The elemental composition of CuLs ligands (C26-35, H23-36, O5-8, N3-4) suggests similarities to breakdown products of the well-known and ubiquitous biomolecule, chlorophyll (C35, H34, O5, N4 for chlorophyllide a). Chlorophyll has a macrocyclic tetrapyrrole ring system of four substituted pyrroles (labeled A-D) connected by methine (meso-carbon) bridges (Fig. ). There is also a ring (E) that is not a pyrrole positioned exocyclic to the macrocycle. Enzymatic catabolism of chlorophylls a and b begins with demetallation (loss of Mg 2+ ) and ester hydrolysis (loss of phytol), followed by oxygenolytic opening of the macrocycle between rings A and D to form a red chlorophyll catabolite (RCC; Fig. ). RCC has an H:C ratio of 1.09, O:C ratio of 0.2, and N:C ratio of 0.11, values that all fall within the ranges observed for the CuLs produced by P. tricornutum. Subsequent catabolism of RCC produces di-, tri-, and tetrapyrroles decorated with a suite of different functional groups. These catabolites have characteristic fragmentation pathways that include: (1) the loss of CH4O from a methoxycarbonyl functional group on ring E, (2) loss of H2O and CO, (3) fragmentation at the meso-carbon positions with loss of pyrroles and (4) decarboxylation reactions (loss of CO2, CH2O2) (Fig. ). Therefore, the corresponding metal-free ligands were targeted for fragmentation. The loss of C7H7O3N and C8H7O3N is also observed in the MS 2 spectrum of the metal free ligand G (Fig. ) as fragment ions at m/z 434.20745, [C₂₅H₂₈O₄N₃] + and 422.20745, [C₂₄H₂₈O₄N₃] + (Table ). In contrast to the MS 2 spectrum of the CuL G, the MS 2 spectrum for the metal-free ligand was dominated by fragment ions arising from the loss of N-containing moieties, supporting the hypothesis that coordination with Cu stabilizes the structure. The N:C ratio of the fragment ions (0.12 to 0.13) is the same or similar to the parent CuL ion (0.13), which indicates that the nitrogen is dispersed across the molecule rather than localized, which is consistent with a tetrapyrrole structure. 2.57 A major fragmentation pathway for metal-free ligand G is the sequential loss of CO2 to produce the fragment ion with nominal m/z 543, followed by the loss of a terminal pyrrole (C7H7ON) to produce nominal m/z 422, followed by the loss of a second pyrrole (C8H11O2N) to produce m/z 269 (Fig. ). The loss of CO2 with the loss of pyrroles has been seen when using higher fragmentation energies (like that used in this study) on bilin tetrapyrroles, heme catabolites that are structurally similar to chlorophyll catabolites, though they lack ring E. In order to confirm the sequential loss of pyrroles from nominal m/z 422, this ion was targeted for further fragmentation. The MS 3 spectrum of m/z 422 (SI Fig. ) yielded a fragment ion at m/z 269, confirming the sequential loss of pyrroles from metal-free ligand G. Fragmentation of ion m/z 269 (SI Fig. ), yielded fragment ions at m/z 241.0 ([M -CO] + ) and 226.0 ([M -CHON] + ), indicating the presence of a carbonyl and a cyclic amide. Based on the fragmentation patterns and the similarities to chlorophyll catabolites, a draft structure for metal-free and Cu-bound ligand G is proposed (Fig. ). The extracted ion chromatograms (EICs) for CuLs A-K yielded unique peaks for each CuL. In contrast, the corresponding EICs of the metal-free ligands exhibited multiple peaks (SI Fig. ). This further supports the stabilization of the ligand structure by complexation to Cu and suggests that there may be different isomers of each metalfree ligand. MS 2 spectra were obtained for all metal-free ligands except for C and I, and all Cu-bound ligands except A, B, and J. However, structures of these ligands can be inferred based on systematic differences stemming from their molecular formulae. Ligand C differs from ligand B by the addition of a CH2 and elutes slightly later in the LC-ESI MS chromatogram (Fig. ). Therefore, we infer that ligand C is a homologue of ligand B. Similarly, ligand I differs from ligand H by an H2, and from ligand J by an O. Given this, we infer that ligands H, I, and J have the same parent structure with ligand I having an additional double bond compared to H and an alcohol functionality compared to J. This is supported by the MS 2 spectra of metal-free ligands H and J which have nine fragment ions in common (SI Fig. ). While the MS 2 spectrum of CuL J was not acquired, spectra for CuLs H and I show five fragment ions in common and four fragment ions that differ by an H2 (SI Fig. ).