THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 12, Issue of March 23, pp. 8643–8647, 2001
Printed in U.S.A.
Isolation and Characterization of a Urobilinogenoidic Chlorophyll
Catabolite from Hordeum vulgare L.*
Received for publication, October 11, 2000, and in revised form, December 11, 2000
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M009288200
Fosca Gattoni Losey and Norbert Engel‡
From the Institute of Organic Chemistry, University of Fribourg, Chemin du Musée 9, CH-1700 Fribourg, Switzerland
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}-homoand 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.
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 (6).
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.
EXPERIMENTAL PROCEDURES
1
Metabolic disappearance of the chlorophylls (Chls) 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 (1, 2).
The first structures of Chl catabolites isolated from a green
alga and a higher plant were published in 1991 (3, 4). Several
Chl catabolites have been isolated since then (Fig. 1). The
similarity of the structures of the red Chl catabolites (3, 4)
isolated from the green alga Chlorella protothecoides with the
colorless catabolites isolated from higher plants (5–7) suggest a
close relationship in the basic skeleton. Biologists regard members of the phylum Chlorophyta as progenitor of the higher
plant cell (9, 10). 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 (6, 11,
12). 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 (13–15). The remarkable allylic pyrroline/pyrrole rearrangement was studied in
detail (11, 16). Most recent in vivo deuterium labeling has
shown that in higher plants Chl b is degraded via Chl a (17).
* This work was supported by the Swiss National Science Foundation
(Project No. 2000-50725.97/1). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 41-26-3008785; Fax: 41-26-300-9739; E-mail: [email protected].
1
The abbreviations used are: Chl(s), chlorophyll(s); MPLC and
HPLC, medium and high pressure liquid chromatography, respectively.
This paper is available on line at http://www.jbc.org
General—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 ␮m) 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 (␦) 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, ␭max (log ⑀) in nanometers. CD spectra were
measured on a Jobin-Yvon Auto Dichograph Mark V, ⌬⑀ [l mol⫺1 cm⫺1].
Plant Matertial—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 (17).
Isolation of the Chlorophyll Catabolite—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 M potassium phosphate (KP)
buffer, pH 6.8:acetone:MeOH (1:1:1). Work-up was essentially as described previously (17). Aliquots of the aqueous phase were injected into
a Lobar® LiChroprep® RP-18 (40 – 63 ␮m) column and eluted (4 bar, 22
ml/min) with a solution of 30 volume % MeOH in 0.01 M KP 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 SepPack® 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
0.01 M 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 SepPack® 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.
8643
8644
Urobilinogenoidic Chlorophyll Catabolite
RESULTS
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-(2,3dihydroxyethyl)-4-methyl maleimide and hematinic acid imide
(cf. Ref. 17).
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 (4). The second
product, which was isolated in about 15 mg, was spectroscopi-
FIG. 1. Structures of chlorophyll a and b (1, 2) and of the
catabolites derived from the green alga C. protothecoides (3, 4)
(Ref. 5), barley (H. vulgare) (5) (Ref. 6), Liquidambar spec. (6)
(Ref. 7), C. japonicum (6) (Ref. 8), and rape (Brassica napus) (7)
(Ref. 6). Distinct chlorophyll a and b catabolites are excreted from
green algae, whereas higher plants degrade chlorophyll a and b to the
same catabolite (see Introduction).
FIG. 2. 500.13 MHz COSY-45 contour
plots of the main catabolite 8 in D2O.
The spectrum was recorded with the gradient-accelerated spectroscopy (GRASP)
technique. Left side, overview spectrum;
right side, enlarged region of the spectrum showing the correlation of the
methyl groups with the protons of the side
and the bridging chains.
cally analyzed. Mass spectrometric analysis at high resolution
showed two molecular ions at m/z 705.2530 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 ␦H 2.7 (Ha(5) and Ha(15)) and ␦H 3.6 (H2(32) and
Hb(182)) in which two and three protons overlap, respectively
(Table I). Four separated methyl groups resonance at high field
␦H 1.45 H3(21), ␦H 1.82 H3(131), ␦H 1.91 H3(171), and ␦H 1.97
H3(71). The fifth methyl group, which absorbs at ␦H 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 ␦H 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 ␦H 4.16 H(16), ␦H4.32 H(4), and ␦H 4.44 H(181),
respectively. At ␦C 35.35 a weak CH cross-peak was found in
the {1H,13C}-correlation spectrum with the H(10) at ␦H 4.74
ppm.
