Dedicated to Professor Franz-Peter Monforts on the occasion of his 70th birthday.
Keywords antioxidants - bilin - catabolite - chlorophyll - fluorescence - glycoside - heterocycles
- pigments - porphyrinoids - phyllobilin - senescence - tetrapyrroles
1
Introduction
The appearance of the fall colors is an enchanting and puzzling phenomenon. The widespread
disappearance of chlorophyll (Chl) in autumn and the re-greening of the vegetation
in spring are probably the most visual signs of life, observable on Earth from outer
space.[1 ] Indeed, the annual apparent ‘recycling’ of the green pigment Chl is a massive biological
process involving about 1000 million tons, worldwide, and occurring by seasonally
alternating de novo Chl biosynthesis and Chl degradation.[2 ] Strikingly, until about 30 years ago Chl seemed to disappear without leaving a trace,
as non-green products of Chl breakdown remained elusive.[2 ]
[3 ] In fact, all searches for genuine Chl catabolites were futile, since they concentrated
on the detection of colored remains of the Chls. However, as is now well known, Chl
breakdown products in higher plants accumulate as colorless linear tetrapyrroles,
primarily.[1,4–7 ] All the same, the bilin-type Chl catabolites, named phyllobilins (PBs),[8 ] turn out to be remarkably related, structurally, to bilins,[9 ] the colored products from heme breakdown.[10 ]
Discovery and Structure Elucidation of a First Non-Green Chl Catabolite
2
Discovery and Structure Elucidation of a First Non-Green Chl Catabolite
Chl breakdown has for a long time been considered to play a particular role in the
recuperation of nitrogen.[3 ] First traces of non-green Chl breakdown products were traced by Matile, Thomas and
coworkers by comparing the pigment pattern of senescent leaves of wild type Festuca pratensis with those in ‘stay green’ mutants of this grass.[11 ] Remarkably, colorless compounds could be spotted in the wild type that were absent
in the green mutant, and which were presumed to represent Chl catabolites. These polar
compounds were called ‘rusty pigments’, as they rapidly converted into rust-colored
products.[12 ] We learned to isolate one such ‘rusty pigment’ without degradation and color formation,
and elucidated its chemical constitution in 1991 with the help of heteronuclear NMR
spectroscopy and soft ionization mass spectrometry.[4 ]
[13 ] It turned out to be fruitful, to solve the desired structure directly by purely
spectroscopic means,[4 ] rather than submitting this polar Chl catabolite to chemical modifications, e.g.,
in order to decrease its polarity.
Scheme 1 Structural formulae of chlorophylls a and b and of Hv -NCC-1 (1 )
The colorless ‘rusty pigment’ from de-greened leaves of barley (Hordeum vulgare ) was unambiguously revealed to be a bilane-type linear tetrapyrrole that carried
structural hallmarks of the Chls, such as their characteristic ring C/E-portion.[4 ] It was classified as a nonfluorescent Chl catabolite (NCC) and given the phenomenological
name Hv -NCC-1 (1 ).[14 ] More recently, it was classified semisystematically as a 1-formyl-19-oxo-16,19-dihydrophyllobilane,[5 ]
[8 ] see Scheme [1 ]. The structure of 1 indicated oxidative ring opening of the Chl macrocycle at the ‘northern’ meso position, between C4 and C5, thus, representing a 4,5-secophytoporphyrinoid.[4 ]
[13 ] This cleavage site contrasted with all expectations from Chl chemistry,[15 ] but turned out to be strikingly reminiscent of the site of ring opening in heme
breakdown.[16 ] However, in contrast to the typical colored bilins from heme breakdown,[9 ]
[16 ] the meso -carbon C5 of Chl a was retained in the formyl group of the NCC 1 , and its three remaining meso positions were saturated. Additional peripheral polar functionality of 1 pointed to puzzling catabolic reactions in its formation, raising further questions
to its biochemical formation and to the general relevance of its structure for the
general problem of Chl breakdown in higher plants.
