor cavity provides a good quality result. Practically, monosaccharide (glucose, mannose or galactose) reacts with a 3-30-fold excess of alcohol (methanol, ethanol, butanol or octanol) in the presence of sulphuric acid catalyst. Nearly quantitative yields are recovered after 20-60 min of MW heating at 60-140°C, as a function of the nature of the alcohol. Further assays, using continuous flow methods, afford quantitative yields with a MW chamber heated at 110-140°C. More recent work highlights the use of Brönsted acids immobilised on silica as convenient solid catalysts with advantages in manufacturing scale synthesis (Fig. 9). The Fischer-type glycosylation of Dglucose with *n*-decanol in solvent-less conditions leads to the expected adduct in 82% yield as an equimolar mixture of and anomers (Richel, 2011b). This protocol offers workup, economic and environmental advantages as catalyst can be recovered by filtration and

An alternative three-step protocol (complete acylation-glycosylation-deprotection) is proposed under MW. Due to the increased thermal stability of acylated compounds, MW Helferich-type reactions are expected to be more effective (Fig. 10). Indeed, addition of decanol on per-*O*acetylated glucose offers a 74% yield (mainly the -pyranose form) after a running time of 3 min at 113°C using zinc chloride as the catalyst. Extended MW exposure times have a detrimental effect over the yield (25% after 300 min) due to decomposition of decyl glycoside. No rationalisation between the furanose-pyranose (and anomeric) distribution and processing conditions is found at this stage (Limousin, 1997). The first elements of answer are proposed by Kovensky and Ferlin in 2008. A microwave temperature of 115°C is found as a good compromise to obtain high glycosylation rates and low decomposition products contents. Both yields and anomeric distributions are dependent on the boiling/melting points and the polarity of the selected alkyl alcohol. Polar alcohols with low boiling/melting points lead to the formation of the corresponding thermodynamic adduct, while long chain alcohols provide preferentially the kinetic anomer in less than 5 min. For each alcohol, an optimum reaction time, beyond which the formed alkyl glycoside is deteriorated, is estimated (Ferlin, 2008).

**Figure 10.** Helferich-type glycosylation of per-*O*-acetylated glucose with alkyl alcohols. Influence of the chain length on both yields andratio.

**Figure 11.** MW-assisted trans-glycosylation of several carbohydrates acceptors and propane-1,2-diol.

Enzymatic methods under microwave conditions are also explored as convenient option for glycosylations. Using -glucosidase, the synthesis of several glycosides by trans-glycosylation is reported with either phenyl -D-glucoside or cellobiose as donors and propane-1,2-diol as acceptor (Fig. 11). The process is performed in dry-media conditions, with reagents adsorbed on alumina in an open vessel system. This microwave reaction, achieved at controlled temperature (between 80 and 110°C), leads to noticeable advantages, in terms of yields and selectivity, when compared to classical heating. In particular, trans-glycosylations go to completion after less than 2h, while hydrolysis is lowered to 10% (Gelo-Pujic, 1997).

