Basic Carotenoid Chemistry

Synnove Liaaen-Jensen

Norwegian University of Science and Technology, Trondheim, Norway

I. INTRODUCTION

This chapter deals with the chemical foundation for the other chapters in this book. For more detailed treatments reference is made particularly to comprehensive recent treatments, including the Carotenoids book series published by Birkhauser, namely Vol. 1A, Isolation and Analysis (1); Vol. 1B, Spectroscopy (2); Vol. 2, Synthesis (3); and Vol. 3, Biosynthesis and Metabolism (4). Other useful references are the Key to Carotenoids (5) and the Carotenoid Handbook (6), giving structures and references to all known naturally occurring carotenoids. Whereas the former Key (5) is exhaustive, the new Carotenoid Handbook (6) gives a critical evaluation of published data and selected, recommended references to diagnostic evidence for established structures.

In this chapter selected carotenoid chemistry is treated that is relevant to the other topics covered in this book. Examples are chosen from carotenoids present in sources of importance in nutrition and health. Such sources include fruits, vegetables, algae, chicken, eggs, and fish. Furthermore, relevant to humans are the carotenoids in serum and retina.

II. CAROTENOIDS: GENERAL STRUCTURE A. Structure and Properties

Carotenoids are usually yellow-red isoprenoid polyene pigments widely distributed in nature. The majority are tetraterpenes formally composed of eight isoprene units, typically b-carotene (1). Also known are so-called higher carotenoids with C45 and C50 (e.g., bacterioruberin, 2) carbon skeletons. Carotenoids with fewer than 40 C atoms are classified as C30 carotenoids or diapocarotenoids (actually triterpenes such as 3), as apocarotenoids that are formally in-chain oxidized carotenoids, or as norcarotenoids where carbon atoms are formally removed from the skeleton. Hydrocarbons are referred to as carotenes and oxygenated derivatives as carotenoids. Elements other than carbon, hydrogen, and oxygen are not directly attached to the carbon skeleton in naturally occurring carotenoids.

The most characteristic structural feature of a carotenoid is the conjugated polyene chain. The polyene chain represents a chromophore responsible for the characteristic colors, going from colorless (phytoene, 4), to yellow (4,4;-diaponeurosporene, 3), orange (b-carotene, 1), red (paprika pigment capsanthin, 5), pink (bacterioruberin, 2) and blue with an increasing number of conjugated double bonds. When associated with proteins as carotenoproteins a dark blue color is obtained, as, for example, crustacyanin in lobster shells (7,8). Unstable reaction intermediates such as carotenoid radical ions, monocations and dications (6) absorb light in the near-infrared (NIR) region (800-1000 nm) (9). The polyene chain is also responsible for the general instability of carotenoids towards air oxidation, strong acids, oxidizing reagents, heat and light, necessitating particular precautions during isolation processes. Work with carotenoids must therefore take place under subdued light in an inert atmos-phere (N2) in the absence of strong acids and peroxides.

p-Carotene (1)

p-Carotene (1)

Carotene Functional Groups
4,4'-Diaponeurosporene (3)

Phytoene (4)

Acylated Cardiac Glycosides
|3-Carotene dication (6)

Furthermore the polyene chain is the basis for cis-trans isomerism, a very characteristic phenomenon in the carotenoid field, discussed in Part V.

Carotenoids may also contain acetylenic bonds and allenic bonds. Other functional groups in carotenoids are oxygen functions such as hydroxyl, methoxy, cyclic ethers, keto, aldehyde, carboxylic acids, lactones, acyl esters, glycosides, glycosyl esters, and sulfates. The reader is referred to the useful Key to Carotenoids (5) and the updated Carotenoid Handbook (6) for complete surveys. An example of a more complex structure is pyrrhoxanthin (7 ex dinoflagellates). It is a C38 norcarotenoid containing an acetylenic bond, an acyl ester (acetate), a butenolide-type lactone, cyclic ether (epoxide), and a secondary hydroxyl group.

Pyrrhoxanthin (7)

Pyrrhoxanthin (7)

Carotenoids that are important in health and nutrition have in general rather simple structures. Discussed in this chapter is particularly b-carotene (1), zeaxanthin, lutein, astaxanthin, lycopene, prolycopene, and neoxanthin.

It is the functional groups that are mainly responsible for the degree of polarity of the various carotenoids, as well as for their solubilities and chemical behavior.

B. Nomenclature

The approved numbering of the carotenoid skeleton is included for b-carotene (1) below. Most carotenoids have been assigned short trivial names useful in oral communication. In addition, IUPAC/IUB has published a semirational nomenclature system (10), defining the chemical structure in an unambiguous way, including the stereochemistry (three-dimensional structure) as treated below. In this system, b-carotene (trivial name) is b,b-carotene (semirational), thus denoting the two b end groups.

