Nonfluorescent Chlorophyll Catabolites in ... - ACS Publications

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Nonfluorescent Chlorophyll Catabolites in Loquat Fruits (Eriobotrya japonica Lindl.) José Julián Ríos, María Roca, and Antonio Pérez-Gálvez* Food Phytochemistry Department, Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Avda. Padre García Tejero, 4, Sevilla 41012, Spain S Supporting Information *

ABSTRACT: Nonfluorescent chlorophyll catabolites (NCCs) and nonfluorescent dioxobilane chlorophyll catabolites (NDCCs) are the terminal compounds of the chlorophyll degradation pathway that may display beneficial properties to human health related to their antioxidant properties, which were recently shown. A profile of NCCs/NDCC of the loquat fruit Eriobotrya japonica Lindl. is described. From the 13 known different NCC structures described to date, three have been identified in loquats. Two new structures not defined so far were characterized in loquat fruits: Ej-NCC2, which corresponds to the methyl ester at C132 of Bn-NCC1 and in very low amount Ej-NDCC1, the only NDCC found in loquats. Keto−enol tautomerism at the C131 position in NCCs is described for the first time as a regular process in chlorophyll catabolism, probably through a nonspecific mechanism since almost all the chlorophyll catabolites structures detected in fruits of loquat present keto and enol tautomers. The results obtained have been possible through a high-performance liquid chromatography coupled with electrospray ionization ion trap and quadropole time-of-flight mass spectrometry fitted with a powerful postprocessing software. KEYWORDS: chlorophyll catabolites, chlorophyll degradation, Eriobotrya japonica Lindl., fruits, HPLC-MS, isomerism, loquat, NCC, NDCCs

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INTRODUCTION Plant food tissues that are or were green contain terminal chlorophyll catabolites, nonfluorescent chlorophyll catabolites (NCCs) accumulated in the vacuoles that are ingested daily. These catabolites are the consequence of the senescent chlorophyll degradation pathway,1 a pathway that has been elucidated thanks to (totally or under certain circumstances) stay-green organisms.2−5 Before reaching the final NCCs structure, the chlorophyll a molecule undergoes several enzymatic-catalyzed modifications to produce pheophorbide a.1 Then, pheophorbide a is oxygenolytically opened to produce red chlorophyll catabolite (RCC), that is, reduced stereospecifically to a primary fluorescent chlorophyll catabolite (pFCC) (Figure 1). The pFCC, once exported from the chloroplast, is prone to several modifications only at three peripheral positions of the molecule. The first position is at C82 (Figure 1), where hydroxylation, glucosylation, or malonylation could take place. The second modifiable position is O134 where a reaction of demethylation is possible, allowing the occurrence of esterified or de-esterified possible structures. This enzymatic modification of pFCC is carried out by MES16, the unique enzyme involved on pFCC route described so far.6 Finally, at C3, FCCs can be dihydroxylated or not at the vinyl group of pyrrole A (Figure 1). Only some combinations of these yield the different FCCs described7 that will experiment tautomerization in the weakly acid environment of the vacuole and instantly turn into their corresponding NCCs.8 Since they were first described,9 different NCC structures have been reported in vegetal species, most in leaves and recently in fruit tissues (Supporting Information, S1). An additional chlorophyll catabolite family present in senescent leaves is NDCCs, nonfluorescent dioxobilane chlorophyll catabolites (Figure 1). © XXXX American Chemical Society

NDCCs have the same peripheral configuration as their corresponding NCCs except for the formyl group at C6 that they lack,10 a function present in all known NCCs. Several hypotheses have been outlined to explain the significance of the chlorophyll catabolism and the meaning of different structural rearrangements leading to different NCCs/NDCCs. Actually, the more plausible is that an appropriate chlorophyll breakdown to nonphototoxic metabolites is necessary to ensure cell viability and resourceful mobilization of nutrients (nitrogen) to other tissues,7 being the refunctionalization, a method to increase the polarity of the catabolites to be accumulated in vacuoles. Though the assessment of chlorophyll catabolism was developed almost exclusively in senescent leaves, some findings denote that the onset of chlorophyll breakdown in ripening fruits deserves more attention. The first report11 about the presence of NCCs in pears and apples allowed the identification of two NCCs that are part of the pool of dietary phytochemicals from fruits. The peels of banana Musa acuminata (Cavendish) present a complex and low NCC profile and a higher accumulation of their precursors (FCCs), and although they accumulate in a nonedible portion of the fruit, their intriguing structural conformation could be reproduced in other fruits.12 Recently, four chlorophyll breakdown products (two NCCs and two NDCCs) have been characterized in ripened quince fruits (Cydonia oblonga, Miller) by high-performance liquid chromatography coupled Received: July 28, 2014 Revised: October 3, 2014 Accepted: October 8, 2014

