Chemical enzymatic synthesis of C14-bit functional steroids

On April 17, 2023, the team of Qianghui Zhou of Wuhan University and the team of Xudong Qu of Shanghai Jiao Tong University published important research progress in the field of steroid synthesis in the journal Nature Synthesis, entitled “Chemoenzymatic synthesis of C14-functionalized steroids”.

In this study, a new steroid C14αhydroxylase was identified through strain screening and gene mining, and two mutants with high catalytic region selectivity were obtained by further combining enzyme engineering and Saccharomyces cerevisiae expression system. With this biotransformation platform, a series of C14-bit functional steroids were synthesized in a variety of functional steroids, and the efficient synthesis of two strong steroid natural products was completed.

The co-first authors of the paper are Song Fuzhen and Zheng Mengmeng; The co-corresponding authors are Zhou Qianghui and Qu Xudong.

Steroidal drugs are the second largest class of drugs that protect people’s lives and health after antibiotics. Existing studies have shown that C14 functionalized steroids generally exhibit good biological activity and have potential for clinical drug development. However, the synthesis of C14 functionalized steroids presents considerable challenges due to the reaction inertness of the C14-bit C(sp3)-H bond of the steroid and the large steric hindrance. The existing chemical synthesis strategies to prepare C14 functionalized steroids usually need to be carried out with the assistance of specific functional groups, the range of substrates is very limited, and the reaction conditions are also relatively harsh, which seriously hinders the research of relevant steroid new drugs. Therefore, it is urgent to develop an efficient and universal synthesis strategy for the preparation of C14 functional steroids.

In this work, the authors isolated and identified a novel C14αhydroxylase (CYP14A) from Curvularia lunata CGMCC 3.3589, performed de novo calculations on its structure using RoseTTaFold and predicted its binding pattern to steroidal substrates, and subsequently identified four key amino acid residues related to their selectivity in the catalytic region by alanine scanning and whole-cell catalysis. Then, the saturation mutation and iterative saturation mutation strategies were used to screen the four sites for combined mutations, and finally two mutant strains I111L-V124W and I111L-M115K with excellent performance were obtained, and the efficient biocatalysts showed good catalytic efficiency and excellent regional selectivity for six C17-position steroid substrates with side chain substituents.

Figure 1: Analysis of catalytic activity of CYP14A and its mutant strains.

After the introduction of a hydroxyl group at the C14 position of the steroid, the hydroxyl group is eliminated to obtain a C14-C15 double bond. With this as the conversion unit, the hydrogen functionalization and bifunctional grouping of Δ14 unactivated olefins were realized by free radical addition and electrophilic addition reaction, and a variety of important functional groups, including β-OH, F, Cl, N3, SePh, etc., were successfully introduced, further increasing the diversity of C14 functional steroids.

Figure 2: Synthesis of C14-bit functional steroids

In addition, based on this chemoenzymatic synthesis strategy, they completed the efficient synthesis of cardiac glycosides natural products periplogenin, (+)-digitoxigenin and their three diastereomers in only 6-7 steps, fully demonstrating the powerful application potential of this strategy in complex steroid synthesis. Therefore, this chemoenzymatic strategy not only provides a new idea for the synthesis of new C14 functional steroids, but also paves the way for the development of related steroid new drugs.

Figure 3: Synthesis of the natural products periplogenin and (+)-digitoxigenin

The research work was supported by the National Key Program Project, the special fund of the basic scientific research business fund of the central universities and the start-up fund of Wuhan University. (Source: Science Network)

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