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/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, while 9- and 10-NO2OA significantly upregulated Hmox1 and Gclm at all oxygen levels. 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 the biological activity of NO2FAs is dependent on oxygen level, which could be used in future research of other oxidative stress-dependent pathways.

Department of Cell Biology and Radiobiology Institute of Biophysics of the Czech Academy of Sciences Kralovopolska 135 612 00 Brno Czech Republic

Department of Chemical Biology Faculty of Science Palacky University Šlechtitelů 27 CZ 783 71 Olomouc Czech Republic

Department of Medical Chemistry and Biochemistry Faculty of Medicine and Dentistry Palacky University Hnevotinska 3 775 15 Olomouc Czech Republic

Department of Organic Chemistry Faculty of Science Palacky University tř 17 listopadu 1192 12 CZ 771 46 Olomouc Czech Republic

Laboratory of Growth Regulators Palacky University and Institute of Experimental Botany AS CR Šlechtitelů 27 CZ 783 71 Olomouc Czech Republic

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