Examples of more complex rational names are fucoxanthin (8) present in Japanese algal food, and citranaxanthin (9) obtained from citrus fruits. Fucoxanthin (8) has several functional groups, including sec. and tert. hydroxyl, epoxy, keto, allene, and is a natural acetate. The R/S designation denotes the chirality (absolute stereochemistry) (see below). Citranaxanthin is a C30 apocarotenoid with an abbreviated carbon skeleton.

Iupac Beta Carotene

It has been recommended that the semirational name should be given at least once in all carotenoid papers. Unfortunately, this has not been implemented in a consistent manner.

C. Occurrence

Carotenoids are synthesized de novo by all photosynthetic organisms, including phytoplankton, algae, higher plants, and phototrophic bacteria. In addition, they are produced by some other bacteria, yeasts, and fungi. Carotenoids are selectively absorbed in the various food chains, where they may undergo metabolic structural changes. For further information the reader is referred to chapters on occurrence (5,11,12) and on chemosystematics (4,11,12) elsewhere. Sources of carotenoids are also treated in Chapter 11 of this book.

III. ANALYSIS

In short the quantitative analysis of carotenoid mixtures involves suitable handling of the biological material; solvent extraction; optional saponification for removal of chlorophylls, fats, and so forth; chromatography on columns (large samples); thin-layer chromatography (TLC) and high-performance liquid chro-matography (HPLC), taking the general precautions for work with carotenoids (1,2). This requires manipulations in an inert atmosphere or at reduced pressure, in subdued light, at the lowest possible temperature and in the absence of acids and of peroxides in the solvents. Quantification is based on known or approximate extinction coefficients for visible light absorption (1). Because exact extinction coefficients are frequently not known, e.g., for unknown components, for cis isomers (see Sec. IV), etc., caution should be made in quoting too exact results. The author discourages stating relative percentage composition with decimals.

General comprehensive treatments on isolation and analysis are available (1,2). Analysis of carotenoids in humans (Chapter 4) and HPLC analysis of human carotenoids (Chapter 5) are treated in detail in this book.

IV. SPECTROSCOPY

For comprehensive treatment of spectroscopic methods employed in studies of carotenoids, the reader is referred to a recent monograph (2). In the following the key information on carotenoid structure obtained by each of the most commonly used spectroscopic methods is considered.

A. Absorption Spectra in Visible Light

Electronic absorption spectra in the visible (VIS) region are indispensible for work on carotenoids. Carotenoids have strong light absorption (a high extinction coefficient) in visible light, and the VIS spectrum is used for quantitative determination of the carotenoid content in solutions. Exact extinction coefficients are available for most all-trans carotenoids. Extinction coefficients for cis isomers are generally lower and not readily available. Consequently, the content of cis isomers is frequently underestimated by using the extinction coefficient of the all-trans isomer. For unknown mixtures an approximate A1%%m = 2500 in hexane or acetone may be used.

The exact position of the absorption maxima and the spectral profile of the VIS spectrum provide most useful information. The absorption profiles are included for each structurally identified naturally occurring carotenoid in the new Carotenoid Handbook (6) and in a survey of algal carotenoids (13). The spectral fine structure may be expressed as %III/II (Fig. 1).

The length and type of chromophore and any conjugated carbonyl functions are reflected by the VIS spectrum. In entirely aliphatic systems (polyene chain not extending into terminal rings) the main middle Amax increases in wavelength in hexane solvent from 397 nm (heptaene chromophore), 424 nm (octaene), 439 nm (nonaene), 454 nm (decaene), 472 nm (undecaene), 481 nm (dodecaene), to 493 nm (tridecaene) with the highest spectral fine structure for the nonaene system with nine conjugated double bonds.

Chromophore Beta Carotene Spectrum

Figure 1 Explanation of terms used in expressions for describing the shape of the absorption spectrum in visible light (14).

Figure 1 Explanation of terms used in expressions for describing the shape of the absorption spectrum in visible light (14).

Conjugated double bonds in the terminal rings contribute less to the bathochromic shift (to longer wavelength), and the spectral fine structure is reduced due to steric conflict between the ring and the polyene system. Conjugated carbonyl groups usually result in considerable reduction of spectral fine structure, particularly in a solvent such as methanol.

The presence of cis double bonds (see Sec. V) generally results in hypsochromic shifts (to lower wavelength), roughly by about 4 nm for one cis bond and more for two cis bonds. This change is accompanied by the appearance of a so-called cis peak at a position about 142 nm hypsochromically displaced from the longest wavelength maximum of the all-irons isomer in hexane solvent. For aliphatic systems the cis peak is double. The intensity of the cis peak may be expressed as %DB/DII (Fig. 1). The relative intensity of the cis peak increases toward the center of the carotenoid molecule in this manner cis-15 > cis-13 > cis-9.

