High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses.

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

Remdesivir triphosphate can efficiently inhibit the RNA-dependent RNA polymerase from various flaviviruses.

Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.

Reactivity of boraguanidinato germylenes toward carbonyl compounds and isocyanides: C-O, C-F and C-N bond activation.

The reactions of two equivalents of germylene [(i-Pr)2NB(N-2,6-Me2C6H3)2]Ge (1) with carbonyl compounds RC(O)R' resulted in carbonyl functionality activation and the formation of 4-(R,R')-1,2-digerma-3-oxa-cyclobutanes (R/R' = Ph/CF3 (2) or C6F5/H (3)). Surprisingly, the analogous reaction of 1 with C6F5C(O)Me led to the insertion of the germanium atom into the C-F bond of the perfluorophenyl group, thus producing a spiro compound (4) with a germanium atom sharing 1,2-digerma-3,5-diaza-4-bora-cyclopentane and 1-germa-2,4-diaza-3-boracyclobutane rings. Furthermore, the reaction of 1 with 2e- donors was investigated. In the case of 4-dimethylaminopyridine (DMAP), an expected complex [(i-Pr)2NB(N-2,6-Me2C6H3)2]Ge(DMAP) (5) was isolated, but using t-BuNC resulted in the formation of germanium(iv) cyanide [(i-Pr)2NB(N-2,6-Me2C6H3)2]Ge(CN)(t-Bu) (6) as a result of C-N bond activation in the starting isocyanide. In contrast, mixing other isocyanides RNC (R = Cy or Ad) with 1 in solution led only to an equilibrium between the starting compounds and most probably the corresponding complexes [(i-Pr)2NB(N-2,6-Me2C6H3)2]Ge(CNR) (R = Cy (7a) or Ad (8a)) based on NMR studies. From these equilibrium mixtures, fortuitously, single crystals of digerma-spiro-complexes (7 and 8) containing two germanium atoms (one of them coordinated to a particular isocyanide) were obtained and structurally authenticated by the X-ray diffraction technique.

Diverse reactivity of a boraguanidinato germylene toward organic pseudohalides.

The reactions of the boraquanidinato germylene (i-Pr)2NB(NDmp)2Ge (1) (Dmp = 2,6-Me2C6H3) with RN3 and RNCS produced rare examples of Ge2N and Ge3S rings, while the treatment of 1 with RNCO led to an insertion into the N-Ge bond leading to a novel type of germylene stabilized within a six membered ring, i.e. [N(R)C(O)N(Dmp)B(N(i-Pr)2)N(Dmp)]Ge (R = t-Bu or Ad).

Facile activation of alkynes with a boraguanidinato-stabilized germylene: a combined experimental and theoretical study.

A boraguanidinato-stabilized germylene, [(i-Pr)2NB(N-2,6-Me2C6H3)2]Ge, reacts with alkynes RC[triple bond, length as m-dash]CR selectively in a 2 : 1 molar ratio to afford 3,4-R,R'-1,2-digermacyclobut-3-enes 1a-e as the products of formal [2 + 2 + 2] cyclization [R/R' = Me/Me (1a), Ph/Ph (1b), Ph/H (1c), t-Bu/H (1d) and Cy/H (1e)]. Ferrocenyl-substituted alkynes react similarly, yielding the corresponding ferrocenylated 3,4-R,R'-1,2-digermacyclobut-3-enes 2a-d [where R/R' = Fc/H (2a), Fc/Me (2b), Fc/Ph (2c), and Fc/Fc (2d); Fc = ferrocenyl]. By contrast, only one of the triple bonds available in conjugated diynes RC[triple bond, length as m-dash]CC[triple bond, length as m-dash]CR is activated with the germylene, while the second one remains intact even in the presence of an excess of the germylene. The exclusive formation of 3,4-R,(C[triple bond, length as m-dash]CR)-1,2-digermacyclobut-3-enes 3a-c [R = Ph (3a), t-Bu(3b), and Fc (3c)] was ascribed to a steric repulsion around the second triple bond. On the other hand, the reaction of the germylene with more flexible dialkyne fc(C[triple bond, length as m-dash]CPh)2 (fc = ferrocene-1,1'-diyl) proceeded in the expected manner, producing compound 4, where both triple bonds are transformed into 1,2-digermacyclobut-3-ene rings by reaction with four equivalents of the germylene. All compounds were characterized by multinuclear NMR spectroscopy, Raman and IR spectroscopy, and in the case of 1a-c, 2a, 2c, 3a, 3b and 4, also by single-crystal X-ray diffraction analysis. The ferrocenyl substituted compounds were studied by cyclic voltammetry (CV). Finally, the plausible reaction pathway was studied for a model reaction of [(i-Pr)2NB(N-2,6-Me2C6H3)2]Ge with MeC[triple bond, length as m-dash]CMe using DFT computations.