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About this book

This unique book covers the latest developments in coupling and decoupling of biomolecules containing functionalized carbohydrate components, being one of the first collections in this important area of applied medicinal chemistry.

Connecting molecules, often referred as bio-conjugation, has become one of the most often performed procedures in modern medicinal chemistry. Sometimes, when the connected molecules are not useful anymore, they must be disconnected. The molecules that must be connected (coupled) may belong to both small and large molecules and include such constructs as glycoproteins, glycopeptides and glycans.

In this work, more than 15 experts address a comprehensive range of potential and current uses of in vitro and in vivo bio-conjugation methodologies, leading to a variety of glycoconjugates. The analytical aspects of bio-conjugation are also here discussed. Medicinal and organic chemists from graduate level onwards will understand the appeal of this important book.

Table of Contents

Frontmatter

Synthesis of N-Linked Glycopeptides Using Convergent Enzymatic Glycosylation Combined with SPPS

Glycosylation of peptides and proteins has emerged as a promising strategy to improve the pharmacokinetic profile of peptide- and protein-based therapeutics. The synthesis of pure homogeneous N-linked glycopeptides and glycoproteins is a challenging task, and efficient routes to access them are in high demand. Endo-β-N-acetylglucosaminidise catalysed glycosylation of N-acetylglucosamine-tagged peptides, using activated oligosaccharide oxazolines as donors, has recently attracted attention due to the relative simplicity by which the process convergently affords glycoconjugates with complete control of stereo- and regioselectivity. Herein, a brief review of some examples of recent enzyme-mediated N-glycosylation used to synthesise glycopeptides with therapetic potential is provided.
Renata Kowalczyk, Harveen Kaur, Antony J. Fairbanks, Margaret A. Brimble

Synthetic Antitumor Vaccines Through Coupling of Mucin Glycopeptide Antigens to Proteins

The requirements for coupling reactions of carbohydrate molecules very much depend upon the biological recognition processes that should be investigated and upon the target structures of the desired carbohydrate ligand. If the carbohydrate conjugate itself is the recognized ligand, as for example, the binding site of a P-selectin ligand comprising sialyl-LewisX and a specific peptide sequence, the natural glycoside bond must be installed. A stereoselective and regioselective block glycosylation between a sialyl-LewisX trichloroacetimidate and a partially deprotected Thomsen–Friedenreich antigen derivative was developed to achieve this aim. In contrast, the coupling reactions by which glycopeptides from tumor-associated glycoproteins are conjugated to immune stimulating components in order to afford efficient vaccines can entail artificial linkages as long as they do not interfere with the immune reactions. For example, the coupling of glycophorin glycopeptides to bovine serum albumin was successfully achieved by carboxylic activation with a water-soluble carbodiimide in the presence of a supernucleophilic additive. This conjugation method is only recommendable if the glycopeptide does not contain several carboxylic and/or amino functions. The photochemically or radical initiator promoted thiol-ene coupling succeeded in couplings of MUC1 glycopeptide antigens to bovine serum albumin, however, is accompanied by oxidative disulfide formation. The conjugation of glycopeptide antigens from the tandem repeat region of the tumor-associated mucin MUC1 to bovine serum albumin or tetanus toxoid is efficiently accomplished using diethyl squarate as the coupling reagent. The intermediate squaric monoamide esters can be isolated and characterized, and then applied to a mild connecting process to the carrier proteins. The MUC1 glycopeptide-tetanus toxoid conjugates proved to be particularly useful vaccines. They induce extraordinarily strong immune responses in mice. The induced antibodies are prevailingly of the IgG1 isotype and show efficient binding to the glycoproteins exposed on epithelial tumor cells.
Markus Glaffig, Horst Kunz

Recent Advances in the Stereochemical Outcome of Multicomponent Reactions Involving Convertible Isocyanides