Scheme 2 Structural formulae of Pheo a (6 ), p FCC (5 ), and of NCCs 2 (R = malonate), 3 (R = 1’-β-d -glucopyranose), and 4 (R = OH) from senescent leaves of oil seed rape (Brassica napus )
Structure Elucidation of Fleetingly Existent Blue-Fluorescent Chl Catabolites
3
Structure Elucidation of Fleetingly Existent Blue-Fluorescent Chl Catabolites
The repeated observation of fleetingly existent blue-fluorescent compounds alongside
of the NCCs in extracts of senescent leaves suggested their relevance in Chl breakdown.[17 ] In vitro experiments by Hörtensteiner and Matile showed the requirement for molecular
oxygen and identified the Mg-free and dephytylated Chl derivative pheophorbide a (Pheo a , 6 ) as a precursor for the presumed Chl catabolites.[18 ] From a preparation with Pheo a as the substrate by using an enzyme active extract of senescent leaves of oil seed
rape (Brassica napus ), a rather instable blue-fluorescent compound became available in small quantities,
believed to be a Chl catabolite. By spectroscopic means its structure was, again,
revealed as a linear tetrapyrrole, [19 ] closely related to 1 , and to three polar Brassica napus NCCs that had meanwhile been found in senescent cotyledons of oil seed rape[20 ] (typical UV/Vis absorption spectra are collected in the reviews[5 ]
[8 ]). The fluorescent compound was, therefore, phenomenologically named Bn -FCC-2 (5 ) and classified as a fluorescent Chl catabolite (FCC) (see Scheme [2 ]).[19 ] The chemical constitution of 5 supported its hypothetical role as a biological precursor of the Bn -NCCs 2 –4 . The molecular formula of the FCC 5 also indicated a close relationship to 6 , involving the mere formal incorporation of two oxygen atoms and four hydrogen atoms
in the course of the presumed formation of 5 from 6 . Hence, the moderately polar FCC 5 was deduced to represent a straight forward (‘primary’) product of the breakdown
of 6 , and was assigned the specific role as ‘primary’ FCC (p FCC).[19 ] As the relative configuration of the three stereocenters of p FCC (5 ) at C82 , C12, and C13 was the same as that in Pheo a (6 ), their absolute configuration was also inferred to be retained. Hence, p FCC (5 ) is a 82
R ,12S ,13S ,10Z ,16n -1-formyl-19-oxo-19,12,13,16-tetrahydrophyllobilene-b .[5 ]
[8 ]
However, in extracts of senescent leaves of sweet pepper (Capsicum annuum ) a different fluorescent Chl catabolite (Ca -FCC-2) was observed, which was identified as an isomer of p FCC (5 ). Ca -FCC-2 was deduced to display the same (relative) stereochemistry at C82 , C12, and C13, but to differ from p FCC (5 ) by the configuration at C16. Hence, Ca -FCC-2 was named epi-p FCC (
epi- 5 ), with the semisystematic name 82
R ,12S ,13S ,10Z ,16epi -1-formyl-19-oxo-12,13,16,19-tetrahydrophyllobilene-b .[5 ]
[21 ] The absolute configuration of C16 of p FCC (5 ) or of epi-p FCC (
epi- 5 ) is not established, so that it is classified provisionally as ‘normal = n ’ in 5 , or as ‘epimeric = epi ’ in
epi- 5 , and in their respective catabolic descendants.[5 ]
The structural analysis suggested the formation of ‘primary’ FCCs from Pheo a (6 ) to involve more than one enzyme and to require the existence of an intermediate,
probably less saturated at its ‘western’ meso position than 5 or
epi -5 .[19 ] In analogy to the red excretion products of Chl breakdown of the alga Auxenochlorella protothecoides , characterized structurally in Gossauer’s group,[22 ] related red linear tetrapyrroles were now taken into consideration as so far elusive
intermediates of the Chl breakdown in higher plants (see Scheme [3 ]).[19 ]
[23 ]
Scheme 3 Structural outline of key steps of Chl breakdown. Oxidative cleavage of Pheo a (6 ) by Pheo a oxygenase (PAO) generates red Chl catabolite (RCC, 7 ) in enzyme-bound form, which is reduced by RCC reductases (RCCRs) to ‘primary’ fluorescing
Chl catabolites. RCCRs-1 furnish p FCC (5 ) stereoselectively, RCCRs-2 epi-p FCC (
epi -5 ).
The Red Chl Catabolite – Key Ring-Opened Tetrapyrrole Accessed by Partial Synthesis
4
The Red Chl Catabolite – Key Ring-Opened Tetrapyrrole Accessed by Partial Synthesis
Since a red Chl catabolite was unknown in higher plants, we set out to prepare the
likely candidate by partial synthesis from the methyl ester of Pheo a (6 ), in order to help test the suggested intermediacy of such a red bilin-type Chl catabolite
in higher plants. Our synthesis of the presumed red Chl catabolite (RCC, 7 ) roughly followed a methodology, developed by Iturraspe and Gossauer, for the preparation
of the corresponding analogs from the green alga.[24 ] For this purpose, 6 was converted into the green Cd(II)-methyl-pheophorbidate 8 , which was subsequently subjected to photo-oxidation at –40 °C, furnishing the corresponding
4,5-secoporphyrinoid Cd complex 9 in about 32% yield (see Scheme [4 ]).[23 ] Reduction of the latter with sodium borohydride at room temperature and work-up
of the reaction mixture with dilute hydrochloric acid furnished the red 4,5-secoporphyrinoid
Me-7 (as a 3:1 mixture of 82 -epimers) in 72% total yield. Under these conditions, (i) the electrophilic meso position between the ‘eastern’ rings of the 4,5-secoporphyrinoid Cd complex 9 was reduced by the hydride reagent selectively, and (ii) the subsequent acid treatment
removed the Cd ion quantitatively. The main isomer Me-7 was isolated by semipreparative HPLC and shown to exhibit the natural β-configuration
at C82 , as deduced from NOE investigations.[23 ] The semisynthetic dimethyl ester Me-7
[23 ] was also identified with a dimethyl-ester isolate of a red Chl catabolite from Auxenochlorella protothecoides .[25 ] By using pig liver esterase, Me-7 was hydrolyzed highly regioselectively to the remarkably stable RCC (7 ),[23 ] the (then) elusive key red catabolite and precursor of p FCC (5 ) and epi -p FCC (
epi -5 ).
Scheme 4 Photooxydation of the Cd-pheophorbidate 8 furnishes the secoporphyrinoid Cd complex 9 , from which the RCC methyl ester Me-7 was obtained by NaBH4 reduction. Selective enzymatic hydrolysis of Me-7 furnished RCC (7 ) nearly quantitatively.
