A safer and more sustainable by design perspective in biocatalytic amide-bond coupling of in silico controlled chemical space

Amide bond synthesis is ranked as the second most important challenge in key green chemistry research areas identified by the ACS Green Chemistry Institute. Generating amides by chemical synthesis typically relies on toxic chemicals, the use of organic solvents, and stoichiometric amounts of coupling reagents, which is associated with generation of considerable amounts of waste. While developing more sustainable amide-bond forming reactions has been in focus, significantly less attention has been given to toxicity, sustainability and environmental aspects of the underlying amine and acid substrates and their corresponding coupled products. Here, we explore biocatalytic amide bond formation from a Safer and more Sustainable by Design perspective, for which a controlled chemical space was navigated based on potential human and environmental toxicity of the substrates and related products. In silico filtering of 15374 amines and 25994 acids generated 188 amine and 54 acid building blocks that could be classified as safe by the threshold we established, referred to herein as “safechems”. Based on the match of the known substrate scope of the marinacarboline amide bond synthetase McbA from Marinactinospora thermotolerans SCSIO 00652 with the structures of safechems, we generated a panel of robust and promiscuous ancestral ATP-dependent amide bond synthetases (ABS) using McbA as template. Ancestral ABS enzymes exhibited an increased thermostability of up to 20 °C and generated complementary biocatalysts to the extant enzyme with respect to amine and acid specificities in the coupling of a representative safechem subset of 17 amines and 16 acids (responding to 272 possible amides). Out of the 272 possible amides considered experimentally from the subset of safechems that were tested in biocatalytic coupling, 38 amides were detected by UPLC-MS out of which 32 were novel structures. Finally, the pool of safechems and their corresponding amides were evaluated by USEtox (the UNEP-SETAC toxicity model), analyzing not only the intrinsic properties of the compounds but also performing a risk assessment based on fate and exposure. The amides were in general predicted as more toxic compared to the starting acids and amines, emphasizing the importance of starting from low-toxicity building blocks. Even when restricting the pool of amines and acids based on their predicted toxicity, it did not prevent the discovery of new amides with potentially potent applications as exemplified here by substructures that are part of drug candidates for e.g. cancer treatment. Safer and more sustainable by design is capable of generating an array of novel products within a controlled chemical space.