Chlorophyll Catabolites in Senescent Leaves of the Lime Tree (Tilia cordata)

In cold extracts of senescent leaves of the Lime tree (Tilia cordata), two colorless nonfluorescent chlorophyll catabolites (NCCs) were identified, named Tc-NCC-1 and Tc-NCC-2, as well as a polar yellow chlorophyll catabolite (YCC), named Tc-YCC. The constitution of the two NCCs was determined by spectroscopic means. In addition, a tentative structure was derived for Tc-YCC. The three chlorophyll degradation products exhibited tetrapyrrolic structures, as are typical of NCCs or YCCs, and turned out to be rather polar, due to a glucopyranosyl group at their 82-position. At their 3-positions, the more polar Tc-NCC-1 carried a 1,2-dihydroxyethyl group and the less polar Tc-NCC-2 a vinyl group. Tc-YCC was identified as the product of an oxidation of Tc-NCC-1.

Introduction. -Chlorophyll biosynthesis and chlorophyll breakdown are fascinating natural life processes on earth [1], which can be observed from outer space [2]. Indeed, an estimated 10 9 tons of chlorophyll are formed and degraded every year on earth [3] [4]. Strikingly, chlorophyll breakdown and the appearance of autumnal fall colors have remained a stunning mystery [4 -6] until about twenty years ago, when a first colorless tetrapyrrolic chlorophyll catabolite 1a (Hv-NCC-1) was identified in senescent primary leaves of barley [7] [8]. Nonfluorescent chlorophyll catabolites (NCCs) were subsequently also found in other plants, and they were assumed to represent the final stages of chlorophyll catabolism in senescent leaves [2] [6] [9] [10]. Since then, over a dozen NCCs were detected in higher plants, and their structures were analyzed (Scheme and Table).
About ten years ago, two urobilinogenoidic chlorophyll catabolites were discovered in extracts of senescent primary leaves of barley [23], i.e., linear tetrapyrroles, which were considered to represent putative products of further breakdown of Hv-NCC-1 (1a) by an oxidative deformylation at ring B. Evidence for another type of further oxidative transformation of NCCs was also provided more recently by the yellow chlorophyll catabolites (YCCs) and pink chlorophyll catabolites (PiCCs) detected in senescent leaves of the Katsura tree (Cercidiphyllum japonicum), which were identified as dehydrogenation products of the tetrapyrrolic NCC 3b (Cj-NCC-1) [24] [25]. All of these findings were consistent with an essentially linear path of chlorophyll breakdown in higher plants (see the Scheme) [26].
However, several recent observations suggested the existence of divergent pathways of chlorophyll breakdown in higher plants. A strikingly contrasting stereo-chemical variant of the urogenobilinoidic catabolites from barley was found in naturally de-greened leaves of Norway Maple (Acer platanoides), classified as dioxobilanes, and indicating a different path to these tetrapyrroles [27]. Structurally divergent, hypermodified blue fluorescent chlorophyll catabolites (FCCs) were observed as remarkably persistent breakdown products in banana (Musa acuminata) fruits [28] and leaves [29], as well as in senescent leaves of the peace Lily (Spathiphyllum wallisii), a tropical evergreen [14].
Here, we describe an investigation of chlorophyll breakdown products in senescent leaves of the lime tree (Tilia cordata), a first representative of the genus Tilia (Malvaceae) to be investigated in this respect. Lime trees are well-known deciduous trees native to the forests of the northern hemisphere in Europe, Asia, and Eastern North America and Central America [30]. A reference to the importance of Tilia sp. in central Europe can be found in the Middle High German Nibelungenlied, where a lime tree leaf (linden leaf) covered Siegfrieds back during his bath in the blood of a wounded dragon and gave rise to his vulnerable spot 1 ). Beside a special medicinal relevance of lime trees (colds, cough, fever, etc. are often treated with extracts of this plant [31]), representatives of these deciduous trees became increasingly important in municipal parks of cities and play a central role as avenue trees [30].
