2.2. Synthesis of allysine

N-Acetyl-L-AAS (allysine) was synthesized from N-acetyl-l-lysine using lysyl oxidase activity from egg shell membrane following the procedure described by Akagawa et al. [15]. Briefly, 10 mM N-acetyl-l-lysine was incubated at constant stirring with 5 g egg shell membrane in 50 mL of 20 mM sodium phosphate buffer, pH 9.0 at 37 °C for 24 h. The egg shell membrane was then removed by centrifugation and the pH of the solution adjusted to 6.0 using 1 M HCl. The resulting aldehydes were reductively aminated with 3 mmol p-amino-benzoic acid (PABA) in the presence of 4.5 mmol sodium cyanoborohydride (NaBH₃CN) at 37 °C for 2 h with stirring. Then, PABA derivatives were hydrolyzed by 50 mL of 12 M HCl at 110 °C for 10 h. The hydrolysates were evaporated at 40 °C in vacuo to dryness. The resulting allysine-PABA was purified by using silica gel column chromatography and ethyl acetate/acetic acid/water (20:2:1, v/v/v) as elution solvent. The purity of the resulting solution and authenticity of the standard compounds obtained following the aforementioned procedures were checked by using MS and ¹H NMR [16].