Pyro‐Phyllobilins: Elusive Chlorophyll Catabolites Lacking a Critical Carboxylate Function of the Natural Chlorophylls

Abstract A β‐keto ester grouping is a characteristic of ring E of the chlorophylls (Chls). Its presence has also reinforced the original identification of nonfluorescent Chl catabolites (NCCs) as colorless, amphiphilic phyllobilins (PBs). Polar NCCs were also detected in higher plants, in which a free carboxyl group replaced the ring E ester group. Such NCCs are surprisingly resistant to loss of this carboxyl unit, and NCCs lacking the latter, that is, pyro‐NCCs (pyNCCs), have not been reported. Intrigued by the question of the natural occurrence of pyro‐phyllobilins (pyPBs), we have prepared a representative pyNCC by decarboxylation of a natural NCC. We also converted the pyNCC into its yellow oxidation product, a pyro‐YCC (pyYCC). The solution structures of pyNCC and of pyYCC, and a crystal structure of the pyYCC methyl ester (pyYCC‐Me) were obtained. pyYCC‐Me features the same remarkable H‐bonded and π‐stacked dimer structure as the corresponding natural yellow Chl catabolite (YCC) with the ring E methyl ester group. Indeed, the latter substituent has little effect on the structure, as well as on the unique self‐assembly and photoswitch behavior of yellow PBs.


Introduction
In higherp lants chlorophyll (Chl) breakdown [1,2] producesb ilintype linear tetrapyrroles,n amedp hyllobilins (PBs). [3][4][5] The PBs have as imilar structure to the ubiquitous heme-derived bilins. [6,7] However,P Bs carry ac haracteristically substituted "extra" cyclopentanone moiety,d erived from ring Eo fC hl. [3,5] Hence, PBs are congesteda ttheir "southern" meso-position, a source,p robably,o fe xtra reactivity and of unusualc onformational control, [8] as is found in highly substituted porphyrin(oid)s. [9,10] The Chl-derived methyl ester (or carboxylic acid) group at ring Eo fn aturalP Bs affects their properties and may be relevant to their possible biological functions. Indeed, in higher plants ap hyllobilin lackingt he carboxylate function at ring E, that is, ap yro-phyllobilin (pyPB), remains to be identified. [6] The naturalf ormation of PBs from Chls implies an oxidative openinga tt he "northern" (a) meso-positiono ft he macrocycle. [11,12] This key step is achieved by pheophorbide ao xygenase (PaO), which specifically degradesp heophorbide a (Pheo a) to the red Chl catabolite (RCC). [11][12][13] This red 1-formyl-19-oxobilin is the precursor of further colorless products of Chl breakdown in higher plants alongt he commonP aO/phyllobilin (PaO/PB)p athway. [4,14] Based on about 80 PB structures delineated over the years, [6] severalo ther key enzymes of the PaO/PB-path have been identified in recenty ears. [14] Al arge part of the relativelya bundant and colorless "nonfluorescent" Chl catabolites (NCCs), [15] such as the NCC 1,a nd pFCC, a "fluorescent" Chl catabolite (FCC), carry the original, Chl derived b-keto ester substituent at their ring Em oiety [6] (see Scheme1). Alternatively, colorless PBs may be functionalized there by a b-keto-carboxylica cid function. [6,16,17] Ac ytosolic methyl esterase, first identified in Arabidopsis thaliana leaves, hydrolyzes the Chl-derived methyl ester function. [18] In earlier work, in extracts of the green alga Chlamydomonas reinhardtii [19,20] pyro-pheophorbide a (pyPheo a)w as detected undera naerobic conditions, and a" decarboxymethylase" was suggested to be relevant in Chl breakdown in this alga (see Scheme2). Alternatively,t his product of an apparent loss of the entirem ethyl ester group of Pheo a mayr esult from a two-step sequence via spontaneousd ecarboxylation of the first-formed 13 2 -carboxyl-pyro-pheophorbide a. [21,22] Likewise,a red pyro-RCC (pyRCC)w as described as an adventitiousd ecarboxylation product of its endogenous b-keto-carboxylic acid precursor,s ecreted by the bleached green alga Auxenachlorella protothecoides. [23,24] By chemical means this pyRCC was converted into incompletely characterized colorless linear tetrapyrroles devoid of the carboxylic acid group. [24] We have become interested in the question of the existence of pyPBs in senescent plants, andi nt he consequence of the lack of the carboxylate functiono nt he structure and chemical reactivity of pyPBs. The polar colorless PBs with a b-keto-carboxylic acid group, such as the NCC 2,t urned out to be remarkably resistant to decarboxylation. We reporth ere 1) the partial synthesis of the pyro-type NCC (pyNCC) 3 from NCC 1, 2) oxidation of 3 to the yellow pyro-type YCC (pyYCC) 4,a nd 3) structural, spectroscopic, and photochemical properties of these novel pyPBs.

