L val

Commercial synthetic γ‐D‐Glu‐L‐Trp, γ‐D‐Glu‐γ‐D‐Glu‐L‐Trp, γ‐L‐Glu‐L‐Trp, γ‐L‐Glu‐γ‐L‐Glu‐L‐Trp, γ‐D‐Glu‐L‐Phe, γ‐D‐Glu‐L‐Met and γ‐D‐Glu‐L‐Val were obtained from Apeptides (Shanghai, China). D‐Gln, L‐Gln, L‐Val, L‐Trp, L‐Met, L‐Phe, Gly‐Gly, and γ‐glutamyl‐p‐nitroanilide (γ‐GpNA) were purchased from Sigma‐Aldrich (Shanghai, China). L‐Glutaminase of Bacillus amyloliquefaciens was acquired from Amano Enzyme China Ltd. (Shanghai, China). The HPLC grade acetonitrile and formic acid were purchased from CapitalBio Corporation (shanghai, China).

Sclerin (1) (200 μg) and cyclosenegalin A (2) (200 μg) were hydrolyzed in 200 μL 6 M HCl at 50 °C for 18 h. After, the residual HCl was removed in vacuo, and then 100 μL of an acetone solution containing 0.1 M of NaHCO₃ and 25 μg of 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (l-FDAA) was added to the residue. The solution mixture was heated at 75 °C for 4 h. Next, the reaction mixture was cooled, neutralized with 2 M HCl (50 µL) and dissolved in MeOH (200 μL). About 10 µL of each solution of FDLA derivatives was analyzed by HPLC. On the other hand, authentic standards of l-Dab, l-Pro, l-Ile, l-Leu, l-Ala, l-Val, l-Thr, l-Tyr, l-Ser (Sigma-Aldrich, St Louis, MO, USA) were treated with l-FDAA, as described above. The l-FDAA derivative of l-amino acid standard were analyzed by HPLC–UV, and the retention times of l-Dab (2.6), l-Pro (4.2), l-Ile (20.1), l-Leu (5.5), l-Ala (4.7), l-Val (9.8), l-Thr (3.0), l-Tyr (6.4), l-Ser (4.6) were compared with the Marfey’s derivative of 1 and 2. HPLC conditions: a 5 μM column X-Terra MS C-18 (150 × 3.0 mm) maintained at 25 °C was eluted at 1 mL/min with 40% MeOH/H₂O containing 0.01% HCOOH for 25 min.