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
␦H 3.88 H(82). This proton couples with a frequency of 3.35 Hz
with a proton at ␦H 4.65 H(10). The {1H,13C}-correlation spectrum showed in addition a cross-peak indicating a tertiary
carbon atom at ␦C 65.44 C(82), which correlates with the H(82)
proton at ␦H 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 catabolite 5 was measured
in D2O (20).
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 heteronuclear
correllation (HETCOR) spectrum, they provided the reference
and indicated the starting points of the specific side chains and
Urobilinogenoidic Chlorophyll Catabolite
1
H and
Atom
TABLE I
C NMR signals of the major catabolite 8; connectivities through space were assigned by 1H{1H} NOE difference experiments
13
␦C (D2O)
1
2
21
3
31
175.89
129.37
7.16
154.67
28.74
2
59.40
58.48
26.99
3
4
5
8645
6
7
71
8
81
82
83
85
9
10
11
12
121
122
123
13
131
14
15
132.73
112.31
8.28
123.92
191.77
171.27
52.70
160.19
35.35
122.44
119.06
20.08
38.61
181.98
114.50
7.95
122.59
26.99
16
17
171
18
181
182
60.54
159.75
11.46
128.36
66.24
63.22
19
173.96
␦H (D2O)
NOE effects
Assignement in
formula 8 (Fig. 3)
% Enhancement
1.54 s
2.4 ddd (14.2/5.65/5.48) Ha
2.64 ddd (14.2/6.59/7.13) Hb
3.56–3.66 m
4.32 dd (4.76/4.75)
2.68–2.74 dd (15.19/4.75) Ha
3.01 dd (15.19/4.76) Hb
Hb(31)a, H2(32)b
3, 2.8
a, b
H(4), Ha(5), H3(71)
1.7, 1.6, 1.6
c, d, e
1.97 s
Hb(5)
Small
e
4.74 pseudo s
H2(121)
3.1
f
2.45–2.50 m
2.1–2.2 m
H2(122), H3(131)
Small, 0.3c
h, i
2.68–2.74 dd (15.00/4.76) Ha
2.85 dd (15.00/4.58) Hb
4.16 dd (4.58/4.76)
Ha(15), H(16)
Ha(15), Hb(15)
Small, 1.6
1.3, 0.8
m, n
l, n
1.91 s
H(181)
0.13c
o
4.44 dd (4.59/4.39)
3.34 dd (11.73/4.59) Ha
3.56–3.66 m Hb
H3(171), Ha(182), Hb(182)
Hb(182), H(181)
Small, 1, 0.9
8.9, 4
o, p, q
r, p
3.69 s
1.82 s
a
Triplet.
b
Doublet.
c
Sharp.
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 ␦H 4.74 H(10) was irradiated the signal group at ␦H
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 ␦C 191.77 and ␦C 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-trihydroxy85-methyl-81-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
FIG. 3. Proton connectivities of the main catabolite 8 through
space. Connections were assigned by homonuclear Nuclear Overhauser effect difference experiments and are indicated by arrows (see
Table I).
slight differences in chemical shifts. Two methyl groups (21)
and (171) are shifted relative ␦ 0.079 ppm to lower field and ␦
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).
8646
Urobilinogenoidic Chlorophyll Catabolite
FIG. 4. Constitutions of the chlorophyll catabolites (5 and 8) isolated in this work from yellow leaves of H. vulgare. The
configurations of the chiral centers (dots) are not determined. The putative ␣-hydroxypyrrole derivative 9 formed by oxidative deformylation of 5
can be protonated on both diastereotopic faces, giving rise to the formation of diastereomers at C(4). Note that two different nomenclature systems
are applied in parallel in this work. If all carbon atoms of the core macrocycle remain in the catabolite, then the compound is denominated as seco
derivative of the original closed macrocycle in accordance with IUPAC rule and numbering system; if the methine carbon atom is lost from the
former macrocycle, then the resulting tetrapyrrole is denominated and numbered in accordance with the IUPAC rules for the corresponding bile
pigment (18, 19).
