Maintaining target binding affinity is another crucial feature to consider while optimizing PROTACs for improved permeability. As previously mentioned, an amide-to-ester substitution between the linker and the VHL-ligand decreased binding affinity by about 2-fold. In this work, we used a similar fluorescence polarization (FP) competition binding assay to determine if the amide-to-ester substitution between the linker and the POI-ligand mimic, was more tolerated when binding to the VHL protein. Using our second amide (3, 15 -17) and ester (10, 18 -20) series, comprised of varying linker lengths, we found that both amides and esters had FP-derived dissociation constants (Kd) that were broadly comparable to each other at each linker length (SI Figure ). The amides appeared to show slightly better binding at each linker length compared with their ester counterparts, yet the Kd values were roughly within the error of each pair. Interestingly, changes in linker length had a more pronounced effect on VHL-binding than the amide-to-ester modification. The two compounds with alkyl linkers, 3 and 10, had Kd values (119 nM and 136 nM, respectively) more similar to VH032 alone (113 nM). Binding affinity was slightly reduced for all compounds containing PEG-based linkers, with 1-PEG and 2-PEG units (amides 15 -16 Kd ≈ 170 nM and esters 18 -19 Kd ≈ 200 nM, respectively) showing comparable binding affinity. The longer 3-PEG unit compounds (17 and 20) showed slight recovery, with Kd values (138 nM and 144 nM, respectively) closer to their alkyl chain counterparts 3 and 10 (119 nM and 136 nM, respectively) for both amide and ester compounds (SI Figure ). However, the Kd values for all compounds in this linker series (either amide or ester) were within 2-fold of the VH032 ligand alone. Encouragingly, this suggests that, for a given linker, an amide-to-ester substitution away from the E3 ligand will have little to no effect on E3 binary binding.