Chlorophyll Breakdown – On a Nonfluorescent Chlorophyll Catabolite from Spinach

Abstract

Dedicated to Prof. Andre M. Braun on the occasion of his 60th birthday In extracts of senescent leaves of spinach (Spinacia oleracea) that had degreened naturally after the onset of flowering, four colorless compounds, which had characteristic UV/VIS properties of nonfluorescent chlorophyll catabolites (NCCs), were detected by HPLC. From the extracts of 58.7 g of senescent leaves of Sp. oleracea, a two-stage HPLC purification procedure provided ca. 15 mmol of So-NCC-2, the most abundant polar NCC in the leaves of this vegetable. So-NCC-2 was isolated as a slightly yellow powder and analyzed by spectroscopic means. The high-resolution mass spectra indicated that So-NCC-2 has the same molecular formula as Hv-NCC-1 from barley (Hordeum vulgare), the first non-green chlorophyll catabolite from a higher plant to be structurally analyzed. Homo-and hetero-nuclear NMR spectroscopy indicated So-NCC-2 to have the same constitution as its epimer Hv-NCC-1, and to differ from the latter by the configuration at C(1). The catabolite from spinach could be identified with one of the products from OsO 4 dihydroxylation at the vinyl group of the main NCC from Cercidiphyllum japonicum. Chlorophyll breakdown in spinach and in C. japonicum apparently involves an enzyme-catalyzed reduction that occurs with the same stereochemical sense at C(1), but opposite to that in barley.

Breakdown of chlorophyll: A nonenzymatic reaction accounts for the formation of the colorless “nonfluorescent” chlorophyll catabolites

“…The molecular formula of Cj-NCC-2 (1) was determined with highresolution MS as C 35 H 40 N 4 O 7 , based on its pseudomolecular ion at m͞z ϭ 629.2990. This result indicated Cj-NCC-2 (1) has a molecular weight smaller than the one of any known NCC (6,18,(20)(21)(22)(23)(24) and relates to Cj-NCC-1 (3) (18) by its lack of one oxygen atom. The NCC 1 was thus identified as an isomer of a primary FCC, either the primary FCC (Bn-FCC-2) obtained from oil seed rape (Brassica napus) (13) 2 -methoxycarbonyl)-4,5-dioxo-4,5-secophytoporphyrin.…”

“…The NCC 1 is a natural NCC lacking a peripheral oxidative refunctionalization. So far, a hydroxyl group or a polar, oxygen-bearing functional group at the ethyl group extending from ring B was considered to be a characteristic of the natural NCCs (6,18,(20)(21)(22)(23)(24). Such a function was assumed to be required as the basis for further peripheral refunctionalization, to assist the inter-organellar transport from the senescent chloroplast to the vacuole (17,25,26).…”

“…The absolute configuration at C-1 appears not to be altered during the subsequent catabolic transformations and the NCCs are also indicated to fall into the two groups, depending on the stereochemistry of their precursor FCC (22,32): e.g., the NCCs 1 and 3 from leaves of C. japonicum fall into the same stereochemical group as the NCCs from C. annuum and are therefore derived from a primary FCC identical to Ca-FCC-2 (2) (16,22). Despite the indicated stereochemical divergence of the NCCs at C-1, the long wavelength parts of the CD spectra of the known natural NCCs are all similar (see, e.g., Fig.…”

“…Analysis of the nuclear Overhauser effect spectra of various NCCs allowed the relative configuration at C-15 and C-13 2 to be derived as being the same in all of them. As configurational equilibration at the exchange labile C-13 2 produces the more stable trans arrangement of the bulky groups at C-15 and C-13 2 , the configurationally stable chiral center C-15 directs the stereochemical preference at C-13 2 (4,5,18,20,(22)(23)(24). The absolute configuration of the stereocenters C-15 and C-13 2 of the NCCs is thus determined upon generation of the chiral center C-15.…”

“…The NCC 1 (Cj-NCC-2) from extracts of senescent leaves of C. japonicum is a nonpolar natural NCC. The FCC 2 is identical to the unknown primary FCC from C. japonicum (22), the catabolic precursor of the NCC 1. The sample of 2 actually used here was obtained as Ca-FCC-2 from C. annuum (16).…”