Pheophorbids

It is interesting to note that the magnesium or zinc complexes of methyl pheophorbide a (11, M = Mg, Zn R = C02Me) or methyl pyropheophorbide a (11, M = Mg, Zn R = H) are cleaved between positions 20 and 1 by singlet oxygen, whereas in contrast nature cleaves the chlorin at the 4,5-C —C double bond.44-45a h46 The ring fission at the 4.5-C —C double bond can be achieved with the cadmium(II) complex of methyl pheophorbide (11, M = Cd R = C02Me) to produce 12.43i... 

Esterification increases the lipophilic character of the pigments that has been recogiuzed as an important factor for interactions with the peptide chains of proteins. The hydrolysis of this side chain results in chlorophyllides and the concomitant removal of the Mg + ion in pheophorbides. Only a Umited number of natural chlorophylls in plants and photosynthetic organisms has been described and is well... 

The early stages of catabolism correspond to the replacement of Mg by two H atoms under acidic conditions and/or by the action of Mg-dechelatase and the cleavage of the phytol chain by the enzyme chlorophyllase. The still greenish intermediates are pheophytins, chlorophyUides, and pheophorbides with intact tet-rapyrrole rings. - ... 

Hortensteiner, S. et ah. The key step in chlorophyll breakdown in higher plants Cleavage of pheophorbide a macrocycle by a monooxigenase, J. Biol. Chem., 273, 15335, 1998. 

Hortensteiner, S., Vicentini, F., and Matile, P., Chlorophyll breakdown in senescent cotyledons of rape, Brassica napus L. enzymatic cleavage of pheophorbide a in vitro, New Phytol, 129, 237, 1995. 

Not-senescent and fresh-cut plants are almost devoid of degradation products like pheophytins and pheophorbides because chlorophylls associated with caro-... 

FIGURE 4.1.1 Possible chlorophyll degradation pathways in plant tissues or in processed foods. Pheophorbide a monooxygenase is specific for pheophorbide a. RCC = red chlorophyll catabolite. FCC = fluorescent chlorophyll catabolite. NCC = non-fluorescent chlorophyll catabolite. 

In the past, no snitable analytical methodologies were capable of investigating these multiple reactions and even today, the complete extraction and analysis of all the componnds is still a difficult task. The methods for extraction must be optimized for each sample according to the solubility of either phytylated (chlorophylls and pheophytins) or dephytylated (chlorophyllides and pheophorbides) derivatives, often requiring several repeated steps and the use of a single or a mixture of organic solvents. 

Chlorophyll catabolism has been intensively studied in some plants, e.g., rape-seed, barley, spinach, tobacco, Cercidiphyllum japonicum, Lolium temulentum, Liq-quidambar styraciflua and Arabidopsis thaliana, which present all NCC catabolites with similar basic structures. " This suggests a uniform breakdown of chlorophyll in which the oxidative opening of pheophorbide a seems to be a key step. Structural differences among the compounds have been related to at least six basic types of peripheral transformations. Some of them seem to operate either in sequence or in parallel, depending on the plant species, which caused the appearances of different... 

However, this accumulation has not been unequivocally proven. The recent identihcation of urobilinogenoidic linear tetrapyrroles in extracts from primary leaves of barley indicated that further degradation of the v-NCC 1 can take place. While the monoxygenation of pheophorbide a in the earlier phases of chlorophyll breakdown in higher plants appears to be a remarkably stringent entry point, the rather diverse structures of NCCs may indicate that the later phases of the detoxi-hcation process follow less strictly regulated pathways." ... 

The development and reports of methods for colorless chlorophyll derivative (RCCs, FCCs, and NCCs) analysis are relatively recent and the structures of the compounds are being elucidated by deduction from their chromatographic behaviors, spectral characteristics (UV-Vis absorbance spectra), mass spectrometry, and nuclear magnetic resonance analysis. The main obstacle is that these compounds do not accumulate in appreciable quantities in situ and, moreover, there are no standards for them. The determination of the enzymatic activities of red chlorophyll catabolite reductase (RCCR) and pheophorbide a monoxygenase (PAO) also helps to monitor the appearance of colorless derivatives since they are the key enzymes responsible for the loss of green color. ... 

Usually, HPLC analysis resolves four peaks identified by co-chromatography with authentic standards as copper pheophorbide a, Cn(II) chlorin e6, Cn(II) chlorin e4, Cu rhodin g7, and their degradation products, but a sum of other colored components can also be found, for example, native chlorophylls, pheophytins, pheophor-bides, and rodochlorins (free carboxyl forms of pheophorbides) besides epimers, allomers, and degradation products that have been only tentatively identified. 

Chlorophyll-a Pheophytin-a Pyropheophitin- a Pheophorbide-a Pyropheophorobide-a Isofucoxanthin-dehydrate Fucoxanthin dehydrate Fucoxanthin-hemiketal Isofucoxanthin dehydrate pheophorbide a ester Isofucoxanthin dehydrate pheophorbide a ester Isofucoxanthin dehydrate pyropheophorbide a ester 23.5 26.4 28.1 5.0 6.9 10.7 12.0 6.4 24.4 22.9 25.4... 

Fig. 2.130. Elution profile by RP-HPLC of the chlorophyll derivative pigments analysed. The pigments were detected spectrophotometrically at 660 nm and fhiorimetrically using excitation and emission wavelengths at 440 and 660 nm, respectively. Peak identification (numbers in parentheses are retention times in min) 1 = chlorophyllide-b (3.10) 2 = chlorophyllide-a (4.98) 3 = pheophorbide-b (7.44) 4 = pheophorbide-a (8.85) 5 = chlorophyll-b (14.74) 6 = chlorophyll-a (16.40) 7 = pheophytin-b (21.49) 8 = pheophytin-a (23.38). Reprinted with permission from L. Almela et al. [301].

Fig. 2.133. HPLC chromatogram of pigment extracts from table olives cv. Gordal (a) healthy fruits and (b) altered fruits. Peaks 1 = 15-glyoxilic acid pheophorbide-b 2 = 15-glyoxilic acid pheophorbide-a 3 = Cu-15-glyoxilic acid pheophorbide-a 4 = pheophorbide-b 5 = pheophorbide-a 6 = pyropheophorbide-a 7 = 15-glyoxilic acid pheophytin-b 8 = Cu-15-glyoxilic acid pheophytin-b 9 = 15-glyoxilic acid pheophytin-a 10 = Cu-15-glyoxilic acid pheophytin-a 11 = 15 -OH-lactone-pheophytin-b 12 = 15 -OH-lactone-pheophytin-a 13 = 15-formylpheophytin-b 14 = pheophytin-b 14 = pheophytin-b 15 = 15-formylpheophytin-a 16 = pheophytin-a 16 = pheophytin-a 17 = pyropheophytin-b 18 = Cu-pheophytin-a 19 = Cu-15-formylpheophytin-a 20 = pyropheophytin-a 21 = Cu-pyropheophytin-a. Reprinted with permission from B. Ganul-Rojas el al. [304].

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