Based on previous successful selections we chose a concentration of 5 M for the washes. Before performing a full selection we wanted to test our new approach using a model proteinpeptide target pair. PADI4_3 is a cyclic peptide inhibitor of PADI4 that was recently discovered using a RaPID screen. A cryo-electron microscopy structure of this peptide bound to PADI4 revealed that His4 of PADI4_3 bound in the active site of PADI4, in the position normally occupied by the arginine side chain of substrate peptides (PDB ID: 8R8U). We hypothesised that substitution of this residue with 2 would enable covalent inhibition of PADI4, as the warhead should be positioned to react with the PADI4 active site cysteine, Cys645. To confirm this, PADI4_3_H4(2) was synthesised. The linear sequence was synthesised by solid-phase peptide synthesis (SPPS) with an ornithine residue in position 4 of the peptide. The resultant peptide was cyclised, the ornithine selectively deprotected to allow addition of the fluoroacetimadoyl group, before full peptide deprotection, resin cleavage and purification.