A peptide-like self-immolative molecular clip is required for release of active drugs from prodrugs by endopeptidases. Upon cleavage from the carrier, this clip must collapse and release the drug rapidly. A series of aminoacyl-5,5-dimethylthiaproline (Aaa-Dmt) N-(2-(4-nitrophenyl)ethyl) am ides were designed. BOC-L-aminoacyl fluorides were coupled with R-DmtOH to give BOC-L-Aaa-R-DmtOH, which were converted to the BOC-L-Aaa-R-Dmt N-(2-(4-nitrophenyl)ethyl)amides. The L,S diastereomeric series was prepared by the reaction of Boc-Aaa PFP esters with S-DmtOH. The L-Aaa-Dmt N-(2-(4-nitrophenyl)ethyl)amides were allowed to cyclise to diketopiperazines (DKPs) in aqueous buffers, expelling 2-(4-nitrophenyl)ethylamine as a model for amine-containing drugs. Reaction rates were dependant on pH. In the L,R diastereomeric series, increasing steric bulk of the Aaa side-chain (Gly, Ala, Phe, Val) led to decrease in the reaction rate. However, in the L,S series, the greatest rate of reaction was observed for the most bulky amino-acid (Val), with t(1/2)=15 min at pH 8.0. The effects of steric bulk and stereochemistry are rationalised through conformational analysis (NMR and X-ray crystallography) of the starting dipeptide amides, the product diketopiperazines and key analogues. Since the dipeptides are (almost) exclusively in the cis-amide conformation, transcis interconversion is not relevant. The data suggest that steric interactions in the reacting conformations of the dipeptide amides, as they form the tetrahedral intermediates, are the controlling factors. Thus, L-Aaa-S-Dmt amides are shown to be excellent candidates for incorporation into the design of novel prodrugs.