To further expand our diketopiperazine library we exploited the increased promiscuity of CDPSs to produce cHF analogues using a phenylalanyl tRNA synthetase mutant (PheRS-A294G) which has a wider binding pocket allowing interactions with a larger range of related Phe analogues. All but two of the known non-canonical amino acids accepted by PheRS-A294G were incorporated into the ring by ParaCDPS (Fig. ). All halogen para-substitutions in the phenyl ring were easily accepted by the CDPS, but not para substitutions introducing polar groups such as amine, nitro and azido (Supplementary Table ). To produce cHP variants, several halogenated proline analogues reported as substrates for ProRS were also utilised by ParcuCDPS. 4-Bromo-proline was not previously shown to be a substrate for ProRS but it was tRNA pool is a far superior method of producing CDP for both CDPSs. c, generation of a cyclic dipeptide library using ParaCDPS and ParcuCDPS using non-canonical amino acids: histidine derivatives are shown in dark blue; phenylalanine derivatives in dark purple and proline derivatives in red.