Isotopically-labeled carotenoids

E. Solid-State Magic Angle Spinning NMR on Isotopically-Labeled Carotenoids in... 

F. Resonance Raman Spectroscopy on Isotopically-Labeled Carotenoids Incorporated into... 

Resonance Raman spectroscopy carried out on carbon-specific, isotopically-labeled carotenoids can provide an important means to assign the Raman spectral bands and, upon incorporation of the carotenoid into RCs, can aid greatly in elucidating structural features not evident from X-ray diffraction studies and in understanding the effect of the protein on the vibrational activity of the carotenoid. Kok et al. (1994,1997) have examined the resonance Raman spectra of several C- and H-labeled spheroidenes incorporated into Rb. sphaeroides R26.1 RCs. 14- H, 15- H, 15 - 14,15 -2H and 15,15 - H... 

Chemical synthesis has a major part to play in the sophisticated interdisciplinary studies that are now needed to study the biological functions and actions of carotenoids, and the interactions of carotenoids with other molecules such as proteins. Essential roles in photosynthesis have been discovered for several different carotenoids, including specific geometrical isomers. Synthesis is able to provide the pure and, when appropriate, isotopically labelled carotenoids that are required for reconstitution studies, investigation of photochemistry, etc. In the field of medicine it is now clear that the provitamin A activity of p,p-carotene (3) may not be the only beneficial effect of carotenoids. Several carotenoids found in the human diet, especially lycopene (31), lutein (133) and zeaxanthin (119), could also be important in giving protection against serious disorders such as cancer, heart disease, and degenerative eye diseases. Characterization of these effects and elucidation of the mechanisms involved require substantial quantities (g to kg) of pure carotenoids these materials can only be produced by chemical synthesis. 

The various parts of Chapter 3 describe the preparation of polyene synthons and the synthesis of different groups of carotenoids, the partial synthesis of carotenoid glycosides, glycosyl esters and sulphates. Technical synthesis and the synthesis of isotopically labelled carotenoids are also covered. 

AMS is a special type of MS that has been used to determine various parameters of carotenoid absorption, distribution, and metabolism, and the basics of this technique have been reviewed by Buchholz et al. (2000). A radio-labeled carotenoid (generally labeled with " C) is fed to a subject, and biological sample or expired air are collected. Samples may be analyzed directly, or first extracted and analyzed by HPLC, where fractions containing the putative isotopically labeled parent carotenoid(s) and/or carotenoid metabolites are collected. 

The sample (biological sample or collected fraction) is oxidized to produce carbon dioxide which is then converted to graphite (Getachew et ah, 2006). The sample is then ionized, and ions are accelerated to high levels of energy in the MS. The ratio of the rare isotope to the abundant isotope is determined ( C/ C, for example). Because of the extreme sensitivity of AMS (in the attomole range), only a small amount of radio-labeled carotenoid is necessary to study absorption and metabolism. 

Stable isotopes of carotenoids (usually labeled with or H), or of compounds which react with carotenoids, are often used with MS studies. We previously mentioned the use of stable isotopes as internal standards for carotenoid quantitation (especially important when matrix suppression of signal is observed), but there are also many other applications. 

Kurilich, A.C. Britz, S.J. Clevidence, B.A. Novotny, J.A. 2003. Isotopic labeling and LC-APCI-MS quantification for investigating absorption of carotenoids and phyl-loquinone Som kale (Brassica oleracea). J. Agric. Food Chem. 51 4877-4883. 

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. 

Chemical synthesis does allow labelling at specific positions with high isotopic enrichment. There are four constraints on the synthesis of labelled carotenoids. 

From the above discussion it is clear that it is of vital importance to gain easy access to carotenoids specifically labelled with stable isotopes, especially with C. In the next Section, the tools for synthesizing labelled carotenoids from simple starting materials will be discussed. The synthesis of labelled Cio-synthons suitable for preparing any carotenoid labelled in the central part at predetermined positions will be described. Next, the linear synthesis of the spheroidenes labelled with in the central part will be presented, followed by a new convergent synthetic scheme by which it is possible to label all carotenoids with isotopes in the central part of the molecule. 

It is convenient to begin the synthesis of the labelled polyene chain with an aldehyde or a ketone and elongate the chain with either labelled or unlabelled synthons. The products of these reactions are subsequently converted into other aldehydes or ketones, allowing further elongation of the chain. By the use of synthons which can be labelled in any position or any combination of positions, polyene chains can be prepared with isotopic labels on any predetermined positions. In this Section, lycopene (31) is used to illustrate which starting materials and synthons are used to label the corresponding positions in the polyene chain (Fig. 1), but the same scheme can be applied to any carotenoid or polyene. 

From work on the synthesis of isotopically labelled vitamin A and vitamin A derivatives [29-38], P-ionone (64) can be labelled at any position, so the end groups of p,p-carotene (3) can now also be labelled at any position. To label positions 1 and 2 of the phosphonium salt 67 (corresponding to positions 10, 11, 10, and IT in the carotenoid), p-ionone (64) is coupled with the anion of triethyl phosphonoacetate (11), which can be obtained in labelled form as described in Section B.l (Scheme 3). The ester 68 is reduced with DIBAH and the resulting alcohol is converted into the phosphonium salt 67 by reacting it with triphenylphosphine hydrobromide in methanol (Scheme 21). In this way the phosphonium salt 67 can be obtained labelled in any predetermined position. 

As an example of the synthesis of an asymmetrical carotenoid, the preparation of [15- C]-and [15 - C]-spheroidene (97) is given (Scheme 26). The versatility of this method is illustrated by the fact that from one labelled Cio-synthon, namely [4- C]-2,7-dimethylocta-2,4,6-trien-8-alnitrile (29), the two isotopically labelled spheroidenes, can be synthesized [46]. 

Several syntheses of carotenoids isotopically labelled with deuterium have been reported [65-68]. The total synthesis of spheroidenes (97) specifically labelled with deuterium in the central part is based on the synthetic scheme discussed above for the C-labelled spheroidenes [68]. When deuterium-enriched compounds are used, a few modifications are necessary to avoid scrambling and isotope dilution (Scheme 28). 

Some details of the stereochemistry of carotenoid cyclization have been elucidated. In the C40 series labelling with stable isotopes (deuterium) has been used for the first time in studies of carotenoid biosynthesis. A Flavobacterium species in the presence of nicotine accumulated the acyclic precursor lycopene (175). When the cells were washed free from the inhibitor and suspended in H20 cyclization of the lycopene proceeded, initiated by High-resolution n.m.r. 

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. 

All other reported examples of carotenoids, both radioactively labelled ( H and " C) and labelled with stable isotopes ( H, C and 0) have been prepared by biosynthesis and used in studies of the metabolic pathways of carotenoids [19]. 

Over 90% of the vitamin A reserve is found in the liver but substantial amounts are distributed in other tissues. Table VIII gives the concentration of vitamin A and carotenoids in several tissues. Isotope dilution techniques using tritium-labeled vitamin A have been applied to assess total body vitamin A status... 

---