Samples of semisynthetic RCC (7 , 82
R ,12S ,13S ,10Z ,15Z -1-formyl-19-oxo-12,13,16,19-tetrahydrophyllobiladiene-b ,c )[5 ] were tested for their presumed central role in Chl breakdown in leaves. Indeed,
an extract from senescent barley leaves containing active stroma enzymes converted
7 stereoselectively into p FCC (5 ). Predominant conversion of 7 to
epi- 5 was, likewise, observed with a corresponding preparation obtained from sweet pepper.[26 ] Furthermore, traces of RCC (7 ) were now detected in an aerated incubation of Pheo a with chloroplast membranes from senescent sweet pepper cotyledons washed free of
stroma components. These in vitro experiments confirmed the relevance of two enzymatic
steps involved in the conversion of Pheo a to p FCC (5 ), with RCC (7 ) representing the original enzyme-bound intermediate with a 4,5-seco-porphyrinoid
bilin-type structure.[5 ]
[26 ] Meanwhile, the two inferred enzymes have been identified by Hörtensteiner and coworkers
as the non-heme iron-dependent enzyme Pheo a oxygenase (PAO)[27 ] and the cofactor-free ferredoxin-dependent RCC reductase (RCCR).[6 ]
[28 ] In fact, two plant-specific lines of RCCRs exist, classified as RCCR-1 and RCCR-2,
which produce either p FCC (5 ) or epi-p FCC (
epi -5 ), respectively, in a stereoselective way (see Scheme [3 ]).[29 ] The oxygen-dependent ring-opening of Pheo a , catalyzed by PAO, has been scrutinized more closely, by using isotopically labeled
O2 (18,18 O2 ). As shown by mass spectrometry, the isotopic label was only incorporated into the
formyl group of 5 (see Scheme [5 ]), indicating the key cleavage of the macro-ring of Pheo a to occur by insertion of one atom of the O2 molecule, characterizing PAO as a monooxygenase.[30 ] This enzyme and the downstream phyllobilins are the common key elements of Chl breakdown
in higher plants, hence, named the PAO/phyllobilin pathway.[5 ]
[31 ]
[32 ]
Scheme 5 Enzymatic conversion of Pheo a in the presence of 18,18 O2 by the monooxygenase PAO and by the RCC reductase of type RCCR-1 produced 18 O-p FCC.
Synthesis of ‘Primary’ Fluorescent Chl Catabolites by Reduction of Red Chl Catabolite
5
Synthesis of ‘Primary’ Fluorescent Chl Catabolites by Reduction of Red Chl Catabolite
Samples of semisynthetic red Chl catabolite (RCC, 7 ) and its methyl ester Me-7 were also used for exploring their chemical conversion into the corresponding ‘primary’
FCCs or their methyl esters. Electrochemical reduction of semisynthetic Me-7 in a deoxygenated methanolic solution at –1.3 V vs. a 0.1 N calomel electrode produced
two major FCC methyl ester fractions, besides yellow regioisomers. [33 ] The separated fractions of the two pure semisynthetic FCC methyl esters were assigned
the structures of methyl esters of p FCC (5 ) and epi-p FCC (
epi- 5 ), on the basis of NMR-spectroscopic analyses. The free catabolite RCC (7 ) was, likewise, reduced electrochemically at –1.3 V vs. 0.1 N calomel electrode in
a deoxygenated methanolic solution containing phenol, which produced roughly equal
amounts of the two ‘primary’ FCCs, 5 and
epi- 5 , as well as their yellow regioisomers,[34 ] with a chromophore structure reminiscent of functionality present in some heme-derived
bilins[9 ] (see Scheme [6 ]). Because of their tendency to isomerize in weakly acidic solution (see below),
the epimeric FCCs needed to be isolated and stored with due precautions. These electrochemical
experiments not only opened up a short preparative route to FCCs (or phyllolumobilins,
PluBs), but they also represented first model reactions for the puzzling enzymatic
conversion of an RCC (7 ) to a ‘primary’ FCC by the cofactor-free RCCRs. Indeed, our studies suggested a mode
of action of RCCRs requiring an external supply with electrons and protons, as deduced
meanwhile in a range of bilin reductases,[35 ] including RCCR from Arabidopsis thaliana .[36 ]
Scheme 6 Electrochemical reduction of the red Chl catabolite (RCC) in MeOH furnished a roughly
(1:1) mixture of the colorless PBs p FCC (5 ) and epi-p FCC (
epi- 5 ), and a yellow constitutional isomer, as a relevant side product.
Nonfluorescent Chl Catabolites from Isomerization of Fluorescent Chl Catabolites
6
Nonfluorescent Chl Catabolites from Isomerization of Fluorescent Chl Catabolites
Comparison of the core structures of FCCs and of natural nonfluorescent Chl catabolites
(NCCs or phyllobilanes), indicated them to represent pairs of constitutional isomers
and suggested the existence of an isomerization path from FCCs to the corresponding
NCCs. This hypothesis was first tested with a sample of authentic
epi -5 . Indeed, in aqueous solutions the ‘primary’ FCC
epi -5 isomerized stereoselectively to the moderately polar NCC
epi -10 , named 82
S ,10R ,16epi -1-formyl-19-oxo-16,19-dihydrophyllobilane (see Scheme [7 ]). The phyllobilane
epi -10 was identified with a natural NCC isolated in small amounts from senescent leaves
of the deciduous tree Cercidiphyllum japonicum , and named phenomenologically as Cj -NCC-2.[37 ]
The crucial stereochemical outcome of the FCC to NCC isomerization, which installs
the newly saturated C10 with R -configuration, was ascribed to the critical contribution of the propionic acid function
in protonating this prochiral meso -C of typical FCCs by an intramolecular path. This fact, as well as the observed (pH-dependent)
overall rates of isomerization of 5 and
epi- 5 at pH 3.5–7 suggested such a spontaneous chemical process to account for NCC formation
under physiological conditions in the acidic vacuoles of the plant cell.[37 ] A sequence of tautomerization and epimerization steps was proposed to account for
this reaction, which is driven thermodynamically by the isomerization of the characteristic
FCC chromophore into the more stable one of an NCC.[37 ] Interestingly, the saturated meso position C10 in natural NCCs appears to conform to a common R -configuration (as derived from their CD spectra), supporting the general relevance
of the isomerization path activated by the propionic acid function of typical natural
FCCs.[5 ] Indeed, in agreement with their tendency to convert spontaneously to their (more
stable) NCC isomers, typical natural FCCs are only fleetingly existent in senescent
leaves as intermediates of Chl breakdown.[6 ]
Scheme 7 In weakly acidic solution p FCC (5 ) and epi-p FCC (
epi- 5 ) isomerize stereoselectively to the NCCs 10 and
epi -10 , respectively.
