on-line at: www.actabp.pl Partial synthesis of serum carotenoids and their metabolites*

Human serum and tissues contain in excess of 12 dietary carotenoids and several metabolites that originate from consumption of fruits and vegetables. Among these also been characterized in human serum and ocular tissues. Semi-synthetic processes have been developed that separately transform commercially available 1 into 4 via 7 as well as 1 into 8. While 8 is converted into 2 by base-catalyzed isomerization, 7 is transformed into 2 and its (3R,3'S;meso)-stereoisomer (9) by regioselective hydroboration.

Epidemiological and experimental evidence to date suggest hydroxycarotenoids may protect against chronic diseases such as cancer (Van Poppel, 1993), cardiovascular disease (Morris et al., 1994) and age-related macular degeneration (AMD) (Seddon et al., 1994).Therefore, supplementation with these carotenoids in individuals with a low dietary intake of fruits and vegetables is essential.The lack of commercial availability of some of these non-vitamin A active dietary carotenoids has limited the investigation of their metabolism and their biological activity.With the exception of 1 and 2, industrial production of hydroxycarotenoids 3-9 have not yet materialized.While the total synthesis of 1 and four of its stereoisomers has been reported (Khachik & Chang, 2009), the isolation of this carotenoid from marigold flowers (Tagetes erecta) on industrial scale has proven to be the most viable and inexpensive route to this carotenoid (Khachik, 1995;Ausich & Sanders, 1997).In addition, 1 with stereocenters at 3,3',6'-positions serves as an excellent precursor in the partial synthesis of optically active hydroxycarotenoids with ε-and β-end groups.An example of this is (6'R)-α-carotene (10) that could be ac- cessible from two consecutive deoxygenation of 1 via 3 (Fig. 3).
Therefore, several relatively straightforward semisynthetic processes have been developed that transform 1 into 3-10 in excellent yields and high optical purities.These processes provide easy access and alternative routes to optically active carotenoids that are normally prepared by multi-step synthesis.

RESULTS AND DISCUSSION
To accomplish the synthesis of hydroxycarotenoids, (3R,3'R,6'R)-lutein (1) that is commercially available from saponified extracts of marigold flowers (Tagetes erecta) has been employed as the key starting material.The allylic deoxygenation of 1 at the 3-position under mild reaction conditions to 3 was accomplished by a number of reagents under mild conditions in excellent yields (Khachik et al., 2007) (Scheme 1).In another approach, 1 was subjected to ionic hydrogenation with triethylsilane/trif-luoroacetic acid (Et 3 SiH/TFA) that is a known reagent for the reduction of multiple bonds and deoxygenation of single bonds such as C-OH (Kursanov et al., 1974).This resulted in the formation of a mixture of 3 and 4 as well as a mixture of lutein dehydration products that were identified as (3R,6'R)-3-hydroxy-3',4'-didehydro-β,γcarotene [anhydrolutein I] (5), (3R,6'R)-3-hydroxy-2',3'didehydro-β,ε-carotene [anhydrolutein II] (6), and (3R)-3hydroxy-3',4'-didehydro-β,β-carotene [anhydrolutein III] (7) (Khachik et al., 2007).Carotenoids 5, 6, and 7 are the dehydration products of dietary lutein that have been isolated and characterized in extracts from human plasma; these carotenoids are presumably formed in human digestive system in the presence of acids (Khachik et al., 1995).It should be noted that 8 is also a precursor of vitamin A2.Following the course of this reaction, it was revealed that 1 was first dehydrated to 5, 6, and 7 and subsequently these carotenoids were in part converted to 3 and 4. Low temperature acid-catalyzed dehydration of lutein has been shown to yield predominantly 5 as the  major product and 6 and 7 as the minor products.Because 5 and 6 appeared to have been converted to 3 by ionic hydrogenation, this approach resulted in the formation of a complex mixture of products in which 3 was the major product and 4 the minor product.Because the ionic hydrogenation of 7 was most likely responsible for the formation of 4, the low yield of this carotenoid appeared to be due to the low yield of its precursor.
Therefore, in an attempt to transform 1 to 4 a twostep process was developed.In the first step, 1 was first dehydrated to a mixture of 5, 6, and 7 at high temperature to obtain the thermodynamically stable 7 as the major product.This was accomplished in a refluxing solution of 1 in propanol-water (PrOH-H 2 O) at 90°C in the presence of catalytic amounts of hydrochloric (HCl) or sulfuric acid (H 2 SO 4 ) in which anhydroluteins I (5) and anhydrolutein II (6) underwent isomerization to anhydrolutein III (7) within 12-18 h to yield a mixture of anhydroluteins in 85% yield in which the composition of the mixture was determined by HPLC as 7 (86%), 6 (9%), and 5 (5%).The ionic hydrogenation of the resulting mixture of anhydroluteins afforded a mixture of 4 (88%) and 3 (12%) in yields greater than 80%.Following two consecutive crystallizations, the ratio of 4 to 3 could be improved to 97:3 (Scheme 3) (Khachik et al., 2007).
Anhydrolutein III (7) has also been shown to serve as a useful precursor in the synthesis of (3R,3'R)-zeaxanthin (2) and its meso-isomer (9).Hydroboration of 7 with BH 3 -THF prepared in situ from NaNH 4 /CH 3 I afford a mixture of 2 and 9 that were separated by enzymemediated acylation with lipase PS (Pseudomonas cepacia) or lipase AK (Pseudomonas fluorescens) in high diastereomeric ratio (dr) as shown in scheme 4 (Khachik, 2009).

CONCLUSION
The semisynthetic approach to optically active hydroxycarotenoids and their metabolites provides an alternative to total synthesis and can minimize the difficulties associated with multistep synthesis and, in addition, affords a much higher yield of the desired product.Commercially available (3R,3'R,6'R)-lutein (1) has been shown to serve as a key starting material in partial synthesis of (3R,6'R)-α-cryptoxanthin (3) and (3R)-β-cryptoxanthin (4) in two convenient steps via anhydroluteins I -III (5-7) in high yield.In addition, 1 can also be directly converted to 3 in a single step in greater than 90% yield.In another process, 1 has been efficiently converted to (3R,3'R)-zeaxanthin (2) via 3'-epilutein (8) that is a metabolite of 1 and/or 2 identified in human serum, breast milk, and ocular tissues.In an alternative process 1 has been transformed into a mixture of 2 and (3R,3'S;meso)zeaxanthin (9) that were separated by enzyme-mediated acylation.These methodologies provide an easy access to optically active hydroxycarotenoids that have been shown to exhibit biological activities and enables researchers to further study the bioavailability and efficacy of these carotenoids in the prevention of chronic diseases.

Figure 1 .
Figure 1.Structures of dietary hydroxycarotenoids found in human plasma, breast milk and ocular tissues.