Chlorophyll Catabolites in Senescent Leaves of the Plum Tree (Prunus domestica)

In cold extracts of senescent leaves of the plum tree (Prunus domestica ssp. domestica), six colorless non‐fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd‐NCCs. In addition, several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd‐NCC‐32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 32‐position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18‐position. In the polar Pd‐NCC‐32, the latter group is further glycosidated at the terminal 182‐position. Four other major Pd‐NCCs and one minor Pd‐NCC were identified with five NCCs from higher plants known to belong to the ‘epi’‐series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented (formal) oxidation products of two of the observed Pd‐NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.


Results and Discussion
Yellow senescent and green leaves were collected from plum trees (Prunus domestica) and frozen for storage. Five major and nine minor colorless NCCs were provisionally identified in extracts of senescent leaves of plum trees on the basis of their characteristic UV-absorbance properties, using analytical HPLC (Fig. 2). All NCC fractions showed UV/VIS spectra featuring a longest wavelength maximum near 314 nm, characteristic of an a-formyl-pyrrole moiety (ring A), as first reported in the spectrum of Hv-NCC-1. [3] Likewise, minor fractions of two YCCs and a trace of a pink Chlcatabolite (PiCC) were also tentatively identified.
For spectroscopic analysis of the most abundant NCCs in the leaves of P. domestica, 18.7 g of  Table 1 for individual NCCs [8] [9] ). senescent plum tree leaves were extracted with cold MeOH (to avoid significant NCC oxidation, see [28]), and the extract was separated by semi-preparative HPLC. A two-stage purification procedure gave a uniform sample of 0.29 mg of Pd-NCC-32 (1), analyzed by UV/VIS-spectroscopy first (see Fig. 3). CD Spectra of Pd-NCC-32 (1) and of Hv-NCC-1 [4] showed the same basic features, suggesting a common (R)-configuration at the stereogenic C(10). [9] A high resolution MALDI-MS spectrum of Pd-NCC-32 (1) [42] (see Fig. 4) displayed its pseudo-molecular ion [ [31] the loss of ring D (see [42]) or of a second sugar moiety, respectively.
In a 600 MHz 1 H-NMR spectrum of Pd-NCC-32 (1) in CD 3 OD at 10°C (see Fig. 5) signals of 47 of the 48 C-bound H-atoms were observed. Among these signals there were a singlet for the formyl H-atom (H-C (20)) at low field, four Me group singlets at high field and a singlet for the methyl ester group at 3.75 ppm. The typical signals for a peripheral vinyl group were not observed. From 1 H, 13 C-heteronuclear (HSQC and HMBC) and 1 H, 1 H-homonuclear NMR-correlations (COSY and ROESY) of Pd-NCC-32 (1) in CD 3 OD, assignment of the signals of 47 H-atoms and 45 13 C-nuclei could be achieved (see Fig. 6). In addition to the signals of the NCC-core, those of 14 H-atoms were observed in the intermediate field of the 1 H-NMR spectrum. 1 H, 1 H-COSY and 1 H, 13 C-HSQC correlations indicated two hexopyranose units, with closely similar 1 H-and 13 C-shifts in both sugar moieties. Only for atoms at or close to the anomeric centre, H-C(1 0 ) (4.17 ppm) and H-C(1″) (4.33 ppm), as well as H-C(2 0 ) (3.17 ppm) and H-C(2″) (3.21 ppm), the chemical shifts of the pairs of signals differed significantly (for atom numbering: see Experimental Section, Fig. 9).
The molecular formula of Pd-NCC-56 (4) was determined as C 41   group of ring A of Pd-NCC-56 (4) and a vinyl group at C (18) of ring D. This indicates a common chemical constitution of 4 and of Nr-NCC-2 [34] (see Fig. 7).