During isolation work with carotenoids frequent recording of VIS spectra is recommended to monitor the stability.

B. Mass Spectrometry

Mass spectrometry (MS) is essential for the identification of carotenoids, requiring only microgram quantities. Of the various methods now available, electron impact remains the most common ionization method (2). By high-precision measurements the exact elemental composition is obtained. By common low-resolution measurements the molecular ion gives the molecular weight without decimal figures. The identification of the molecular ion should be supported by reasonable fragment ions such as M-92 and M-106 from the polyene chain and M-H2O for carotenols. Detailed consideration of the fragmentation pattern of pure carotenoids provides useful structural information (2).

C. Nuclear Magnetic Resonance

Proton magnetic resonance (1H NMR) may, in the hands of experts, serve as the single spectroscopic method for complete structural determination of a pure carotenoid, including relative stereochemistry. This requires the application of suitable two-dimensional methods now available, such as WH COSY, 2D ROESY, and HMBC techniques. In combination with 13C NMR additional information is obtained. In modern structural studies of carotenoids, complete 1H NMR and 13C NMR assignments serve to prove the structure unequivocally. The position of cis bonds in carotenoids is readily established by 1H NMR spectroscopy (2).

The structural result of a modern NMR analysis of a complex carotenoid, namely, the microalgal carotenoid pyrrhoxanthin (7), referred to in Sec. II as an

Beta Carotene Structure
Figure 2 Structural assignment of pyrrhoxanthin (7) based on 1H NMR chemical shifts (top) and coupling constants, 13C NMR chemical shifts (middle), and 2D ROESY data (bottom) showing in-space interactions (15).

example of a carotenoid with complex structure, is illustrated in Fig. 2 (15). Further treatment is beyond the scope of this book.

D. Infrared Spectra

Infrared (IR) spectra, less commonly used nowadays, serve in structural determinations to identify functional groups, particularly different types of carbonyl functions, allenes, acetylenic bonds, and so forth, including functionalities such as sulfate that are not directly evident in NMR spectra.

E. Circular Dichroism

NMR spectroscopy serves to define relative configuration only. Absolute configuration (chirality; see Sec. VI) may in principle only be determined by direct comparison with a synthetic carotenoid of known stereochemistry or by X-ray analysis. Only some simple carotenoids have been successfully analyzed by X-ray spectroscopy due to problems in obtaining suitable crystals. However, certain key carotenoids have been degraded, and defined degradation products containing the chiral centers have been successfully studied by X-ray analysis and chiralities assigned.

Circular dichroism (CD) serves as a useful method for stereochemical correlation of carotenoids of known chirality with carotenoids under investigation. For a detailed treatment one can refer to a recent overview (2). The structural requirement for obtaining a CD spectrum is treated in Sec. VI. CD spectra can be obtained on microgram quantities of chromatographically pure carotenoids. Unfortunately, CD instruments are less available than the other spectrometers mentioned here.

It should be noted that for certain carotenoids exhibiting so-called conservative CD spectra (with several positive and negative maxima of high intensity, integrating to about zero), the presence of a cis bond results in mirror image CD. Hence, the presence of contaminating cis isomers causes problems. The intensity of the CD spectra is temperature dependent. Weak CD at room temperature is enhanced at a lower temperature (— 180oC).

With reference to Sec. VI, enantiomeric carotenoids (with mirror image structures) exhibit mirror image CD spectra. Not all chiral centers influence the CD spectrum to the same extent. For instance, the absolute configuration of the 3'-hydroxy group of lutein (10) is not reflected in its CD spectrum, which is determined by the chiralities at C-3 and C-6'.

Lutein (10)

Lutein (10)

An additivy hypothesis, whereby the Cotton effect (CD contribution) of two chiral end groups in carotenoids of the same chromophore is additive (2,16), has been useful.

An example of conservative CD spectra is reproduced in Fig. 3, displaying (3S,30S)-astaxanthin (11) and its enantiomer (12). The negligible CD of the in

Spectra Chiral Molecules

Figure 3 CD spectra in dichloromethane of (3S,30S)-astaxanthin (11,-----), its enantiomer (3R,30R)-astaxanthin (12,-), and the (3R,30S) meso form (13, ) (17).

Figure 3 CD spectra in dichloromethane of (3S,30S)-astaxanthin (11,-----), its enantiomer (3R,30R)-astaxanthin (12,-), and the (3R,30S) meso form (13, ) (17).

principle optically inactive meso (13) form is also illustrated (17). For a closer treatment of chirality, see Sec. VI.

