Fig. 5. Design and biochemical evaluation of covalent peptides targeting the PHD3 domain. (A) H3K4me3 6mer peptides bearing covalent lysine-reactive warheads. (B) PHD3 (10 μM) was treated with peptides 27–32 at 50 μM, and the extent of labelling was monitored over time by intact protein LCMS. (C) Comparison of cross-linking efficiency between linear (29) and cyclic (C-33 and D-34) peptides. (D) Structures of covalent cyclic peptides C-33, D-34, and D-35. (E) Comparison of peptide-PHD3 adduct formation between WT and mutant PHD3 after 5 h incubation time. Triazole probe D-35 was used for this experiment. (F) HCD product ion spectrum of the triazole probe (D-35)-labeled PHD3 domain following tryptic digestion, corresponding to precursor ion at m/z = 678.879⁵⁺. C* indicates carbamidomethylated Cys. The structure of the probe adduct following digestion is shown and the spectrum indicates K1620 as the site of modification.