A Modular Chemoenzymatic Approach to C14-Functionalized Steroids

Abstract

C14-functionalized steroids belong to a unique class of steroids with important biological activities. However, the lack of efficient methods to access C14-functionalized steroids impede related steroidal drug discovery. Herein we report a modular chemoenzymatic approach to access diversified C14-functionalized steroids. We first identified a novel C14α-hydroxylase (CYP14A) from Cochliobolus lunatus with high catalytic efficiency and substrate promiscuity. Protein engineering of CYP14A generated three variants I111A, M115K and V124A that greatly improved the C14-hydroxy regioselectivity. Based on this efficient biocatalytic method, a range of C14α-OH steroids with C17 side chain were prepared in good yields, which was then transformed into Δ14 olefins through a facile elimination. The newly formed Δ14 olefin served as a versatile handle to install diversified functional groups (e.g. epoxide, β-OH, F, Cl and N3) at C14 position through hydrofunctionalization. Furthermore, the synthetic utility of this powerful chemoenzymatic methodology was demonstrated by performing a 7-step semisynthesis of periplogenin and the diversity-oriented synthesis of cardenolide (+)-digitoxigenin and its three diastereomers in a concise manner.

Selective Hydroxylation of C(sp3)−H Bonds in Steroids

“…Interestingly, changing R from methyl to ethyl resulted in a diastereoselectivity switch, with selective cis-16β hydroxylation being favoured, likely due to steric interactions between the N substituent and the 13β methyl group which consequently affects the conformation of the ligand in the metal complex. Yields of bidentate ligands (85)(86)(87)(88) were typically better than the corresponding tridentate ligand (89), and the yields for the aminomethylpyridino ligands (n = 1) were slightly higher than the corresponding aminoethylpyridino ligands (n = 2).…”

“…A variant containing a single point mutation at M115K, expressed in S. cerevisiae, was able to convert deoxycorticosterone 49 into 50 on gram-scale, which allowed for the total synthesis of a range of cardenolide steroids 51-53 (Scheme 9A). [89] It was found that a broth culture of T. cucumeris expressing TcP450-1 was able to directly hydroxylate the C19 position of 54 to form 55 with 80 % conversion, bypassing the alternative lengthy and low yielding chemical synthesis (2 % over 13 steps). [90] This methodology was conducted on large-scale to yield over 5 g of 55, which was further elaborated to provide a range of 19-OH steroids 56-58 (Scheme 9).…”