Abstract
Influenza virus is the cause of major respiratory illness in human populations, and the trimeric viral hemagglutinin (HA) and neuraminidase (NA) proteins are the two most prominent antigens targeted by the host immune system. The design and synthesis of NA inhibitors is a subject of intense research owing to both the recent outbreaks of avian influenza and the emergence of viruses that are highly resistant to widely used drugs. In this study, a series of acylguanidine-modified zanamivir analogs (~40 analogs) were synthesized, and their inhibitory activities against the NAs of avian influenza viruses (H1N1 and H3N2) were evaluated. In particular, zanamivir derivative 3j, with a hydrophobic naphthalene substituent, exhibits the best inhibitory activity against group-1 NA, with an IC50 of 20 nM. Our results show that the average IC50 values of these zanamivir derivatives are in the nanomolar range, indicating the importance of both the carbonyl group, which can generate additional contacts through hydrogen bonding with the E119 and D151 residues of NA, and the hydrophobic group, which may interact with the adjacent 150 cavity. Our results provide information that may aid in the development of more potent neuraminidase inhibitors. Enzymatic synthesis is suitable for synthesizing complex oligosaccharides. However, tedious purification step often lead to a low overall yield and costs a lot of time. Therefore, a simple and effective method, synthetically viable to complex glycan structures circumventing the need of labor-intensive and time-consuming purification steps is highly desirable. To address this, here, we have developed a new purification technique of carbohydrates in combination with photolysis and enzymatic synthesis by incorporating a fluorous tag at the reducing end of glycans. In this study, we are successful to synthesize oligosaccharides, such as Sialyl Lewis X (SLex) and poly-LacNAc. We first synthesized the fluorous tag containing N-acetylglucosamine by chemical synthesis, and the carbohydrate chains were elongated at the non-reducing end by employing transferases, such as thymidylyltransferase (RmlA), N-acetylhexosamine-1-kinase (NahK), β-1,3-N-acetyl-glucosaminyltransferase (HpGnT), β-1,4-galactosyltransferase (NmGalT), α-1,4-galactosyltransferase (LgtC), and α-2,3-sialyltransferase (Cst I), and α-1,3-fucosyltransferas (FucT). Furthermore, we also developed a diverse glycan library through the fluorous tag.