Multi-component reactions (MCRs) have become an integral part of organic synthesis as short and very efficient routes to molecular diversity with included varying stereochemistry. MCRs have evolved rapidly in terms of the components used in the reactions and their role on the stereo chemical outcomes. This chapter focuses on covering recent contributions towards MCRs including targeted asymmetric control. Furthermore, advances in MCRs, as reported by many researchers, covering the utility of the convertible isocyanides, stereochemical advances of the Passerini and Ugi reactions and finally, the use of carbohydrates as chiral auxiliaries are examined and discussed.
Krishnakant Patel, Peter R. Andreana

Glycoconjugate-Based Inhibitors of Mycobacterium Tuberculosis GlgE

Tuberculosis (TB) is the leading cause of death globally as a result of a single infectious disease. A staggering 6 million new cases were reported in the 2014. In order to eradicate the ongoing threat of TB and combat rising rates of TB drug resistance new therapeutics must be developed. In this chapter, we briefly review the history of TB, Mycobacterium tuberculosis (Mtb) cell wall structure, the enzymes involved in synthesizing cell wall, and the trehalose utilization pathways (TUP). We focus on the recent discovery of enzyme Mtb GlgE, a glycosyl hydrolase-like phosphorylase, which has been found to be essential for Mtb viability and the ongoing efforts to design inhibitors against this target.
Sri Kumar Veleti, Steven J. Sucheck

Selective Transformations of the Anomeric Centre in Water Using DMC and Derivatives

2-Chloro-1,3-dimethylimidazolinium chloride (DMC) and its derivatives are useful for numerous synthetic transformations, which involve selective activation of the anomeric centre of unprotected reducing sugars in water. This chapter summarises research reported to date using DMC and derivatives, such as 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADMP). DMC has been successfully employed for the synthesis of glycosyl oxazolines, 1,6-anhydro-, 1-azido-, and a variety of thioglycosides. The use of ADMP allows the one-pot synthesis of glycosyl triazoles in water via the Cu-catalysed azide-alkyne Huisgen cycloaddition reaction. This latter methodology can be applied to a wide variety of carbohydrates and is also amenable to convergent glycopeptide synthesis in which oligosaccharides are directly conjugated to peptides that contain propargyl glycine residues. Such protecting group free methodologies, particularly when applied to complex oligosaccharides isolated from natural sources, may allow ready access to a wide variety of biologically interesting glycoconjugates.
David Lim, Antony J. Fairbanks

[3, 3]-Sigmatropic Rearrangement as a Powerful Synthetic Tool on Skeletal Modification of Unsaturated Sugars

The development of practical, atom-economical and stereocontrolled routes to modify monosaccharides occupy a central importance in synthetic carbohydrate chemistry. This review provides an account on carbon–carbon and carbon–heteroatom bond formation as a skeleton modification on monosaccharides, through [3, 3]-sigmatropic rearrangement reactions on unsaturated sugar synthons, the generality of the reaction conditions and synthetic utilization of the resulting functionalized sugar building blocks. Major emphasis is laid on thermal rearrangement reactions, namely, Claisen, Ireland–Claisen, aza–Claisen and Johnson–Claisen rearrangements on carbohydrate-derived allyl vinyl ethers, silyl ketene acetals, allylic trichloroacetamidates and allylic orthoesters, respectively. These reactions offer a very promising prospect and permit a straightforward approach to access a large variety of biologically important, densely functionalized and novel carbohydrate mimetics. Further, examples of bioactive complex natural products, secured in high yields and profound stereoselectivity through such thermal rearrangement reactions on monosaccharides are described herein.
Gour Chand Daskhan, Malyasree Giri, Narayanaswamy Jayaraman

Recognition of Thiols in Coupling Reactions to Organic and Carbohydrate Acceptors

Reactions of thiols in thio-click coupling processes with various reactive systems (including carbohydrates) are compiled. A selection of simple and complex thiols in stereoselective and non-stereoselective approaches recognizing their reactivity is also reviewed. Solvents, employed in the discussed processes including water, are briefly discussed as well.
Zbigniew J. Witczak, Roman Bielski