Results and Discussion. -In a cold MeOH extract of yellow (senescent) fall leaves of a lime tree (Tilia cordata), two polar colorless and nonfluorescent chlorophyll catabolites (NCCs) and a yellow chlorophyll catabolite (YCC) were provisionally identified by analytical HPLC, on the basis of their characteristic UV-absorbance properties (see Fig. 1) [24] [32]. To inhibit adventitious oxidation of NCCs to yellow catabolites such as Tc-YCC (13; Fig. 2) [24] during isolation and workup, the freshly picked senescent leaves were immediately frozen with liquid N 2 and directly analyzed by HPLC. The different HPLC retention times on the stationary reversed phase (t R (Tc-NCC-1 (5)) 31.8 min, t R (Tc-YCC (13)) 33.8 min, and t R (Tc-NCC-2 (4b)) 47.8 min) reflected the different polarities of the three catabolites.
For further structural analysis, 100 g (wet weight) of senescent lime tree leaves were extracted according to a three-stage purification procedure based on a cold extraction, followed by separation by MPLC and by preparative HPLC (for details, see the Exper. Part) to give 5.1 mg (6.1 mmol) of Tc-NCC-1 (5), 1 mg (1.2 mmol) of the less polar Tc-NCC-2 (4b), and 0.2 mg of the yellow catabolite Tc-YCC (13) (determined by UV/VIS spectroscopy).
Mass spectrometry was utilized to derive a tentative molecular formula of Tc-NCC-1 (5) as C 41  A 500-MHz 1 H-NMR spectrum of a solution of Tc-NCC-1 (5) in CD 3 OH revealed signals of 44 of the 52 H-atoms: a singlet (at low field) of the formyl H-atom, four singlets (at high field) of the four Me groups attached at the b-pyrrole positions, and a singlet at 3.74 ppm (due to a methyl ester function). In addition, the signals of the Hatoms HÀN(21), HÀN (24), and HÀN(23) were detected between 8.0 and 11.3 ppm, and could be assigned with the help of COSY, ROESY, and HMBC data. However, in contrast to the 1 H-NMR spectrum of Tc-NCC-2 (4b), the typical signals for a peripheral vinyl group in the intermediate field range were absent. 1 H, 13 C-Heteronuclear NMR correlations (from HSQC and HMBC spectra [33] of Tc-NCC-1 (5) in CD 3 OH) allowed assignment of the signals of a 1,2-dihydroxyethyl side chain. 1 H, 1 H-Homonuclear correlations from ROESY spectra and 1 H, 13 C-heteronuclear correlations from HMBC spectra [33] indicated C(8 2 ) as the site of the attachment of the sugar  moiety. The shifts of the 1 H and 13 C signals for the CH 2 (8 2 ) group were consistent with an O-bridge to the peripheral sugar substituent. The latter was identified as a bglucopyranoside unit by comparing the 1 H and 13 C chemical shifts with the spectra of methyl b-d-glucopyranoside [34], as well as by comparing the NMR spectra of Tc-NCC-1 (5) with those of Bn-NCC-2, Nr-NCC-2, and Zm-NCC-1, where a peripheral bglucopyranosyl group at C(8 2 ) had also been found [18] [19] [22]. The signal of HÀC(15) appeared as a doublet (J(H,H) ¼ 2.5 Hz, in CD 3 OH) due to coupling with HÀC(13 2 ). Therefore, a relative trans-configuration of HÀC(13 2 ) and HÀC (15), which is typical for stable isomers of NCCs [13] [32], in Tc-NCC-1 was derived using the Karplus relation [34]. ESI-MS in the positive-ion mode was also used to