Conversion of Carbohydrates Under Microwave Heating 31

Beside the benchmark Fischer- and Helferich-glycosylations, novel strategies under microwave heating are nowadays emerging in the peer-reviewed literature. Some examples are proposed in Fig. 12. Most of them concern the catalytic conjugation of alcohols (designated as acceptors) to protected 4-pentenyl glycosides or methyl glycosides (donors), notably for the production of di- and oligosaccharides. As an illustration, the synthesis of disaccharides is described in acetonitrile starting from 4-pentenyl donors and is achieved in high yields after less than 25 min at 100°C in closed vessel conditions without side-products formation (Fig. 12a) (Mathew, 2004). The use of more effective Lewis acids, like ytterbium(III) triflate, affords high yields for the anchorage of long alkyl chains on benzoylated glucose derivatives (Fig 12b). This acetal-exhange type glycosylation is performed in dichloromethane below 100°C in closed vessel conditions. This constitutes the main advantage of microwave over conventional heating techniques (Yoshimura, 2005). Application of very low temperatures (-10°C) within the MW cavity, attained by simultaneous cooling, offers a convenient approach for the synthesis of valuable saccharides and Lewis X oligosaccharides (Fig. 12c). The process involves a fucosyl acetate donor and a glucosamine acceptor in about 2h in ether. Yields culminate at about 80-90% without side-products formation, whilst the expected disaccharide is not obtained using classical protocols (Shimizu, 2008). Another strategy demonstrates the usefulness of microwave heating, in synergy with heterogeneous acid catalysts, for the regioselective functionalisation of D-glucuronic acid. Conversions are quantitative when performed in solvent-less conditions with an excess of alcohol under microwave exposure. Less than 10 min of heating at 85°C in closed vessel conditions affords selectively the corresponding -glucurono-6,3-lactone adduct. When heating in an oil bath, only the disubstituted adducts, as a mixture of -furanoses and -pyranoses, are formed through competitive esterification and *O*-glycosylation processes (Fig. 12d). Comparative yields are recorded after 6 to 24 h at 85°C, evidencing that MW accelerates reactions and imparts over the selectivity. This methodology is eco-friendly as water in the only by-product and the solid catalyst can be reused for consecutive batch MW runs (Richel, 2012). These monosubstituted glucuronolactones are not easily attainable using other conventional protocols and required multi-step synthesis. These original products find special relevance in

the field of surface-active agents and emulsifiers (Razafindralambo, 2011).

Glycosamines are valuable compounds as glyco-amino acid building blocks for glycopeptides synthesis. Their preparation is traditionally achieved using a Kochetkov reductive amination procedure. Practically, a treatment at room temperature for 6 days of saccharides in aqueous solution with 50 equiv of ammonium bicarbonate affords the expected -glycosaminated adducts in high yields. Another route involves the heating at 42°C for 36 h of mono- or disaccharides with an aqueous solution of ammonia in the presence of one equivalent of ammonium hydrogen carbonate. However, -glycosamines can be deteriorated under aqueous solutions, even with accurate pH control. Nowadays, the preparation of these glycosamines is typically performed under microwave conditions, at 40°C for 90 min, by selective amination of reductive carbohydrates using reduced quantities

*4.1.3. Glycosamines synthesis*

**Figure 12.** Novel microwave-assisted glycosylations.

Beside the benchmark Fischer- and Helferich-glycosylations, novel strategies under microwave heating are nowadays emerging in the peer-reviewed literature. Some examples are proposed in Fig. 12. Most of them concern the catalytic conjugation of alcohols (designated as acceptors) to protected 4-pentenyl glycosides or methyl glycosides (donors), notably for the production of di- and oligosaccharides. As an illustration, the synthesis of disaccharides is described in acetonitrile starting from 4-pentenyl donors and is achieved in high yields after less than 25 min at 100°C in closed vessel conditions without side-products formation (Fig. 12a) (Mathew, 2004). The use of more effective Lewis acids, like ytterbium(III) triflate, affords high yields for the anchorage of long alkyl chains on benzoylated glucose derivatives (Fig 12b). This acetal-exhange type glycosylation is performed in dichloromethane below 100°C in closed vessel conditions. This constitutes the main advantage of microwave over conventional heating techniques (Yoshimura, 2005). Application of very low temperatures (-10°C) within the MW cavity, attained by simultaneous cooling, offers a convenient approach for the synthesis of valuable saccharides and Lewis X oligosaccharides (Fig. 12c). The process involves a fucosyl acetate donor and a glucosamine acceptor in about 2h in ether. Yields culminate at about 80-90% without side-products formation, whilst the expected disaccharide is not obtained using classical protocols (Shimizu, 2008). Another strategy demonstrates the usefulness of microwave heating, in synergy with heterogeneous acid catalysts, for the regioselective functionalisation of D-glucuronic acid. Conversions are quantitative when performed in solvent-less conditions with an excess of alcohol under microwave exposure. Less than 10 min of heating at 85°C in closed vessel conditions affords selectively the corresponding -glucurono-6,3-lactone adduct. When heating in an oil bath, only the disubstituted adducts, as a mixture of -furanoses and -pyranoses, are formed through competitive esterification and *O*-glycosylation processes (Fig. 12d). Comparative yields are recorded after 6 to 24 h at 85°C, evidencing that MW accelerates reactions and imparts over the selectivity. This methodology is eco-friendly as water in the only by-product and the solid catalyst can be reused for consecutive batch MW runs (Richel, 2012). These monosubstituted glucuronolactones are not easily attainable using other conventional protocols and required multi-step synthesis. These original products find special relevance in the field of surface-active agents and emulsifiers (Razafindralambo, 2011).

