The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-l-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-l-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

The absence of orthogonal aminoacyl-tRNA synthetases that accept non-L-a-amino acids is the primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers. Here we report PylRS enzymes that accept a-hydroxy acids, a-thio acids, N-formyl-L-a-amino acids, and a-carboxyl acid monomers (malonic acids) that are formally precursors to polyketide natural products. These monomers are all accommodated and accepted by the translation apparatus in vitro. High-resolution structural analysis of the complex between one such PylRS enzyme and a meta-substituted 2-benzylmalonate derivative reveals an active site that discriminates pro-chiral carboxylates and accommodates the large size and distinct electrostatics of an a-carboxyl acid substituent. This work emphasizes the potential of PylRS for evolving new enzymes capable of encoding diverse non-L-a-amino acids in synergy with natural or evolved ribosomes.