Results and Discussion
The semisynthetic pyNCC 3 was selected as af irst model pyNCC (see Scheme 3). The naturalp olar NCC 2 represents a rational substrate for the preparation of 3 by decarboxylation.
NCC 2 (named So-NCC-3 originally as it was first isolated from senescent leaves of spinach) features two free carboxylic acid functions. [6,25] In spite of the identification of the polar NCC 2 in av arietyo fp lants, [25,26] so far,a ne fficient natural supply for it has not been found. Hence, the abundant NCC 1 (a 1-formyl-3 2 -hydroxy-19-oxo-16,19-dihydro-16-epi-phyllobilane) served as precursor of 2 and as the effective startingm aterialf or the semi-synthesis of the pyNCC 3 (Scheme 3). NCC 1 wasf irst isolated from de-greened leaves of the deciduous Katsura trees (Cercidiphyllum japonicum) [27] and named Cj-NCC-1. [28] For the preparation of the NCC 2 with ac arboxylic acid group at the 8 2 -position (96 %yield), the NCC 1 was treated with porcine esterase (163Umg À1 )a t3 8 8Ci na queous phosphate buffer (pH 7.9) in the dark. Spectral analysisc onfirmed the formation of 2,which wasidentified separately with So-NCC-3. [25] At room temperature the polar NCC 2 was rather stable, even at pH 4.0. However,w ith 20 mm aqueous H 2 SO 4 at 80 8C, the decarboxylation reaction of 2 proceeded within hours. To preparep yNCC 3,adeoxygenated solution of the NCC 2 (680 mg, 1.08 mmol) in aqueous 20 mm H 2 SO 4 (310 mL) was heated under Ar at 80 8Cf or six hours.A fter workup (see the Experimental Section), about 560 mg of ar aw mixture containing about 50 %p yNCC 3 were isolated by precipitation. A 100 mg sample of the mixture of raw 3 was furtherp urified using preparative HPLC,f urnishing two pure fractionsw ithU V/ Vis absorption and CD characteristics typical of natural NCCs. pyNCC 3 was obtained as an off-white powder (33 mg, 56 mmol), as were 8mg( 14 mmol) of an isomer of 3,t entatively identified as the C16-epimer of 3.I na ddition, am inor fraction with UV/Vis absorption and HPLC properties of the pyYCC 4 was also isolated.
In senescent leaves yellow Chl catabolites( YCCs) have been observed. [29] As discovered recently, [30] homogenates of Spathiphyllum wallisii (S. wallisii)l eaves oxidizeN CC 1 to corresponding 15-hydroxy-and/or 15-methoxy-substituted NCCs. These polar NCCs undergo acid-induced elimination of H 2 Oo rM eOH at C15 and C16, respectively,p roducing YCC, identicalw ith Cj-YCC-2, first found in extracts of senescent leaves of Cercidiphyllum japonicum (C. japonicum). [29] Homogenates of S. wallisii leaves were tested here for the analogous preparative oxidation of the pyNCC 3 to the corresponding pyYCC 4 (Scheme 4). In this case, as ample of 3 (8.2 mg, 14 mmol) in 6mLo fa1:1 mixtureo fM eOH and aqueous phosphate buffer pH 5.2 was treated with freshly ground leaf material from 25 cm 2 of yellow-green S. wallisii leaves.T he slurry was stirred for 22 hours at 23 8Cu nder O 2 in the dark. Workup of the resulting reactionm ixture (see the Experimental Section) furnished 4.8 mg (8.2 mmol, 59 %y ield) of yellow pyYCC 4,i dentified by its spectral features as described below.
