A Chemoenzymatic Strategy for the Synthesis of Steroid Drugs Enabled by P450 Monooxygenase-Mediated Steroidal Core Modification

Abstract

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.

Cytochrome P450 Monooxygenases Catalyse Steroid Nucleus Hydroxylation with Regio‐ and Stereo‐Selectivity

“…From another point of view, these trace products may help deduce the enzymatic mechanism and provide instructions for engineering. In a recent screening, LG‐23 showed slightly high 11α selectivity (90% and 78%) for 22 and 31 , and the selectivity was essentially switched from 7β to 11α, likely because of the presence of an additional double bond between 22 and 4 [50] . MD simulation identified the key residue, T438, which affected the LG23‐catalysed 11α‐hydroxylation of 22 .…”

“…In a recent screening, LG-23 showed slightly high 11α selectivity (90% and 78%) for 22 and 31, and the selectivity was essentially switched from 7β to 11α, likely because of the presence of an additional double bond between 22 and 4. [50] MD simulation identified the key residue, T438, which affected the LG23-catalysed 11α-hydroxylation of 22. Therefore, the key residue T438 was targeted and subjected to NNK-based site-saturation mutagenesis.…”

“…Variant LG-23/T438S was identified with improved 11α selectivity (92%) and substrate conversion (> 95%) and served as the key step to achieve the synthesis of steroidal drugs trenbolone and trenbolone acetate at a (nearly) gram scale with 83-91% isolated yields. [50] The application of scanning alanine mutation inspired the engineering of steroid hydroxylases, and The fifteen residues which were undertake a focused mutagenesis are labeled as W47, W72, K76, Y77, L78, R79, D80, F81, L82, S88, T177, Q185, L188, F205, and T209. The triple mutant F87G/A328G/A330W is served as the starting template.…”

Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3

“…Cytochrome P450 enzymes (P450s) are a superfamily of heme proteins involved in drug metabolism, xenobiotic detoxification, and steroid biosynthesis [ 1 , 2 ]. P450s are promising biocatalysts for organic synthesis, drug discovery, and bioremediation because of their versatile catalytic oxyfunctionalizations of a variety of substrates [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. However, one of the limitations of applying P450s as in vitro biocatalysts is that most P450s require reduced nicotinamide cofactors NAD(P)H and a complex system of electron transport partners (redox partners) to activate molecular oxygen for the formation of active species.…”

A Modular Chemoenzymatic Approach to C14-Functionalized Steroids

“…[24][25][26][27][28][29] It harnesses the power of both biocatalysis and chemical synthesis, thus can enormously shorten the synthetic routes and increase the overall efficiency. Inspired by the elegant chemoenzymatic studies, [30][31][32][33] we envision a similar strategy to prepare the C14-functionalized steroids, which involves the identification of a versatile C14αhydroxylase to prepare diversified C14α-OH steroids 34 with a C17 substitution directly and the following chemical manipulations of the C14α-OH group to access other C14-functionalized steroids (Fig. 1c).…”