Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for K48 polyubiquitin and ISG15 originating from certain species. The PLpro of BtSCoV-Rf1.2004 was used as a tool alongside PLpro of CoV-1 and CoV-2 to design 30 novel noncovalent drug-like pan subgroup 2b PLpro inhibitors that included determining the effects of using previously unexplored core linkers within these compounds. Two crystal structures of BtSCoV-Rf1.2004 PLpro bound to these inhibitors aided in compound design as well as shared structural features among subgroup 2b proteases. Screening of these three subgroup 2b PLpros against this novel set of inhibitors along with cytotoxicity studies provide new directions for pan-coronavirus subgroup 2b antiviral development of PLpro inhibitors.

The preparation of glycosyl dibutyl phosphates in the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) and pseudaminic acid series and their application to the formation of C-glycosides are described. Both donors were obtained from the corresponding thioglycosides by treatment with dibutylphosphoric acid and Niodosuccinimide. As with the thioglycosides, both donors adopted very predominantly the strongly electron-withdrawing tg conformation of their side chains, which is reflected in the excellent equatorial selectivity of both donors in the formation of exemplary O-glycosides. With respect to C-glycoside formation on the other hand, contrasting results were observed: the KDO donor was either relatively unselective or selective for the formation of the axial C-glycoside, while the pseudaminic acid donor was selective for the formation of the equatorial C-glycoside. These observations are rationalized in terms of the greater electron-withdrawing ability of the azides in the pseudaminic acid donor compared to the corresponding acetoxy groups in the KDO series, resulting in a reaction through tighter ion pairs even at the S N 1 end of the general glycosylation mechanism. The contrast in the axial versus the equatorial selectivity between C-and O-glycosylation cautions against the extrapolation of models for S N 1-type glycosylation with weak nucleophiles for the explanation of O-glycosylation.