Isolation and Characterization of a Urobilinogenoidic Chlorophyll Catabolite from Hordeum vulgare L.

Abstract

A new type of chlorophyll catabolite was isolated from extracts of de-greened primary leaves of barley (Hordeum vulgare cv. Lambic). Its constitution was elucidated by one-dimensional and two-dimensional {1H,13C}-homo- and heteronuclear NMR spectroscopic techniques and by high resolution mass spectroscopy. The isolated catabolite, a water-soluble, colorless, and nonfluorescent linear tetrapyrrole, resembles urobilinogen in which one of the propionic side chains forms a five membered isocylic ring system, indicating its origin from the chlorophylls. A new type of chlorophyll catabolite was isolated from extracts of de-greened primary leaves of barley (Hordeum vulgare cv. Lambic). Its constitution was elucidated by one-dimensional and two-dimensional {1H,13C}-homo- and heteronuclear NMR spectroscopic techniques and by high resolution mass spectroscopy. The isolated catabolite, a water-soluble, colorless, and nonfluorescent linear tetrapyrrole, resembles urobilinogen in which one of the propionic side chains forms a five membered isocylic ring system, indicating its origin from the chlorophylls. chlorophyll(s) medium and high pressure liquid chromatography, respectively Metabolic disappearance of the chlorophylls (Chls)1 in phototrophic organisms indicates programmed close down of photosynthesis. Although several linear tetrapyrrolic Chl catabolites were isolated during the last decade from green algae and higher plants, the metabolic pathway of tremendous amounts of the Chls is still under question. What products follow after the familiar formybilinones? What are the ultimate products of Chl degradation? Degradation of the Chls occurs in light as well as in darkness. During senescence cellular components are hydrolyzed and metabolized; liberated rare elements such as [N], [P], [S] and metal ions are relocated (1Stahl E. Stahl E. Zur Biologie des Chlorophylls, Laubfarbe und Himmelslicht. G. Fischer Verlag, Jena, Germany1909Google Scholar, 2Peoples M.B. Dalling M.J. Noodén L.D. Leopold A.C. Senescence and Aging in Plants. Academic Press, New York1988: 181-217Google Scholar). The first structures of Chl catabolites isolated from a green alga and a higher plant were published in 1991 (3Engel N. Jenny T.A. Mooser V. Gossauer A. FEBS Lett. 1991; 293: 131-133Crossref PubMed Scopus (104) Google Scholar, 4Kräutler B. Jaun B. Bortlik K. Schellenberg M. Matile P. Angew. Chem. Int. Ed. Engl. 1991; 30: 1315-1318Crossref Scopus (172) Google Scholar). Several Chl catabolites have been isolated since then (Fig. 1). The similarity of the structures of the red Chl catabolites (3Engel N. Jenny T.A. Mooser V. Gossauer A. FEBS Lett. 1991; 293: 131-133Crossref PubMed Scopus (104) Google Scholar, 4Kräutler B. Jaun B. Bortlik K. Schellenberg M. Matile P. Angew. Chem. Int. Ed. Engl. 1991; 30: 1315-1318Crossref Scopus (172) Google Scholar) isolated from the green alga Chlorella protothecoides with the colorless catabolites isolated from higher plants (5Gossauer A. Engel N. J. Photochem. Photobiol. B Biol. 1996; 32: 141-151Crossref Scopus (76) Google Scholar, 6Kräutler B. Matile P. Acc. Chem. Res. 1999; 32: 35-43Crossref Scopus (132) Google Scholar, 7Iturraspe J. Moyano N. Frydman B. J. Org. Chem. 1995; 60: 6664-6665Crossref Scopus (38) Google Scholar) suggest a close relationship in the basic skeleton. Biologists regard members of the phylum Chlorophyta as progenitor of the higher plant cell (9Campbell N.A. Campbell N.A. Biology. The Benjamin/Cummings Publishing Company, Inc., Redwood City, CA1990: 566-567Google Scholar, 10van den Hoek C. Mann D.G. Jahns H.M. van den Hoek C. Mann D.G. Jahns H.M. Algae. An Introduction to Phycology. Cambridge University Press, 1995: 483-512Google Scholar). This information triggered research activities in several disciplines to elucidate the apparently unique catabolic pathway of the Chls in the green plant lineage. The studies range from the elucidation of chemical and enzymatic reaction mechanisms to molecular biological research (6Kräutler B. Matile P. Acc. Chem. Res. 1999; 32: 35-43Crossref Scopus (132) Google Scholar, 11Engel N. Curty C. Gossauer A. Plant Physiol. Biochem. 