In-peptide amino acid racemization via inter-residue oxazoline intermediates during acidic hydrolysis

Isopedopeptins are antibiotic cyclic lipodepsipeptides containing the subsequence L-Thr—L-2,3-diaminopropanoic acid—D-Phe—L-Val/L-3-hydroxyvaline. Acidic hydrolysis of isopedopeptins in D(2)O showed the D-Phe residues to racemize extensively in peptides with L-3-hydroxyvaline but not in peptides with L-Val. Similarly, one Leu residue in pedopeptins, which are related peptides containing the subsequence Leu—2,3-diaminopropanoic acid—Leu—L-Val/L-3-hydroxyvaline, was found to racemize in peptides with L-3-hydroxyvaline. Model tetrapeptides, L-Ala—L-Phe—L-Val/3-hydroxyvaline—L-Ala, gave the corresponding results, i.e. racemization of L-Phe only when linked to a L-3-hydroxyvaline. We propose the racemization to proceed via an oxazoline intermediate involving Phe/Leu and the L-3-hydroxyvaline residues. The 3-hydroxyvaline residue may form a stable tertiary carbocation by loss of the sidechain hydroxyl group as water after protonation. Elimination of the Phe/Leu H-2 and ring-closure from the carbonyl oxygen onto the carbocation results in the suggested oxazoline intermediate. The reversed reaction leads to either retained or inversed configuration of Phe/Leu. Such racemization during acidic hydrolysis may occur whenever a 3-hydroxyvaline residue or any amino acid that can form a stable carbocation on the C-3, is present in a peptide. The proposed mechanism for racemization was supported by incorporation of (18)O in the 3-hydroxyvaline sidechain when the acidic hydrolysis was performed in H(2)O/H(2)(18)O (1:1). The 2,3-diaminopropanoic residues of isopedopeptins and pedopeptins were also found to racemize during acidic hydrolysis, as previously described. Based on the results, the configuration of the Leu and 2,3-diaminopropanoic acid residues of the pedopeptins were reassigned to be L-Leu and D-Leu, and 2 × L-2,3-diaminopropanoic acid. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00726-021-02951-7.