Glycine-Containing Flaxseed Orbitides

Five new orbitides, cyclolinopeptides <b>21</b>–<b>25</b>, were identified in flaxseed oil (<i>Linum usitatissimum</i>) extracts. Their HPLC-ESIMS quasimolecular ion peaks at <i>m</i>/<i>z</i> 1097.7 (<b>21</b>), 1115.6 (<b>22</b>), 1131.6 (<b>23</b>), 1018.6 (<b>24</b>), and 1034.6 (<b>25</b>) [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]-G<i>O</i>LIPPFFVI, [1–9-NαC]-GMLVFPLFI, and [1–9-NαC]-G<i>O</i>LVFPLFI for cyclolinopeptides <b>21</b>–<b>25</b>, respectively. Previously reported orbitides, [1–9-NαC]-ILVPPFFLI (<b>1</b>), [1–9-NαC]-MLIPPFFVI (<b>2</b>), [1–9-NαC]-<i>O</i>LIPPFFVI (<b>3</b>), [1–8-NαC]-MLVFPLFI (<b>7</b>), and [1–8-NαC]-<i>O</i>LVFPLFI (<b>8</b>), were also present in flaxseed oil. The precursors of orbitides <b>21</b>, <b>22</b>, and <b>24</b> also produced orbitides <b>1</b>, <b>2</b>, and <b>7</b> by alternative cyclization. Cyclolinopeptides <b>3</b>, <b>8</b>, <b>23</b>, and <b>25</b> contain MetO (<i>O</i>) and are not directly encoded, but are products of post-translational modification of the Met present in <b>2</b>, <b>7</b>, <b>22</b>, and <b>24</b>, respectively. Sufficient cyclolinopeptide <b>23</b> 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 <b>21</b>, <b>22</b>, <b>23</b>, <b>24</b>, and <b>25</b>, respectively.