The synthesis of steroid drugs by multistage modifications of the steroidal core is challenging since site-specific and selective modification is essentially required, which is often difficult or complicated for chemocatalysis. For example, the synthesis of Trenbolone (3), a versatile anabolic–androgenic steroid, relies on a four-step chemical procedure on its core modifications of estra-4,9-diene-3,17-dione (1). Here, we have designed a two-step chemoenzymatic strategy that includes a biocatalytic one-pot C11-hydroxylation/17β-ketoreduction of 1 with a computationally designed P450 monooxygenase and an appropriate 17-ketosteroid reductase to generate 11α-OH-9(10)-dehydronandrolone (2a) as an intermediate followed by chemical dehydration to introduce the double bond at carbons 11 and 12 with the formation of Trenbolone (3). To obtain a highly active and C11-selective enzyme, molecular dynamics simulations were performed, uncovering a crucial role of water molecules for substrate recognition and targeted hydroxylation of steroids. Moreover, Trenbolone is further subjected to esterification to produce Trenbolone acetate (9) that has been widely used in veterinary medicine. Finally, our approach enables the regio- and stereoselective synthesis of both steroid drugs 3 and 9 on a (nearly) gram scale with 83–91% isolated yields, showing great potential for industrial applications.

中文翻译：

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P450 单加氧酶介导的类固醇核心修饰实现类固醇药物合成的化学酶策略

通过甾体核心的多级修饰合成甾体药物具有挑战性，因为基本上需要位点特异性和选择性修饰，这对于化学催化来说通常是困难或复杂的。例如，群勃龙 ( 3 ) 是一种多功能合成代谢-雄激素类固醇的合成，其核心修饰 estra-4,9-diene-3,17-dione ( 1 )依赖于四步化学过程。在这里，我们设计了一个两步化学酶策略，包括生物催化一锅 C11-羟基化/17β-酮还原1，使用计算设计的 P450 单加氧酶和适当的 17-酮类固醇还原酶生成 11α-OH-9(10) -去氢诺龙（2a) 作为中间体，然后进行化学脱水以在碳 11 和 12 处引入双键，形成群勃龙 ( 3 )。为了获得高活性和 C11 选择性酶，进行了分子动力学模拟，揭示了水分子在底物识别和类固醇靶向羟基化中的关键作用。此外，群勃龙进一步酯化生产醋酸群勃龙（9），已广泛用于兽药。最后，我们的方法能够在（近）克规模上区域选择性和立体选择性合成类固醇药物3和9，分离产率为 83-91%，显示出巨大的工业应用潜力。