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dx.doi.org/10.1021/jf503619s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Figure 1. Proposed chlorophyll degradation pathway in senescent loquats fruits (Eriobotrya japonica Lindl.).

fraction, only one publication17 studied it considering its bioactive potential with medical purposes. Currently, numerous scientific studies point to tetrapyrroles as significant bioactive components related to cancer prevention, antioxidant and antimutagenic activity, mutagen trapping, modulation of xenobiotic metabolism, and induction of apoptosis.18 Measurement of antioxidant capacity of NCCs brought new insights for the assignment of functionality to these molecules. It has been shown that NCCs display a similar antioxidant capacity to that of bilirubin, which points to a possible role of NCCs, to soften the stress induced by senescence.11 This hypothesis is even

with electrospray ionization and mass spectrometry (HPLC− ESI−MS) with a couple of NCC/NDCC ratios equal to the catabolites identified in senescence leaves of maize (Zea mays L.) and barley (Hordeum vulgare L.), respectively, and a duo of two new structures.13 Autumn or early winter is time for loquats (Eriobotrya japonica Lindl.) to flower, and fruits are ripe in the market in late winter or early spring when few competitive fruits are available, so loquat fruits can be sold at higher prices.14 The scientific literature describes the phenolics and antioxidative potential of loquat fruits,15,16 but in relation to the chlorophyll B



Article

magnitude range to ensure good quality fragmentation spectra. The instrument control was performed using Bruker Daltonics HyStar 3.2. Data Analysis. An in-house mass database created ex professo comprised monoisotopic masses, elemental composition, and, if known, retention time and characteristic product ions for 13 NCCs and the five NDCCs.10,11,13,21−31 Additionally, elemental composition of new NCCs/NDCCs not described so far was included in the database. These new chlorophyll catabolites were generated holding two rules: (1) only those substituents already described in literature are allowed to enter in the basic NCC/NDCC structure and in the same carbon atom positions; and (2) dioxobilin-type chlorophyll catabolites are generated from all NCC structures (both known and new ones) by deformylation at C6 in ring B. Thus, some of the new NCC structures were the de-esterified form of Nr-NCC1 and the esterifed form of Bn-NCC1. Data evaluation was performed with Bruker Daltonics DataAnalysis 4.0. From the HPLC/TOF-MS acquisition data, an automated peak detection on the extracted ion currents (EICs) expected for the protonated molecule of each compound in the database was performed with Bruker Daltonics TargetAnalysis 1.2 software. The software performed the identification automatically according to mass accuracy and in combination with the isotopic pattern in the SigmaFit algorithm. This algorithm provides a numerical comparison of theoretical and measured isotopic patterns and can be utilized as an identification tool in addition to accurate mass determination. The calculation of mSigma values includes generation of the theoretical isotope pattern for the assumed protonated molecule and calculation of a match factor based on the deviations of the signal intensities. Only those hits with mass accuracy and mSigma values within the tolerance limits, which were set at 5 ppm and 50, respectively, are included in the final report list that was carried out using a Microsoft EXCEL-based script. This software has been successfully applied for screening of some other phytochemicals.32,33 The interpretation of the MS2 spectra was performed using the SmartFormula3D module included in the DataAnalysis software. This module includes an algorithm that estimates whether a formula for a product ion is a subset of a formula for the precursor ion. On the basis of expected chemistry, the elements carbon, hydrogen, oxygen, nitrogen, bromine, and iodine were allowed. Sodium and potassium were also included for the calculation of adduct masses. The number of nitrogen atoms was limited to an upper threshold of ten. The number of rings plus double bonds was checked to be chemically meaningful (between 0 and 50). For each NCC/NDCC detected in the sample, the module shows the original MS and MS2 data as peak lists. From all possible formulas for the precursor ion, only one should fit with the elemental composition expected for the protonated molecule and satisfy thresholds for mass accuracy and mSigma values. Once the correct formula is selected, the module displays the formulas and neutral losses in the MS2 spectrum fitting to the boundary conditions for the precursor ion, and they should be consistent with the MS2 data peak list. The SmartFormula3D checks the consistency and highlights the monoisotopic peaks with formula suggestion and the related isotopic peaks. On the basis of this combined data evaluation, a fragmentation pattern for each NCC/NDCC can be generated to support its identification in the sample. Mass Frontier 4.0 is a software package for the management, evaluation, and interpretation of mass spectra, including the automated generation of possible fragments and rearrangement mechanisms, starting from a user-supplied chemical structure. With this feature of the software, we can check the consistency between a chemical structure and its mass spectrum and recognize the structural differences between spectra of closely related compounds. The program generates a fragmentation scheme for the drawn molecular structure using fragmentation rules of mass spectrometry known in the literature as well as the selected ionization mode and the number of fragmentation steps. The program parameters used in this study were ESI ionization method, inductive cleavage, and five as the maximum number of reaction steps. The fragmentation reactions were selected to include hetero- and homolytic cleavage, neutral losses, and hydrogen rearrangements. Other parameters were left as their default values.

