Stereochemical determination of hydroxylated derivatives of progesterone using DFT NMR calculations and DP4+...

Hydroxylated derivatives of progesterone are biologically relevant compounds obtained through regio- and stereoselective biotransformations, but determining their stereochemical configuration is challenging due to subtle NMR chemical shift differences. In this study, monohydroxylated (prog-15-OH) and dihydroxylated (prog-7,15-di-OH) progesterone derivatives produced by Penicillium oxalicum CBMAI 1996 were analyzed. Quantum NMR calculations combined with statistical validation allowed the unambiguous identification of the 15β-OH and 7β,15β-di-OH isomers, with DP4+ probabilities of 100% and 99.8%, respectively. These findings confirm the stereochemical outcome of enzymatic hydroxylation and demonstrate the reliability of integrating computational NMR with experimental spectroscopy for resolving stereochemical ambiguities in complex steroidal frameworks.

Conformational searches were performed using the MMFF94 force field, followed by DFT geometry optimizations at the B3LYP/6-311G(2d,p) level with IEFPCM (chloroform). NMR shielding tensors were computed by the GIAO method at the mPW1PW91/6-311G(d,p) level with PCM (chloroform), and chemical shifts were referenced to TMS. Experimental and calculated ¹H/¹³C shifts were compared using correlation coefficients (R²), mean absolute deviation (MAD), root mean square deviation (RMSD), and DP4+ probability analysis. All calculations were performed with Gaussian 16.