The molecular formula of Pd-NCC-40 (3) could be deduced tentatively as C 35 H 42 N 4 O 10 by ESI mass spectrometry, which showed the experimental base peak [M + H] + at m/z 679.2. In the mass spectra, characteristic fragment-ion peaks at m/z 647.2 and 522.1 were also detected, which corresponded to the loss of MeOH and to the loss of ring D (from [M + H] + ). Accordingly, the catabolite Pd-NCC-40 (3) (see Fig. 7) was deduced to have the same chemical constitution as So-NCC-2 from spinach. [21] [22] A positive-ion-mode ESI-MS spectrum of Pd-NCC-35 (2)  According to their fragmentation pattern, [42] Pd-NCC-35 (2) (see Fig. 7) and Zm-NCC-1 [35] show the same chemical constitution.
The molecular formula of Pd-NCC-71 (6) was determined as C 35 H 40 N 4 O 7 with a pseudo-molecular ion at m/z 629.2. Fragments at m/z 597.2 and 506 indicate the loss of MeOH and ring D. Pseudo-molecular ion and fragment-ions are consistent with a chemical constitution of 6, as previously found for Cj-NCC-2 (Fig. 7). [13] Identity of Pd-NCC-71 (6) and of Cj-NCC-2 was supported by a common retention time of 6 and Cj-NCC-2 in a HPLC co-injection experiment.
Analysis of a minor NCC (tentatively named Pd-NCC-54) by LC/ESI-MS revealed a pseudo-molecular ion at m/z 661.2 ([M + H] + ), consistent with the molecular formula of C 35 H 40 N 4 O 9 . We suspected Pd-NCC-54 as product of the formal addition of an O-atom to Pd-NCC-60 (5) from an endogenous oxidation process. Indeed, as shown recently, [28] NCCs may undergo C (15) hydroxylation by endogenous, as well as by additional efficient adventitious oxidation during preparation of leaf homogenates and their extracts. From NCCs hydroxylated at their C(15) position, H 2 O may eliminate easily, resulting in corresponding YCCs. [28] Indeed, a YCC was detected in the fresh plum leaf extracts, named Pd-YCC-67, which showed mass spectral data (pseudo-molecular ion with m/z 643.2) consistent with its formation as the formal product of an oxidative dehydrogenation of Pd-NCC-60 (5). A further minor fraction, classified as YCC from a prominent absorption maximum near 420 nm, was also subjected further to ESI-MS analysis. The latter data suggested Pd-YCC-61 (m/z 805.1) to represent a YCC derived from oxidation of the glucosylated Pd-NCC-56 (4). When extracts were prepared after storage of senescent leaves of the plum tree at room temperature for 7 min, an increase of the content of both YCCs (Pd-YCC-61 and Pd-YCC-67) was observed, as well as the formation of 15-OH-Pd-NCC-60, identified by comparison with its analogue from the established oxidation of Cj-NCC-1. [28] However, this hydroxylated NCC differed (in its retention time) from Pd-NCC-54. Clearly, work-up and preparation of extracts of cold senescent leaves need to be done swiftly, in order to avoid oxidation artefacts.

Conclusions
Extracts of naturally senescent leaves of the plum tree (Prunus domestica ssp. domestica) were shown to contain a range of NCCs, two YCCs, and, in traces, a PiCC, all members of the 'type I' phyllobilin family. In spite of the absence of DCCs, [7][8] a remarkable structural diversity of Chl-catabolites was, thus, indicated. The polar NCC Pd-NCC-32 (1) showed a previously unknown structure and is functionalized with two glycopyranose moieties on the 'distant' pyrrole rings A and D. The structure of Pd-NCC-32 (1) also provided the first (indirect) evidence for enzymatic glycosidation of an FCC at the 18 2 -position (a primary alcohol function resulting from dihydroxylation of the corresponding vinyl group of the precursor FCC). [7][8] Five more NCCs were tentatively identified with known catabolites based on their matching UV/VIS-and mass spectroscopic features. Further identification by HPLC of Pd-NCC-60 (5) and Pd-NCC-71 (6) with corresponding Cj-NCCs, indicated the plum NCCs to belong to the C(16)-epi series, as well. [8][9] [26] Additional investigations will be required to secure the structures of several minor NCC-and of the YCC-containing fractions. Based on the deduced structures of the plum NCCs, a tentative pathway of their formation in the senescent leaves of the plum tree could be derived (see Fig. 8).