more significant once it was shown that NCCs are resistant to digestion and absorbed by human intestinal epithelium.19 The aim of this work was to determine NCCs/NDCCs profile in loquat fruits, a perennial species that may present an intricate process of chlorophyll catabolism, and produce a heterogeneous catabolites profile in comparison to the regular profile described in deciduous plant fruits described to date. To achieve this aim, a high-performance liquid chromatography coupled with electrospray ionization ion trap and quadropole time-of-flight mass spectrometry (HPLC/ESI-IT/qTOF-MS) method was applied to reach a high specificity for description of those already known NCCs and also to screen new structural rearrangements to expand knowledge of the chlorophyll breakdown route.

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MATERIALS AND METHODS

Plant Material. Eriobotrya japonica Lindl. trees (ca. 10 years old) were cultivated in an open field with a Mediterranean climate (Seville, Spain). Determination of the NCC profile was performed in naturally yellow fruits. Leaves of spinach (Spinacea oleracea L.) were bought at local market, and they were allowed to senescence by dark-incubation in distilled water in Petri dishes at 25 °C for 5−7 days.20 Reagents. Potassium phosphate was provided by Sigma-Aldrich Chemical Co. (Madrid, Spain). HPLC LC/MS grade solvents were supplied by Panreac (Barcelona, Spain). The deionized water used was obtained from a Milli-Q 50 system (Millipore Corp., Milford, MA, U.S.A.). Na (NaCOOH) (10 mM NaOH in 300 μL of formic acid) was used for calibration. NCCs Extraction. Fresh material (ripened loquat fruits and senescent leaves of spinach) was homogenized in liquid nitrogen and extracted into four volumes of 20 mM potassium phosphate buffer (KPi), pH 7.0/methanol (1:3, v/v). The extract was centrifuged at 14 000 × g for 5 min, and the supernatant was concentrated21 by a C18 column (Bakerbond SPE, 500 mg/6 mL, J.T. Baker, Deventer, Holland). The SPE column was activated with two volumes of water and two volumes of methanol. Then, the extract, previously diluted to 80% with water, was applied to the column. The SPE with the sample was cleaned with four volumes of water for desalting, and the NCCs fraction was eluted with 1 mL of 20 mM KPi pH 7.0/methanol (1:9, v/v).19 Liquid Chromatography/Electrospray Ionization Ion Trap/ Time-of-Flight Mass Spectrometry. The liquid chromatograph system was Dionex Ultimate3000RS U-HPLC (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Chromatographic separation was performed as described earlier22 but with modifications as follows. The eluent components were 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in methanol (B). The proportion of B was increased from 20% to 32% in 10 min, to 40% in 15 min, to 60% in 30 min, to 100% in 10 min, and held at 100% for 18 min. Initial conditions were reached in 2 min, and the equilibrium time was 10 min. The injection volume was 30 μL, and the flow rate was 1 mL/min. A stainless steel column (20 cm × 0.46 cm i.d.), packed with 3 μm C18 Spherisorb ODS-2 (Teknokroma, Barcelona, Spain), was used. A split postcolumn of 0.4 mL/min was introduced directly on the mass spectrometer electrospray ion source. The HPLC/ESI-qTOF operated for mass analysis using a micrOTOF-QII High Resolution Time-of-Flight mass spectrometer (UHR-TOF) with qQ-TOF geometry (Bruker Daltonics, Bremen, Germany) equipped with an ESI interface. The HPLC/ESI-IT system operated for MS2 analyses and selected an ion of a particular m/z value to obtain exclusively its daughter ions. In this case, the instrument was operated in positive ion mode using a scan range from m/z 50−1200. Mass spectra were acquired in MS fullscan mode, and data were used to perform multitarget-screening using TargetAnalysis 1.2 software (Bruker Daltonics, Bremen, Germany). MS2 spectra were acquired in auto-MS2 mode (data-dependent acquisition) and were used for structural confirmation of the compounds detected. Collision energy was estimated dynamically based on appropriate values for the mass and stepped across a ± 10% C



