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

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 N-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 N⁶-carboxymethyl lysine (CML), glyoxal lysine amide (GOLA), glyoxal lysine dimer (GOLD), and N⁶-glycolyl lysine (GALA). Here, we introduce a novel amidine cross-link structure N¹,N²-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 N²-t-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.