The UV/Vis spectrumo ft he pyYCC 4 in MeOH showed maximaa t4 27 and 310 nm (see Figure 1), very similart ot hose of YCC, [29] which suggests the identityo ft heir chromophores. Retention of configuration at the C10-position was verifiedb y the basically similar CD spectrao f4 and of the YCC in MeOH. The molecular formula of 4 (C 33     of HC15 at d = 6.21 ppm. A 1 H, 1 H-ROESY spectrum confirmed the Z configuration of the double bond C15=C16. Am ain differenceb etween the 1 HNMR spectra of pyYCC 4 and YCC was due to H 2 C8 2 ,w hich gave rise to two signals at d = 2.89 and 3.24 ppm in the spectrum of 4,c oupling with each othera nd HC10 in a 1 H, 1 H-COSY spectrum.I nt he 1 H, 13 C-HSQC spectrum of pyYCC 4 these protonsc orrelated with carbona toms C8 2 at d = 50.5 ppm and C10 at d = 32.4 ppm, whereas the corresponding carbon atoms of YCCg ave signals at d = 67.3 and 37.1 ppm, respectively. [26] Due to the lack of the substituent at C8 2 ,t he 13 C-signals of C10 and C8 2 of 4 movedt oh igherf ield, when compared with those of the spectrum of YCC. Treatment of 2.7 mg (4.6 mmol) of the pyYCC (4)w itha n excess of (benzotriazol-1-yl-oxy)-tris(dimethylamino)-phosphonium hexafluorophosphate( BOP,4mg, 9.0 mmol) and triethylamine (TEA, 5 mL) in MeOH under Ar at 23 8Cp roduced the pyYCC methyl ester (pyYCC-Me, Z4-Me,S cheme 5). Purification and crystallization from CHCl 3 /n-C 6 H 14 furnished 2.3 mg of yellow microcrystals of Z4-Me (83 %yield).
The UV/Vis absorption and CD spectra of pyYCC-Me (Z4-Me) and of pyYCC( 4)i nM eOH were very similar,i ndicating common chromophores and an R configuration at the C10 position. The derived molecular formula of Z4-Me (C 34 H 38 N 4 O 6 ) was confirmed in an ESI-mass spectrum,w ith pseudo-molecular ions [M+ +H] + and [M + Na] + at m/z:599.3 and 621.3.
The structure of the pyYCC-Me Z4-Me was first established by its NMR spectra. A5 00 MHz 1 H-NMR spectrum of pyYCC-Me at 25 8Ci n [ D 6 ]DMSO( Figure 3) revealed the signals of all its 38 hydrogen atoms. At low field five signals of af ormyl and of four pyrrole NH atoms were present, the signature of ap eripheral CH=CH 2 group and the singlet of HC15. From thorough NMR analyses ( 1 H, 1 H-COSY, 1 H, 1 H-ROESY, 1 H, 13 C-HSQC,a nd 1 H, 13 C-HMBC) the complete assignment of the 1 Ha nd 13 Cs ignals of Z4-Me was achieveda nd the Z configurationo ft he C15=C16double bond was confirmed.
Single crystalso fp yYCC-Me Z4-Me grew from CHCl 3 /hexane at ambient temperature. Z4-Me crystallized in the monoclinic space group PÀ2 1 .T he crystal structureo fZ4-Me confirmed its earlier NMR derived structure, including, the lactam functiono f ring D, as well as aZconfiguration and ah igh double-bond charactero ft he C15=C16 bond (see Figure 4). The absolute configuration at C10 of pyYCC-Me Z4-Me was assigned on the basis of the CD spectra of Z4-Me and YCC-Me, the absolute structure of which has been deduced by crystallography. [31] In the crystal,t wo molecules of pyYCC-Me (Z4-Me)f orm a hydrogen-bonded and p-stacked homodimer in a" doubledecker"a rrangement, separated by the ring B/E-section as a spacer,a sr ecently found for YCC-Me (see Figure 5). [31] In these C2-symmetric dimers, the short (C15 = C16) bonds of two Z4-Me molecules are positioned nearly on top of each other.T he ("bilirubin"-type) ring C/D moiety of Z4-Me is nearly planar, with am eand eviation of 0.056 ,s imilar to the situation in YCC-Me (0.041 ). [31] Likewise, the dihedrala ngles between the nearly coplanarr ings Ca nd Do fZ4-Me (9.18)a nd of YCC-Me (6.28)w ere similarly small. The dihedrala ngle between rings E Scheme5.Preparation of pyYCC-Me (Z4-Me)bye sterificationo fpyYCC 4 (BOP = (benzotriazol-1yl-oxy)tris(dimethylamino)phosphonium-hexafluorophosphate, TEA = triethylamine).  In Z4-Me C10 is movedb elow the plane of the other ring E carbon atoms (C8, C8 1 ,C 8 2 ,a nd C9) by 0.306 ,c ompared to 0.361 in YCC-Me.C arbon C8 2 is positioned 0.304 above the plane of the other carbon atoms of ring E( C8, C8 1 ,C 9, and C10) in Z4-Me versus 0.335 for that in YCC-Me. Hence ring E of Z4-Me is twisted aroundt he bond between the sp 3 -hybridized carbon atoms C82 and C10, buts lightly less than in YCC-Me, al ikely consequence of its methoxycarbonyl group at C8 2 .
