Apocarotenoids

Condensation of the lactone 50 with the phosphonate 51 provided the half ester 52, which was converted into the acid chloride 53. Subsequent reduction with lithium tri-r-butoxyaluminium hydride gave the (2 , 4Z)-C7-aldehyde ester 54. 

In this part of the Chapter, the synthesis of carotenoids labelled with stable isotopes at predetermined positions is described. These labelled molecules are extremely valuable for research on the structure and function of carotenoids at the atomic level in protein complexes. 

In order to understand fully the biological processes in which protein-bound carotenoids are involved, detailed information is needed about the structure and electronic surroundings of the carotenoid in the carotenoprotein. A three-step strategy has been developed by the authors in collaboration with others to obtain information at the atomic level on intact and functionally active proteins [4]. 

The great advantage of this strategy is that resolution at the atomic level is achieved with intact and functionally active proteins. No changes in steric and electronic properties are introduced by isotopic labelling natural protein complexes also contain carotenoids with the heavy isotope at the natural abundance level (e.g. for 1.1%). 

As can be concluded from the above outline, access to carotenoids isotopically labelled with and at predetermined positions is an essential part of this strategy. Labelled carotenoids can be prepared in two ways, namely by biosynthesis or by chemical synthesis. For the preparation of specifically labelled carotenoids biosynthesis is not suitable the labelled carotenoids are obtained by growing bacteria or yeasts on media containing simple isotopically labelled precursors such as sodium acetate [19]. The position of labelling can often not be predetermined and multiple labelling often occurs. Another major disadvantage is that isotopic dilution occurs for studies of carotenoid-protein complexes, carotenoids labelled at specific positions with high isotopic enrichment (preferably 99%) are needed. 

Carotenoids occur in the leaves, shoots and roots of all higher plants (content up to 

1 % of dried plant materials). They serve as color filters for photosynthesis in the leaves of plants, giving rise to the yellow and red color of the leaves during fell because they are more slowly degraded than the green chlorophyll. Many Suits such as paprika Capsicum annuum, Solanaceae Table 8) contain various carotenoids. As colors of flowers, carotenoids play a minor role when compared with anthocyanidines and flavonoids nevertheless, they contribute to yellow and red shades in the blossoms and Suits of Rosaceae and Liliaceae. 

P-carotenoid astaxanthin (Table 8). p,p-Carotene and some other carotenoids are vitamin A active as they are degraded to vitamin A aldehyde in the human and mammal organism trans-cis isomerization of vitamin A aldehyde bound to the protein opsin in rhodopsin in the retina of the eyes is the key step of the visual process (section 4.2). As non-toxic natural compounds giving no cause for concern, P,P-carotene and some other carotenoids are used as coloring agents for foods and cosmetics, and/or as vitamin A precursors and antioxidants in medicines. 

Terpenoids formally arising from carotenoids by separation of terminal fragments are referred to as apocarotenoids The position of separation is indicated according to the numbering system of carotenoids (section 7.1). In keeping wife this, p-carotenal isolated from orange peel and egg yolk is systematically referred to as 8 - 

Correspondingly, neurosporaxanthin from the microorganisms Neurospora crassa and N. sitophila is denoted as 4 -apo-p, /-caroten-4 -oic acid. 

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Plant apocarotenoids have a wide variety of structures and functions. As expected, there is a small gene family of CCDs with different cleavage sites and somewhat promiscuous substrate selection. Some CCDs are stereo-specific, for example, 9-cis epoxycarotenoids are the substrates for NCEDs (9-cis expoxy dioxygenases) that produce the precursor of ABA biosynthesis, xanthoxin. Both linear carotenoids (lycopene) and cyclic carotenoids are substrates for cleavage at various double bonds including the central 15-15 and eccentric 5-6, 7-8, 9-10, 9 -10, and 11-12 bonds. Some CCDs cleave both linear and cyclic carotenoids and may cleave the same molecule twice, e.g., both 9-10 and 9 -10 positions. 

The aroma and red color of the spice saffron are partly due to the style-specific accumulation of carotenoid cleavage products produced by both enzymatic and thermal degradation. M. Giaccio reviewed the renewed interest in saffron as a colorant, spice, and nutraceutical. " Crocetin is a C20 apocarotenoid derived from zeaxanthin (Figure 5.3.4B). ... 

Little is known of how the biosynthetic metabolon is assembled, what mechanisms control the membrane-specific targeting, and how the conversions to apocarotenoids occur. Yet the current approach to drive import of bacterial or plant genes is to use transit sequences of a stromal protein that may limit the effectiveness of the transgene. In addition, for specific applications of controlling carotenoid composition, we need to better understand the interactions of the various enzymes,... 

Castillo, R., Fernandez, J.A., and Gomez-Gomez, L., Implications of carotenoid biosynthetic genes in apocarotenoid formation during the stigma development of Crocus sativus and its closer relatives, Plant Physiol. 139, 674, 2005. 

Walter, M.H., Fester, T., and Strack, D., Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the yellow pigment and other apocarotenoids. Plant J. 21, 571, 2000. 

Auldridge, M.E., McCarty, D.R., and Klee, H.J., Plant carotenoid cleavage oxygenases and their apocarotenoid products, Curr. Opin. Plant Biol. 9, 315, 2006. 

Moraga, A.R. et al., Glucosylation of the saffron apocarotenoid crocetin by a gluco-syltransferase isolated from Crocus sativus stigmas, Planta 219, 955, 2004. 

Mercadante, A.Z., Steck, A., and Pfander, H., Isolation and identification of new apocarotenoids from annatto (Bixa orellana L.) seeds, J. Agric. Food Chem., 45,1050, 1997. 

FIGURE 11.1 Chemical structures of carotenoid oxidation products occurring in nature apocarotenoids 10 -apolycopen-lO -oic acid (504.4), apo-lO -violaxanthal (502), diapocarotenoid rosafluin (547.2), and seco-carotenoid 3-carotenone (562). The compound number corresponds to those in Britton et al. (2004). 

CCO enzymes is frequently a regulated process with additional biochemical steps that create the biologically active apocarotenoid (Iuchi et al. 2000, Simkin et al. 2004a,b, Rodrigo et al. 2006). In the following section we will discuss the different types of CCOs that have been isolated and characterized over the last decade. 

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