Previous research from our group demonstrated the use of small molecules containing a dinitrobenzyl ester coupled to an amino acid (aa-DBE) as substrates for CDPSs. These minimal substrates are useful tools to investigate substrate specificity, and binding to P1/P2, while also presenting an alternative to using aatRNA as substrates. Prior to this work, hybrid reaction conditions using a combination of aa-DBE and aatRNA as substrates were not performed.