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Semipreparative HPLC Isolation of NCC Product(s). To obtain enough quantity of Ej-NCC4, this catabolite was isolated from a NCC concentrated sample of loquat by semipreparative HPLC (injection volume, 500 μL; flow rate, 4.5 mL/min). Solvent A, 0.1% formic acid in water; solvent B, methanol; solvent composition (A/B) as a function of time (0−180 min), 0−5, 80:20; 5−120, 80:20 to 35:65; 120−180, 35:65 to 0:100. A stainless steel column (25 cm × 1 cm i.d.) packed with 3 μm C18 Spherisorb ODS-2 (Thermo Scientific Easy, Barcelona, Spain) was used. A split postcolumn of 0.30 mL/min was introduced directly on the mass spectrometer electrospray ion source using the same acquisition conditions described above. Three consecutive semipreparative HPLC runs were performed, and fractions containing Ej-NCC4 were collected at 59−61 min. For desalting, the aqueous solution was applied to a C18 SPE column (Bakerbond SPE, 1 g/12 mL, J.T. Baker, Deventer, Holland) activated with two column volumes of methanol and preconditioned with two column volumes of water, washed with 20 mL of H2O, and eluted with 4 mL of MeOH. The solvents were removed using a rotary evaporator. Chemical Derivatization of Ej-NCC4 Product. Isolated compound was subjected to chemical derivatization for the identification of functional groups. Specific methylation of the hydroxyl function at the propionic acid group was performed with diazomethane.34 An aliquot of the sample was dried with nitrogen and redissolved in methanol. Fresh diazomethane was generated from an ethereal solution of N-methyl-N-nitroso-p-toluene-sulfonamide in the presence of KOH and MeOH. A gentle stream of N2 was bubbled from the reaction vessel, where diazomethane is generated, to the one containing the Ej-NCC4 solution to achieve the methylation reaction upon development of a light yellow color of the solution. For silylation, an aliquot of the isolated compound was dried with nitrogen and redissolved in 200 mL of bis(trimethylsilyl)trifluoroacetamide (BTSFA) in pyridine; the solution was heated at 90 °C for 20 min. Afterward, the final volume was evaporated with a nitrogen stream to 10 μL of MeOH for analysis by MS.35

different molecular formulas corresponding to NCCs and one to NDCC. Following the nomenclature convention (initials from latin species name followed by a number that indicates order in HPLC chromatogram),36 these catabolites are designed as Ej-NCC1−Ej-NCC4 and Ej-NDCC1. Table 1 contains the mass spectroscopic characteristics of the NCC/ NDCC detected in senescent fruits of loquat. Considering the elution order, Ej-NCC1 (peaks 1 and 3 in Figure 2) is the first NCC present in loquat fruits with a predicted elemental composition equal to C35H42N4O10, and with an experimental m/z value equal to 679.2990 and 679.2974 for the protonated molecule (peaks 1 and 3, respectively). This compound should be equivalent to So-NCC2 described in senescent leaves of spinach,22 and the results obtained for the standard So-NCC2 in our HPLC−MS system. The same elution time (39.4 min) with identical UV spectra and showing the same elemental composition and an experimental m/z value equal to 679.2989 for the protonated molecule confirmed the hypothesis. Additionally, the characteristic product ions of Ej-NCC1 and So-NCC2 obtained by IT−MS2 are the same. A signal at m/z 647 corresponds to the product ion [M+H−CH3OH]+, m/z 591, and m/z 522 [M+H-ring A]+. The high resolution ESI− TOF−MS2 analysis of So-NCC2 from an extract of spinach senescent leaves and of its equivalent in loquat fruits confirms the consistency of the elemental composition and formulas of all product ions, which obtained positive records in all cases for both compounds. Consequently, Ej-NCC1 is the equivalent of So-NCC2 in loquat ripened fruits, which means a dihydroxylation in the vinyl group at C3, hydroxylation at C82, and keeps the esterification at O134 (Figure 1) . Ej-NCC3 is the third NCC compound in elution order (57.7 min and 58.4 min, peaks 7 and 8, Figure 2) with an accurate mass for the protonated molecule of m/z 631.2775 and 631.2762 and a predicted molecular formula of C34H38N4O8, identical to that of So-NCC3. The IT−MS2 fragmentations of Ej-NCC3 are replicated in the corresponding analysis of the standard So-NCC3 in spinach extracts22 and detailed in Table 1. Signals at m/z 625 and 613 corresponding to the loss of H2O and that at m/z 508 corresponding to the loss of ring A (both from the protonated molecule) were observed for Ej-NCC3 and So-NCC3. In addition, the elemental composition and formulas of all product ions were checked with the SmartFormula3D algorithm by high resolution ESI−TOF− MS2 analysis of the Ej-NCC3 observed in loquat fruits and SoNCC3 in spinach, which obtained positive records for all fragments in both cases. The structure proposed for Ej-NCC3 maintained the vinyl group at C3, was hydoxylated at C82 and de-esterified at O134 (Figure 1). The last chlorophyll NCC detected in loquat fruits was EjNCC4 (59.0 min and 60.4 min, peaks 8 and 9, Figure 2). The protonated molecule of this product was observed at m/z 645.2910 in the high resolution ESI−qTOF−MS, with an elemental composition (C35H40N4O8) that fits with that of SoNCC4. The high resolution ESI−qTOF−MS data analysis of So-NCC4 showed the same predicted elemental composition as did Ej-NCC4 and an experimental m/z value equal to 645.2918 for the protonated molecule (data not shown). The observed characteristic IT−MS2 product ions for Ej-NCC4 and SoNCC4 were the same and are detailed in Table 1. The most prominent product ion was the one representing the loss of ring A, a characteristic fragmentation in the mass spectra of NCCs as well as the loss of methanol moiety from the protonated molecule that indicates the presence of a methyl