DISCUSSION
The new catabolites isolated from yellow cotyledons of barley
are colorless, nonfluorescent, and optically active. The constitution resembles Urobilinogen IX␣, 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 ␤-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 (11, 16). 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 (11, 16). It is most likely that both diastereomers arise
from the uniformly configured 5 previously isolated from barley
leaves by a consecutive oxidative process.
Chemically, the cleavage of the formyl group of the main red
chlorophyll catabolite from C. protothecoides (3) was achieved
using H2O2/pyridine at 70 °C. Both possible C(4) diastereomers
of the corresponding biliverdine were obtained from this reaction in a yield of 48% (16) and in a relation of 1:1. A comparable
enzymatic formyl cleavage can be envisioned, for example,
through a Baeyer-Villiger reaction with subsequent hydrolysis
of the formiate ester group or through an oxidative decarboxylation mechanism. Nevertheless, the terminal protonation at
C(4) is irreversible (vide infra) under those conditions (Fig. 4).
Both diastereomers are observed in a relation of 1:0.6 as determined by HPLC. This indicates that this protonation most
likely is nonenzymatically. Instead, the relation reflects the
different rate constants of protonation of both diastereotopic
faces of the double bond C(3)⫽C(4) in 9.
That the catabolite 8 is produced in the leaf tissue of barley
and not during the work-up procedure was demonstrated by an
experiment in which the whole work-up procedure, including
cell opening and HPLC separation, was performed in the presence of heavy water (30 atom % D). 1H and 2H NMR experi-
FIG. 5. Superimposed UV-visible and CD spectra of the major
(solid line) and the minor (broken line) diastereomer of 8. UVvisible and CD spectra were measured in 0.02 M potassium phosphate
buffer, pH 6.8; c ⫽ 8 ⫻ 10⫺5 M.
ments showed that all carbon bond hydrogen atoms remained
unlabeled; exchange occurred, as expected, only at position
C(82). Moreover, a mirror experiment in which the leaves were
bleached in the presence of heavy water (80 atom % D) showed
by 2H NMR that a deuterium atom resides in position C(4) of
compound 8 (data not shown). These results demonstrate that
the catabolite is formed entirely in the plant cell and not during
the work-up procedure and further that H(4) is tautomerizationally stable under those conditions. Skeletal transformation
between the two valence tautomers of urobilin/bilirhodin was
reported to be reversible only under basic conditions (21).
From the Chlorophyte Bryopsis maxima a red, water-soluble
tetrapyrrole was isolated (22), and a portion of structural information was published (23). The proposed constitution, a
biliviolin, lacks the formyl group too and contains a phytol
group and a sugar moiety. Nevertheless, the UV-visible spectrum is in disagreement with the constitution, which remains
to be determined.
In Cercidiphyllum japonicum almost quantitative amounts
of the chls were isolated as catabolite 6 (8), whereas in this
work only about 20% of the tetrapyrrolic catabolites were recovered. As charged from a screening test of several autumnal
plants using the chromic acid degradation method, (tetra)pyrroles are not always present. This raises the questions whether
urobilinogenoids are peculiar or common intermediates in the
catabolic pathway of the chls and whether they are finally
degraded in the plant cell to nonpyrrolic, nitrogen-less
compounds.
Urobilinogenoidic Chlorophyll Catabolite
Most recently, several maleimides were isolated from senescent cotyledones of barley (24). Therefore, it appears that phototrophic organisms are capable to degrade chls to maleimides
derivatives (possibly via urobilnogenoids), a pathway that has
already been discussed by Hendry et al. (25). Maleimides still
contain the nitrogen atoms of the former Chls. Can this nitrogen be re-utilized by the plant? This adaptation would certainly
be of evolutionary advantage for plants, which grow in environments in which nitrogen is in high demand. Nevertheless,
the ultimate fate of the Chls is still a matter of speculation.
Acknowledgments—We appreciate the professional knowledge of F.
Nydegger and F. Fehr to obtain optimal mass and NMR spectra. We are
indebted to Florimond Desprez, Cappelle en Pévèle F-59242, for providing us with H. vulgare cv. Lambic.
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