V. CIS-TRANS ISOMERISM A. General Basis

Carbon-carbon double bonds located in cyclic parts of a carotenoid structure are in a sterically restricted position due to the ring system, e.g., the C-5,6 double bond in b-carotene (1) below. In principle each carbon-carbon double bond in the polyene chain of carotenoids may exhibit cis or trans configuration, as illustrated for lycopene (14) below. Some double bonds are called sterically hindered because the cis configuration leads to a sterically hindered configuration. While still referring to b-carotene (1), this is the case for the C-7,8, and the C-11,12 double bonds. This means that cis bonds are in practice formed under suitable conditions at C-9,10, C-13,14, the central C-15,15', and the C-13',14' and C-9',10' positions. Di-cis and poly-cis configurations are also possible but are energetically less favorable.

p-Carotene (1)

p-Carotene (1)

cis-Carotenoids differ from the parent all-trans isomer in VIS spectra, including the so-called cis peak (see Sec. IV), in adsorption affinity (HPLC, TLC), melting points, and solubility properties. Cis double bonds may also influence drastically the CD spectra of chiral carotenoids as mentioned in Sec. IV.

Assignment of cis configuration is based on VIS and NMR spectra or VIS/HPLC comparison with isomers obtained by total synthesis.

B. Nomenclature

According to the IUPAC nomenclature for carotenoids (10), the cis-trans convention is still used to denote the configuration of the polyene chain. Cis bonds have the largest substituents at each end of the double bond on the same side of the double bond. For a trans bond the largest substituents are on the opposite side. The more recent E/Z designation, based on the priority rules referred to for R/S absolute configuration in Sec. VI, may also be used and is unambiguous. In most cases cis-carotenoids have Z-configuration and trans -carotenoids E configuration. However, for in-chain substituted carotenoids, including pyrrhoxanthin (7), cis bonds represent the E-configuration.

C. Formation and Stability of cis Isomers

The generalization that all-trans carotenoids are usually the naturally occurring stereoisomer and the thermodynamically most stable one still holds, but with an increasing number of exceptions, e.g., the Dunaliella case discussed below. Bacteria living at extreme conditions frequently produce many cis isomers besides the all-trans carotenoid (18,19).

The trans-cis isomerization in solution is a facile process promoted by light, heat, and various catalysts. The common procedure is iodine-catalyzed stereoisomerization in light. Recently the application of diphenyldiselenide as an alternative catalyst has been explored for photochemical isomerization (20). This catalyst will at suitable conditions also isomerize allenic bonds (21), not further discussed here.

Detailed procedures for controlled trans-cis isomerization in the presence of iodine or diphenyldiselenide in appropriate solvents are available (20,21). The analysis is based on an HPLC instrument equipped with a diode array detector, allowing simultaneous recording of VIS spectra employing suitable columns. Irrespective of the starting isomer the same qualitative and quantitative equilibrium is reached. This is also the basis for reversibility tests, whereby an isomer is converted to the same equilibrium mixture, thereby confirming the parent all-trans carotenoid.

Iodine-catalyzed stereoisomerization leads to what was considered as a quasi-equilibrium defined by the conditions employed. However, since the application of diphenyldiselenide results in the same equilibrium, this is now considered to reflect the thermodynamic equilibrium (21).

In most cases, including common dicyclic carotenoids, the all-trans isomer is the dominating isomer in the thermodynamic equilibrium with lesser amounts of the cis isomers. This is, however, not the case for aliphatic carotenoids with very long polyene chains, for acetylenic carotenoids, and for so-called cross-conjugated carotenoids with in-chain aldehyde functions. Isomers with sterically hindered cis bonds, including prolycopene (15), are rare in nature, and are not encountered in this equilibrium mixture. Examples of such natural cis isomers, given below, include prolycopene (15) from a particular tomato variety, 9,9'-di-cis-alloxanthin (manixanthin, 16) from old cultures of diatoms, and rhodopinal (17) from phototrophic purple bacteria.

Alloxanthin
Manixanthin (16, 9,9'-dic/'s-alloxanthin)
Cisisomers

Since cis isomerization is such a facile process, cis isomerization inevitably occurs during the isolation and complicates the analysis of carotenoids. The cis isomers are in fact the most common isolation artifact, as discussed below in Sec. VIII.

D. b-Carotene

A detailed example for the analysis of cis-trans mixtures of b-carotene (1) can be found in (1). Whereas all-trans b-carotene (1) is the single geometrical isomer in most sources, the Dunaliella bardawil (salina) case deserves particular attention. In this green alga b-carotene (1) is produced in very large quantities as a so-called secondary carotenoid under extreme growth conditions (strong light and nitrogen deficiency). In this case the 9-cis isomer is the major isomer in addition to the all-trans isomer. The bent 9-cis isomer crystallizes less readily and is more soluble, which may offer advantages. The provitamin A activity is considered to be lower for the 9-cis than for the all-E isomer. For further comments, see Sec. VIII below.

Cis Beta Karoten

E. Lycopene

Lycopene (14) is a carotene with much current concern as to health aspects and prostate cancer (22). It is an entirely aliphatic carotene with a long conjugated undecaene (eleven) double-bond system, resulting in steric lability. The sterically unhindered mono-cis isomers (14b-e) are depicted below.

