Glycine-Containing Flaxseed Orbitides

Five new orbitides, cyclolinopeptides 21–25, were identified in flaxseed oil (Linum usitatissimum) extracts. Their HPLC-ESIMS quasimolecular ion peaks at m/z 1097.7 (21), 1115.6 (22), 1131.6 (23), 1018.6 (24), and 1034.6 (25) [M + H]⁺ corresponded to the molecular formulae C₅₉H₈₉N₁₀O₁₀, C₅₈H₈₇N₁₀O₁₀S, C₅₈H₈₇N₁₀O₁₁S, C₅₃H₈₀N₉O₉S, and C₅₃H₈₀N₉O₁₀S, respectively. Their structures were elucidated by extensive HPLC-ESIMS/MS analyses, and their presence was confirmed by precursor proteins identified in flax genomic DNA sequence data. The amino acid sequences of these orbitides were confirmed as [1–10-NαC]-GILVPPFFLI, [1–10-NαC]-GMLIPPFFVI, [1–10-NαC]-GOLIPPFFVI, [1–9-NαC]-GMLVFPLFI, and [1–9-NαC]-GOLVFPLFI for cyclolinopeptides 21–25, respectively. Previously reported orbitides, [1–9-NαC]-ILVPPFFLI (1), [1–9-NαC]-MLIPPFFVI (2), [1–9-NαC]-OLIPPFFVI (3), [1–8-NαC]-MLVFPLFI (7), and [1–8-NαC]-OLVFPLFI (8), were also present in flaxseed oil. The precursors of orbitides 21, 22, and 24 also produced orbitides 1, 2, and 7 by alternative cyclization. Cyclolinopeptides 3, 8, 23, and 25 contain MetO (O) and are not directly encoded, but are products of post-translational modification of the Met present in 2, 7, 22, and 24, respectively. Sufficient cyclolinopeptide 23 was isolated for characterization via 1D (¹H and ¹³C) and 2D (NOESY and HMBC) NMR spectroscopy. These compounds have been named as cyclolinopeptides U, V, W, X, and Y for 21, 22, 23, 24, and 25, respectively.