deduce the tentative molecular formula of Tc-NCC-2 (4b) as C 41 H 50 N 4 O 13 (see Fig. 3, b). The peak at m/z 807.37 corresponded to the pseudo-molecular ion C 41  A 600-MHz 1 H-NMR-spectrum of 4b (in CD 3 OD) exhibited signals for 40 of the 50 H-atoms: a singlet (at low field) for the formyl H-atom, four singlets (at high field) for the four Me groups in the b-pyrrole positions, and a singlet at 3.74 ppm (due to the methyl ester function). In addition, the typical signal pattern for a vinyl group was detected around 6 ppm. 1 H, 13 C-Heteronuclear correlations (HSQC and HMBC [33]) allowed the assignment of all 1 H and 13 C signals (see Exper. Part). A sugar moiety was identified as a b-glucopyranosyl group at C(8 2 ) from 1 H, 1 H-ROESY correlations, as well as 1 H, 13 C-heternuclear correlations from HMBC spectra [33]. Again, the indicated site of attachment for the peripheral glucopyranosyl substituent (C( 8 2 )) via a bridging O-atom, O(8 3 ), was consistent with the typical downfield shifts of the 1 H and 13 C signals of CH 2 (8 2 ). The lack of the signal for HÀC(13 2 ) in the 1 H-NMR spectra (in CD 3 OD) of Tc-NCC-2 (4b) indicated the exchange-labile a-position of the b-keto ester functionality at ring E to have undergone H/D exchange. HPLC Co-injection of Tc-NCC-1 (5) and of the constitutionally identical analog from maize, Zm-NCC-1 [19], indicated a common t R of ca. 30 min, suggesting the two NCCs to be identical (see Fig. 4 in the Exper. Part; for details of co-injection experiments, see the Exper. Part).
The CD spectra of the Tc-NCCs 5 and 4b were consistent with the suggested common configuration of C(15) in naturally occurring NCCs from higher plants [7] [26] [32]. The stereogenic center C (15) has been proposed to result from a nonenzymatic, stereoselective isomerization of fluorescent chlorophyll catabolites [35] [36] to the corresponding NCCs [13]. The relative configuration at C(15) and C(13 2 ), with HÀC(15) cis to the COOMe group at C(13 2 ) in (the prevailing epimer of) 5 and 4b is a likely result of a nonenzymatic equilibration reaction at the acidic b-keto ester position C(13 2 ), which adjusts its configuration to that at C (15). The latter process would occur in the vacuoles of senescent plant cells [13] [37], but it also takes place when isolated NCCs are kept in a protic solution.
A tentative molecular formula of Tc-YCC (13) could be deduced as C 41 H 50 N 4 O 15 by LC/ESI-MS in the positive-ion mode (see Fig. 3, c), which showed a prominent peak at m/z 839.1 of the pseudo-molecular ion C 41 Fig. 3), could not be observed in the mass spectrum of Tc-YCC (13), compatible with the C¼C bond at ring A. For further characterization of 13 as oxidation product of Tc-NCC-1 (5), a nearly colorless spot of analytically pure 5 on a silica-gel TLC plate was irradiated for 10 min with a 365nm UV lamp [25], whereupon the spot acquired a brownish-red color (see Exper. Part). A major yellow product fraction formed, which had a UV/VIS spectrum typical for a YCC with (Z)-configuration of the C(1)¼C (20) bond [24] [25], and which was shown by analytical HPLC to co-elute with the yellow catabolite Tc-YCC (13).