In the hydrogen-bonded dimer structure, the ring C/D and ring C'/D' moieties are situated above each other in nearly parallel planes (with ad ihedrala ngle of 5.98 for pyYCC-Me and 1.18 for YCC-Me). The mean deviation for the more extended planes of ring C/D/A' is 0.061 for pyYCC-Me (Z4-Me), whereas that for the plane of ring C'/D'/A in YCC-Me is 0.040 .T he intermolecular distances for C15-C16' and C15'-C16 of Z4-Me are 3.97 ,t hat is, approximately 0.3 longer than that in YCC-Me. [31] Likewise, the distance betweent he two parallel planes is 3.55 in Z4-Me,t hat is, also slightly longer than in YCC-Me,w here it amounts to 3.47 . [31] Hence,i ntermodular packing in (YCC-Me) 2 ,t he dimero ft he more highly substituted YCC-Me,i sr emarkably more tight than in the dimer (Z4-Me) 2 of the pyro-YCC analogue Z4-Me.
pyYCC-Me (Z4-Me)a nd its analoguew ith the C8 2 ester group, YCC-Me, are remarkably similarly structured both in crystal (see above, Figure 5) andi ns olution. Indeed, 1 HNMR spectra of both compounds overlapt oalarge extent,w hen in the same solvent, for example, in [D 6 ]DMSO or in CDCl 3 .I n [D 6 ]DMSO pyYCC-Me (Z4-Me)d isplays NMR spectral features of amonomeric pyro-phyllobilin. In contrast, 1 HNMR data of solutions of Z4-Me in CDCl 3 ,w ere not compatible with am onomeric structure (see Figure 3). When compared with the corresponding signals in [D 6 ]DMSO, the signals of four NH groups in CDCl 3 shift to lower field with Dd in the range of d = 0.50 to 1.10 ppm, that is, Dd (HN21): d = 1.06 ppm, Dd (HN22): d = 0.5 ppm, Dd (HN23): d = 0.65 ppm, and Dd (HN24): d = 1.1 ppm. Thus, the data of Z4-Me in CDCl 3 indicate the existence of H-bonded dimers with intermolecularH -bonds between Oa nd HN functionalities, as found in the crystal. Highfield shifts of the CHO signal (Dd = À2.13 ppm) and for CH, trans-H A C18 2 and cis-H B C18 2 (of d = 0.58, 0.44, and 0.23 ppm, respectively) of the vinyl group in CDCl 3 may be ascribed to shielding-effects of the pyrrole ring Ao ro ft he conjugated ring C/D framework, respectively,o np rotons of the partner molecule. The solvente ffects in 1 HNMR spectra of Z4-Me and of YCC-Me [31] in [D 6 ]DMSO or in CDCl 3 are similar and compatible with H-bonded double-decker dimer structures in CDCl 3 solution and in the crystal. The vinyl group protons H A C18 2 and H B C18 2 correlate with HN23 or H 2 C1712 1 and H 2 C12 2 ,r espectively,i nt he 1 H, 1 H-ROESY spectra. Such correlations could hardly occur via intramodular couplings, in view of the long distance from the vinyl group at C18 to rings Ba nd Co fa monomer.L ikewise, the signalo fC HO showed correlations with all NH signals, as well as aw eakc orrelation to H A C8 2 . These NOEd ata are consistentw ith as olution dimer of Z4-Me, as seen in the crystal. In fact, as for YCC-Me, the crystal structure of pyYCC-Me (Z4-Me)h elped to rationalize the NMR spectral features of pyYCC-Me (and pyYCC) in CDCl 3 ,a nd the 1 H, 1 Hcorrelationsf it well with the dimer structure of Z4-Me.T herefore, hydrogen-bonded dimers (Z4-Me) 2 also predominate in CDCl 3 ,a sd educed earlier for the analogue YCC-Me. [31] Depending upon the solventa nd its polarity,p yYCC-Me (Z4-Me)p redominantly exists in solution as am onomer or as the hydrogen-bonded dimer (Z4-Me) 2 .U V/Vis, fluorescence and CD spectra reflected this fact consistently (see Figure 6), as did the observed dual path photochemistry of YCC-Me. [31] UV/Vis-spectra of Z4-Me were solvent dependent, and they were slightly different in MeOH and in CHCl 3 .A na bsorption "tail" is seen at low transition energies in the latter solvent (see the Supporting Information, Figure S4). Likewise, CD spectra of Z4-Me are also strongly medium-responsive, as observed earlier for YCC-Me. [31] Solutionso fZ4-Me in MeOH exhibit weak CD-effects near 340 nm. In contrast, they display as trong and "positive" Cotton-effect near 340 nm in CHCl 3 ,d iagnostic of aP -type arrangemento fr ings Ao fh ydrogen-bonded dimer (Z4-Me) 2 ,a s found in the crystal.
