The Michael addition of thiols to 13-oxo-octadecadienoate (13-oxo-ODE) with implications for LC-MS analysis of glutathione conjugation

William E Boeglin; Donald F Stec; Saori Noguchi; M Wade Calcutt; Alan R Brash

doi:10.1016/j.jbc.2024.107293

The Michael addition of thiols to 13-oxo-octadecadienoate (13-oxo-ODE) with implications for LC-MS analysis of glutathione conjugation

J Biol Chem. 2024 Apr 16;300(5):107293. doi: 10.1016/j.jbc.2024.107293. Online ahead of print.

Authors

William E Boeglin¹, Donald F Stec², Saori Noguchi¹, M Wade Calcutt³, Alan R Brash⁴

Affiliations

¹ Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA.
² Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA; Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA.
³ Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA.
⁴ Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA; Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA. Electronic address: alan.brash@vanderbilt.edu.

PMID: 38636660
DOI: 10.1016/j.jbc.2024.107293

Abstract

Unsaturated fatty acid ketones with αβ,γδ conjugation are susceptible to Michael addition of thiols, with unresolved issues on the site of adduction and precise structures of the conjugates. Herein we reacted 13-keto-octadecadienoic acid (13-oxo-ODE or 13-KODE) with glutathione (GSH), N-acetyl-cysteine, and β-mercaptoethanol and identified the adducts. HPLC-UV analyses indicated none of the products exhibit a conjugated enone UV chromophore, a result that conflicts with the literature and is relevant to the mass spectral interpretation of 1,4 versus 1,6 thiol adduction. Aided by the development of an HPLC solvent system that separates the GSH diastereomers and thus avoids overlap of signals in proton NMR experiments, we established the two major conjugates are formed by 1,6 addition of GSH at the 9-carbon of 13-oxo-ODE with the remaining double bond α to the thiol in the 10,11 position. N-acetyl cysteine reacts similarly, while β-mercaptoethanol gives equal amounts of 1,4 and 1,6 addition products. Equine glutathione transferase catalyzed 1,6 addition of GSH to the two major diastereomers in 44:56 proportions. LC-MS in positive ion mode gives a product ion interpreted before as evidence of 1,4-thiol adduction, whereas here we find this ion using the authentic 1,6 adduct. LC-MS with negative ion APCI gave a fragment selective for 1,4 adduction. These results clarify the structures of thiol conjugates of a prototypical unsaturated keto-fatty acid and have relevance to the application of LC-MS for the structural analysis of keto-fatty acid glutathione conjugation.

Keywords: (1)H-NMR; HPLC; UV-Vis spectrometry; keto fatty acid; mass spectrometry; oxo fatty acid.