Conclusions. -In the present work, non-green chlorophyll catabolites were analyzed in fresh extracts of naturally de-greened lime tree (Tilia cordata) leaves, a first representative of the genus Tilia to be investigated in this respect. Two colorless nonfluorescent chlorophyll catabolites (NCCs) were identified in the senescent leaves, Tc-NCC-1 (5) and Tc-NCC-2 (4b), and their structures were characterized. The basic build-up of 4b and 5 was found to be the same as known for NCCs from higher plants. The available NMR data revealed the functionalization at C(8 2 ) of the Tc-NCCs to imply a common b-glucopyranosyl moiety, as in Bn-NCC-2 (12) from oilseed rape (Brassica napus), in Nr-NCC-2 (4b) from tobacco, and in Zm-NCCs 4b and 5 from senescent maize leaves (Zea mays). Furthermore, the spectroscopic data indicated the tetrapyrrolic cores of the two Tc-NCCs 5 and 4b to have a common relative configuration at the stereogenic centers C(1), C(13 2 ), and C(15). The absolute configuration at C(1) was deduced to be the same in the Tc-NCCs 5 and 4b as in the glucosylated Nr-NCCs 4b and 6, and Zm-NCCs 4b and 5. Therefore, the absolute configuration at C(1) was indicated to be opposite to that in Bn-NCC-2 (12). NCCs (and FCCs) fall into two classes with respect to the configuration at C(1) due to the evolution of two types of RCC-reductases (RCCR) in higher plants [36] [38 -40]. Hence, it was of interest to note the structural relationships with respect to the type of peripheral functionalization for the NCCs and of their configuration at C(1). The indicated availability of two closely related NCCs, Tc-NCC-1 and Tc-NCC-2, in senescent lime tree leaves parallels the occurrence of NCCs in other senescent plants, where a range of peripheral groups was observed [32] [41]. Our studies on the Tc-NCCs provide further support for the view that the main constitutional variations of the chlorophyll catabolites in various higher plants involve enzyme catalyzed, peripheral refunctionalization reactions. The peripheral adaptions with polar functions have been suggested to be of relevance for the transport and for the final deposition of chlorophyll catabolites in the vacuoles [6] [10] [37] [42].
A yellow chlorophyll catabolite Tc-YCC (13) was identified in fresh extracts of lime tree leaves, and it was suggested to be a product of a naturally occurring oxidation reaction of 5 during senescence. Our study, therefore, provides (further) evidence for the notion that NCCs may not generally be the final products of chlorophyll in senescent plants [41]. All these findings strengthen the view that, while the pathways of chlorophyll catabolism in various higher plants may be closely related, they follow divergent branches, when including functional details. Indeed, the natural formation of the colored and photoactive YCCs such as of 13 would not be consistent with the role of chlorophyll breakdown as a mere detoxification process either.
We would like to thank C. Kreutz for acquiring the NMR-spectra, and Hans-Jçrg and Hildegard Patscheider for giving us access to the Hotel Linde lime tree. Financial support by the Bundesministerium für Wissenschaft und Forschung (BM.W_F, Project SPA/02 -88/Recycling the Green to T. M.) and by the Austrian Science Foundation (FWF, Project No. L-472 to B. K.) is gratefully acknowledged.
Five-g and 1-g Sep-Pak-C18 cartridges were from Waters Associates. The pH values were measured with a WTW Sentix 21 electrode connected to a WTW pH535 digital pH-meter.
HPLC: Dionex Summit HPLC system with manual sampler, P680 pump, online degasser and diode array detector, 1-ml or 20-ml injection loop. Data were collected and processed with Chromeleon V6.50. Analysis of Chlorophyll Catabolites in Senescent Leaves by Anal. HPLC. Freshly picked lime tree leaves were collected from a lime tree grown near the Hotel Linde (Innsbruck; see illustration for the Table of Contents) and immediately stored in liquid N 2 . A leaf (with the area of ca. 20 cm 2 ) was grounded in a mortar and extracted with 2 ml of MeOH. The resulting suspension was centrifuged 5 min at 13,000 rpm. The methanolic supernatant was diluted with 50 mm aq. potassium phosphate (pH 7.0) 80 : 20 (v/v). After centrifugation for 5 min at 13,000 rpm, 1 ml of the extract was injected into the anal. HPLC system, to be analyzed with parallel detection at 320 and 420 nm (see Fig. 1).