Fluorescences pectra of Z4-Me were also strongly solvent dependent. In polar solvents at 23 8C, the fluorescenceo fZ4-Me was weak, characteristico fadeactivation of the excited singlet state by ar apid twist and eventual Z/E-isomerization aroundacritical( C =C) bond. The fluorescencee mission in MeOH had ap ronounced maximum near 490 nm, but near 630 nm in CHCl 3 .T he former situation suggests emission from the monomeric state of Z4-Me,t he latter excimer-emission from the preformed noncovalent dimer(Z4-Me) 2 .
Irradiation of as olution of pyYCC-Me (Z4-Me)i nM eOH with the light of the fluorescent lamp causede fficient conversiont o the E isomer E4-Me reaching as teady state near 50 %c onversion. The photochemically produced E isomer E4-Me was fully characterized spectroscopically (see the Supporting Information, Figures S7-S9). In particular, Z to E isomerization is reflected by changes of the relative absorption intensitiesi nt he UVand Vis-regions of the UV/Vis-spectra of E4-Me compared to that of Z4-Me (see Figure 7), consistentw ith the earlier observations made with the corresponding E/Z-isomericv ersions of YCC.
In marked contrast to the situation in MeOH,e xposure of a solutiono fZ4-Me in CDCl 3 to the light of the fluorescent lamp at 0 8Cc aused bleachingo ft he characteristic4 20 nm absorption (see Figure 8) and resulted in clean, regio-and stereoselective conversion to the colorless octapyrrolic photodimer 5-Me (> 95 %), as derived from extensive NMRa nalysis( see Scheme 6a nd Figure 9).    The covalent C 2 -symmetric [2+ +2]-photodimer 5-Me was rather stable at 0 8Ca nd its structure could be thoroughly investigated, based on homo-andh eteronuclear NMR spectra. The two halves of the octapyrrole 5-Me are connected via acyclobutanec ore, characterizedb ym ethine resonances at d = 4.33 ppm ( 1 H) and at d = 43.5 ppm ( 13 C), as well as by a 13 Csignal of the quaternary carbon center at d = 73.2 ppm. The hydrogen-bonding contacts seen in the crystal and in CDCl 3 solution of the homodimer (Z4-Me) 2 appear to be kept in 5-Me.T he UV/Vis-spectrum of the dimer 5-Me exhibited an absorptionm aximum near 320 nm (due to its formyl-pyrrole unit), and is consistent with the interruption of the main chromophoreo fZ4-Me at the C15-mesoposition. The strong Cotton effect near 330 nm in the CD-spectrum of 5-Me (see Figure S13, Supporting Information) suggested as imilar P-type arrangement of the rings A/A' of 5-Me,a ss hown in the crystal for the structure of the H-bonded dimer (Z4-Me) 2 .
Thermolytic [2+ +2]-cycloreversion of the thermally labile covalent dimer 5-Me occurred slowly at room temperature in CDCl 3 and furnished the hydrogen-bonded dimer (Z4-Me) 2 (see Scheme6and Figure 10). In acid-free CHCl 3 the decomposition of the colorless, covalent photodimer 5-Me displayed firstorder kinetics with ah alf-life of about 18 min at 50 8Ca nd about 700 min at 23 8C. Using the Eyring equation to analyze the decomposition kinetics, enthalpy and entropyo fa ctivation were calculated as 103.2 kJ mol À1 and 11.9 JKmol À1 ,r espectively.F rom ac orresponding Arrheniusa nalysis, the activation energy of this ring-opening reaction was determined as 105.8 kJ mol À1 and ar emarkably high frequencyf actor (7.3 10 13 s) was calculated (see Figure 10 and the Supporting Information, Figures S14 and S15). Hence,o pening of the cyclobutane ring of 5-Me in CHCl 3 was more rapid by about eight times than that observed for the cycloreversion of the corresponding photodimero fYCC-Me on the basis of the half-life at 50 8C. [31] The structures of the monomeric pyro-type phylloxanthobilins E4-Me and Z4-Me (see the Supporting Information, Figure S16), as well as of the hydrogen-bonded p-stacked homodimer (Z4-Me) 2 and the covalentp hotodimer 5-Me (see Figure 11 andt he Supporting Information, Ta ble S5) were mod-eled with density functionalt heory (BP86/def2-TZVP/D3;f or details see the Experimental Section).