In contrast to p FCC and epi-p FCC, their FCC methyl esters Me-5 and Me-epi -5 were persistent and required strong acid (trifluoroacetic acid), in order to promote
their rather stereounselective isomerization to NCC methyl esters. The theoretically
complete set of four stereoisomers was obtained, i.e. Me-10 and Me-epi -10 , and their C10 epimers Me-ent -10 and Me-ent -epi -10 . Thus, the methyl esters of the so far unknown type of enantiomeric NCCs (Me-ent- 10 and Me-ent -epi -10 ) was obtained, as well as the methyl ester (Me-epi -10 ) of the natural NCC named Cj -NCC-2 (see Scheme [8 ]).[34 ] As observed elsewhere, the chiroptical properties of the NCCs were dominated by
the effect of the absolute configuration at C10.[34 ]
Scheme 8 The isomerization of the synthetic methyl esters Me-5 and Me-epi -5 of p FCC (5 ) and epi-p FCC (
epi -5 ) to NCCs requires strong acid and shows insignificant stereo-selectivity.
On the basis of the list of roughly thirty structurally different natural NCCs, known
at present,[5 ] a corresponding number of natural FCCs would, hence, be expected to occur in senescent
plants as short-lived intermediates of Chl breakdown. So far only six of the rather
unstable natural FCCs have been characterized structurally that carry the critical
free propionic acid function.[5 ] However, the existence of two epimeric ‘primary’ FCCs, 5 and
epi- 5 , opens up a path to the two corresponding epimeric lines of the natural NCCs.[5 ] The stereochemical classification of a range of NCCs and of other downstream phyllobilins
(PBs) as ‘n ’ or ‘epi ’ was achieved by the identification of key NCCs, which were first screened by mass
spectrometry[38 ] and UV/Vis spectroscopy[39 ] with known NCC reference compounds (further NMR-spectral and HPLC-based comparison).
Thus, the assignment of the structure, e.g., of NCCs from spinach leaves as belonging
to the ‘epi ’-series was secured by identifying the polar So -NCC-2 (
epi -1 ) as C16-epimer of 1 (Hv -NCC-1).[13 ]
[40 ]
[41 ] For this purpose, an additional stereounselective synthesis of a pair of reference
compounds with the common 1,2-dihydroxyethyl side chain of the two NCCs was carried
out. It was achieved by regioselective OsO4 oxidation of the exocyclic vinyl group[41 ] of Cj -NCC-1 (
epi -11 , 82
S ,10R ,16epi -32 -hydroxy-1-formyl-19-oxo-16,19-dihydrophyllobilane), the ‘model’ NCC of the ‘epi -series’ (see Scheme [9 ]).[42 ]
[43 ]
Scheme 9 Stereounselective dihydroxylation of Cj -NCC-1 (
epi -11 ) with OsO4 furnished a roughly 1:1 mixture of So -NCC-2 (
epi -1 ), the C16-epimer of Hv -NCC-1 (1 ), and its uncharacterized 181 -epimer.
In the three NCCs from senescent oil seed rape leaves (the Bn -NCCs 2 –4 ) a β-keto-carboxylic acid function (from eventual hydrolysis of the original β-keto
methyl ester function of p FCC) turned out to be surprisingly stable and spontaneous decarboxylation did not
occur with any one of them.[20 ] Furthermore, from enzymatic hydrolysis of the methyl ester function of
epi -11 the semisynthetic carboxylic acid analogue
epi- 12 was obtained, also found in senescent leaves of spinach as So -NCC-3.[40 ] Decarboxylation of the β-keto-carboxylate
epi- 12 was remarkably slow and required heating with dilute sulfuric acid to give the corresponding
‘pyro’-NCC
epi -13 (see Scheme [10 ]), a 10R ,16-epi -32 -hydroxy-82 -decarbomethoxy-1-formyl-19-oxo-16,19-dihydrophyllobilane.[44 ]
Scheme 10 Preparation of py NCC (
epi -13 ) by forced decarboxylation of
epi -12
Persistent Fluorescent Chl Catabolites and Blue- Luminescent Bananas
7
Persistent Fluorescent Chl Catabolites and Blue- Luminescent Bananas
In the peels of ripening bananas a variety of strikingly persistent natural FCCs accumulates
and give the ripe banana a blue glow.[45 ]
[46 ]
[47 ] Spectroscopic analysis of such banana FCCs revealed the presence of propionate esters
as their new structural feature, as, e.g., in Ma -FCC-56 (
epi -14 ) (see Figure [1 ]).[45 ]
[47 ]
Figure 1 Structural formulae of Ma -FCC-56 (
