Attempts at the synthesis of a novel sialic acid ABPP probe for viral neuraminidases

Abstract

The influenza virus causes a common acute respiratory infection, affecting around 1 billion humans annually and resulting in 290-650 thousand respiratory deaths yearly. The viruses use glycoproteins hemagglutinin and neuraminidase to enter and spread throughout cells of the host. Targeting the neuraminidases with activity-based protein profiling (ABPP) can give us great insight into the virus’s workings. ABPP uses chemical probes to specifically label active enzymes of interest. Incorporation of a recognition element in the ABPP achieves specificity, which drives the probe to the target. For viral neuraminidases substrate sialic acid, a nine-carbon monosaccharide often found on the terminus of glycoconjugates and highly important for the entry and spread of the viral particles, is an appropriate recognition element. Incorporating a reactive group that covalently binds the target allows for stable and long-lasting binding of the probe. Installing a difluoro motif on the sialic acid causes the covalently bound intermediate to be trapped in the active site of viral neuraminidases. Finally, the probe contains a clickable tag that acts as a latent reporter element. Although ABPP probes for difluoro sialic acid have been developed, they feature a clickable element that compromises crucial interactions within the neuraminidases' active site. Consequently, our emphasis in this study is on incorporating the clickable tag at the C7 position of sialic acid, which has no interactions within the neuraminidase active site. N-acetylated sialic acid was first modified to accept the difluoro motif by installing a double bond between the C2 and C3 positions. Simultaneously, an azide was installed on the C4 position and converted into a Boc protected amine. Next, the C8 and C9 positions were selectively protected by an isopropylidene acetal. Installing an azide directly on the C7 position, either via a Mitsunobu conversion or an SN2 nucleophilic substitution proved highly difficult. Therefore, installing a small spacer molecule onto the C7 position with a terminal azide was attempted. However, both an ether-bond linker and a carbamate linker could not be successfully installed. A C7-azide neuraminidase ABPP probe could not be successfully synthesized. Alternative approaches of obtaining the C7-azide clickable ABPP probe were theorized. For instance, further optimization of the spacer attachment reactions or applying a chemoenzymatic approach.