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.

接受非-1 -α-氨基酸的正交氨酰基转移RNA（tRNA）合成酶的缺乏是阻碍序列定义的异源寡聚物和生物材料体内翻译的主要瓶颈。在这里，我们报道吡咯赖氨酰-tRNA合成酶（PylRS）和某些PylRS变体接受α-羟基、α-硫代和N-甲酰基-1 -α-氨基酸，以及作为聚酮化合物天然产物前体的α-羧酸单体。这些单体在体外被翻译装置容纳和接受；那些具有反应性亲核试剂的物质在体内掺入蛋白质中。对一种 PylRS 酶和一种m-之间形成的复合物进行高分辨率结构分析取代的 2-苄基丙二酸衍生物揭示了一个活性位点，该活性位点可以区分前手性羧酸盐并适应 α-羧基取代基的大尺寸和不同的静电。这项工作强调了 PylRS 衍生酶用单体酰化 tRNA 的潜力，该单体的 α-取代基与蛋白质氨基酸的 α-胺有很大不同。这些酶或其衍生物可以与天然或进化的核糖体和/或翻译因子协同作用，产生不同的序列限定的非蛋白质杂聚物。