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RESULTS AND DISCUSSION NCCs/NDCCs Profile in Senescent Fruits of Loquat. Figure 2 depicts the HPLC 320 nm chromatogram obtained

Figure 2. HPLC chromatogram of loquat senescent fruit extract acquired at 320 nm, showing the target compounds described in Table 1. Inserts correspond to UV spectrum of Ej-NCC4 (peak 9). A, artifact peak.

from the extract of ripened fruits of loquat and shows the NCCs/NDCCs identified through the application of the dataprocessing procedure from the acquired HPLC/ESI-TOF-MS data. Additionally, the UV−vis spectrum of the major NCC found (peak 9, Ej-NCC4) is included with the characteristic maximum absorbance peak at 320 nm as a consequence of the α-formyl pyrrole moiety at ring B.22 In ripened fruits of loquat, the nonfluorescent chlorophyll catabolite profile contains four D



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Table 1. Nonfluorescent Chlorophyll Catabolites (NCCs and NDCC) Composition in Ripened Fruits of Eriobotrya japonica Lindl. Determined by HPLC/ESI-IT/TOF-MS time-of-flight compound Ej-NCC1

Ej-NCC2 Ej-NDCC1 Ej-NCC3

Ej-NCC4

ion trap

peak number

tR (min)

error (ppm)

SigmaFit value

molecular formula

[M + H]+ (m/z)

1

39.4

2.7

23.3

C35H42N4O10

679.2990

3 2 4 5 6 7

41.9 40.1 42.2 50.4 50.8 57.7

0.9 0.2 1.9 5.9 2.9 0.1

19.2 39.7 42.4 45.9 5.6 46.4

8 9

58.4 59.0

2.0 1.0

46.5 14.0

10

60.4

1.5

11.0

C38H42N4O11 C34H40N4O8 C34H38N4O8

C35H40N4O8

679.2974 731.2880 731.2878 633.2882 633.2919 631.2775 631.2762 645.2910 645.2919

group at O134 as it has been previously described.13,24,29 An additional product ion was observed corresponding to the loss of the propionic moiety at C17 (m/z 571). This fragmentation has been described for chlorophyll derivatives37 but not for NCCs so far. It is logical to assume that the fragmentation pattern of chlorophylls and their derivatives (NCCs) may display common product ions as in this case. To be sure that the signals obtained in the MS2 analyses correspond to the predicted fragments, we checked the elemental composition and formulas of all product ions with the SmartFormula3D algorithm. Consistency of the monoisotopic peaks and the related isotopic peaks was determined by high resolution ESI− qTOF−MS2 analyses, which obtained positive records for all fragments. The structure of Ej-NCC4 (and So-NCC4) is the simplest possible since it only implies the hydroxylation at C82, which maintains the vinyl group at C3 and the esterification at O134. In addition, a chromatographic peak of the HPLC analysis (Rt: 55.8 min) of ripened fruits of loquat showed spectroscopic properties that match with those of a NCC (Supporting Information, S2). The MS spectrum of this “new” NCC indicated an elemental composition of C36H42N4O9, which means a molecular weight of 674.2951. The MS2 spectrum, obtained by ESI−qTOF−MS to guarantee the interpretation with SmartFormula3D (Supporting Information, S3), shows two fragments corresponding to neutral losses of one or two molecules of methanol from the protonated molecule at m/z 643.2762 [M-CH3OH+H]+ and at m/z 611.2515 [M-2 × CH3OH+H]+, respectively. In addition, the characteristic product ions corresponding to NCCs were also present, including the product ion at m/z 552.2337 ([M-ring A+H]+) and the product ion at m/z 520.2105 [M-ring A-CH3OH+H]+ ion. With these data, the new NCC structure was tentatively assigned to the one presented in S3 of the Supporting Information. The compound derives from Ej-NCC4, to which a methoxy function was added at the C82 position. To confirm that the new NCC is an artifact produced during solvent extraction of the plant material from Ej-NCC4, ripened fruits of loquat were extracted with ethanol, and the HPLC gradient composition was changed by acetonitrile. A new compound was formed, with a protonated molecule at m/z 689.3181, that is, the derivatization of Ej-NCC4 with ethanol; therefore, the new NCC is really an artifact produced during solvent extraction of the plant material. An FCC artifact formed during solvent