Chemical Approaches Towards Neurodegenerative Disease Prevention: The Role of Coupling Sugars to Phenolic Biomolecular Entities

Polyphenols are natural molecular entities exhibiting a wide variety of bioactivities including anticholinergic and/or antiamyloidogenic activities. Their low solubility is recognized as a key factor for bioavailability and their glycosylation is indeed relevant to improve the bioaccess to these molecules. In this chapter, chemical and enzymatic syntheses of glycosylated flavonoids, stilbenoids, phenylethanoids and phenylpropanoids are illustrated, covering examples that demonstrate the impact of coupling sugars to bioactive aglycones in their bioavailability and in their pharmacological activity. The chapter is focused particularly on glycosyl polyphenols with promising activities against neurodegenerative impairments, given their potential to intervene in biological processes that cause catastrophic diseases, namely the Alzheimer’s disease.
Catarina Dias, Ana M. Matos, Amélia P. Rauter

Probing for Trypanosoma cruzi Cell Surface Glycobiomarkers for the Diagnosis and Follow-Up of Chemotherapy of Chagas Disease

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease in humans. Linear and branched O-glycans with non-reducing, terminal α-galactosyl (α-Gal) glycotopes located on cell surface glycosylphosphatidylinositol (GPI)-anchored mucins of the infective trypomastigote form of the parasite are foreign to humans and elicit high levels of anti-α-Gal antibodies in Chagas disease patients (Ch anti-α-Gal antibodies). These antibodies have the capability to lyse the parasite in a complement-dependent or -independent manner. Ch anti-α-Gal antibodies have a considerably higher reactivity to the parasitic surface α-Gal glycotopes than the normal human serum (NHS) anti-α-Gal antibodies, which are present in every healthy human being. A series of ten mercaptopropyl saccharides with α-Gal moieties at the non-reducing end, all connected to another galactose unit, and five non-α-Gal-containing glycan controls were synthesized, and conjugated to maleimide-derivatized bovine serum albumin. This produced neoglycoproteins (NGPs), which were assembled into glycoarrays for the interrogation with sera of chronic Chagas disease patients and healthy individuals using chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA). This study identified the terminal Galα(1,3)Galβ disaccharide as an immunodominant T. cruzi glycotope and biomarker, which shows a considerable binding differential between Ch and NHS anti-α-Gal antibodies. Therefore, this glycotope is suitable for the diagnosis of Chagas disease, and could also be potentially used for follow-up studies for the effectiveness of chemotherapy in Chagas disease patients.
Nathaniel S. Schocker, Susana Portillo, Roger A. Ashmus, Carlos R. N. Brito, Igor E. Silva, Yanira Cordero Mendoza, Alexandre F. Marques, Erika Y. Monroy, Andrew Pardo, Luis Izquierdo, Montserrat Gállego, Joaquim Gascon, Igor C. Almeida, Katja Michael

Syntheses and Functions of Glycosaminoglycan Mimicking Polymers

Glycosaminoglycans (GAGs) are important polysaccharides in the living system. Though total syntheses of GAGs oligosaccharides have been reported, it is still difficult to obtain GAGs. In this investigation, GAGs mimetics were prepared by polymerization of vinyl sugars instead of total synthesis. Glycopolymers are polymers with pendant saccharides, and exhibit a strong molecular recognition due to the multivalency. GAGs mimicking glycopolymers were prepared by polymerizing acrylamide derivatives carrying sulfated N-acetyl glucosamine (GlcNAc). The polymer interactions with proteins were investigated. The glycopolymer libraries were prepared with varying molecular weight, sugar structure, and sugar ratios. The GAG glycopolymers with biodegradable backbone, dendrimer, and arrays were also prepared. The inhibitory activity of Alzheimer amyloid beta peptides by the glycopolymers was investigated in detail. The glycopolymer with sulfated GlcNAc inhibited the aggregation of amyloid beta (Aβ) and the multivalency of sulfated GlcNAc was the key of the interaction. The activity depends on the chemical structure of glycopolymers. Also, the sulfated saccharide function was correlated to the functions of native GAGs.
Yoshiko Miura, Tomohiro Fukuda, Hirokazu Seto, Yu Hoshino

