The chiral β-hydroxy-α-amino acid, (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-propanoic acid, is a key intermediate in the synthesis of the API (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-1-(pyrrolidin-1-yl)propan-1-one, a developmental drug candidate. Two d-threonine aldolase enzymes were identified to catalyze the aldol addition of glycine and pyridine 4-carboxaldehyde for the synthesis of the β-hydroxy-α-amino acid. The two d-threonine aldolase enzymes have similar properties. Efficient recombinant E. coli fermentation processes were developed for producing the enzymes. The stabilities of the enzymes were significantly improved by addition of divalent cations. An unexpected and beneficial finding was that the β-hydroxy-α-amino acid aldol addition product directly crystallized out from the reaction mixture in high purity and high diastereo- and enantioselectivity, contributing also to high yield and allowing easy isolation, processing, and downstream utilization. The temperature, pH, and amounts of reactants and enzyme were optimized to minimize reaction time and enzyme and raw material usage and maximize amino acid formation. Efficient d-threonine aldolase-catalyzed synthesis and recovery of the β-hydroxy-α-amino acid at the 100 L scale was demonstrated leading to a highly efficient and environmentally friendly process for the production of the API.

中文翻译：

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重组d-苏氨酸醛缩酶制备β-羟基-α-氨基酸

手性β-羟基-α-氨基酸（2 R，3 S）-2-氨基-3-羟基-3-（吡啶-4-基）-丙酸是合成API的关键中间体（2 R，3 S）-2-氨基-3-羟基-3-（吡啶-4-基）-1-（吡咯烷-1-基）丙-1-酮，一种发展中的药物候选物。鉴定出两种d-苏氨酸醛缩酶，以催化甘氨酸和吡啶4-羧醛的醛缩醛加成，以合成β-羟基-α-氨基酸。两种d-苏氨酸醛缩酶具有相似的性质。高效重组大肠杆菌开发了用于产生酶的发酵方法。通过添加二价阳离子，酶的稳定性得到显着改善。一个出乎意料且有益的发现是，β-羟基-α-氨基酸羟醛加成产物以高纯度，高非对映和对映选择性直接从反应混合物中结晶出来，也有助于高收率，并易于分离，加工和下游加工利用率。优化温度，pH值以及反应物和酶的量，以最大程度地缩短反应时间，减少酶和原料的使用并最大程度地形成氨基酸。高效d经证明，苏氨酸醛缩酶催化的100 L规模的β-羟基-α-氨基酸的合成和回收可导致生产API的高效，环保的工艺。