#### *4.1.3. Glycosamines synthesis*

30 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

**Figure 12.** Novel microwave-assisted glycosylations.

 Using -glucosidase, the synthesis of several glycosides by trans-glycosylation is reported with either phenyl -D-glucoside or cellobiose as donors and propane-1,2-diol as acceptor (Fig. 11). The process is performed in dry-media conditions, with reagents adsorbed on alumina in an open vessel system. This microwave reaction, achieved at controlled temperature (between 80 and 110°C), leads to noticeable advantages, in terms of yields and selectivity, when compared to classical heating. In particular, trans-glycosylations go to

completion after less than 2h, while hydrolysis is lowered to 10% (Gelo-Pujic, 1997).

O

Glycosamines are valuable compounds as glyco-amino acid building blocks for glycopeptides synthesis. Their preparation is traditionally achieved using a Kochetkov reductive amination procedure. Practically, a treatment at room temperature for 6 days of saccharides in aqueous solution with 50 equiv of ammonium bicarbonate affords the expected -glycosaminated adducts in high yields. Another route involves the heating at 42°C for 36 h of mono- or disaccharides with an aqueous solution of ammonia in the presence of one equivalent of ammonium hydrogen carbonate. However, -glycosamines can be deteriorated under aqueous solutions, even with accurate pH control. Nowadays, the preparation of these glycosamines is typically performed under microwave conditions, at 40°C for 90 min, by selective amination of reductive carbohydrates using reduced quantities of ammonium bicarbonate (5 equiv) (Fig. 13). Under closed vessels conditions, the reaction is achieved in methanol or in anhydrous dimethylsulfoxide to prevent degradation and side-products formation (Liu, 2010). Yields are of about 70-95%.

Conversion of Carbohydrates Under Microwave Heating 33

O O OH O O

> 63 % 47 %

> 16 % 67 %

> 54 % 62 %

> 45 % 52 %

47 % 25 %

31 % 23 %

+

O H OH

O

CO2H

Rehydration

(a) HCl, H2O, 200°C, 1 sec (b) H2SO4, H2O, 200°C, 5 min

("microwave hot compressed water process"), while 5-HMF yield and fructose conversion are respectively of 12 and 27% after an identical runtime using a sand bath heating. Zirconium oxide demonstrates great performances for the catalytic dehydration of glucose, favouring isomerisation of glucose to fructose prior dehydration (Qi, 2009). When using an acid ion-exchange resin in an acetone/dimethylsulfoxide system, improvement in 5-HMF selectivity is encountered together with high yields (98% fructose conversion with 5-HMF selectivity of 92% for 10 min MW assisted reaction at 150°C in closed vessel conditions). Addition of low-boiling point acetone improves products separation and reduces adverse environmental impacts. The same results are published for strong ion-exchange resin in acetone-water systems, providing a truly eco-friendly process (Qi, 2008b). The common Lewis acid catalyst, aluminium(III) chloride, promotes also efficiently the dehydration of carbohydrate substrates, in water-organic solvents and in water. 5-HMF yield is higher when using an organic solvent than in pure water. Involvement of ionic liquids as convenient media for the conversion of D-glucose in 5-HMF is effective using chromium(III) chloride as a catalyst. With microwave heating at 140°C for 30 sec, a 5-HMF yield of 71% is obtained for 96% glucose in 1-butyl-3-methyl imidazolium chloride as the solvent (Qi, 2010). Lignin-based solid catalyst, produced by carbonisation and sulfonation of raw lignocellulosic materials, seems nowadays to be the most active catalyst for the dehydration of carbohydrates. In synergy with MW, 84% 5-HMF yield is obtained with 98% fructose conversion rate at 110°C for 10°C. A mixture of dimethylsulfoxide-ionic liquid has a beneficial effect over the selectivity of the process. Interestingly, the catalyst can be

