Chemo-enzymatic Synthesis of O-Acetylated Sialosides and Their Unnatural Analogs to Probe Virus-Glycan Interactions

Abstract

The transmission of viruses from animal reservoirs to humans poses great threats to public health. Preparedness for future zoonotic outbreaks requires a fundamental understanding how viruses of animal origin have adapted to binding to a cell surface component/receptor of the new host. In Chapter 2, we describe such specificities of human and animal viruses that engage with O-acetylated sialic acid. Key to these studies was the development of a chemo-enzymatic methodology that can provide almost any sialate-acetylation patterns. The collection of O-acetylated sialoglycans was printed as microarray for receptor specificity determination. It showed host-specific patterns of receptor recognition and revealed that three distinct human respiratory viruses bind 9-O-acetylated α2,8-linked disialoside. Immunofluorescence and cell entry studies support that such glycotope as part of a ganglioside is a functional receptor for human coronaviruses. In Chapter 3, the HE-mediated regioselective de-O-acetylation was extended to the preparation of a panel of O-acetylated N-glycolylneuraminic acid oligosaccharides. The resulted compound library was printed as glycan microarray to investigate receptor binding specificities of viral envelope glycoproteins, including spike proteins and HEs, from animal and human coronaviruses. We have found that the binding pattern of these viral proteins to N-glycolylated sialosides differ considerable from that to N-acetylated counterparts; and that the ability of the spike proteins to tolerated N-glycolyl modification varies among viruses targeting different hosts, whereas the corresponding HEs showed a decrease or loss of binding to N-glycolylated sialosides. In Chapter 4, we describe a chemoenzymatic approach that can readily provide sialoglycan analogs in which acetyl esters at C4 and/or C7 are replaced by stabilizing acetamide moieties. The analogs and their natural counterparts were used to examine ligand requirements of the lectin domain of coronaviral hemagglutinin-esterases (HEs). It revealed that HEs from viruses targeting different host species exhibit different requirements for 7-O-acetylation. It also showed that ester-to-amide perturbation results in decreased or loss of binding. STD-NMR and molecular modeling of the complexes of the HE of BCoV with the acetamido analogs and natural counterparts revealed that binding is governed by the complementarity between acetyl moieties of the sialosides and hydrophobic patches of the lectin. The precise spatial arrangement of these elements is important, and perturbations such as O-to-N change results in substantial loss of binding. Induction of broadly and durably protective response has been the ultimate goal of prophylactic vaccine development. The use of toll-like receptor agonists has been increasingly pursued to augment such immune responses. Furthermore, attachment of these immunomodulating agents to immunogens offers several advantages over combined administration thereof as separate entities. However, there is a lack of general method to conjugate TLR agonists to complex immunogens without compromising important neutralization epitopes. In Chapter 5 we describe a one-step enzymatic conjugation approach which allows for covalent attachment of small molecule immune activators to N-linked glycans of a native-like HIV envelope trimer immunogen. The drug-to-protein ratio can be readily quantified chromatographically. Binding studies with several broadly neutralizing antibodies reveal that the glycan-targeted covalent modification of the glycoprotein immunogen minimally affects its neutralization epitopes.