Regio- and Stereoselective Synthesis of Nitro-fatty Acids as NRF2 Pathway Activators Working under Ambient or Hypoxic Conditions

Nitro-fatty acids (NO₂FAs) are endogenously produced electrophiles and NRF2 activators with therapeutic potential. In this work, we developed a synthetic protocol combining a Henry reaction and base-promoted β-elimination, yielding ultra-pure regio/stereoisomers of nitro-stearic (NO₂SA), nitro-oleic (NO₂OA), and conjugated/bis-allylic nitro-linoleic (NO₂LA) acids. These were tested for NRF2 pathway activation in bone marrow cells under different oxygen conditions. We observed that 9- and 10-NO₂OA, and 10-NO₂LA increased NRF2 stabilization under hypoxia, whereas only 9- and 10-NO₂OA significantly upregulated Hmox1 and Gclm at all oxygen levels. Both 9- and 10-NO₂OA enhanced HO-1 and GCLM proteins independently of oxygen, while 10-NO₂LA was oxygen-dependent, boosting HO-1 under hypoxia and GCLM under ambient conditions. Moreover, 10-NO₂OA and 10-NO₂LA induced NRF2 nuclear translocation. In contrast, the saturated 10-NO₂SA, which has lower electron-acceptor ability, was inactive. In summary, these findings suggest that the biological activity of NO₂FAs is dependent on oxygen level, which could be used in future research of other oxidative stress-dependent pathways.