Marfey assay for chiral analysis of amino acids.

For determination of the configuration of 4-hydroxyphenylglycine 6 (Hpg6) and 3-OH-Asn9, a sample of ca. 0.1 mg 8D1-1 was hydrolyzed in 2 ml 6 N HCl in a sealed tube at 120°C for 24 h. The hydrolysate was dried under reduced pressure at 50°C in a Buchi R210 rotary evaporator, dissolved in 20 μl double-distilled water (ddH₂O), and transferred to a 1.5-ml Eppendorf tube, and 40 μl of 1% acetone solution of N-(5-fluoro-2, 4-dinitrophenyl)-d-alaninamide (FDAA [Marfey's reagent]) and 8 μl of 1 M NaHCO₃ solution were added to the hydrolysate for derivatization. The reaction mixture was heated at 40°C with frequent shaking for 1 h over a hot plate and then cooled to room temperature. The reaction was quenched by adding 4 μl of 2 N HCl and diluted with 200 μl MeOH for LC-MS analysis (33). To determine the configuration of Trp3, a sample of 0.1 mg 8D1-1 was hydrolyzed in a 2-ml solution of trifluoroacetic acid-HCl at 1:2 (5% thioglycolic acid) in a sealed tube at 166°C for 0.5 h (34). The hydrolysate was then dried under reduced pressure at 60°C and treated by the same procedure as above. Standards (l/d-Hpg, 3-OH-l-Asp, and l/d-Trp) were derivatized using the same procedure.