Figure 2. Development of the bis-electrophilic warhead 1.

(a) Deactivation of Coxsackie virus 3C protease by reaction with bis-electrophilic warhead 1. Reaction scheme. (b) Inhibition of the protease in the FRET-based enzyme activity assay at various concentrations of 1. (c) Deconvoluted ESI-mass spectrum of the reaction of the protease with 1. The mass of the unmodified protein, 21,471.5 Da, is shifted to a main peak at 21,588.5 Da, corresponding to the mass of the protein +1 after cleavage of the ethyl ester. The peak at 21,755.5 corresponds to the protein mass +1 +Tris (buffer)–water. (d) Deactivation of the protease is quantified as relative inhibition, the observed deactivation rate, k_obs, plotted against inhibitor concentration. The slope of the linear regression curve of the k_obs values plotted against the corresponding concentration value yields the k_inact/K_I-value for compound 1 and equals 2.4±0.1 M⁻¹ s⁻¹.