Carotenoid 5,6, 5,8-and 3,6-epoxides

The isolation, semisynthesis and structure elucidation of carotenoid 5,6, 5,8-and 3,6-epoxides as well as their derivatives are discussed in detail. This review gives a brief summary of the plausible biosynthetic transformations of the 5,6- epoxy-β -ring. A wide variety of carotenoid end-groups can be obtained from 5,6-epoxy carotenoids. In order to get information on the enzyme activities and regulation of the biosynthetic pathway of carotenoids some of the intermediate steps of these transformations were investigated (e.g. formation of allenic, κ - and γ -end-groups as well as ring opening reactions). The ring opening of the 3-hydroxy-5,6-epoxy end group, resulting in the formation of 3,5,6-trihydroxy compounds, was studied in detail in order to obtain evidence for the proposed reaction mechanisms of the two different biosynthetic routes (acid or enzyme catalysis) which also explain the configurations at the C(5) and C(6) atoms.


Introduction
Carotenoids are among the most common natural pigments, and more than 600 different compounds have been characterized.Carotenoids are responsible for many of the red, orange, and yellow colors of plant leaves, flowers and fruits, as well as the colors of some birds, insects, fish and crustaceans.Only plants, bacteria, fungi, and algae can synthesize carotenoids, but many animals incorporate them from their diet. 1 The biological properties of carotenoids can be divided into functions, which are essential to the well-being of organisms, and actions or associations, which are either responses to the administration of carotenoids, or are phenomena which still lack evidence of causal relationship to the presence of carotenoids. 2Carotenoids have two major functions in photosynthesis: they serve as accessory pigments for light harvesting and in the prevention of photooxidative damage.Carotenoids can also have antioxidant properties (e.g. in animals), and the so-called provitamin A carotenoids are used as sources of vitamin A.
The unique structure of carotenoids determines their potential biological functions and actions.Most carotenoids can be derived from a 40 carbon basal structure including a system of conjugated double bonds.The central chain may carry cyclic end-groups that can be substituted with oxygen-containing functional groups.Based on their composition, carotenoids are divided into two classes, carotenes containing only carbon and hydrogen atoms, and oxocarotenoids (xanthophylls) carrying one or more oxygen atoms.Hydroxy groups are common, particularly in position 3 or 4.Many carotenoids possess an epoxy group, usually in positions 5,6 or 5,8 (the 5,8-epoxides are often referred to as furanoids or furanoid oxides), though some 1,2-epoxides, 3,6-epoxides and 4,5-epoxides have been reported also.Carotenoids containing aldehyde, keto, carboxy, carbomethoxy, and methoxy groups, acetates and lactones are also encountered. 3his review gives a summary concerning the carotenoid 5,6-, 5,8-and 3,6-epoxides as well as the isolation, semisynthesis and structure elucidation of the above mentioned carotenoids and their derivatives, paying special attention to the results obtained in our laboratories.

31
The 5,6-epoxides of cyclic carotenoids are readily rearranged into 5,8-epoxides in the presence of acid catalysts.The mechanism of the epoxide-furanoid rearrangement was proposed by Eugster (Scheme 1). 20he reaction leads to a shortening of the chromophore by one conjugated double bond, causing a characteristic hypsochromic shift of the UV/VIS spectrum of ca.20 nm for each 5,6epoxy group.This chemical reaction is the most useful microscale test for the fast identification of carotenoid 5,6-epoxides.

34
The isolation of cucurbitaxanthin A (35) and cucurbitaxanthin B (36) from pumpkin, 24 and of cucurbitaxanthin A (35) and capsanthin 3,6-epoxide (37) from red paprika 25 was successful at about the same time.In 1990, Eugster and Gmünder 26 published an attempt to synthesise the cycloviolaxanthin (38) containing two 5-hydroxy-3,6-epoxy groups.Unfortunately, the last reaction step resulted in a compound with heterocyclic rings, instead of cycloviolaxanthin.At the same time, cycloviolaxanthin (38) and cucurbitaxanthin B (36) could be isolated in our laboratory from red paprika in very small amounts.