recovered after the reaction and reused for consecutive batch reactions (Guo, 2012).

 Overview of MW-assisted production of 5-HMF from lignocellulosic biomass materials.

**Figure 13.** Synthesis of 1-amino-1-deoxy--D-lactoside by microwave-assisted Kochetkov amination of unprotected lactose.

In summary, reactions of the first category, involving hydroxyls manipulation appear generally faster under MW conditions than their "classical" counterparts. Expected products are indeed obtained in good yields after only a few minutes. The use of eco-friendly and non-toxic (heterogeneous) and milder catalysts in synergy with microwaves, affords ideal "green chemical" conditions and minimise workup. Microwave allows also the formation of specific protected products, not easily accessible under conventional "heating" conditions.

#### **4.2. Microwave-assisted production of platform chemicals (category 2)**

The production of 5-HMF (5-hydroxymethylfurfural), a five-membered ring compounds ranging in the US Top 10 of most valuable chemicals issued from biomass, is currently extensively investigated worldwide. Most research concerns its production via thermallyinduced acid-catalysed dehydration of fructose, typically in dimethylsulfoxide, in the presence of homogeneous acid catalysts (HCl, H2SO4, H3PO4, oxalic or levulinic acids) (Fig. 14). Reactions produces selectively 40-60% yields of 5-HMF with complete conversion of starting sugar materials but suffers from drawbacks in terms of acids recovery and equipment corrosion. Heterogeneous catalysts offer workup and environmental advantages associated to high 5-HMF selectivity but provide low fructose conversions (30-60% only after up to 2h). Microwave has progressively emerged as an attractive heating source allowing energy savings and improved yields and selectivity. Recent developments in the field have been reviewed by Richel in 2011 (Richel, 2011b). They include the use of heterogeneous catalysts in various media (organic solvents, water, ionic liquids or biphasic systems).

**Figure 14.** Catalytic dehydration of fructose for the production of 5-HMF.

Titanium and zirconium oxides, as sulphated zirconia, are effective catalysts for this dehydration, especially under microwave conditions. Yields in 5-HMF reach 35% for a fructose conversion of 78% after only 5 min heating at 200°C in closed vessels conditions ("microwave hot compressed water process"), while 5-HMF yield and fructose conversion are respectively of 12 and 27% after an identical runtime using a sand bath heating. Zirconium oxide demonstrates great performances for the catalytic dehydration of glucose, favouring isomerisation of glucose to fructose prior dehydration (Qi, 2009). When using an acid ion-exchange resin in an acetone/dimethylsulfoxide system, improvement in 5-HMF selectivity is encountered together with high yields (98% fructose conversion with 5-HMF selectivity of 92% for 10 min MW assisted reaction at 150°C in closed vessel conditions). Addition of low-boiling point acetone improves products separation and reduces adverse environmental impacts. The same results are published for strong ion-exchange resin in acetone-water systems, providing a truly eco-friendly process (Qi, 2008b). The common Lewis acid catalyst, aluminium(III) chloride, promotes also efficiently the dehydration of carbohydrate substrates, in water-organic solvents and in water. 5-HMF yield is higher when using an organic solvent than in pure water. Involvement of ionic liquids as convenient media for the conversion of D-glucose in 5-HMF is effective using chromium(III) chloride as a catalyst. With microwave heating at 140°C for 30 sec, a 5-HMF yield of 71% is obtained for 96% glucose in 1-butyl-3-methyl imidazolium chloride as the solvent (Qi, 2010). Lignin-based solid catalyst, produced by carbonisation and sulfonation of raw lignocellulosic materials, seems nowadays to be the most active catalyst for the dehydration of carbohydrates. In synergy with MW, 84% 5-HMF yield is obtained with 98% fructose conversion rate at 110°C for 10°C. A mixture of dimethylsulfoxide-ionic liquid has a beneficial effect over the selectivity of the process. Interestingly, the catalyst can be recovered after the reaction and reused for consecutive batch reactions (Guo, 2012).

