Phyllobilins

Phyllobilins (PBs) are linear tetrapyrroles generated by the metabolic breakdown of chlorophyll in plants. They are products of the pheophorbide a oxygenase/phyllobilin (PAO/PB) pathway of natural chlorophyll breakdown, and are distantly related to the bilins resulting from heme breakdown, such as bilirubin. Chlorophyll breakdown is a massive phenomenon, most visual when the fall colors arise, producing each year about 109 tons of PBs worldwide. Strikingly, despite its obvious importance, chlorophyll breakdown has long remained enigmatic, as the search for its expected tetrapyrrolic products has been futile. By 1991, organic chemist Bernhard Kräutler, botanist Philippe Matile, and their teams succeeded in unambiguously identifying a first chlorophyll catabolite from plants (then called RP-14) and elucidating its striking structure. This first, colorless PB from de-greened cotyledons of barley (Hordeum vulgare) was classified as a non-fluorescent chlorophyll catabolite (NCC) and given the provisional name Hv-NCC-1 (fig. 1). A colorless, but fluorescent PB found in oil seed rape, subsequently characterized and named "primary" FCC (pFCC), is a formal precursor of Hv-NCC-1, and has a structure more similar to pheophorbide a (Pheo a). The pFCC-structure highlights the critical cleavage of the porphyrinoid macrocycle of chlorophyll (fig. 1) in the course of chlorophyll breakdown in plants.

Nomenclature

Original names of the PBs have been phenomenological, short acronyms, specifying their plant source and structure-type, such as Hv-NCC-1. A structure-based nomenclature named the tetrapyrrolic chlorophyll catabolites as 4,5-dioxo-4,5-seco-phytoporphyrinates, i.e., as between C4 and C5 ring-opened derivatives of Pheo a. Accordingly, pFCC is a 31,32-didehydro-1,4,5,10,17,18,20-(22H)-octahydro-132-methoxycarbonyl-4,5-dioxo-4,5-seco-phytoporphyrinate. Since 2014, when linear tetrapyrroles from Chl-breakdown were classified as "phyllobilins" (PBs) by Kräutler, previous names were replaced by a phyllobilane-based semi-systematic nomenclature (fig. 2), as recommended for linear tetrapyrroles by the International Union of Pure and Applied Chemistry (e.g., for the heme-derived bilins). In consequence, pFCC is a (10Z)-1-formyl-19-oxo-12,13,16,19-tetrahydro-phyllobilene-b. With the discovery of two 1,19-dioxo-phyllobilins that were structurally related to Hv-NCC-1, a puzzling altered type of phyllobilins became known (2001, 2011), to be established (only in 2013) as first representatives of an important second branch of chlorophyll breakdown in some plants. Since then, PBs are classified either as type-I PBs (1-formyl-19-oxo-phyllobilins), or as type-II PBs (1,19-dioxo-phyllobilins). Nowadays, over 80 different natural PBs (including their synthetic metal complexes) are known.

Colorless phyllolumibilins and phylloleucobilins The two basic types of the important colorless phyllobilins in plants are the (nonfluorescent) phylloleucobilins and (fluorescent) phyllolumibilins. They carry a remarkable variety of functional groups at the periphery of their tetrapyrrole cores, typically increasing their polarity (fig. 3 and table). The colorless PBs represent the major group of the known natural PBs. A hydroxyl group attached at C32 is the first one of the substituents introduced at the stage of the primary FCCs (pFCCs), giving the corresponding secondary FCCs (sFCCs). Further modifications are deduced to furnish a variety of modified FCCs (mFCCs, fig. 3). In fact, typical FCCs are only fleetingly existent catabolites and isomerize non-enzymatically in aqueous environment to the corresponding NCCs, 82S,10R-isomers, as, e.g., Hv-NCC-1. Hence, with few exceptions, the peripheral modifications were only detected in NCCs (figs. 1, 3).

When pFCC was identified, its configuration at C16 could not be characterized, and was later classified provisionally as n. In fact, the "primary" FCCs are generated plant-specifically, either as pFCC, C16n (fig. 1), or as epi-pFCC, with epi-configuration at C16, and both C16-epimeric FCCs exist naturally. Hydroxylation at C32 of the respective primary FCC occurs irrespective of its C16 configuration: sFCC is derived from pFCC, epi-sFCC from epi-pFCC. NCCs inherit their configuration at C16 from their precursor FCCs; hence, Hv-NCC-1 is C16n, as it is derived from pFCC via sFCC. Oxidative removal of the 1-formyl function of the sFCC was deduced to generate the sDFCC, the cryptic natural methyl ester that is rapidly demethylated enzymatically (in Arabidopsis thaliana) to At-DFCC-33, a first DFCC, actually isolated from a leaf (fig. 3). At-DFCC-33 isomerizes rapidly to the corresponding DNCC, opening up access to further downstream 1,19-dioxo-phyllobilins in A. thaliana. In view of the existence of two important breakdown paths, the PBs are now classified either as type-I PBs (1-formyl-19-oxo-phyllobilins) or as type-II PBs (1,19-dioxo-phyllobilins).

In contrast to typical FCCs (fig. 3), so called hypermodified fluorescent FCCs (hmFCCs), first discovered in ripening bananas as, e.g., Mc-FCC-56, (fig. 4) are uniquely persistent. They accumulate as blue fluorescent natural "optical brighteners" in some ripening fruit, a particularly intriguing feature of de-greened banana fruits, and in senescent banana leaves. Among the hmFCCs, lipophilic derivatives have been found, such as Ma-FCC-114 that contains parts of a main chloroplast membrane constituent. An exceptional hmFCC variant is represented by phyllolumibilins with a bicyclo-glycoside appendage, such as Vv-FCC-55, isolated from grapevine leaves. In this phyllolumibilin, a 1',6'-glucose link is "bridging" between its carboxyl-O124 and hydroxyl-O33 in a remarkable macrocycle (fig. 4 right).

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