Determination of Chlorophyll in Green and Senescent (yellow) Lime Tree Leaves by UV/VIS Spectroscopy, of Nonfluorescent and of Yellow Chlorophyll Catabolites (NCCs and YCC, resp.) in Senescent (yellow) Leaves by Anal. HPLC. Chlorophyll a and b in Green Leaves. A total area of 9 cm 2 was cut out of a green lime tree leaf. The leaf was frozen in liquid N 2 , pulverized in a mortar, and extracted with MeOH. The slurry was filtered through a sintered glass filter, and the residue was grounded in a mortar and extracted with MeOH. The procedure was repeated, until the residue was colorless. The MeOH extracts were combined and diluted with MeOH to 100.00 ml in a volumetric flask. The extracts were analyzed by UV/VIS spectrometry. In green Tilia cordata leaves, 72.09 AE 3.93 mg · cm À 2 (80.37 AE 4.38 nmol · cm À 2 ) of chlorophyll a and b were found (n ¼ 4), the data analysis was based on [44].
Chlorophyll a and b in Senescent (Yellow) Leaves. A total area of 99 cm 2 was cut out of eleven senescent lime tree leaves. The extraction and the UV/VIS analysis were performed as described above. Yellow Tilia cordata leaves were found to contain 0.55 AE 0.10 mg · cm À 2 (0.61 AE 0.11 nmol · cm À 2 ) of residual chlorophyll a and b.
Collection, Isolation and Structure Elucidation of Tc-NCCs. Senescent Tilia cordata leaves were collected at the main campus of the University of Innsbruck in October 2009 and stored at À 808. Senescent (yellow) leaves (100 g (wet weight)) were frozen in liquid N 2 , crushed into small pieces, and freeze-dried in vacuo for 3 d, resulting in a dry-weight of ca. 40 g. The dried leaves were pulverized using liquid N 2 and a 300-W blender, and extracted with 70 ml of MeOH. The suspension was filtered with suction over a Büchner funnel. The extraction was repeated using another 70 ml of MeOH. The combined 140 ml of MeOH extracts were added portionwise to 350 ml of Et 2 O to precipitate a raw product that was enriched in chlorophyll catabolites. The raw product was cooled in an ice-bath for 15 min. After filtration with suction the white precipitate was dried in vacuo (dry weight: 1.77 g) and stored at À 808 for further purification by MPLC: The crude product was dissolved in 48 ml of aq. potassium phosphate buffer soln. (50 mm ; pH 7) using an ultrasonic bath. After filtration with a Sartorius filter two aliquots of the filtrate (24 ml of clear red-brownish solutions) were injected into the MPLC system (flow rate: 10 ml min À 1 ); solvent A: 50 mm aq. potassium phosphate (pH 7.0); solvent B: MeOH; solvent composition (A/B) as function of time (0 -120 min): 0 -90, 70 : 30; 90 -120, 70 : 30 to 0 : 100.
The fractions containing Tc-NCC-1 and Tc-NCC-2 were collected after 50 and 105 min, resp. The collected fractions were concentrated using a rotary evaporator, freeze-dried in vacuo, and stored at À 808 for further purification.
The (more-polar) fraction containing Tc-NCC-1 (5) was dissolved in 0.2 ml MeOH and 2.5 ml of H 2 O using an ultrasonic bath. After filtration of the suspension through a Sartorius filter the sample was divided in three aliquots and applied to prep. HPLC; injection vol., 1 ml; flow rate, 7 ml min À 1 ; solvent A: The (less-polar) Tc-NCC-2 (4b) fraction was likewise dissolved in 0.6 ml of MeOH and 2.4 ml of H 2 O using an ultrasonic bath. After centrifugation for 3 min at 13,000 rpm, prep. HPLC (injection vol., 1 ml; flow rate, 5 ml min À 1 ) was applied for three aliquots; solvent A: 50 mm aq. potassium phosphate   -1, b) of Zm-NCC-1, and c) of a 1 : 2 mixture of the two NCCs. In the samples of Tc-NCC-1 and Zm-NCC-1, a second fraction (ca. 10%) occurred in addition to the main fraction (presumably, the minor fraction is the 13 2 -epimer of the main NCCs, due to epimerization at C(13 2 ) of the b-keto ester functionality).