Methods for the High Resolution Analysis of Glycoconjugates

Glycans and their conjugates form the largest and most diverse class of biological molecules found in nature. These glycosides are vital for numerous cellular functions including recognition events, protein stabilisation and energy storage. Additionally, abnormalities within these structures are associated with a wide range of disease states. As a result, robust analytical techniques capable of in depth characterisation of carbohydrates and their binding partners are required. This chapter provides an overview of currently used analytical techniques, focussing on chromatographic and mass spectrometry-based methods.
Christopher Gray, Sabine L. Flitsch

Masking Strategies for the Bioorthogonal Release of Anticancer Glycosides

Significant progress in the bioorthogonal field has resulted in the advent of a new type of prodrug: bioorthogonal prodrugs, i.e. metabolically stable precursors of therapeutic agents that are specifically activated by non-native, non-biological, non-perturbing physical or chemical stimuli. The application of such unique drug precursors in conjunction with their corresponding activating source is under preclinical experimentation as a novel way to elicit site-specific activation of cytotoxic drugs, with particular emphasis on anticancer glycosides. In this chapter, the strategies developed for the masking and bioorthogonal release of cytotoxic nucleosides using benign electromagnetic radiations, biocompatible click chemistry and bioorthogonal organometallic (BOOM) catalysis will be discussed in detail.
Belén Rubio-Ruiz, Thomas L. Bray, Ana M. López-Pérez, Asier Unciti-Broceta

Example of Sacrificial Unit Using Two Different Click Reactions in Coupling and Decoupling (CAD) Chemistry

An example of a specific coupling and decoupling (CAD) chemistry is described. It takes advantage of propargyl acrylate as a sacrificial unit (SU). The addition of a selected compound representing a molecular unit equipped with an azide functionality to the terminal triple bond of the SU and another compound acting as a molecular unit equipped with a thiol functionality to the conjugated double bond of the SU proceeded at very good yields. The construct containing two molecular units can be decoupled using a few different reactions and the decoupling can take place at two positions.
Roman Bielski, Zbigniew J. Witczak, Donald Mencer

Increased Efficacy of NKT Cell-Adjuvanted Peptide Vaccines Through Chemical Conjugation

Through vaccination infectious diseases such as smallpox, polio, measles, and tetanus have either been eradicated or significantly restricted. However, there remain many diseases for which no effective vaccine exists, and therefore new vaccine approaches are still needed. Current vaccine approaches that generate strong immune responses are often based on ill-defined immunogens such as heat-killed or live-attenuated biological products that suffer from concerns related to safety, stability, and lengthy or complex manufacturing processes. For these reasons, there is a strong push toward vaccines that elicit immune responses to defined structures within the targeted pathogen or tissue, which can be achieved by injecting defined antigenic proteins or peptides. On their own, proteins or peptides are generally poorly immunogenic and they must be combined with immune stimulants known as adjuvants to drive antigen-specific immune responses. Recent studies have shown that the direct conjugation of adjuvant compounds to protein or peptide antigens can enhance the magnitude and quality of induced immune responses. In this chapter, we will discuss the chemical approaches our group has used to synthesize a new class of vaccines based on conjugation of peptides with lipid structures that activate innate-like T cells. The stimulatory milieu created by these structures helps drive potent T cell-mediated immune responses that can prevent infectious disease, or can act therapeutically in noncommunicable conditions as diverse as cancer and allergy.
Colin M. Hayman, Ian F. Hermans, Gavin F. Painter
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