Cis Beta Carotin

The 5-cis isomer was overlooked until recently because of a difficult chromatographic separation from the all-trans isomer. Moreover, the all-trans and 5-cis isomers have identical VIS spectra and the cis-isomer with its terminal cis double bond has no characteristic cis peak.

However, the 5-cis isomer may now be successfully separated by HPLC analysis (23) and it now appears that when isomerized lycopene is present, the 5-cis isomer is always a dominating isomer besides the all-trans, e.g., in human blood (24).

In elegant synthetic work as many as 14 different cis isomers of lycopene (14) have been prepared by stereoselective total synthesis or isomeri-

zation and fully characterized by VIS spectra, HPLC, and NMR (23). This includes all sterically unhindered mono-cis isomers (14b-e) and the sterically hindered 7-cis isomer as well as several di-cis and tri-cis isomers and prolycopene (15). Prolycopene (7-cis, 9-cis, 7'-cis, 9'-cis) is naturally occurring in a particular tomato variety and has four cis bonds, two of which are sterically hindered.

An example of the state of the art is shown in an HPLC diagram (Fig. 4) of a lycopene mixture obtained by isomerizing all-trans lycopene (14) in refluxing heptane in the absence of catalyst and after filtration (24). The mixture consequently does not represent the thermodynamic equilibrium. In the particular

Bipolar Trans Carotenoids
Figure 4 HPLC separation of a cis-trans mixture of lycopene on a Nucleosil 300-5 column developed with hexane containing 0.05% ethyldiisopropylamine (23). For peak identification, see text.

system developed for this HPLC separation the dominant mono-cis isomers [peak 2 = 5-cis (14b), peak 10 = 9-cis (14c), peak 11 = 13-cis (14d)] were well separated and exhibit shorter retention times than most of the di-cis isomers (peak 5 = 5,5'-di-cis, peak 8 = 9,9'-di-cis, peak 13 = 9,13'-di-cis). Peak 1 represents the all-irons (14) isomer.

VI. CHIRALITY A. Definitions

We shall here define the terms chiral, achiral, chiral center, chiral carbon atom, chiral axis, enantiomer, meso form, diastereomer, epimer, racemization, and the requirements for optical activity and CD.

A chiral compound has a nonidentical mirror image. Thus, a sphere is not chiral because it is identical to its mirror image. It is achiral. The most popular example of a chiral subject is a hand. The right hand is the mirror image of the left hand. They are clearly not identical, as proved by trying to put a right-hand glove on the left hand.

The majority of the known carotenoids are chiral. The chirality is due to the presence of chiral centers, in most cases one or more chiral carbon atoms; for chiral axis, see below. A chiral carbon has four different substituents, e.g., H, OH, CH, CH2.

The nonidentical mirror form is called an enantiomer. Enantiomers have identical physical properties, except for optical properties. Enantiomers can therefore not be separated by common chromatographic methods. A chiral chromatographic system (expensive columns) is required. Typical examples of enantiomers are the two astaxathins 11 and 12, where the two chiral centers are both reverted (see Sec. IV.E). If only one chiral center is reverted in astaxanthin we have a special case, called a meso form (13). Since the two chiral centers in the the meso form are opposite we have a so-called internal compensation. This results in no optical activity of the meso form 13 in contrast to 11 and 12 which have opposite optical activities. Lack of optical activity also results for a 1:1 mixture of enantiomers. A process whereby a chiral center is converted to the opposite configuration (chirality) is called racemization.

Diastereomers contain two or more chiral centers. They differ in configuration at one or more, but not all chiral centers. Diastereomers have different physical properties and may in principle be separated upon chromatography.

Epimers represent a special case of diastereomers. In epimers only one chiral center has an opposite configuration. Typical examples of epimers are lutein (10) and epilutein (18) with opposite configuration at C-3' only.

Allenes (with two adjacent carbon-carbon double bonds connected by the same carbon), provided they are substituted in an appropriate way (two different

Optical Activity Allenes

substituents at the two ends of the system), also represent a chiral center, actually a chiral axis. An example is neoxanthin (19 and 20) with different chirality for the allene in these two structures (hydrogen pointing up or down at the allene). Naturally occurring neoxanthin has structure 19.

Allene Structures

Optical activity can be measured by simple optical rotation, by optical rotatory dispersion (ORD) or by CD. For application of the CD method a substrate with a chromophore (unsaturated system) close to a chiral center is required. Carotenoids, containing a long polyene chain, lend themselves particularly well for this purpose; for CD, see Sec. IV E. Enantiomers have opposite CD curves, (Fig. 3), whereas diastereomers have less predictable CD spectra.