epi -14 ), a hypermodified FCC (hm FCC), and of Vv -FCC-55 (
epi- 15 ), a bicyclo-FCC (bc FCC) with 1’β,6’-d -glucopyranosyl bridge
Indeed, this type of natural ester modification deactivates FCCs against their isomerization
to NCCs and makes such ‘hypermodified’ FCCs (hm FCCs) persistent, so that they may accumulate in ripening fruit, [45 ]
[48 ] as well as in senescent leaves of some evergreens.[49,50 ] A new basic variant of the hm FCCs was discovered recently in Vv -FCC-55 (
epi- 15 ) isolated in senescent leaves of grapevine (Vitis vinifera ). It exhibited two-fold attachment of a sugar unit furnishing a ‘bicyclo’-FCC (bc FCC) with a β-glycopyranosyl linker bridging the propionate side chain extending from
C12 and the hydroxyethyl substituent extending from C3 of the phyllobilin core.[51 ] Formation of hm FCCs and bc FCCs indicates biosynthetic investments in ripening fruit and in senescent leaves
that suggests still unknown biological roles of such PBs in the plants.[5 ]
[51 ]
[52 ]
Discovery, Structure Elucidation, and Biological Formation of Dioxobilin-Type Chl
Catabolites
8
Discovery, Structure Elucidation, and Biological Formation of Dioxobilin-Type Chl
Catabolites
The absorption of colorless PBs at around 320 nm, which is due to the characteristic
formyl-pyrrole unit of the PBs identified until about 2008,[53 ] allowed on-line detection of PBs by routine HPLC analyses of extracts of senescent
leaves,[13 ]
[20 ]
[40 ]
[41 ]
[54 ] of vegetables,[40,55 ] and ripe fruit.[56,57 ] Fluorescence at 450 nm was also used for on-line identification in more recent analyses.[45 ]
[49 ] The analytical setup was extended to detection at 250 nm, when Losey and Engel reported
on two epimeric nonfluorescent ‘urobilinogenoidic’ Chl catabolites (or DNCCs, see
below) from senescent leaves of barley that absorbed little at 320 nm.[58 ] Likewise, in extracts of senescent leaves of Norway maple, NCCs were absent and
the colorless and nonfluorescent dioxobilin-type NCC (DNCC) 16 was found instead.[59 ] Interestingly, the maple DNCC 16 was a chiroptically distinct stereoisomer (possibly the enantiomer) of one of the
earlier described ‘urobilinogenoidic’ Chl catabolites from barley leaves (see Figure
[2 ]).[58 ] This curious finding contrasted with the presumed formation of DNCCs from NCCs,[58 ] and raised the question of the biological formation of dioxobilin-type PBs.
Figure 2 Formulae of the DNCC 16 from senescent leaves of Norway maple, of the key FCC, 32 -hydroxo-p FCC (17 ), and of At -4HM-DNCC-41 (18 ), a remarkable, 4-hydoxymethylated DNCC from senescent leaves of A. thaliana
In collaboration with the Hörtensteiner lab, dioxobilin-type NCCs (DNCCs) were established
as the dominant colorless PBs in senescent leaves of A. thaliana , and the cytochrome-P450 enzyme CYP89A9 was shown to be crucial for their formation.[60 ] CYP89A9 is a new (heme-dependent) P450-type oxygenase that deformylates the ring
A α-formyl-pyrrole unit of relatively apolar FCCs oxidatively, generating fleetingly
existent dioxobilin-type FCCs (DFCCs or 12S ,13S ,10Z -1,19-dioxo-1,4,12,13,16,19-hexahydrophyllobilenes-b ).[60 ] Dioxobilin-type PBs have, hence, been classified as type-II PBs, as they are generated
from the first produced formyl-oxobilin-type PBs, or type-I PBs, in the course of
the PAO/phyllobilin pathway of Chl breakdown of higher plants.[8 ] The oxidative deformylation by CYP89A9 was first tested with the p FCC (5 ) itself and resulted in a mixture of fluorescent dioxobilin-type PBs (FDCCs).[60 ] Amazingly hydroxymethylated iso -DNCCs, such as the 4HM-DNCC 18 (see Figure [2 ]), were among the downstream dioxobilin-type products generated from p FCC (5 ) in vivo, as deduced by heteronuclear NMR spectroscopy.[61 ]
Scheme 11 The HO-p FCC (17 ) is generated from 5 by the hydroxylase TIC55, and is deformylated by CYP89A9 to the DFCC Me-19 , which is hydrolyzed enzymatically (by the methylesterase MES-16) to the fleetingly
existent DFCC 19 . The DFCC 19 isomerizes spontaneously in weakly acidic medium to DNCC 20 .