32 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

side-products formation (Liu, 2010). Yields are of about 70-95%.

unprotected lactose.

of ammonium bicarbonate (5 equiv) (Fig. 13). Under closed vessels conditions, the reaction is achieved in methanol or in anhydrous dimethylsulfoxide to prevent degradation and

**Figure 13.** Synthesis of 1-amino-1-deoxy--D-lactoside by microwave-assisted Kochetkov amination of

80%, only

MW 40°C, 90 min

(NH4)2CO3 DMSO or MeOH

In summary, reactions of the first category, involving hydroxyls manipulation appear generally faster under MW conditions than their "classical" counterparts. Expected products are indeed obtained in good yields after only a few minutes. The use of eco-friendly and non-toxic (heterogeneous) and milder catalysts in synergy with microwaves, affords ideal "green chemical" conditions and minimise workup. Microwave allows also the formation of specific protected products, not easily accessible under conventional "heating" conditions.

The production of 5-HMF (5-hydroxymethylfurfural), a five-membered ring compounds ranging in the US Top 10 of most valuable chemicals issued from biomass, is currently extensively investigated worldwide. Most research concerns its production via thermallyinduced acid-catalysed dehydration of fructose, typically in dimethylsulfoxide, in the presence of homogeneous acid catalysts (HCl, H2SO4, H3PO4, oxalic or levulinic acids) (Fig. 14). Reactions produces selectively 40-60% yields of 5-HMF with complete conversion of starting sugar materials but suffers from drawbacks in terms of acids recovery and equipment corrosion. Heterogeneous catalysts offer workup and environmental advantages associated to high 5-HMF selectivity but provide low fructose conversions (30-60% only after up to 2h). Microwave has progressively emerged as an attractive heating source allowing energy savings and improved yields and selectivity. Recent developments in the field have been reviewed by Richel in 2011 (Richel, 2011b). They include the use of heterogeneous

**4.2. Microwave-assisted production of platform chemicals (category 2)**

catalysts in various media (organic solvents, water, ionic liquids or biphasic systems).

Titanium and zirconium oxides, as sulphated zirconia, are effective catalysts for this dehydration, especially under microwave conditions. Yields in 5-HMF reach 35% for a fructose conversion of 78% after only 5 min heating at 200°C in closed vessels conditions

[H+] - 3H2O

**Figure 14.** Catalytic dehydration of fructose for the production of 5-HMF.

H

**Figure 15.** Overview of MW-assisted production of 5-HMF from lignocellulosic biomass materials.

Scaling up of the MW-assisted fructose dehydration process has been recently evaluated using a stop-flow MW reactor system and compared to a continuous cartridge-based reactor. Results highlight that 5-HMF yield can attain 86% yield after 2min of MW exposure (91% using a cartridge-base reactor) with a 5-HMF productivity of 0.72 g/h (versus 2.07 g/h for the continuous process). Even if quite less productive, the MW-assisted methodology is more selective and cleaner as no side-product is detected in the final mixture (Schoen, 2011).

Conversion of Carbohydrates Under Microwave Heating 35

Bozell, J. J.; Petersen, G. R. (2010). Technology development for the production of biobased products from biorefinery carbohydrates—the US Department of Energy's "Top 10"