We began by synthesizing a small set of disulfide-containing Homo-PROTACs as starting compounds for the later-developed DCC assay and for biological and biophysical profiling. The initial Homo-PROTAC set was designed to contain both degrading and non-degrading PROTACs to represent the wide range of outcomes of early-stage DCC-derived PROTACs. We leveraged our previously revealed SAR of VHL Homo-PROTACs, with compound CM11 (Figure ), featuring an 18-atom PEG-5 linker, forming the most stable VHL2:CM11 ternary complex and being the most potent degrader in cell, while analogous compounds with shorter linkers, e.g. PEG-3 and PEG-4, exhibited poorer performance in the same assays. We therefore designed ten disulfide-containing Homo-PROTACs, featuring either the same VHL ligand as in CM11 (namely, VH032) or its benzyl-methylated analog, and alkoxyether or alkyl linkers of various length (Figure ). In line with previous SAR of VHL-targeting Homo-PROTACs, the 18-atom linkers were expected to perform better than those with shorter linkers. To be able to decipher the role of the underexplored disulfide motif, we also designed and synthesized analogs featuring the benzyl-methylated derivative of VH032 as VHL ligand on each side with the same linker as in CM11 (MeCM11) and an 18-atom alkyl chain equivalent as linker with VH032 as VHL ligand on either side (alCM11) (Figure ). All symmetric Homo-PROTACs were assembled from established VHL ligands and disulfide-containing linkers in a 2:1-ratio using HATU-mediated amide couplings (Scheme 1A). Two general strategies were employed to synthesize the disulfide-containing linkers. If the corresponding thiols were commercially available, such as 2-mercaptoacetic acid, the disulfides were formed via oxidative dimerization using hydrogen peroxide as oxidant. Alternatively, disulfide linkers were synthesized from corresponding alkyl halide precursors by reaction with thiourea to form S-alkylisothiouronium salts, which were then oxidatively hydrolyzed to the desired disulfides (Scheme 1B).