B. Nomenclature

Chirality is denoted by the symbols R and S according to the Cahn-Ingold-Prelog convention, involving priority rules for the four substituents at a chiral carbon atom, and looking at the direction you move when keeping the lowest priority substituent (usually H) in the back and going from the substituent with the highest to the next lowest priority. If it is clockwise you have R configuration, whereas an anticlockwise direction is denoted by S. Rules are defined for assigning R or S configuration to a chiral allene (25). R/S designation has already been given on carotenoid structures in Secs. IV and VI.

C. Examples

In the following are selected some particular carotenoid examples where chirality is important in a biological context.

1. Astaxanthin

Let us consider astaxanthin. Astaxanthin is present in all salmonid fishes, in various marine animals, as well as in some flowers. Astaxanthin is responsible for the pink color customers require from a healthy salmon. It was mentioned above that astaxanthin in principle can exist as three optical isomers, the 3R,3'R form 11; its enantiomer, the 3S,3'S form 12; and the optically inactive meso form, the 3R,3'S isomer 13.

Astaxanthin (11) 3R,3'R

Astaxanthin (12) 3S,3'S

Astaxanthin (13) 3R,3'S (meso)

In red flowers (Adonis annua) the optically pure (3S,3;S) isomer 12 is present. At a time it was expected that carotenoids always were present in the biological material in pure optical form. When a nature-identical astaxanthin was desired by chemical synthesis for feed additive purpose upon salmon farming, it was anticipated that an expensive enantioselective synthesis of a single isomer was required. However, it turned out that in shrimp the three

Meso Isomers

Astaxanthin (11) 3R,3'R

Astaxanthin (12) 3S,3'S

Astaxanthin (13) 3R,3'S (meso)

isomers occur naturally as a 1: 2 (meso form): 1 mixture. In salmon also the three isomers are present, but the ratio differs. Reported values are (3S,3'S) 75-85% of total astaxanthin, (3R,3'S, meso) 2-6% and (3R,3'R) 12-17% (26). Today the synthetic astaxanthin marketed is a 1 : 2 (meso) : 1 mixture, whereas that produced by the yeast Pfaffia rhodozyma is the optically pure (3R,3'R) isomer 11. The third common source, the alga Haematococcus sp., provides the optically pure (3S,3'S) isomer 12. It has been demonstrated that all three isomers are equally well resorbed by the fish and that no metabolic interconversion is observed in the flesh (27). It may also be mentioned that all three isomers are equally well bound in the blue crustacyanin astaxanthin complex present in lobster (28). Apparently a racemization process occurs in certain crustaceans including zooplankton on which the wild fish is feeding (26). The separation at the three astaxanthin isomers may be effected on a chiral HPLC column or after derivatization to diastereomeric camphanates on an achiral HPLC column (1).

2. Zeaxanthin

Our next example is zeaxanthin. The structure differs from astaxanthin only by lacking the two carbonyl groups in 4,4' positions. Zeaxanthin has two chiral centers (C-3,30) where the hydroxyl groups are located and may exist as the (3R,30R) isomer 21, the (3S,30S) enantiomer 22, and the (3R,30S) meso form 23.

Zeaxanthin (21) 3R,3'R

Zeaxanthin (22) 3S,3'S

In a mixture these three optical isomers are best separated after derivatization to diastereomeric carbamates on a common, achiral column. In nature the (3R,3'R) isomer 21 dominates by far, e.g., in maize and egg yolk, the (3S,3'S) isomer 22 is very rare, and the meso form (23) is occasionally encountered in animal sources,

Isomers Lutein

Zeaxanthin (22) 3S,3'S

e.g., in human retina. The formation of the meso form in retina is tentatively formulated via lutein (16); see below.

3. Lutein

Let us proceed with a structurally more complex example, lutein. Lutein is a major carotenoid in all green leaves, in some various marine animals, and in human serum and retina.

Lutein has two different end groups with a total of three chiral carbons (C-3, C-30, and C-60). Since each chiral carbon atom in principle can assume the (R-) or (S-) configuration, lutein can exist as eight different optical isomers, consisting of four pairs of enantiomers. Of two suggested systems for the denomination of the lutein isomers the systematic semirational, one is cited below (29).

a ent-a

The common green leaf lutein is lutein ent-d (10) and the so-called epilutein from flowers of Calthapalustris is ent-a (18). Other alternatives are 24-29. When mentioning lutein the chirality needs specification by the R/S nomenclature. It is interesting to note that five of these eight isomers have been claimed to occur in natural sources and six of them have been prepared by chemical synthesis (25). CD and NMR data are essential for the identification of the individual isomers. By :H NMR the C-3',6'-trans (hydroxy group and polyene chain on opposite side) or alternative cis relationship is readily established from relevant chemical proton shifts. It appears that lutein ent-d (10) and, to a much lesser extent, lutein ent-a (18) are synthesized de novo in nature, whereas the other lutein isomers are metabolic products in certain animals.

