Hartman et al. previously determined the ability of aaRSs to accept non-canonical amino acids and some CDPSs accept these as substrates to form CDPs. Consequently, we explored the capability of ParaCDPS and ParcuCDPS in accepting amino acid analogues as substrates for cyclic dipeptide production. Using a range of commercially available non-canonical analogues with the tRNA pool (Fig. ), a library of diverse CDPs was produced from just two CDPSs (Fig. alanine. Since all analogues employed were previously shown to be substrates for histidinyl tRNA synthetase (HisRS) , the CDPS enzyme is posing an additional sieve for substrate selection.