The optimized structureso ft he monomers E4-Me and Z4-Me were calculated (see their stereo projections in the Supporting Information, Figure S16), reproducing the highers tability of Z4-Me compared to E4-Me (DG = À18.7 kJ mol À1 ). Both monomers have-in their lowest-energy conformation in the gas phase-two intramolecular hydrogenb onds, resulting in 'tucked-in" conformations. Among the H-bonds is ac ommon one between the nitrogeno fr ing Ba nd the carbonyl oxygen atom of the methyle ster substituent in ring C. The second Hbond is formed between the terminal OH-group at the ethylsubstituent of ring Aa nd, for Z4-Me,t he NH of ring C, or,f or E4-Me,t he lactam carbonyl oxygen atom of ring D. In the case of E4-Me,t he calculated "tucked-in" H-bonded conformation helps explain qualitativelyt he noteworthy observed 1 Figure S1 for atom numbering and ring nomenclature).
Stereoprojections of calculateds tructures of (Z4-Me) 2 and 5-Me are depictedi nF igure 11.Q uantum chemical calculations confirmed the integrity of the H-bondedn etwork in 5-Me as found for the previously reported 2-Me.T he computed structural models of pyYCC-Me (Z4-Me) 2 and of 5-Me featured intramolecular (inter-and intramodular)d istances that were consistent with the observedN OEs and with the proposed network of eight hydrogen bonds that appearst obe conserved during the interconversion of (Z4-Me) 2 and 5-Me.The optimized structures of the homodimer pairs (Z4-Me) 2 and (YCC-Me) 2 ,a sw ell as the main framework of the corresponding photodimerp airs 5-Me and 2-Me are calculated to be highly similar( Supporting  Information, FiguresS17 and S18).
The cyclobutane unit of 5-Me shows remarkable differences in the calculated CÀCb ond lengths and the newly formed C15ÀC16' andC 16ÀC15' are 0.08 longer than C15ÀC16 and C16'ÀC15'.T hise ffecti sm ore pronouncedf or 5-Me than was found for 2-Me.C oncerning the p-p stacking of the a-formyl pyrrole rings A, the intermodular distances between these rings are little affectedb yt he formation of the cyclobutane ring in 5-Me anda re very similara si nt he H-bonded dimer

Conclusions
This paper introducest he pyNCC 3 as ar epresentative of the pyro-phyllobilins, so far unknown PB analogues, whichl ack the carboxylate function at their ring Em oiety.T ypical NCCs carry a b-keto carboxylate grouping at ring E, which is inherited from their Chl precursors, and whichr enders their C8 2 asymmetric and prone to solvent-assisted loss of ap roton. [5,6] Hence, natural NCCs (as well as YCCs and Chls) exist as C8 2 -epimeric pairs exhibiting significantly different relative stabilities. [28,31,32] However, as shown here, the carboxylate substituent at C8 2 has little effect on the flexible structure of NCCs and on the photoreactivity of YCCs. Thus, pyPBs are close biosimilars of the abundantn atural PBs, and it may be rewarding, indeed, to search routinely for pyNCCsi nextracts of plant materials.
Spontaneousl oss of carbon dioxide from 8 2 -carboxy-NCCs (such as 2)i sr emarkably slow. [25] This behavior of 2 and of related polar NCCs [16,17,25] contrasts with the situation with 13 2carboxy-pyro-pheophorbide a [33] and knownc orresponding RCC forms, [24] in which decarboxylation at C8 2 to pyPheo a and pyRCC, respectively (see Scheme 2), occurs easily and is activated by extensive conjugative stabilizationo ft he critical decarboxylation intermediates. Along thesel ines, the origin of remarkable Chl-derived linear tetrapyrroles that lack ac arboxylate group at their rings Ei sa lso intriguing, which represent the bioluminescent emitter in Krill (Euphausia pacifica) [34] and luciferin of dinoflagellates. [35] These bilin-typenatural products may be derived from ap yro-pheophorbide precursor or from decarboxylation of an unknown later breakdowni ntermediate.