1996; 34: 77-83Google Scholar, 12Rosybel D. Hörtensteiner S. Donnison I. Bird C.R. Seymour G.B. Physiol. Plant. 1999; 107: 32-38Crossref Scopus (51) Google Scholar). Previous labeling experiments with oxygen isotopes and heavy water showed a high regio- and stereoselectivity of the oxidative ring opening mechanism and the involvement of a monooxygenase in the ring cleaving step (13Curty C. Engel N. Gossauer A. FEBS Lett. 1995; 364: 41-44Crossref PubMed Scopus (42) Google Scholar, 14Hörtensteiner S. Wüthrich K.L. Matile P. Ongania K.-H. Kräutler B. J. Biol. Chem. 1998; 273: 15335-15339Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 15Curty C. Engel N. Plant Physiol. Biochem. 1997; 35: 707-711Google Scholar). The remarkable allylic pyrroline/pyrrole rearrangement was studied in detail (11Engel N. Curty C. Gossauer A. Plant Physiol. Biochem. 1996; 34: 77-83Google Scholar, 16Curty C. Chlorophyll Catabolism in Plants: Chlorella protothecoides and Cercidiphyllum . Ph.D. thesis No. 1143. University Fribourg, Switzerland1996Google Scholar). Most recent in vivo deuterium labeling has shown that in higher plants Chl b is degraded via Chl a (17Folly P. Engel N. J. Biol. Chem. 1999; 274: 21811-21816Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Chl breakdown has long been considered as a detoxification process in which photodynamic active Chls convert to colorless tetrapyrrolic products, all of them carry a characteristic formyl group (Fig. 1). Those products were regarded as the final products of Chl breakdown in senescent plants, which apparently do not cause further cleavage into smaller fragments (6Kräutler B. Matile P. Acc. Chem. Res. 1999; 32: 35-43Crossref Scopus (132) Google Scholar). This work focuses on the isolation and determination of the constitution of a new type of chlorophyll catabolite by spectroscopic methods and discusses the origin and relevance in the catabolic pathway of the chlorophylls in plants. Chemicals were reagent grade; all solvents were distilled before use. HPLC solvents were supplied from Fluka (Buchs, Switzerland). HPLC columns and Nucleosil 100-7 C8 VP 250/10 were from Macherey-Nagel (Oensingen, Switzerland), and MPLC columns Lobar® LiChroprep® RP-18 (40–63 μm) were purchased from Merk (Darmstadt, Germany). 35cc Sep-Pack® Vac C-18, 10 g, were provided from Waters (Milford, MA). Homo- and heteromagnetic resonance experiments were performed on a Bruker Avance DRX-500 spectrometer operating at the frequencies of 500.13 MHz for 1H and 125.75 MHz for 13C. Chemical shifts (δ) are given in parts/million downfield from the solvent used and coupling constants (J) in Hertz (Hz). Mass spectra were obtained with a Bruker FTMS 4.7T BioAPEXII, using electrospray ionization technique in the positive mode. UV-visible spectra were recorded on a Perkin-Elmer Lambda 40 spectrometer, λmax (log ε) in nanometers. CD spectra were measured on a Jobin-Yvon Auto Dichograph Mark V, Δε [l mol−1 cm−1]. Barley seeds (Hordeum vulgare L. cv. Lambic) were a gift from Florimond Desprez. The seeds were germinated in high density (5 seeds/cm2) in moist garden soil and grown under natural light conditions. The primary leaves were harvested when they reached about 10–15 cm in height. The greening and de-greening procedure of barley leaves was essentially as described previously (17Folly P. Engel N. J. Biol. Chem. 1999; 274: 21811-21816Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). 