While the first Chl-catabolites in Rosaceae crops were found in leaves and fruits of apple and pear trees, [17] which belong to the Pyreae tribus, here a stone fruit (that is part of the Amygdaleae tribus) was studied for the first time. The findings with senescent leaves of the plum tree are consistent with the related earlier studies with leaves of apple and pear trees. [17] With members like apples, pears, peaches, strawberries, raspberries and many others, the Rosaceae family belongs to the six most economically important crop families worldwide. [43] Thus, this study suggests the conserved PaO/phyllobilin pathway of Chl breakdown to NCCs to operate in senescent leaves of the Spiraeoideae subfamily of the Rosaceae.

Analysis of Chl-Catabolites in Senescent Leaves by HPLC
Senescent plum tree leaves were harvested in November 2013 from a commercial orchard in Aldino (South Tyrol). They were immediately frozen in a freezer (À80°C) and transported in a cold box (À20°C) to Innsbruck, where they were stored cold (À80°C).
A leaf segment (with the area of about 20 cm 2 ) was frozen in liquid N 2 , grounded in a mortar and extracted with 1 ml of MeOH. The resulting suspension was centrifuged for 3 min at 13,000 g. Five hundred microliter of the MeOH supernatant were diluted with 2 ml of 50mM aq. potassium phosphate buffer (pH 7.0). After centrifugation for 3 min at 13,000 g, 200 ll of the extract was analyzed by HPLC (see Fig. 2).
Isolation and Structure Elucidation of Pd-NCC-32 (1). Yellow-greenish senescent plum tree leaves (18.7 g) were frozen in liquid N 2 , pulverized to a fine powder and extracted with 60 ml of MeOH. The suspension was centrifuged for 5 min at 4000 g. Forty-two milliliter of the supernatant were diluted with 168 ml of 50mM aq. potassium phosphate buffer (pH 7.0). After centrifugation for 5 min at 4000 g, the soln. was extracted two times with hexane. The MeOH extract was diluted with 300 ml of 50mM potassium phosphate buffer (pH 7.0) and applied to a pre-conditioned 5 g SepPak cartridge. This was washed with 35 ml of H 2 O and the NCC-containing fraction was eluted with 30 ml of MeOH. The solvents were removed by using a rotary evaporator. The residue was dissolved in 1 ml of MeOH and 4 ml of 50mM aq. potassium phosphate buffer (pH 7.0) using an ultrasonic bath. After centrifugation for 3 min at 13,000 g, the sample was divided in four aliquots and applied to semi-prep. HPLC; injection volume, 1.25 ml; flow rate, 0 -5 min: 1 -4 ml min À1 , 5 -90 min: 4 ml min À1 ; solvent A: 4mM aq. AcONH 4 (1) was collected between and diluted with 20 ml of 50mM aq. potassium phosphate buffer (pH 7.0). For de-salting, the aq. soln. was applied to a pre-conditioned 5 g SepPak cartridge, washed with 15 ml of H 2 O and eluted with 5 ml of MeOH. After removal of the solvents using a rotary evaporator, the sample was dried under high vacuum and a uniform sample of 0.29 mg of Pd-NCC-32 (1) was obtained.
Isolation of Raw Pd-NCCs for Structural Analysis. 12 anal. extracts were prepared, combined and diluted with 95 ml of 50mM aq. potassium phosphate buffer (pH 7.0). This was applied to a pre-conditioned 5 g SepPak cartridge, washed with 30 ml of H 2 O and the NCC-containing fraction was eluted with 30 ml of MeOH. The fraction was dried under reduced pressure and the precipitate was dissolved in 400 ll of MeOH and 1.6 ml of 4mM aq. AcONH 4 . After centrifugation for 3 min at 13,000 g, the sample was divided in two aliquots and applied to semi-prep. HPLC; injection volume, 1.00 ml; flow rate, 0 -5 min: 1 -4 ml min À1 , 5 -90 min: 4 ml min À1 ; solvent A: 4mM aq. AcONH 4  Spectroscopic data (for atom numbering)