Carbohydrates are the most abundant group of natural products found on the earth. Aside from their important roles in supplying energy to cells and structural support to plants, carbohydrates are implicated in a vast array of biological processes. These include hormonal activities, fertilization, embryogenesis, neural development, and many other cellular processes such as cell-cell recognition, cell proliferation, cellular transport, viral infection, bacterial adhesion, and tumour cell metastasis.106 Thus, it is not surprising that
the efficient synthesis of saccharides and their incorporation into various systems to obtain specific biological effects has attracted much attention in the past few decades. One such example is the tremendous effort that has been devoted to enhancing the multivalency of carbohydrates by incorporating them onto multivalent architectures such as dendrimers. It is known that multivalent interactions are prevalent in biology, such as in the adhesion of viruses and bacteria to cell surfaces and in the binding of cells to other cells.107 Many of these processes involve the interactions of carbohydrates with protein receptors called lectins. While the interactions of individual carbohydrate ligands with lectins is often weak, multivalency provides a means of significantly increasing the strength of the interaction.107
A wide variety of nanoscale materials such as linear and HBPs, nanoparticles, and polymer assemblies can be used as backbones to present carbohydrates in a multivalent manner,108-111 but the well-defined nature of the dendrimer backbone provides advantages. For example, the number of carbohydrate ligands present on a given molecule can be precisely determined, allowing advancements in the fundamental understanding of carbohydrate-lectin interactions. In addition, the product monodispersity and reproducibility in its synthesis is advantageous for the development of a clinical therapeutic. A wide range of saccharides including mannose, galactose, glucose, lactose, maltose, xylose, N-acetylneuraminic acid (Neu5Ac) (sialic acid), and other oligosaccharides have been conjugated to various dendrimer peripheries via different linkages such as amide, hydrazide, amine, thioether, thriourea, and triazole linkages.112 Unlike dendrimer-drug conjugates, in which fine-tuned lability of the linkage is essential for controlled release of the drug, carbohydrates typically do not need to be released from the dendrimer periphery in order to exhibit activity. Thus, although the linkages can have modest effects on the binding affinities of multivalent carbohydrates, the choice of linkage is determined primarily by synthetic requirements. In this section, the conjugation of Neu5Ac, which is relevant to this thesis, to the peripheries of various dendrimer backbones will be discussed and the biological properties of the resulting bioconjugates will be briefly introduced. This discussion provides representative examples of the different conjugation chemistries that can be for coupling of dendrimers with carbohydrates.
1.2.2.2.1
Dendrimer-N-Acetylneuraminic Acid Conjugates
Neu5Ac is the most abundant sialic acid found in mammalian cells. This negatively charged molecule is found in complicated glycans on mucin and in glycoproteins that are embedded in cell membrane. It is known that all types of influenza viruses interact with Neu5Ac residues on the host cell surface through their trimeric lectin hemagglutinin (HA), and this is followed by endocytosis of the virus into the cell.107 Monovalent Neu5Ac can inhibit this interaction at millimolar concentrations, but there is significant interest in the development of multivalent Neu5Ac derivatives in order to obtain higher binding affinity. For this purpose, various dendrimer-Neu5Ac conjugates have been developed. Different strategies to construct such bioconjugates is shown in Figure 1.11.
Thus far, the formation of a thioether linkage is the most commonly reported approach for the conjugation of Neu5Ac to the peripheries of dendrimers.113-120 To construct these conjugates, a thiol must be installed on either sialic acid or on the dendrimer periphery. Because unprotected thiols tend to dimerize readily, a process that is facilitated on the dense peripheries of dendrimers, the introduction of the thiol to Neu5Ac has been a more viable approach. For example, Roy and coworkers have prepared a protected 2-α- thioacetyl-Neu5Ac and reacted it with three types of N-chloroacetylated dendrimers including polypeptide,113 PAMAM,114,115 and gallic acid-oligoethylene glycol dendrimers.116 The acetyl groups on the sugars were then removed under basic conditions to yield the unprotected dendrimers. An enzyme-linked lectin inhibition assay using human α1-acid glycoprotein as the coating antigen and horseradish peroxidase-labeled Limax flavus agglutinin (LFA) for detection purposes was performed. Their results showed that the globular dendrimer with a valency of 12 exhibited a 182-fold increase in inhibitory potency compared to the reference monomeric Neu5Ac.115
To investigate different spacers between Neu5Ac and dendrimers, Matsuoka and coworkers prepared a library of brominated carbosilane dendrimers with different types of spacers on their peripheries.118,119 The thiol-functionalized Neu5Ac derivative was similar to that employed by Roy and coworkers but with an additional 5-carbon aliphatic spacer between the sugar and the thiol. Introduction of this molecule onto different brominated carbosilane dendrimers with either normal, ether elongated, or amide elongated peripheral groups was accomplished by initially treating the thioacetic acid- functionalized sugar and bromide terminated dendrimer mixture with sodium methoxide (NaOMe)/methanol (MeOH) in N,N-dimethylformamide (DMF) followed by addition of acetic anhydride/pyridine. Fully deprotected dendrimers were obtained by treatment of these dendrimers with NaOMe/MeOH and 0.1 M sodium hydroxide solution. Biological evaluations of these glycodendrimers showed that all of the ether- and amide-elongated compounds had inhibitory activities for the influenza sialidases in the millimolar range. Surprisingly, the glycodendrimers having normal aliphatic linkages did not exhibit any activities except for a dendrimer with a valency of 12.119
Hawker and coworkers recently reported the glycosylation of a 4th generation dendrimer via a free-radical thiol-ene coupling reaction between thiol-functionalized carbohydrates including Neu5Ac, mannose, glucose, and lactose and a dendrimer having peripheral alkene moieties.120 This reaction results in the formation of a thioether linkage in high yield.
McReynolds and coworkers have investigated amide linkages between Neu5Ac and PAMAM dendrimers.120 They have constructed their bioconjugates with or without spacers between the dendrimer and Neu5Ac. In the case without any spacers, commercially available Neu5Ac was directly conjugated to the amine peripheral groups of the dendrimers using (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP). Alternatively, to minimize steric congestion between Neu5Ac and the dendrimer, a bifunctional spacer molecule was first conjugated to the carboxylic acid functionality of Neu5Ac. After deprotection of the other terminus of the spacer, which resulted in the formation of a free amine, it was coupled to the periphery of acid-functionalized PAMAM dendrimers. Subsequent sulfation of the conjugates was accomplished by reacting the obtained dendrimers with an SO3-pyridine complex. When evaluated for inhibition of Human immunodeficiency virus (HIV)-1 infection, the sulfated Neu5Ac-PAMAM glycodendrimer bearing 16 Neu5Ac moieties with 11 sulfate groups was found to inhibit all four HIV-1 strains tested in the low micromolar range.
Finally, thiourea conjugates of Neu5Ac and dendrimers have also been prepared. An acetate protected p-isothiocyanatophenyl derivative of Neu5Ac was prepared and was coupled to the peripheral amines of PAMAM dendrimers to give the protected Neu5Ac dendrimers in high yields (71-100%).121,122 Complete deprotection was accomplished by sequential ester hydrolysis in first NaOMe/MeOH followed by 50 mM NaOH solution to hydrolyze the acetyl followed by methyl ester groups. By performing a competitive enzyme-linked lectin assay, it was demonstrated that these dendrimers exhibited a substantial 210-fold increase in the inhibitory activity compared to monomeric Neu5Ac.121