L 2 aminoadipic acid

Supernatants obtained from centrifuged cultures were filtered through 0.2 μm polytetrafluoroethylene (PTFE) filters (VWR, Barcelona, Spain). BA were derivatized with diethyl ethoxymethylenemalonate (DEEMM) (Sigma–Aldrich). 100 μl of sample were mixed with 175 μl of 1 M borate buffer (1 M boric acid neutralized with NaOH until pH 9.0), 75 μl of methanol (Merck), 2 μl of L-2-aminoadipic acid as internal standard (2 g/L) (Sigma–Aldrich) and 3 μl of DEEMM. The mixture was incubated at 30°C in an ultrasound bath (Selecta, Barcelona, Spain) for 45 min. Samples were then heated at 70°C for 2 h to allow the complete degradation of excess DEEMM and reagent by-products. Samples were filtered through 0.2 μm PTFE membranes (VWR) before injection into the chromatograph system. Samples were diluted, when necessary, with 0.1 N HCl (Merck). BA were separated and quantified by ultra high performance liquid chromatography (UHPLC) system (Waters, Milford, MA, United States) with an UPLC^®BEH C18 1.7 μm column (Waters), following the method previously described (Redruello et al., 2013 (link)). Empower 2 software (Waters) was used to control the system and to analyze the data. Standards were prepared with agmatine, putrescine dihydrochloride (Acros Organics, Geel, Belgium), tyrosine and tyramine in Milli-Q water. The BA concentrations provided are the average of three independent cultures.

DL-threo-β-methylaspartic acid, β-glutamic acid, DL-3-aminobutanoic acid, L-lysine and L-2-aminoadipic acid (2AAA) were obtained from Sigma-Aldrich; L-β-lysine from Enamine (Riga, Latvia) and (E)-6-aminohex-2-enoic acid (6-AHEA) from Toronto Research Chemicals (North York, Canada).