4. Neoxanthin

The structurally most complex carotenoid to be addressed here in conjunction with our consideration of chirality is neoxanthin. Neoxanthin has five chiral carbon atoms (C-3,5,3',5',6') and a chiral axis (the allene). All naturally occurring allenic carotenoids exhibit (R)-configuration for the allene, although the natural occurrence of (S)-allene at a time was claimed (30).

Of the several theoretically possible optical isomers of neoxanthin only the (3S,5R,6R,3'S,5'R,6'S) isomer 19 is encountered in nature (6R specifies the chirality of the allene). However, the allenic (6S) isomer 20 (see Sec. VI) has been prepared synthetically from the (6R) isomer by allenic isomerization in light, employing diphenyldiselenide as catalyst (21).

Abscisic Acid Biosynthesis

The relative configuration between the 3'-hydroxy group and the epoxide is trans (on opposite sides of the ring) in neoxanthin (19) as in all other naturally occurring carotenoids with this particular structural element. The epoxide-

furanoid rearrangement reaction is treated in Sec. VII.

COOH

COOH

Grasshopper ketone

Abscisic acid

Epoxide carotenoids are abundant in fruits and flowers. The best source of neoxanthin (19) is spinach. Neoxanthin (19), as its 9'-cis isomer, serves in green leaves as the biosynthetic precursor of the growth hormone abscisic acid (30). The ant-repelling allenic grasshopper ketone (31) is also most likely a metabolic product of neoxanthin (19) (25).

VII. CHEMICAL REACTIONS A. Partial Synthesis

Chemical reactions involving only functional group modifications are generally considered as partial syntheses of carotenoids. Examples are simple derivatization reactions such as acylation of carotenols or complex metal hydride reduction of ketocarotenoids. In such reactions the carotenoid skeleton is intact. However, some other simple chemical reactions that result in carbon skeletal changes may also be considered as partial synthesis, e.g., aldol condensation of carotenals with acetone to provide products of citranaxanthin (9) type with prolonged carbon skeleton, or oxidative cleavage of carotenoids to products with shorter carbon skeletons.

Derivatization reactions of carotenoids are still useful in characterization and identification of carotenoids, particularly in the absence of complete spectral data. Chemical reactions of carotenoids have been treated in detail elsewhere (31). This is also the case for microscale reactions useful for identification (1).

Three examples of partial synthesis are chosen here, which are relevant for topics treated in this book. The first example demonstrates the possible conversion of lutein (10) to epilutein (18) via the 3'-keto derivative 32. Selective allylic oxidation provides the ketone 32 which by complex metal hydride reduction gives an approximate 1: 1 mixture of lutein (10) and epilutein (18). Since these products are diastereomers they may be separated by chromato-

graphy. The keto derivative 32 has been encountered as a metabolite in both chicken and in eggs. Epimerization of lutein (10) in animal tissues to epilutein (18) is likely to occur by a similar set of enzymatic reactions.

Lutein Mechanism

The second example also deals with lutein (10), namely, its conversion to meso-zeaxanthin (23) by isomerization of the double bond of the s ring into conjugation by means of strong base (33). Since this reaction does not influence the chirality of the C-3' hydroxy group, (3R,3'S, meso)-zeaxanthin (23) is obtained. The meso compound is in principle optically inactive, as has been verified in this case by lack of optical activity (no CD) (33). The technical conversion of lutein (10) ex Tagetes flowers to zeaxanthin is conducted by a process based on this reaction, and actually leads to meso-zeaxanthin (23).

The formation of meso-zeaxanthin in animal tissue is likely to proceed by a similar enzymatic reaction.

Meso Zeaxanthin

The final example shows the reaction mechanism for epoxide-furanoid rearrangement of common carotenoid 5,6-epoxides of neoxanthin (19) type by the influence of weak acid.

Compounds Carotene
8 R 8 s v J

It has been proved that the chirality at C-5 is unchanged during this reaction (34). Since the reaction proceeds via a planar cation both C-8 diastereoisomers will result, in approximately the same amount. Like diastereomers in general, the two C-8 diastereomeric products may be separated chromatographically and characterized. Had this reaction occurred in nature via a stereospecific enzyme only one furanoid product would be obtained. Consequently, when two C-8 diastereomeric furanoid products are isolated upon analysis of a biological material it is taken as a strong indication that it deals with artifacts obtained by acid-catalyzed rearrangement of a natural carotenoid 5,6-epoxide during the isolation procedure (see Sec. VIII. B).

B. Total Synthesis

In contrast to the partial synthesis discussed above, the total synthesis of carotenoids is considered as a series of chemical reactions serving to build up the desired carotenoid skeleton with the functional groups in correct positions.

