3.4. Determination of the Absolute Configuration of the Amino Acids by Marfey’s Method

In the case of 1, one sample (0.5 mg in 1mL of acetone at 0 °C) was oxidized with Jones reagent prior to the hydrolysis. Compounds 1, oxidized-1, and 2 (0.5 mg each) were hydrolyzed in 6 N HCl (1 mL) and analyzed by Marfey’s method [13]. The reaction mixture was maintained in a sealed glass bomb at 104 °C for 18 h. The acid was removed in vacuo, and the residue was re-suspended in 250 μL of H₂O. FDAA solution (1-fluoro-2,4-dinitrophenyl)-5-l-alanine amide in acetone (0.03 M, 20 μL per each amino acid in the peptide) and NaHCO₃ (1 M, 20 μL per each amino acid) were added to each reaction vessel. The reaction mixture was stirred at 40 °C for 2.5 h in the dark. HCl (2 M, 10 μL per each amino acid) was added to each reaction vessel, and the solution was evaporated in vacuo. The FDAA-amino acids derivatives from hydrolysate were dissolved in 1 mL CH₃CN and compared with standard FDAA-amino acids by an HPLC analysis: LiChroCART RP-18 column (5 μm, 250 × 4.6 mm); flow rate 1 mL/min; ultraviolet (UV) detection at 340 nm; linear gradient elution from 0.1% aq. TFA buffer (pH 3) to 6:4 MeCN:0.1% aq. TFA buffer (pH 3) within 60 min. The absolute configuration of each amino acid was determined by spiking the derivatized hydrolysates with a d,l-mixture of the standard derivatized amino acids.

Compound 2 was also analyzed by the advanced Marfey method [19]. Two 0.5 mg portions were hydrolyzed as described above and derivatized, one with l-FDAA and the other with d-FDAA. The two samples of l- and d-FDAA derivatives were analyzed by ESI LC MS. The analysis was performed on a Waters Acquity UPLC coupled with a UV detector (Waters Acquity-TUV detector) (Waters, Milford, MA, USA) and mass spectrometer (Waters Xevo TQD) (Waters, Milford, MA, USA) on a C18 (1.7 μm, 2.1 Å, ~100 mm) column (Waters Milford, MA, USA) The mobile phase compositions were (A) 95:5 H₂O/MeCN + 0.1% formic acid (FA) and (B) MeCN + 0.1% FA. The elution gradient was as follows: 1 min of 100% A, linear gradient to 40% B over 25 min, and hold for 4 min. Samples of 10 μL were injected, and the flow rate was 0.5 mL/min. The UV detector was set to 340 nm, and the mass spectrometer was operated in both negative and positive ion modes, scanning between 200 and 650 mass units. The interpretation of the data was conducted after the run on both positive and negative ion modes by MassLynx software (v4.1, Waters Laboratory Informatics).