150 g of de-greened yellow leaves of H. vulgare (fresh weight) were homogenized in a blender with 300 ml of a solution consisting of 0.1 mpotassium phosphate (KP) buffer, pH 6.8:acetone:MeOH (1:1:1). Work-up was essentially as described previously (17Folly P. Engel N. J. Biol. Chem. 1999; 274: 21811-21816Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Aliquots of the aqueous phase were injected into a Lobar®LiChroprep® RP-18 (40–63 μm) column and eluted (4 bar, 22 ml/min) with a solution of 30 volume % MeOH in 0.01 mKP buffer, pH 6.8. Two fractions positive in the chromic acid degradation assay were sampled. MeOH was evaporated in vacuo. The polar fraction contained both urobinlinogens, the less polar fraction contained formylbilinone 5. The resulting aqueous phases were again concentrated on a 35cc Sep-Pack®RP-18 cartridge. Separation and purification of the urobinlinogens were achieved on a HPLC Nucleosil 100–7 C8 VP 250/10 column eluted isochratically (flow rate: 5 ml/min) with a solution of MeOH in 0.01m KP buffer, pH 6.8 (13 volume %). The fractions with a retention time of 13 min and of 14 min, respectively (DAD-UV detection at 250 nm) were collected at 0 °C and stored under argon. After removal of the volatile solvent in vacuo, each product was de-salted on a 35cc Sep-Pack® C-18 cartridge by first washing with 200 ml of distilled water and afterward eluted with aqueous acetone (50 ml of 50 volume %). The gross solvent was eliminated in vacuo, and the remaining aqueous solution was lyophilized giving 15 mg and 10 mg, respectively, of a slightly yellow powder. A triad of tetrapyrrolic compounds was isolated by HPLC from yellow cotyledons of barley. After chemical degradation all showed on TLC plates the same characteristic maleimide fragments, namely 3-(2-hydroxyethyl)-4-methyl maleimide, 3-(2,3-dihydroxyethyl)-4-methyl maleimide and hematinic acid imide (cf. Ref.17Folly P. Engel N. J. Biol. Chem. 1999; 274: 21811-21816Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The less polar compound isolated in about 7 mg was spectroscopically identical in all aspects with catabolite 5 (Fig.1) previously isolated from H. vulgare cv. Gerbel (4Kräutler B. Jaun B. Bortlik K. Schellenberg M. Matile P. Angew. Chem. Int. Ed. Engl. 1991; 30: 1315-1318Crossref Scopus (172) Google Scholar). The second product, which was isolated in about 15 mg, was spectroscopically analyzed. Mass spectrometric analysis at high resolution showed two molecular ions at m/z705.2530 atomic mass units (100%) and m/z 743.2093 atomic mass units (10%). This corresponds exactly with a molecular ion [C34H41KN4O10 + H]+ (calculated: 705.2532; error: 2.5 × 10−4) and [C34H41KN4O10 + K]+ (calculated: 743.2091; error: 1.8 × 10−4), respectively, establishing the bulk molecular formula to be C34H41KN4O10. Details on the constitution of this metabolite were deduced from the analysis of the one-dimensional and two-dimensional {1H,13C}-homo- and heteronuclear NMR spectroscopy (Fig. 2). The compound when dissolved in D2O shows 33 carbon-bound hydrogen atoms and 33 carbon atoms. Seven protons are bound to hetero-atoms, which rapidly exchange for solvent. Attached proton test spectra show 5 primary, 7 secondary, 4 tertiary, and 17 quaternary carbon atoms. All protons resonate largely as isolated systems and are well resolved, except the signal group at about δH 2.7 (Ha(5) and Ha(15)) and δH 3.6 (H2(32) and Hb(182)) in which two and three protons overlap, respectively (TableI). Four separated methyl groups resonance at high field δH 1.45 H3(21), δH 1.82 H3(131), δH 1.91 H3(171), and δH 1.97 H3(71). The fifth methyl group, which absorbs at δH 3.69 H3(85), is assigned to a methyl ester group, because of its chemical shift, line sharpness, and intensity. The highest resonance frequency was observed at δH 4.74 H(10); this excludes the presence of an aldehyde group. Two-dimensional {1H,1H}- and {1H,13C}-correlation spectra show the presence of two ethylene groups and three AMX systems, the X portions of which have resonances at δH 4.16 H(16), δH4.32 H(4), and δH 4.44 H(181), respectively. At δC 35.35 a weak CH cross-peak was found in the {1H,13C}-correlation spectrum with the H(10) at δH 4.74 ppm.Table I1H and 13C NMR signals of the major catabolite8; connectivities through space were assigned by1H{1H} NOE difference experimentsAtomδC(D2O)δH(D2O)NOE effects% EnhancementAssignement in formula 8 (Fig. 3)1175.892129.37217.161.54 