Formerly total synthesis of optically inactive carotenoids was considered satisfactory, even if chiral centers were present. However, today enantioselective synthesis with a single carotenoid product with the correct chirality at all chiral centers and correct configuration of all double bonds in the polyene chain is the ultimate goal. The art of total synthesis has been dealt with in extensive overviews (3,35). Highlights in the total synthesis of carotenoids may be illustrated by the total synthesis of several individual cis isomers of all-trans lycopene (14) (23), which is achiral, and total synthesis of (3S,5R,6S,30S,50R,60R)-peridinin (33) with six chiral centers (36).

Peridinin (33)

VIII. SOME USEFUL LESSONS A. Minimal Identification Criteria

In microscale isolation and analytical work a carotenoid is rarely identified by all spectroscopic data to the extent that an identification is unequivocal. Supplementary data by diagnostic chemical reactions may strenghten the identification. However, frequently identifications are based on HPLC data alone, which is not satisfactory evidence for a safe conclusion. A set of minimal identification criteria has been defined for a reasonably safe identification (1). These are:

1. VIS spectrum in one, preferably two, defined single solvents (for identification of the chromophore).

2. Chromatographic evidence in two defined chromatographic systems, preferably including HPLC (for establishment of relative polarity determined by functional groups), and direct co-chromatography with the authentic reference carotenoid.

3. Mass spectra of at least the quality giving the molecular ion and some supporting fragmentations, e.g., loss of toluene or xylene from the polyene chain, H2O from carotenols, etc. (2).

These criteria may suffice for an achiral carotenoid. For determination of the absolute stereochemistry of chiral carotenoids, a combination of 1H NMR and CD data is usually required.

If some of the above criteria are not fulfilled a tentative identification may in some cases be justified. In such a case the ending "-like" should be added. Thus, zeaxanthin-like would be an appropriate tentative identification of a carotenoid with ^-carotene (1)-type visible spectrum and polarity compatible with a carotenoid diol in the absence of additional data.

B. Artifacts

The definition of carotenoid artifacts has been discussed (3). Artifacts are here considered as unwanted products of defined chemical structure arising during an unintended chemical reaction. Artifacts have conveniently been divided into two types as discussed in the following sections.

1. Pre-Extraction Artifacts

These may often be ascribed to improper handling of the biological material prior to extraction and are frequently overlooked. Included are enzyme-catalyzed reactions, acid-catalyzed reactions, and miscellaneous effects. The observation of chlorophyll degradation products in fresh extracts of photosynthetic tissues indicates that pre-extraction artifacts may be present.

2. Isolation Artifacts

Isolation artifacts are produced upon and after extraction by improper handling of the extracts. The type of reactions involved are light- and heat-induced reactions, acid-catalyzed reactions, base-catalyzed reactions, e.g., during saponification conditions, oxidations by air or peroxides (in ether solvent), reactions on active surfaces, thermal reactions, and miscellaneous effects. The most common artifacts are caused by trans-cis isomerization during the isolation procedure caused by exposure to daylight and slightly elevated temperature. In order to prove that a cis isomer is naturally occurring, fast extraction at low temperature followed by fast HPLC analysis in a suitable system is required (37). During standard isolation conditions some trans-cis isomerization is unavoidable.

Other very common artifacts are the so-called furanoxides or carotenoid 5,8-epoxides, formed from natural 5,6-epoxides such as neoxanthin (19) and peridinin (37) by the influence of weak acids, as shown in Sec. VII. The reaction leads to furanoid products with shorter chromophore. Since a new chiral center is created at C-8 both C-8 epimers (8R and 8S) are formed. These may frequently be separated chromatographically in approximately 4: 6 ratio, thus representing evidence for the artifactual character of the products. Carotenoid epoxides are common in plant tissues, and precautions should be taken to avoid this complication during the isolation.

Another artifact is the base-catalyzed aldol condensation taking place with acetone and carotenals (aldehydes) during saponification conditions. The presence of acetone must be strictly avoided during saponification which by standard conditions are carried out in diethyl ether-ethanol containing about 5% sodium hydroxide. As an example the reaction of the C30 carotenal 34 with acetone leading to the methyl ketone citranaxanthin (9) is shown below, the natural occurrence of which may be questioned.

C30-carotenal (34)

C30-carotenal (34)

Astaxanthin and its esters present in salmonid fishes readily undergo another base catalysed reaction of the a-ketol functional group with molecular oxygen (traces of air) at alkaline saponification conditions. This leads to the achiral enolised a-diketone astacene (35) shown below. Hence astaxanthin extracts should not be submitted to saponification conditions for removal of lipids.

KOH, 02

KOH, 02

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Responses

  • Olo
    What precaution should be taken during circular dichroism precaution?
    6 years ago
  • Carl
    Does b carotene have a chiral carbon?
    4 years ago
  • milly
    Is betacarotene chiral or achiral?
    4 years ago

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