The novel pyro-type YCC (pyYCC), as well as YCCs, the bright yellow bilin-type products of Chl breakdown in higher plants, feature the basic chromophore of the heme-breakdown product bilirubin (BR). [7,36,37] Hence, YCCs and pyYCCs could be considered PB-type analogues of the yellow bile pigmentB R. [6] Whereas ac rystal structure of BR revealed it in an intramolecular H-bonded monomericf orm with a" roof tile" shape, [37] pyYCC-Me, the naturalp hylloxanthobilin YCC and its methyl ester YCC-Me assemble into self-templated dimeric "doubledecker"s tructures, spanned by the characteristicB /E-ring moiety of the PBs. The C2-symmetric dimers constitute a "hand shake" motif, [31] which does not depend significantly on the presence of ac arboxylate function (as in YCC or YCC-Me), or its absence in pyYCC (and in pyYCC-Me),a ss hown here.
Indeed, the lack of ac arboxylate substituent at the C8 2 -position in pyYCC-Me (Z4-Me)h as only as mall effect on the whole structure, most clearly seen in the crystal, in which only slightly differing dihedral angles between the planes of the ring B/E and ring C/D subsystems are observed. Aside of this, the substituentsa tC 8 2 show negligible effects on the structure, the chemicalr eactivity and, photochemistry of both types of yellow phyllobilins.
pyYCC-Me (Z4-Me)r epresents am edium-responsive photoswitch similar to YCC (and YCC-Me). [31] In polar solution, in which YCCs and pyYCCs prefer to be monomeric,t hey display insignificant luminescencea nd undergo clean photochemical Z/E-isomerization. Hence, in such solvents, YCCs and pyYCC show photochemistryr emarkably comparable to that of the natural heme catabolite BR. [37] BR undergoes ar elated, but modestly selective, photoinduced Z to E isomerization, which is considered the basis of the blue-light therapy of neonatal jaundice. [36] However, the photochemistry of YCCs and pyYCCs is strongly medium responsive, and in apolar media, the selfassembled Z isomers YCC, YCC-Me, and pyYCC-Me( Z4-Me)u ndergo aclean and surprisingly efficient photoinduced[ 2 + +2]-cycloaddition to homodimericoctapyrroles, such as 5-Me.
The novel, "non-natural"c olorless pyNCC 3 and the yellow pyYCC 4 may be considered to represent close biosimilars of NCCs and YCCs, their known naturalr elatives. By possessing a unique ring Em oiety,a ll phyllobilins are conformationally restricted bilin-type tetrapyrroles. Hence, their overall structures and their chemical properties deviates ystematically from those of the relatedh eme-derived bilins, which play ar ange of important biological roles: [7,[37][38][39][40] For example, phytochrome with bound phytochromobilin (PFB), when excited by visible light, undergoes fast Z/E-isomerization of the C15=C16 bond. [39] This property of this natural bilin-type photoswitchi s used in higherp lants for their crucial photoregulation. [40] YCC and relatedy ellow phyllobilins also undergo efficient photoinduced Z/E-isomerization of their C15=C16 bond in polar solutions. Coloredp hyllobilins (collectively named phyllochromobilins), such as YCCs and PiCCs (pink oxidation products of YCCs), are also excellent ligands for some transition-metal ions and their metal complexes display af urther spectrum of colors and (photo)chemistry. [41] Furthermore, persistent blue fluorescent FCCs, classified as "hypermodified" FCCs (hmFCCs),o ccur in some fruit and leaves of evergreens (such as bananas), acting aso ptical brighteners in them. [42] Clearly,t he interesting antioxidant properties of some PBs, [43] their bright colors and their photochemistry [29,31] constitute ar ange of chemical qualities that may be essential for the still elusive biological roles of PBs in fruit and leaves. [6] As pyro-phyllobilins (pyPBs) appear to feature similars tructures and (photo)chemistrya st he corresponding natural PBs, they could, indeed, be seen as biosimilars with ar elated potentialf or active or inhibitory participation in physiological processes.

