Further optimization aimed at reducing the equilibration time to a reasonable time frame while maintaining conditions that will be tolerated by VCB. In order to achieve rapid scrambling of the DCL members, we used 1,4-dithiothreitol (DTT) as initiating reagent. Under optimized exchange conditions (100 mM DTT in tris buffer at pH = 8.5 with 10% DMSO under argon atmosphere) the libraries equilibrated at 37 °C within 24 h. With these conditions in hand, we attempted the first templated DCC experiment at the smallest possible library size using only two symmetric Homo-PROTACs, the potent VHL-degrader SH2 and the much less potent Homo-PROTAC SH4, as starting point (Figure ). In this scenario, our DCL would be composed of three Homo-PROTACs, i.e., the two symmetric starting materials along with the unsymmetric scrambling product, along with the two corresponding monomeric thiols. While only small amounts of the unsymmetric DCL-Homo-PROTAC H2-H4 formed in the blank library, considerable larger amounts of H2-H4 were detected in the VCB-templated library after equilibration. Overall, 67% relative enrichment of the novel, unsymmetric Homo-PROTAC H2-H4 was observed.