Toward in-cell acylation, we examined binding profiles of PEG-conjugated LANA-DBM-FITC 28 and LANA-FITC 29 on chromatin in living cells by fluorescence recovery after photobleaching (FRAP) experiments (Figures ). We used 6r (D-Arg), instead of 5R, as DBM to prevent degradation by peptidases in living cells. Compared to the rapid and almost full recovery of 29 (88% recovery at 3 s), 28 recovered slowly (46% at 3s). In the recovered fraction, the recovery t1/2 corresponding to the residence time of 28 (0.87 s) was approximately three times longer than 29 (0.29 s), indicating that LANA-DBM-PEG was the more potent binder than LANA-PEG, to chromatin in living cells. We then synthesized LANA-DBM-PEG-BAHA catalyst 30 (Figure ). As expected, 30 promoted H2BK43 acetylation in HeLa S3 cells (Figures 5g). Taken together, developing the LANA-DBM ligand and precise optimization of the catalytic motif's position on the Gly linker between the LANA and DBM moieties, sharply switched regioselectivity of the histone acylation catalyst from H2BK120/K108 to H2BK43.