Thiolate addition to withaferin A: a GFN2-xTB and DFT study of site-selective glutathione conjugation

Abstract Withaferin A (WFA), the major steroidal lactone of Withania somnifera , alkylates cysteine residues in vimentin, Hsp90 and IKKβ. Glutathione (GSH), the cell’s most abundant thiol, serves as the initial step in detoxifying electrophiles. The carbon that interacts with it determines both the rate of clearance and the extent to which a compound remains to alkylate proteins. It is unquantified at any of WFA’s four electrophilic carbons: the ring-A enone (C3), the side-chain lactone (C24) and the epoxide carbons (C5, C6). This study computes conformer-ensemble free energies with GFN2-xTB in implicit water with quasi-RRHO corrections, scans the C–S approach coordinate at each site, and recalibrates with M06-2X single point calculations on truncated models. Nucleophiles were modelled as thiolates and adducts as anions; the isolable species is the neutral protonated conjugate. Addition is most exergonic at C3-α (ΔG = − 11.7 kcal mol⁻¹), then C6 (− 8.9), C5 (− 8.3) and C24 (+ 0.2 to − 4.4). Site-specific DFT corrections invert this ordering toward the epoxides. A def2-TZVP check shifts every correction by about + 7.8 kcal mol⁻¹, removing the sign difference between reaction classes but, because it is uniform, leaving the ranking intact: the inversion is robust to basis set though the corrections are not. No Hessian-verified saddle was located, so no activation energy is reported; C3 has no interior scan maximum whereas both methods place one at the epoxide, supporting kinetic control without establishing it. The methanethiolate probe reproduced glutathionate’s site preference but inverted its facial preference, α by 7.0 against β by 13.3 kcal mol⁻¹, so minimal-thiol screening ranks sites dependably, faces unreliably.