A HTC protocol for the generation of the reactive fragment libraries was pursued to enable the rapid generation of SF fragments in 384-well plates. This allowed for screening in a D2B format as crude reaction mixtures, circumventing the requirement for purification and thus accelerating reactive fragment library screens. A succinimide-activated (OSu) amide coupling was employed using DMSO and N-ethylmorpholine (NEM) as the solvent and base, respectively. The conditions were initially trialled on a panel of 12 diverse aminefunctionalised fragments by addition of SF reactive moieties (1a, 2a, and 3a) in both dry DMSO, and DMSO:water (9:1) to assess robustness to hydrolysis under the reaction conditions. Reactions with OSu esters 2a and 3a afforded good conversion to the desired products and tolerance of 10% water, while meta-substituted OSu ester 1a gave poorer conversions due to hydrolysis of the SF group to the sulfonic acid (see Fig. &). Two 352-membered SF reactive fragment libraries were subsequently synthesised employing para-substituted reactive moiety 2, and azetidine-linked reactive moiety 3. A set of 352 amine-functionalised fragments were selected from the GSK compound collection by first filtering for fragment-like properties (aromatic ring count ≤2; HBDs/HBAs ≤4; heavy atoms ≤15; 150<MW≤250), and then selecting for maximal chemical diversity by clustering on chemical fingerprints (see Fig. ). LC-MS analysis of six wells selected at random indicated good conversion to the desired products, and conversions were found to be highly reproducible across three library syntheses conducted on separate occasions (see Fig. ).