However, a preferred FCC-substrate for CYP89A9 was 32 -hydroxo-p FCC (17 ),[62 ] which is generated from 5 by the hydroxylase TIC55.[63 ] (see Scheme [11 ]). Deformylation of 17 furnished the intermediate DFCC Me-19 . The stereoselective isomerization of the more polar and hardly detectable fluorescent
type-II hydrolysis product DFCC 19 to the main nonfluorescent DNCC 20 of A. thaliana (named At -DNCC-33), was elucidated by a chemical in vitro experiment. This isomerization was
proposed to occur by the same basic mechanism,[64 ] as described for the analogous FCC isomerization to NCCs (see Scheme [11 ]).[37 ] In senescent leaves of grapevine (Vitis vinifera ) the colorless PBs are of the ‘epi -type’ and the main DNCC in such leaves is a C16-epi analogue of the DNCCs of A. thaliana .[51 ] Indeed, DNCCs functionalized at the 32 -position, such as 20 , appear to be widespread nonfluorescent type-II PBs and have been detected in recent
years in a range of senescent leaves,[51 ]
[59 ]
[60 ]
[65 ]
[66 ] in some vegetables,[55 ] and also in fruit.[67 ] DNCCs more directly derived from p FCC (5 ) by oxidative deformylation and their hydroxymethylated analogs (such as the 4HM-DNCC
18 ) are much less abundant in senescent leaves.[61 ]
[68 ]
Occurrence, Partial Synthesis, and Structure of Phyllochromobilins, the Colored Bilin-Type
Chl Catabolites
9
Occurrence, Partial Synthesis, and Structure of Phyllochromobilins, the Colored Bilin-Type
Chl Catabolites
The original classification of Hv -NCC-1 (1 ) as a ‘rusty pigment’ referred to the readily occurring transformations of NCCs to
colored compounds (typical UV/Vis absorption spectra are collected in the reviews[5 ]
[8 ]). Chemical oxidation of Cj -NCC-1 (
epi -11 ) by DDQ at low temperature and work-up at room temperature with a short treatment
with trifluoroacetic acid produced the yellow bilin-type tetrapyrrole 21Z
as main product, characterized as 82
S ,10R ,15Z -32 -hydroxy-1-formyl-19-oxo-19,N24-dihydrophyllobilene-c , a type-I phylloxanthobilin (PxB) commonly classified as a yellow Chl catabolite
(YCC).[69 ] The yellow pigment 21Z
, and its more polar 15E -isomer 21E
, have also been observed in extracts of senescent leaves of C. japonicum (and were first given the phenomenological name Cj -YCC-2 and Cj -YCC-1, respectively).[70 ] These two PxBs have a chromophore identical to the one present in bilirubin.[69 ]
[71 ] In methanolic solution they interconvert by Z /E -isomerization upon irradiation with day light (see Scheme [12 ]).[70 ]
Scheme 12 The NCC
epi -11 is oxidized by chemical oxidants or by a puzzling ‘oxidative activity’ in leaves
to 15-OH-
epi -11 , from which water is lost readily to furnish the YCC 21Z
. YCC 21Z
photoisomerizes to the YCC 21E
reversibly.
However, 21Z
and its methyl ester (Me-21Z
) exhibit a remarkably medium-responsive photochemistry, which is due to the tendency
of these two type-I 15Z -PxBs to associate to dimers (in a ‘hand shake motif’) in less polar media, e.g.,
when dissolved in CHCl3 or in membrane-mimetic micellar detergent solutions.[72 ] Photolysis of a solution of 21Z
in chloroform led to selective formation of a C
2 -symmetric, unstable [2+2]-cycloadduct that reverted to 21Z
cleanly at room temperature.[72 ] In dimethylsulfoxide solution 21Z
was monomeric and exhibited a weak emission at 493 nm. It bound Zn ions cleanly in
a 2:1 complex, which exhibited a strong red-shifted fluorescence at 538 nm, about
100 times more intense than that of 21Z
itself.[73 ]
Fortunately, the methyl ester Me-21Z
furnished single crystals, suitable for X-ray analysis. The crystal analysis confirmed
the NMR-based structure determination and showed Me-21Z
to associate into H-bonded and π-stacked dimers in the crystal.[72 ] It also allowed for the unambiguous determination of the absolute configuration
of the asymmetric carbons C82 and C10 as S and R , respectively,[72 ] confirming the tentative stereochemical assignment of carbons C82 and C10 in the prevailing epimers of natural NCCs,[43 ] which was derived earlier in the course of the studies of the acid-induced isomerization
of
epi- 5 to
epi- 10 .[37 ]
The repeated observation of YCCs in extracts of senescent leaves and ripened fruit[69 ]
[70 ]
[74 ]
[75 ] has been intriguing. Indeed, aerated aqueous or methanolic extracts of several senescent
leaves converted
epi- 11 (and its C16-epimer 11 ) cleanly to polar NCCs at ambient temperature, which were oxidized at C15 stereo-
and regioselectively. These oxidized NCCs eliminated water (or methanol) under weakly
acidic conditions to furnish the YCC 21Z
selectively and in good yield.[74 ] This finding led us to use a variety of leaf extracts (especially of the evergreen
Spatiphyllum wallisii ) to prepare YCCs from corresponding NCCs by a type of ‘green synthesis’.[44 ] The identity of the ‘oxidative activity’ in leaves is still puzzling.[74 ] However, the ‘green synthesis’ method with leaves of Sp. wallisii was also used for preparative oxidation of the semisynthetic ‘pyro ’- NCC
epi -13 to the corresponding optically active ‘pyro ’- YCC 22Z
(82
S ,10R ,15Z -32 -hydroxy-82 -decarboxymethyl-1-formyl-19-oxo-19,N24-dihydrophyllobilene-c ) (see Scheme [13 ]). The methyl ester form Me-22Z
crystallized readily and exhibited a dimer structure virtually superimposable to
that of the YCC methyl ester Me-21Z
.[44 ]
Scheme 13 Py NCC
epi- 13 is oxidized by aerated leaf extracts to py YCC 22Z
; the methyl ester Me-py YCC Me-22Z
crystallized in H-bonded and π-stacked dimers.