Product ions (m/z) 647 591 522 552 522 601 510 625 613 508 613 522 571

[M+H−CH3OH]+ [M+H-ring A]+ [M+H-malonyl-ring A]+ [M+H−CH3OH]+ [M+H-ring A]+ [M+H−H2O]+ [M+H-ring A]+ [M+H−CH3OH]+ [M+H-ring A]+ [M+H−C3H6O2]+

isolation was reported recently12 in banana fruits (Musa acuminata Colla) consisting of a trans-esterification by methanol at the propionic group. Special attention should be paid to the proper identification of noncolored chlorophyll catabolites since it seems they are prone to experiment modifications during solvent extraction. New NCC/NDCCs in Senescent Loquat Fruits. Screening was not the sole strategy for NCC/NDCC determination; by exploiting the capabilities of this new methodology, we included in the target database elemental composition of new possible structures of NCCs/NDCCs not described so far. Of all of the plausible enzymes implicated in the modifications of FCCs (and consequently NCCs/NDCCs), only MES16, a methylesterase has been described, and taking into account that in loquat senescent fruits most NCCs (Ej-NCC1 and EjNCC4) are esterified, the ratio of esterified/de-esterified should be important in senescent fruits of loquat. Consequently, new theoretical structures were designed in base of de-esterifying or esterifying alternatives in already identified NCCs (not described to date). The methyl ester at the C132 form of BnNCC1 (m/z 730.2850, C38H42N4O11) and the de-esterified forms of Hv-NDCC, Zm-NCC1, and Nr-NCC1 at C132, corresponding to the following masses and elemental compositions: m/z 652.2744, C33H40N4O10; m/z 826.3273, C40H50N4O15; and m/z 878.3222, C43H50N4O16, respectively (Supporting Information, S1), were also included in the target database. Two positive matches (40.1 min and 42.2 min) with NCC UV absorption spectra were detected during the postprocessing procedure, both with an exact mass determined by high resolution ESI−TOF−MS of m/z 731.2880 and 731.2878, corresponding to C38H42N4O11. Taking into account the structure of an NCC and the functional groups described, such an elemental composition could correspond to the ester form of Bn-NCC1, which means a vinyl group at C3, esterified at O134, and a malonyl group at C82 (Figure 1). Table 1 reports chromatographic characteristics of this NCC, designed as Ej-NCC2, following the nomenclature convention.36 The MS2 spectrum analyzed by IT−MS of Ej-NCC2 is shown in Figure 3, panel A. Besides the signal of the protonated molecule ion (m/z 731), two signals of characteristic product ions of BnNCC120 are detected, one at m/z 552 and the other at m/z 522, corresponding to the [M-ring A-malonyl +H]+ ion. Within this strategy of including new possible structures in the target database of elemental composition for the screening E



