The fact that solution phase synthesis did not result in a statistical ratio of more favored HC [8]s, encouraged us to study if the selectivity of mono-biotinylated mixHC [8] formation can be increased via tuning the parameters of the mechanochemical reaction. To investigate the effect of multiple external stimuli on the assembly of 8-membered macrocycles, we utilized RSM for experimental design and the screening of reaction conditions. The HPLC yields of mixHC [8] and cycHC [8] obtained after the aging step were plotted as 3D response surfaces, highlighting the conditions favorable for the assembly of mixHC [8] compared to cycHC [8] (Figure -B, Tables S2-S6, Figures ). The ratio of monomers, loading of aqueous mineral acid (HClO 4 ), milling duration, and aging temperature, which affect CU macrocyclization, 8 were simultaneously explored.