Synthesis of pyNCC (3) by decarboxylation of NCC 2
The polar NCC 2 (680 mg, 1.8 mmol) was dissolved in H 2 O( 116mL) under Ar with assistance of ultrasound. To the above solution, aqueous 20 mm H 2 SO 4 was added (195 mL), and the stirred solution was purged with Ar.A fter 7h at 80 8C, the reaction mixture was cooled down to room temperature and ap recipitate of raw pyNCC 3 formed. The precipitate was separated off by filtration and washed with hexane. After drying, raw 3 (558 mg) was obtained. As ample of raw 3 (100 mg) was purified further by preparative HPLC. The fractions of purified 3 were combined, diluted 1:1 with H 2 Oa nd loaded on aS ep-Pak cartridge. After washing with H 2 O( 40 mL), the product was eluted with MeOH. The solvent was removed under reduced pressure and the sample was dried under high vacuum, yielding pyNCC 3 (33 mg, 29 %y ield). The fractions of as lightly more polar NCC were also collected and worked up, furnishing an NCC (8 mg, 7% yield), characterized as epi-3 (see the following and main text).

Kinetic analysis of the thermolysis of 5-Me to Z4-Me
As tock solution (1.78 10 À4 mol L À1 )o fZ4-Me in acid-free CHCl 3 was prepared. A1mm UV/Vis cell was filled with 0.2 mL of the stock solution and purged with Ar for 1min. The solution was then irradiated at 0 8Cb yt he fluorescent lamp for 120 min, until light absorption at 420 nm was minimal, indicating full conversion to 5-Me.S ubsequently,t he solution of 5-Me was left at 23 8Ci n the darkness for certain time. Light absorption at 420 nm was employed to monitor the decomposition reaction. Twof urther experiments were performed in parallel at 40 8Ca nd at 50 8C, in order to observe the temperature dependence of the decomposition reaction of 5-Me.T he decomposition kinetics were monitored on the basis of the absorption at 420 nm.

Computational details
The experimental X-ray crystal structure of pyYCC-Me (Z4-Me) 2 was used as as tarting structure for the quantum chemical investigation. The structure was fully optimized with density functional theory when employing the BP86 [44] density functional in combination with the triple-zeta basis set def2-TZVP [45] and the resolutionof-identity technique. [46] Incorporation of empirical dispersion corrections of the D3 type by Grimme [47] were required to reproduce the experimental (Z4-Me) 2 structure and obtain the correct distance for the p-p stacking of rings Ca nd D. D3 corrections were used for all structure optimizations. All calculations were performed with the program suite Turbomole. [48] The starting structure of pyro-photodimer 5-Me was created by manual modification of (Z4-Me) 2 in MOE. [49] As no crystal data was available, various conformations were generated by Maestro'sc onformational search tool [50] und subsequently optimized with BP86/ RI/def2-TZVP/D3. The same procedure was applied to pyYCC-Me monomer Z4-Me and E4-Me,w hich were not observed as monomers in apolar solvents.
To ensure all reported structures represent energy minima on the potential energy surface, vibrational spectra were calculated with Turbomole's NumForce module at the BP86/RI/def2-TZVP/D3 level. Solvent effects are neglected because only implicit solvent corrections are feasible and they are expected to be small in apolar solvents. Zero-point energies and thermal corrections to electronic energies were obtained by Turbomole's freeh tool. Reported reaction energies are Gibb's free energies at standard conditions.
Recently published similar structures of the natural yellow phyllobilin dimer YCC-Me named (Z1-Me) 2 [31] and the corresponding photodimer,n amed 2-Me here, as well as the monomer units named Z1-Me and E1-Me,r espectively, [31] have been generated and optimized with the same quantum chemical protocol (for details see Ref. [31]).
Structures were visualized using PyMOL. [51] Overlay structures of pyYCC-Me dimer (Z4-Me) 2 and YCC-Me dimer (Z1-Me) 2 were created by alignment of rings Ca nd D( see the Supporting Information, Figure S18). To determine the angle between rings Ba nd E, Ea nd CD as well as Ba nd CD, Amber's cpptraj tool [52] was used.

X-ray crystal structure analysis
Single crystals of Z4-Me:Asolution of the crystalline Z4-Me (5 mg) was dissolved in CHCl 3 (2.5 mL). The solution was filtered through at ight plug of cotton wool and the filtrate was collected in a5mL vessel. Hexane (2.5 mL) was slowly added above CHCl 3 to form two layers. After ca. 1week, single crystals were obtained. For collection of X-ray data of as ingle crystal of Z4-Me with Mo radiation, aB ruker D8 Quest diffractometer was used controlled by the APEX2 software. Data integration and reduction were performed using the SAINT software. [53] The crystal structure was solved and refined with SHELXT [54] and SHELXL, [55] respectively.F urther details are described in the Supporting Information. CCDC 1581089 contains the supplementary crystallographic data for this paper.T hese data are provided free of charge by The Cambridge Crystallographic Data Centre.