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obtained show that at least Ej-NDCC1 is present as a consequence of a wild-type chlorophyll metabolism. Evidence for Tautomerism in NCCs Metabolism. The description of the chlorophyll catabolites in extracts of fully developed loquat fruits shows a pair of isomers for each NCC/ NDCC. For each catabolite, it detected a second product at a different retention time (see Table 1) with a feature UVspectrum of NCC/NDCC, the same elemental composition, and an equivalent MS 2 fragmentation pattern. These compounds were assigned as isomers of Ej-NCC1, Ej-NCC2, Ej-NDCC1, Ej-NCC3, and Ej-NCC4. The possible effect of formic acid in the isomerization of NCCs was ruled out with a control experiment without formic acid in the HPLC gradient, which checked the presence of isomers in the control assay. This is not the first time that isomers of chlorophyll catabolites are described in senescent tissues. Four fractions of NDCCs produced by the activity of cytochrome P450 CYP89A9 have been described in Arabidopsis leaves,30 and in senescent quince fruits, a pair of isomers for each NCC and two pairs for the NDCCs have been reported.13 Although NCCs display three chiral centers at C1, C132, and C15, occurrence of different configurations is very constrained; thus, the possibility of epimerization at the chiral center C1 is excluded since this conformation is enzymatically originated and constant within the same vegetal species.38 Structural configurations of the chiral centers C132 and C15 are S and R, respectively, for all NCCs described to date. This imposed spatial distribution is a sine qua non condition to carry out the spontaneous acidcatalyzed reaction of the FCC precursor to NCC in the weakly acidic vacuolar environment.3 Consequently, few new structural rearrangements are available and hypothesized. One of those structural rearrangements to explain the NCC isomers is the keto−enol tautomerism at C131. This possible structural conformation is supported in the occurrence of enol forms of chlorophyll a and its derivatives at the isocyclic ring E. In fact, chlorophyll a keto−enol tautomerism was already evidenced by trapping the enol isomer as trimethylsilyl ether derivative.39 By following the experimental approach that demonstrated keto− enol tautomerism in chlorophylls, the chemical derivatives of isolated Ej-NCC4 peaks (both isomers of the main NCC of loquat fruits) were obtained by semipreparative HPLC. To check the number of possible hydroxyl groups in the molecule(s) of Ej-NCC4 (two in the case of the keto tautomer, three for the enol configuration, Figure 4), a silylation reaction was performed with BSTFA, and the derivative compounds were analyzed by high resolution qTOF−MS. At 62.3 min, a derivative product was observed with a feature UV−vis spectrum of NCC, but regarding the original protonated molecule of Ej-NCC4, the atomic mass increased by 216 Da, which corresponds to the incorporation of three trimethylsilyl ether groups (TMSi) on the molecule (see Figure 4 for molecular rearrangement of Ej-NCC4 derivatives). At a different retention time the presence of di-TMSi derivative was also observed as shown in the table inserted in Figure 4 that contains retention time, elemental composition, and experimental atomic mass of the protonated molecules of the derivative products from Ej-NCC4 as well as the mass error and mSigma values from which the consistency of the isotopic pattern was ascertained. The incorporation of three TMSi groups to the Ej-NCC4 molecule is the hint for the presence of the enol isomer. The occurrence of the di-TMSi derivative should arise from the keto isomer. We also proceed to the specific methylation of the hydroxyl function from the

Figure 3. MS2 spectra of (A) Ej-NCC2 and (B) Ej-NDCC1. The protonated molecule ion and the main product ions are shown.

procedure we also added the NDCCs theoretical alternatives that can be obtained from all the NCC structures described so far. NDCCs are generated from all NCC structures (both known and new ones) by deformylation at C6 in ring B. The screening showed the occurrence of Ej-NDCC1 (50.4 min and 50.8 min, peaks 5−6, Figure 2), both with the typical UV absorption spectrum of a NDCC10,27 with broad bands near 237 nm and 274 nm. The only NDCCs detected in loquat fruits present in the high resolution ESI−TOF−MS a protonated molecule ion at m/z 633.2882 and 633.2919, both with a predicted elemental composition of C34H40N4O8. The MS2 spectrum obtained by IT−MS (Figure 3B), besides the signal of the protonated molecule, showed two prominent signals of product ions, one at m/z 601 that corresponds to the neutral loss of methanol [M-CH3OH+H]+ and the other at m/ z 510 in response to the ring A loss, [M-ring A+H]+. Both losses are characteristics of NDDC MS fragmentation.27 On the basis of previous configurations of NDCCs, the elemental composition of Ej-NDCC1 corresponds to the structure proposed in Figure 1, a vinyl group at C3, hydroxylated at C82, and keeping the esterification at O134. This means that EjNDCC1 is the NDCC that corresponds to Ej-NCC4. This compound has been identified previously in Arabidopsis MES16 mutant leaves,31 a mutant that accumulates a different chlorophyll catabolite profile with novel and higher amounts of dioxobylins (FDCCs plus NDCCs, Figure 1). The results F



