Characterization of tryptolinophyllins from the treefrog Boana boans expands the structural and functional diversity of the trypytophyllin family of peptides.

Amphibian skin secretions constitute a rich source of bioactive peptides whose structural diversity is often amplified by post-translational modifications. In this study, we report the discovery and comprehensive characterization of a post-translational modification that has not been described previously in peptides from a vertebrate. Tryptophyllins are a heterogeneous group of short, tryptophan- and proline-containing peptides widely distributed among anuran species and are thought to contribute to skin protection against oxidative and UV-induced damage. Two peptides derived from tryptophyllins, termed tryptolinophyllins, were isolated from skin secretions of the giant gladiator treefrog Boana boans. They are distinguished by the presence of an N-terminal (S)-tryptoline-3-carboxylic acid (Tpi) residue, resulting from the conversion of tryptophan into a tetrahydro-β-carboline moiety via a Pictet-Spengler-type reaction. The primary structures of two tryptolinophyllins were elucidated by high-resolution tandem mass spectrometry and multidimensional NMR spectroscopy, and confirmed through total chemical synthesis of the corresponding diastereoisomers. To our knowledge, this represents the first confirmed tetrahydro-β-carboline post-translational modification in a vertebrate peptide, revealing an unexpected level of chemical innovation in amphibian peptide biosynthesis. Computational analyses of electronic structure identified the Tpi residue as the preferred locus for electron transfer, suggesting a potential role in redox chemistry. ABTS radical-cation scavenging assays demonstrated that the antioxidant activity of the tryptolinophyllins was reduced compared with unmodified tryptophyllins, highlighting a nuanced relationship between post-translational chemical diversification and functional output. Together, these findings expand the known repertoire of naturally occurring peptide modifications in vertebrates and underscore the exceptional chemical plasticity of amphibian skin secretions.