The kinetics of binding were investigated by incubation of the resynthesised and purified SFs 2b-e with CAII at a range of concentrations and analysis by intact protein LC-MS over a 24 h period (see Fig. ). The concentration-response was analysed based on a two-step model of reversible ligand (L) binding and subsequent irreversible covalent modification of the protein (P) (P + L ⇌ P•L → PL) (see Fig. ). Time courses were fit to a single exponential function to give a kobs for each concentration (Fig. ). These were then fitted using a Michaelis-Menten model to determine kinact (corresponding to the rate of covalent bond formation) and KI (corresponding to the recognition of the ligand for the protein pocket) (Fig. &). All fragments were found to have strong reversible affinity (KI), and these were below the minimum concentration screened for 2d and 2e (<5 µM). This is consistent with the strong binding of aryl sulfonamides to the CAII Zn 2+ binding pocket. The SFs exhibited unexpectedly slow rates of covalent modification, with 50% modification achieved between 6.5-10.5 h for all 100 µM conditions. The slow rate of reaction may be attributed to a sub-optimal trajectory of the SF group toward the His64 residue in the reversible bound conformation, and/or low nucleophilicity of the residue. An advantage of the HTC-D2B approach taken with this screening strategy is the opportunity to rapidly explore iterative libraries of compounds which are structurally similar to the original hits identified. Such iteration-based screens allow for faster design-make-test cycles in contrast to traditional methods in early-stage drug discovery, leading to the more efficient identification of potent and selective probes. To explore whether any alternative sulfonamide-containing fragments would give high modification yields at a faster rate, a new library was designed. For this, 96 amine-functionalised fragment analogues of hits 2b-e were selected, coupled to OSu ester 2a, and the resultant library was incubated with CAII for 1 h prior to analysis by intact protein LC-MS. Inspection of the resultant mass spectra after this short incubation revealed many fragment hits that exhibited significantly higher modification yields (>70%, see Fig. ) when compared with the original hits (<15% at 1 h, see Fig. ). This highlights the importance of electrophile trajectory in determining kinact, and demonstrates the utility of the HTC-D2B approach as a means to rapidly optimise kinetic parameters.