s3154.673128.742.4 ddd (14.2/5.65/5.48) HaHb(31)1-aTriplet., H2(32)bDoublet.3, 2.8a, b2.64 ddd (14.2/6.59/7.13) Hb3259.403.56–3.66 m458.484.32 dd (4.76/4.75)526.992.68–2.74 dd (15.19/4.75) Ha3.01 dd (15.19/4.76) HbH(4), Ha(5), H3(71)1.7, 1.6, 1.6c, d, e6132.737112.31718.281.97 sHb(5)Smalle8123.9281191.778283171.278552.703.69 s9160.191035.354.74 pseudo sH2(121)3.1f11122.4412119.0612120.082.45–2.50 mH2(122), H3(131)Small, 0.3cSharp.h, i12238.612.1–2.2 m123181.9813114.501317.951.82 s14122.591526.992.68–2.74 dd (15.00/4.76) Ha2.85 dd (15.00/4.58) HbHa(15), H(16)Small, 1.6m, n1660.544.16 dd (4.58/4.76)Ha(15), Hb(15)1.3, 0.8l, n17159.7517111.461.91 sH(181)0.13cSharp.o18128.3618166.244.44 dd (4.59/4.39)H3(171), Ha(182), Hb(182)Small, 1, 0.9o, p, q18263.223.34 dd (11.73/4.59) HaHb(182), H(181)8.9, 4r, p3.56–3.66 m Hb19173.96a Triplet.b Doublet.c Sharp. Open table in a new tab Although the proton part of the spectra of the compound when measured in Me2SO-d6 as solvent showed considerable lower resolution, the missing proton became clearly visible at δH 3.88 H(82). This proton couples with a frequency of 3.35 Hz with a proton at δH4.65 H(10). The {1H,13C}-correlation spectrum showed in addition a cross-peak indicating a tertiary carbon atom at δC 65.44 C(82), which correlates with the H(82) proton at δH 3.88. This indicates that this carbon-bound proton is in dynamic equilibrium (keto-enol) and rapid exchange with deuterium from the solvent D2O. This “blind spot” has been previously observed when the plant catabolite5 was measured in D2O (20Kräutler B. Jaun B. Amrein W. Bortlik K. Schellenberg M. Matile P. Plant Physiol. Biochem. 1992; 30: 333-346Google Scholar). In the COSY spectrum (Fig. 2), particularly the appearance of six cross-peaks caused by long range couplings of the four methyl groups facilitated the constitutional assignment. Together with the information gathered from the heteronuclearcorrellation (HETCOR) spectrum, they provided the reference and indicated the starting points of the specific side chains and the bridging network for each of the two dipyrrylmethanone units. Nuclear Overhauser effect experiments corroborated the assignment of the fragments and showed, in addition, that both units are interconnected (Fig. 3 and Table I). Thus, when the proton at δH 4.74 H(10) was irradiated the signal group at δH 2.45–2.50 H2(121) was enhanced. When the latter signal was irradiated a methyl group H3(132) and a methylene group H2(122) responded through space. The overall bonding network and chemical shift assignments of the quaternary carbon atoms were deduced from correlation spectroscopy via long-range coupling (COLOC) spectra. The relaxation times used for the evolution of the long range couplings were equivalent to 1H,13C coupling constants of 5, 10, and 20 Hz, respectively. Although two quaternary carbon atoms at δC 191.77 and δC 160.19 showed no correlation, the former value was assigned for chemical shift reasoning to the carbonyl group C(81) and the latter consequently to the remaining aromatic carbon atom C(9). Thus, chemical degradation, mass, nuclear magnetic resonance, and UV spectra are consistent for the constitution of a potassium 31,181,182-trihydroxy-85-methyl-8 1-oxo-82,10-cyclourobilinogen (8) shown in Fig. 4. The third tetrapyrrole was isolated in about 10 mg and showed in the mass spectrometer a molecular mass ion at m/z 705.2534 atomic mass units (100%), indicating the presence of a configurational isomer of the former. 