Along these lines, yellow PBs (phylloxanthobilins, PxBs) have been observed in extracts
of senescent leaves of grapevine[51 ] and of an A. thaliana MES16-mutant,[68 ] and were provisionally classified as type-II PxBs, proposed to represent oxidation
products of the major isomeric DNCCs in each of these leaves. The major DNCC
epi- 23 of the C16-‘epi ’ series from senescent leaves of grapevine (named Vv -DNCC-51[51 ] and the C16-‘n ’-epimer 23 of the A. thaliana mutant[62 ] were used as substrates for selective oxidation by the ‘green synthesis’ method
using leaves of Sp. wallisii. This furnished the corresponding dioxobilin-type YCCs (named DYCCVv and DYCCAt ) in roughly 70% yield (see Scheme [14 ]).[76 ] The oxidation in the aerated leaf extract occurred again at the ‘western’ meso position, with remarkable regioselectivity, as observed earlier in the corresponding
NCC-oxidation processes,[74 ] thus, desaturating the stereo-differentiating asymmetric C16. In fact, the oxidation
of both, the ‘n’ - and ‘epi’ -DNCC, apparently furnished a single DYCC 24 (82
S ,10R ,15Z -32 -hydroxy-1,19-dioxo-1,4,19,N24-tetrahydrophyllobilene-c ), suggesting the asymmetric C4 to have the same absolute configuration in both DNCCs
and their oxidation products. Hence, in the course of the oxidative deformylation
of the corresponding epimeric FCC precursors by CYP89A9, C4 would be installed with
the same absolute configuration in both DNCCs.[60 ] On the basis of NMR-spectral analysis, these type-II PxBs were assigned their structures
as 15Z -isomers.[76 ] Interestingly, the CD- and luminescence spectral properties of 24 suggest a low tendency to associate into (homo)dimers even in apolar solvents,[76 ] indicating the availability of the formyl-pyrrole unit in type-I PxBs (such as in
the YCCs 21Z
and 22Z
) to be a crucial factor in stabilizing their H-bonded and π-stacked dimers.[44 ]
[72 ]
Scheme 14 DNCCs 23 and
epi -23 are oxidized by chemical oxidants and by a puzzling ‘oxidative activity’ in leaves
(e.g.) of Sp. wallisii at the ‘western’ moiety to the apparently common DYCC 24 .
The blue-light-absorbing chromophore of YCCs is prone to further oxidation in the
presence of air to pink-colored Chl catabolites (PiCCs), or type-I phylloroseobilins
(PrBs), and PiCC 25 is a side product of the formation of the YCCs 21Z
and 21E
by low-temperature DDQ or light-induced oxidation of NCCs.[69 ]
[70 ] Traces of 25 are also found in extracts of senescent leaves of C. japonicum .[70 ] The most useful preparative method for the synthesis of 25 from 21Z
turned out to be a two-step procedure encompassing (i) complexation of the YCC 21Z
with Zn(II) ions in dimethylformamide solution,[73 ] which, upon exposure to air, led to the clean formation of the blue Zn(II) complex
26 of the PiCC 25 ,[77 ] followed (ii) by removal of the Zn ion of 26 by treatment of its solution in acetonitrile with 20 mM aqueous phosphate buffer
(pH 4.7), furnishing 25 in an overall yield of 91%.[77 ] The structure of 25 was determined as a 10Z ,15E -phyllobiladiene-b ,c (see Scheme [15 ]), on the basis of its NMR-spectral NOE data. A crystal structure analysis of 25 (as the potassium salt) confirmed this assignment of the molecular structure completely
and showed 25 to crystallize as H-bonded and π-stacked pairs of enantiomers (25 is a racemate from equilibration at its acidified asymmetric 82 -position).[77 ] Complexation of the effectively tridentate 25 by divalent transition-metal ions [such as Zn(II), Ni(II), Cu(II), Cd(II)] occurs
readily and induces the isomerization of the effective double bond C10=C11 to the
Z -configuration in the blue complexes M(II)-26 with the four divalent metal ions M(II).[77 ]
[78 ] Solutions of the blue complexes Zn-26 and Cd-26 with the closed shell ions Zn(II) and Cd(II), respectively, exhibit an intense red
luminescence, allowing for the quantitative analysis of Zn(II) and Cd(II) ions down
to the nM range[77 ] (typical UV/Vis absorption spectra are collected in the reviews[5 ]
[8 ]).
Scheme 15 YCC 21Z
slowly oxidizes with air in methanolic solution to PiCC 25 . In the presence of air and Zn(II) ions 21Z
is rapidly complexed and oxidized to the blue Zn complex Zn-26 , from which Zn ions are removed by dilute phosphoric acid, furnishing the pink PiCC
25 in high yield.
Scheme 16 DYCC 24Z
slowly oxidizes with air in methanolic solution to DPiCC 28 . Zn(II) ions rapidly complex 24Z
and furnish the oxidized blue Zn complex Zn-27 in the presence of air. Zn ions are removed from Zn-27 by dilute phosphoric acid, furnishing the pink DPiCC 28 .
In exploratory experiments, the dioxobilin-type PxB (DYCC) 24 was likewise revealed to have a pronounced tendency to oxidize in the presence of
Zn(II) ions to the blue Zn(II) complex 27 , from which the chiral and optically active type-II PrB (DPiCC) 28 was set free with dilute phosphate buffer (pH 4.7) in 92% yield (see Scheme [16 ]).[79 ] The weakly luminescent 28 bound Zn(II) ions with high affinity and furnished the red-luminescent Zn(II) complex
27 in practically quantitative yield.
