Novel Amidine Protein Cross-Links Formed by the Reaction of Glyoxal with Lysine

Abstract

One crucial aspect of the Maillard reaction is the formation of reactive α-dicarbonyl structures like glyoxal, which are prone toward further reactions with proteins, e.g., the <i>N⁶</i>-amino group of lysine. The initially formed labile glyoxal-imine was previously established as a key intermediate in the formation of the advanced glycation end products <i>N</i>⁶-carboxymethyl lysine (CML), glyoxal lysine amide (GOLA), glyoxal lysine dimer (GOLD), and <i>N</i>⁶-glycolyl lysine (GALA). Here, we introduce a novel amidine cross-link structure <i>N</i>¹,<i>N</i>²-bis-(5-amino-5-carboxypentyl)-2-hydroxy-acetamidine (glyoxal lysine amidine, GLA), which is formed exclusively from glyoxal through the same isomerization cascade. After independent synthesis of the authentic reference standard, we were able to quantitate this cross-link in incubations of 40 mM <i>N</i>²-<i>t</i>-Boc-lysine with glyoxal and various sugars (40-100 mM) under mild conditions (pH 7.4, 37 °C) using an HPLC-MS/MS method. Furthermore, incubations of proteins (6 mg/mL) with 50 mM glyoxal confirmed the cross-linking by GLA, which was additionally identified in acidic hydrolyzed proteins of butter biscuits after HPLC enrichment.