4.4. Preparation and Analysis of the Marfey Derivatives

About 0.5 mg of the synthesized cycloheptapeptide 8 was hydrolyzed by heating in 1 mL of 6 M HCl at 110 °C for 24 h. After cooling, the solution was evaporated to dryness and redissolved in 50 μL of water. Then 100 μL of a 1% w/v solution of FDAA (Marfey’s reagent) in acetone was added to the acid hydrolyzate solution (or to 50 μL of a 50 mM solution of the respective amino acid). After addition of 20 μL of the 1 M NaHCO₃ solution, the mixture was incubated for 1 h at 40 °C. The reaction was stopped by the addition of 10 μL of 2 M HCl. Finally, the solvents were evaporated to dryness and the residue was dissolved in 1 mL of MeOH. An aliquot of this solution (20 μL for the cyclopeptide 8 and 10 μL for the standards) was analyzed by HPLC (Phenomenex Luna C18, 4.6 × 250 mm, 5 μm, solvents: (A) water + 0.05% TFA, (B) MeCN, linear gradient: 0 min 35% B, 30 min 45% B, 1 mL min⁻¹, 25 °C). The retention times (min) of the FDAA amino acid derivatives used as the standards were as follows: gly (18.24), l-ile (28.92), d-ile (33.32), l-tyr (20.51), d-tyr (33.08), l-leu (27.25), d-leu (25.05), l-phe (17.41), d-phe (22.32), l-pro (17.45), and d-pro (18.39). Retention times (min) and relative peak area (%) of the observed peaks of the FDAA derivatized hydrolysis products of cyclopeptide 8 were as follows: gly (18.27, 2.39%), l-leu (27.23, 2.48%), l-ile (28.90, 3.67%), l-tyr (20.54, 5.24%), l-phe (17.44, 7.33%), d-phe (22.36, 6.26%), and l-pro (17.42, 7.82%).