10
Conclusion and Outlook
Chl breakdown has for a long time been considered to play a particular role in the
recuperation of scarce plant nutrient, such as bioavailable forms of nitrogen.[3 ] However, the earlier assumption that the plant would go after the four nitrogen
atoms of the Chl molecules has received no support from the structures of the known
PBs.[5 ] Once Chl is broken down to the colorless PBs the further fate of these products
of Chl degradation depends upon a variety of factors. In general, the colorless PBs
found in extracts of senescent leaves do not account for the amount of Chl present
in green leaves (see e.g. [65 ]) and known PBs do not accumulate for longer times in the tissue of senescent leaves
and ripened fruit.[47 ] The deduced in vivo disappearance of PBs may reflect their further degradation,
physiological use involving further metabolic transformations, or extracellular transport
processes. Typical PBs are excellent antioxidants (such as NCCs and YCCs) that are
prone to undergo oxidation reactions. Their oxidation products, like the phyllochromobilins,
may have a tendency to undergo further transformations. Indeed, the pink-colored PiCC
25 was not stable to storage in methanolic solution at ambient temperature. In exploratory
experiments, 25 underwent a retro-Dieckmann reaction opening the β-keto-ester function of ring E
to the corresponding tetrapyrrolic yellow dimethyl ester 29 (a 15Z -32 -hydroxy-1,19-dioxo-1,4,19,N24-tetrahydro-81 ,82 -secophyllo-bilene-c ) (see Scheme [17 ]), which slowly decomposed to still uncharacterized further products.[80 ] The relevance of such reactions for intracellular processes occurring in senescent
leaves remains to be established. However, the further eventual cleavage of downstream
PBs into bicyclic and monocyclic pyrrole derivatives would explain the observation
in the Shio lab of apparently Chl-derived maleimides.[81 ]
Scheme 17 The pink Chl catabolite 25 undergoes retro-Dieckmann reaction at the Chl-derived ring E moiety readily, furnishing
the yellow 81 ,82 -seco-PB 29 .
Because of the availability of the chemical structures of key bilin-type Chl catabolites[1 ]
[5 ] several of the critical enzymes directly required in the breakdown process have
been identified, establishing the common PAO/phyllobilin pathway of Chl breakdown
in the so far studied angiosperms.[6,32,82 ] The regulation of these important biochemical processes in the plants has attracted
much interest and receives broad attention, currently, in order to obtain further
insights into the plant’s way of adjusting the cellular availability of Chl to the
presence and absence of light, to biotic and biological external and internal stress,
etc.[31,32 ] Interestingly, in such studies, the occasional earlier bioinformatics-based miss-assignments
of enzymes have been corrected, such as the one of the p FCC-hydroxylase TIC55.[63 ] However, the ubiquitous enzymes active in the downstream part of the pathway, involved
in attaching sugar units once or twice by glycosylation or esterification, are still
unknown.[6 ]
[51 ]
Most recent studies in an ongoing collaboration with Thomas Müller have turned to
investigating the structures of phyllobilins in Innsbruck, that occur in de-greened
tissue of some gymnosperms, ferns, and other ‘exotic’ higher plants, such as Ginkgo biloba .[83 ] Interestingly, we have been finding structural evidence from detailed spectroscopic
work for unprecedented and intriguing deviations of Chl breakdown from the PAO/phyllobilin
pathway in these families of higher plants.[83 ] Bioinformatics methods have identified genes coding for activities related to those
of PAO in green alga,[32 ]
[84 ] and to those of PAO and RCCR in ferns and gymnosperms.[84 ] The results of the structural and spectroscopic studies will, again, invite follow-up
investigations dealing with elucidating the corresponding new biochemical processes
and their key regulatory processes.
An ever intriguing question in the field of Chl breakdown has concerned the plant
biological ‘why’.[6 ]
[85 ] Earlier views on Chl breakdown considered the degradation of the potentially phototoxic
Chl, and its removal from the plant cells, to represent a mere detoxification[14,85 ] (metabolism of Chl has been deduced to have the same purpose in some herbivores,
see e.g.[86 ]). In contrast, phyllobilins have become a topic of interest in their own right,
lately, because of their possible hypothetical physiological roles in plant cells.[5 ]
[45 ] Indeed, the chemical features of phyllobilins suggest a wide range of specific biological
roles: (i) persistent fluorescent Chl catabolites, such as hm FCCs, bc FCCs, and bc DFCCs, act as optical brighteners in fruit and leaves, making them blue fluorescent[5 ]
[45 ]
[47 ] – this may represent a signal to frugivorous animals;[45 ] (ii) FCCs are effective sensitizers for the light-induced generation of singlet
oxygen,[87 ] a relevant cellular molecular signal;[88 ] (iii) some phyllochromobilins bind transition metals very effectively, suggesting
a possible cellular role in heavy metal detoxification.[78 ] (iv) phyllochromobilins and their metal complexes are intensely colored compounds,[78 ] adding to the list of pigments in plant cells;[89 ] (v) NCCs and YCCs are very effective amphiphilic antioxidants[56 ] that extend the repertoire of plant cells in their control and neutralization of
reactive oxygen species; (vi) bicyclo-PBs (bc PBs)[51 ]
[90 ] and other ‘persistent’ hm PBs[45 ] are heterocyclic natural products with structures that may make them attractive
as anti-infective agents in plants and in other applications.[51 ] Furthermore, phyllochromobilins feature structures strikingly related to those of
some (heme-derived) natural bilins, specifically when taking into account some colored
type-II or dioxobilin-type PBs.[5 ]
[76 ] Hence, phyllochromobilins may play crucial roles as inhibitors of bilin-dependent
enzymes, e.g., in photoregulation,[91 ] or as effectors, in their own right, in such enzymes. Clearly, the discovery of
the presumed, but still elusive plant-biological roles of phyllobilins is an exciting
scientific quest, as is the possible use of phyllobilins in pharmacological applications.[5 ]