Transformation of 5,6-epoxy-carotenoids
The number of naturally-occurring carotenoids whose structures have been elucidated now numbers more than 600.However, these carotenoids show a wide diversity of structural features and modifications to the basic end-groups, and virtually nothing is known about the biosynthetic reactions that lead to the formation of these structures.It has been known for a long time that the 5,6-epoxy structure can give rise to a wide variety of carotenoid end-groups and Scheme 2 summarizes the plausible biosynthetic transformations of the 5,6-epoxy-β-ring (with or without a 3-hydroxy group) to give diverse structures such as the cyclopentane (κ-ring) carotenoids capsanthin and capsorubin, the retro-carotenoids eschscholtzxanthin and rhodoxanthin, allenic carotenoids (eg.neoxanthin, fucoxanthin), 5,6-diols (eg.karpoxanthin, latoxanthin), 3,6-epoxides as in eutreptiellanone and cucurbitaxanthin, the 6-hydroxy-γ-ring structure found in prasinoxanthin and nigroxanthin, as well as the 5,6-seco-5,6-dione-end-group of semi-βcarotenone. 33Although these proposed routes are, on paper, very reasonable, in general there is no experimental evidence to support them.The intermediate steps in the carotenoid biosynthetic pathway were postulated several decades ago by standard biochemical analyses using labeled precursors, specific inhibitors and characterization of mutants.In recent years, the moleculargenetic approach to carotenogenesis study has provided a wealth of information and new perspectives of both the enzyme activities and the regulation of the pathway. 34

Formation of the allenic end-group
As shown in Scheme 3 the allenic group originates from proton abstraction from C (7), neighbouring one of the 5,6-epoxy-5,6-dihydro-β rings of violaxanthin, followed by rearrangement of the epoxy group to the 5-hydroxy-allenic end group. 35This reaction is catalysed by the enzyme neoxanthin synthase isolated and characterized in 2000. 36 HO O HO CH 3 H Scheme 3

Formation of the κ-end-group
The proposal that the κ -end group is formed as shown in Scheme 4 has received support from experiments that have demonstrated the incorporation of radioactive antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively, by chromoplasts of Capsicum annuum. 37The capsanthin-capsorubin synthase (CCS) enzyme that catalyses the conversion of 5,6-epoxy-end groups into κ-end groups was isolated and characterized in 1994. 38Certain similarities of CCS were observed with the C. annuum lycopene cyclase, the enzyme catalyzing the cyclization of lycopene.

Scheme 4
The fact that CCS also exhibits lycopene cyclase activity, is likely to be related to similarities in the chemical mechanisms leading to the formation of β-rings in β-carotene (Scheme 4a) and κrings in capsanthin and capsorubin (Scheme 4b).In both mechanisms, an intermediate carbenium ion at C(5) forms 40 and, in addition, both reactions probably are initiated by protonic attack on either a double bond or an epoxy group.

Scheme 6
The proposed mechanism also involves the formation of 3,6-epoxy-end group.The nucleophilic attack of the 3-hydroxy group to C(6) results in the 3,6-epoxy-5-hydroxy-β-end group.
In the plants which do not contain carotenoids with the κ-end group (e.g.Rosa foetida, ripe hips of Rosa pomifera), the ring opening of carotenoid-5,6-epoxides probably is acid catalysed and follows the mechanism given in Scheme 6.During the acid-catalysed hydrolysis of carotenoid-5,6-epoxides, the configuration at C(6) may change, whereas that at C(5) remains unchanged.The different biosynthetic routes may explain the differences between the configurations of carotenoids with the 3,5,6-trihydroxy-β-end group isolated from different sources.

Formation of the γ-end group
Liaaen-Jensen and co-workers isolated prasinoxanthin 51 (69) and preprasinoxanthin 52  In our laboratory, during the isolation of cycloviolaxanthin 27 from paprika, several unknown carotenoids were obtained by column chromatography.Further investigation of the chromatographic zone on the CaCO 3 column between cucurbitaxanthin A and B furnished a new carotenoid (71) for which the name nigroxanthin was proposed.Nigroxanthin was isolated and identified as (all-E)-3',4'-didehydro-β,γ-carotene-3,6'-diol. 53The configuration at C(6') at that time was unknown.Several years later, another carotenoid containing the γ-end group, namely the prenigroxanthin (72), was isolated from red spice paprika (C.a.var.longum).The constitution of this carotenoid was identified as β,γ-carotene-3,3',6'-triol.As the configuration at C(6') of 71 and 72 had not yet been clarified by modern spectroscopic methods, the biosynthetic pathway of paprika carotenoids was taken into consideration.Compounds 71 and 72 may be formed from antheraxanthin (5) and their occurrence is interrelated with the biosynthesis of the κ-end group.The probable biosynthetic route for the formation of the γ-end group is as follows: enzymatic opening of the 5,6-epoxy ring results in a carbenium ion at C(5) in compliance with Scheme 9.According to three different routes, this intermediate can be stabilized by the formation of: a) a κ-end group ; b) a 3,6-dihydroxy-γ-end group or c) a 3,6-dihydroxy-ε-end group.It was assumed that the latter end group was not stable and it could be rearranged easily into the 6-hydroxy-3,4-didehydro-ε-end group by elimination of water.In these reactions, the configuration at C(6) remains unchanged, thus, strongly supporting the proposed structures with the 6'S configuration for nigroxanthin (71) and prenigroxanthin (72).