To test if hybrid reaction conditions would yield product, we synthesised His-DBE; Pro-DBE, and Phe-DBE. Reactions containing only minimal aa-DBE substrates were highly unfavourable and yielded very little CDP. In contrast, hybrid reactions using all available combinations for aa-DBE and aatRNA substrates (Fig. ) revealed that more product was formed using a combination of the aa-tRNA/aa-DBE substrates, albeit with lower overall yield when compared to the natural tRNA substrates (Fig. ). It is important to note that aa-DBE has a limited half-life, which also impacts reaction yields. In these hybrid reaction conditions, we hypothesised that the small aa-DBE substrate would be turned over to generate a cyclic dipeptide product when occupying the deeper narrower P1 pocket, while P2 was occupied by an aatRNA, and not the other way around since aa-DBE occupying P2 would result in unproductive binding conformations, leading to aa-DBE hydrolysis and not cyclic dipeptide formed.