1H,13C NMR spectroscopic investigation corroborates a diastereomeric relationship. A 1:1 mixture of both diastereomers shows in 1H NMR only slight differences in chemical shifts. Two methyl groups (21) and (171) are shifted relative δ 0.079 ppm to lower field and δ 0.0134 ppm to higher field, respectively, the rest of the spectrum remains nearly identical. The CD spectrum shows that this compound too is optically active and that both diastereomers are chiroptically almost indistinguishable (Fig. 5). The new catabolites isolated from yellow cotyledons of barley are colorless, nonfluorescent, and optically active. The constitution resembles Urobilinogen IXα, a common metabolite of heme catabolism in warm-blooded organisms. The similarities in the peripheral substitution pattern of both catabolites indicate that the new catabolite derives from catabolite 5 by an oxidative process. The configurations of the five asymmetric centers cannot be deduced from the spectroscopic data. Suitable crystals are not available yet. Only one stereocenter at C(82) has its origin from the Chls, but due to the β-keto ester functional group, it is prone to epimerization. When measured in Me2SO-d6, a coupling of only 3.35 Hz between H(10) and H(82) indicates anticonfiguration. As found previously, proton H(15) in formula 5 arrives from the protic solvent during acid-catalyzed pyrroline/pyrrole rearrangement of both geometric isomers of the red chlorophyll catabolite from C. protothecoides (11Engel N. Curty C. Gossauer A. Plant Physiol. Biochem. 1996; 34: 77-83Google Scholar, 16Curty C. Chlorophyll Catabolism in Plants: Chlorella protothecoides and Cercidiphyllum . Ph.D. thesis No. 1143. University Fribourg, Switzerland1996Google Scholar). Intensive mechanistic-chemical studies showed a remarkable high stereoselectivity for this rearrangement. Because of the stereochemical consequence of these experiments, this position was tentatively assigned an R configuration. The proton at this position is highly resistant, even in boiling acetic acid-d1 it does not exchange (11Engel N. Curty C. Gossauer A. Plant Physiol. Biochem. 1996; 34: 77-83Google Scholar,16Curty C. Chlorophyll Catabolism in Plants: Chlorella protothecoides and Cercidiphyllum . Ph.D. thesis No. 1143. University Fribourg, Switzerland1996Google Scholar). It is most likely that both diastereomers arise from the 5 previously isolated from barley leaves by a oxidative process. the cleavage of the formyl group of the red chlorophyll catabolite from C. protothecoides was achieved using at diastereomers of the were obtained from this reaction in a of C. Chlorophyll Catabolism in Plants: Chlorella protothecoides and Cercidiphyllum . Ph.D. thesis No. 1143. University Fribourg, Switzerland1996Google Scholar) and in a of A enzymatic formyl cleavage be for through a reaction with of the ester group through an oxidative the at is under (Fig. diastereomers are observed in a of as by This indicates that this most likely is the the constants of of both of the in the catabolite 8 is in the of barley and not during the procedure was by an in which the cell opening and HPLC was performed in the presence of heavy water atom % 1H and NMR experiments showed that all carbon hydrogen atoms exchange as only at position a in which the leaves were in the presence of heavy water atom % showed by NMR that a deuterium atom in position of compound 8 not that the catabolite is in the plant cell and not during the procedure and further that is under conditions. between the two of was to be only under basic A. H. D. The Academic Press, New Scholar). the a tetrapyrrole was isolated M. M. K. H. M. M. Plant Physiol. and a of information was published K. M. H. K. M. Plant Physiol. 1995; Scholar). The a the formyl group too and a group and a the UV-visible spectrum is in with the which remains to be In Cercidiphyllum almost amounts of the were isolated as catabolite C. Engel N. 1996; Scopus Google in this work only about of the tetrapyrrolic catabolites were As from a test of several plants using the chromic acid degradation are not This the are common in the catabolic pathway of the and they are degraded in the plant cell to Most several were isolated from senescent of barley Plant Physiol. 1999; Scopus Google Scholar). it that phototrophic organisms are to to via a pathway that has been by New 107: PubMed Scopus Google Scholar). still the atoms of the former this be by the This be of for plants, which in in which is in high the ultimate of the Chls is still a of the of and to mass and NMR spectra. are to Florimond for with H. vulgare cv.