Evidence of Isomerization in the Michael-Type Thiol-Maleimide Addition: Click Reaction between L-Cysteine and 6-Maleimidehexanoic Acid

Abstract

The reaction between L-cysteine (Cys) and 6-maleimidohexanoic acid (Mhx) in an aqueous medium at different levels of pH was analyzed via RP-HPLC, finding the presence of two reaction products throughout the evaluated pH range. By means of solid-phase extraction (SPE), it was possible to separate the products and obtain isolated profiles enriched up to 80%. The products were analyzed individually through mass spectrometry, DAD-HPLC, NMR 1H, 13C, and two-dimensional evidence of isomerization between the hydrogen atoms of the α-amino and the thiol group present in the cysteine. Thus, it was concluded that the products obtained corresponded to a mixture of the isomer Cys-S-Mhx, where the adduct is formed by a thioether bond, and the isomer Cys-NH-Mhx, in which the union is driven by the amino group. We consider that the phenomenon of isomerization is an important finding, since it has not previously been reported for this reaction.