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. We developed a synthetic protocol combining a Henry reaction and base-promoted β-elimination, yielding ultrapure regio/stereoisomers of nitro-stearic (NO₂SA), nitro-oleic (NO₂OA), and conjugated/<i>bis</i>-allylic nitro-linoleic (NO₂LA) acids. These were tested for NRF2 pathway activation in bone marrow cells under different oxygen conditions. We observed that <b>9-</b> and <b>10-NO</b>_<b>2</b><b>OA</b>, and <b>10-NO</b>_<b>2</b><b>LA</b> increased NRF2 stabilization under hypoxia, while <b>9-</b> and <b>10-NO</b>_<b>2</b><b>OA</b> significantly upregulated <i>Hmox1</i> and <i>Gclm</i> at all oxygen levels. <b>9-</b> and <b>10-NO</b>_<b>2</b><b>OA</b> enhanced HO-1 and GCLM proteins independently of oxygen, while <b>10-NO</b>_<b>2</b><b>LA</b> was oxygen-dependent, boosting HO-1 under hypoxia and GCLM under ambient conditions. Moreover, <b>10-NO</b>_<b>2</b><b>OA</b> and <b>10-NO</b>_<b>2</b><b>LA</b> induced NRF2 nuclear translocation. In contrast, the saturated <b>10-NO</b>_<b>2</b><b>SA</b>, which has lower electron-acceptor ability, was inactive. In summary, these findings suggest the biological activity of NO₂FAs is dependent on oxygen level, which could be used in future research of other oxidative stress-dependent pathways.