Abstract
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 D2O 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 18O in the 3-hydroxyvaline sidechain when the acidic hydrolysis was performed in H2O/H218O (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.
Highlights
Molecules produced by the metabolism of living organisms frequently contain one or more stereogenic carbon atom in their structures
We present data supporting that the observed racemization occurs via interresidue oxazoline intermediates, which may be formed in the presence of an amino acid, e.g. L-3-hydroxyvaline (OHVal) that can form a stable carbocation
When isopedopeptin B and D were subjected to acidic hydrolysis in D2O, and analysed by the advanced Marfey’s method (Harada et al 1995; Fujii et al 1997), LCMS peaks from 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) derivatives of L-Thr, L-Asp, L-OHVal, L-Leu, L- and D-Phe, L- and D-diaminopropanoic acid (DAPA), as well as L-2,4-diaminobutanoic acid (L-DABA) were obtained (Fig. 2a)
Summary
Molecules produced by the metabolism of living organisms frequently contain one or more stereogenic carbon atom in their structures. Isopedopeptin A-H are antibacterial cyclic lipodepsipeptides (Fig. 1) which are produced by the bacterium Pedobacter cryoconitis UP508 (Nord et al 2020) These peptides have the general structure cyclo[(R)-3-hydroxyalkanoyl—L-2,3-diaminopropanoyl—L-Leu—L-2,4-diaminobutanoyl—(Z)-2-amino-2-butenoyl—L-Thr—L-2,3-diaminopropanoyl—D-Phe—L-3-hydroxyvaline/L-Val—L-Asp] (Fig. 1) (Nord et al 2020). When characterizing these peptides, using the advanced Marfey’s method (Harada et al 1995; Fujii et al 1997), we noticed that L-2,3-diaminopropanoic acid (DAPA) racemized to a large extent during acidic hydrolysis of the peptides (Nord et al 2020), in accord with previous findings (Kjær and Olesen Larsen 1959). We present data supporting that the observed racemization occurs via interresidue oxazoline intermediates, which may be formed in the presence of an amino acid, e.g. L-3-hydroxyvaline (OHVal) that can form a stable carbocation
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