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including Zea mays L., although no enzymatic determination has been developed. A third and new level of structural modification is the conjugation of the hydroxyl group at the C82 position with malonyl directly from Ej-NCC4, which means a complement to a previous proposal.22 This new compound formed, Ej-NCC2, has a noncommon refunctionalization on C82 in plants, since the only vegetal species in that has been identified at the moment is Brassica napus.20 In fact, Brassica napus, L., has three different NCCs, two of them with the same modifications in C82 as in loquat fruits. Nevertheless, no data are available in relation to the esterases enzymes implicated in the FCC modifications at C82. At this point, the identification of the enzymes responsible of such modifications will be very important for a better understanding of chlorophyll catabolism. An alternative pathway (Figure 1) is the deformylation at C6 from pFCC by CYP89A96, which originates pFDCC. The hydroxylation at C82 is the reaction responsible for the formation of Ej-FDCC1. The esterase responsible for such hydroxylation should be very potent in loquat fruits since it is the dominating reaction in the refunctionalization of NCCs (EjNCC4, also hydroxylated in C82 is the major NCC). Later, EjFDCC1 is imported to the vacuole, where the low pH isomerizes to Ej-NDCC1. In loquats, CYP89A9 activity seems to be not very active, FDCC formation is very low and consequently so is Ej-NDCC1. The ratio among the sum of the UV-absorbance areas of NCCs (at 320 nm) and the sum of the UV-absorbance areas of NDCC (at 250 nm) in loquat fruits is about 1500. Clearly, there are two types of senescent tissues under the point of view of chlorophyll degradation, the NCCs species (most of the species analyzed so far) and the NDCCs species (Norway maple and Arabidopsis thaliana leaves). Recently, NDCCs were described for the first time in fruits,13 and it was found that quince fruits have a dominated NDCC metabolism. The results obtained for loquats demonstrate that their major chlorophyll metabolism yields to NCC and consequently that the existing divergence between NCC or NDCC dominant metabolism in senescent leaves is also a reality in ripened fruits. This fact is another parallelism between chlorophyll catabolism in fruits and leaves.

Figure 4. Scheme and results of Ej-NCC4 derivatization. Localizations for silylation (---) and methylation (······) reactions are shown. Cal., calculated; meas., measured.

propionic group at C17 via diazomethane (see Figure 4), and then the silylation reaction was performed. In this case, we only observed the derivative products corresponding to the incorporation of one or two TMSi groups in the keto and enol isomer, respectively (see insert in Figure 4). Although specific analyses are required to definitively establish the structures of isomers found in senescent tissues, keto−enol tautomerism is a plausible possibility that should be elucidated in future research. Proposed NCC/NDCC Pathway in Loquat Senescent Fruits. Figure 1 depicts a scheme for the structural rearrangements that originate the wide profile of FCCs in senescent loquat fruits by considering the pathway proposal outlined before.22 It must be noted that the modification at C3, C8, and C132 takes place exclusively on the FCC/FDCC structure; however, FCC/FDCCs are very unstable, so we analyzed the NCC/NDCC structures. The first structural modification of pFCC is the hydroxylation at the terminal carbon of the ethyl group at C8, which yields a secondary FCC (sFCC) that later is structurally modified by different catabolic enzymes,6 which introduces new functions in the peripheral of the molecule and yields modified FCCs (mFCCs). In senescent loquat fruits, the major NCC is Ej-NCC4, which arises from a hydroxylation at C82 (resembles the rest of the structural features of FCC) and represents 80% of total NCCs. In parallel, three refunctionalization routes are available from Ej-NCC4.22 First, the dihydroxylation at the vinyl group of ring A; second, the de-esterification process at ring E (C132). Certainly, esterified NCCs represent 95% of total NCCs in loquat senescent fruits. Remarkably, the only enzyme involved in the refunctionalization of NCCs that has been identified to date is MES16,6 a member of the methylesterase protein family, which localizes to the cellular cytosol and specifically demethylates FCCs. It could be hypothesized that senescent fruits of loquat do not display a significant activity of MES16 as do some other vegetal species

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ASSOCIATED CONTENT

* Supporting Information S

Table with descriptions of the NCCs described up to now, MS chromatogram of Ej-NCC4 methanol derivative, and MS2 chromatogram of Ej-NCC4 methanol derivative. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +00 34 954 691054. Fax: +00 34 954 691262. Funding

This work was supported by the Comisión Interministerial de Ciencia y Tecnologiá (CICYT-EU, Spanish and European Government, AGL 2012−39714) and by Junta de Andaluciá (AGR 6271−2011). Notes

The authors declare no competing financial interest. G



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ABBREVIATIONS USED FCC, fluorescent chlorophyll catabolite; NCCs, nonfluorescent chlorophyll catabolites; NDCC, urobilinogenoidic chlorophyll catabolite.

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