The Chemistry of Novel Xanthophyll Carotenoids

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The Chemistry of Novel Xanthophyll Carotenoids

Henry Jackson, PhD,a Cristi L. Braun, BS,a and Hansgeorg Ernst, PhDb,*

Natural product isolates are typically not developed as drug candidates because of thedifficulty in obtaining the desired stable molecular orientation (ie, stereochemistry),purity, and scale required to meet pharmaceutical industry standards. Recent ad-vances in medicinal and process chemistry have played key roles in transforming aclass of dietary natural products— carotenoids—into potential medical therapeutics.Carotenoids are natural pigments derived from the acyclic C40 isoprenoid lycopene,which can also be classified as a tetraterpene. Carotenoids are classified on theirchemical composition as either carotenes or xanthophylls. There are 5 C40 carote-noids manufactured synthetically on an industrial scale, including lycopene, ß,ß-carotene, and canthaxanthin (which are achiral compounds); zeaxanthin (producedin enantiopure form, as the 3R,3=R enantiomer); and astaxanthin (produced asmixture of configurational isomers) for use as nutritional supplements and for animalfeed additives in poultry farming and aquaculture that are essential for the animals’growth, health and reproduction. The xanthophyll astaxanthin shows pharmaceuticalpotential, but the configurational complexity has thus far made it difficult to synthe-size an enantiopure form on a large scale. Astaxanthin has 2 identical asymmetriccarbon atoms (position 3 and 3=) and can therefore exist in 4 different configurations,providing 3 different configurational isomers: (3S,3=S) and (3R,3=R), which are en-antiomers, and (3R,3=S) and (3S,3=R), which are identical (a meso form). An enan-tiopure industrial scale synthesis of astaxanthin (3S,3=S) has recently been developedby BASF AG. The desired stereochemistry (chirality) is introduced early in thesynthetic process by a proprietary catalytic reaction using an intermediate of theexisting technical astaxanthin production process as a substrate. By controlling thisessential process, it is possible to produce pharmaceutical quality astaxanthin inquantities large enough to support drug development programs for medicaltherapies. © 2008 Elsevier Inc. All rights reserved. (Am J Cardiol 2008;101[suppl]:
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arotenoids are widespread in nature and found in plants,nimals, and microorganisms.1 These intensely coloredolecules are responsible for the yellow, orange, and red

olors of various fruits, vegetables, flowers, birds, fish, andrustaceans (Figure 1).2 In all, �750 carotenoids have beensolated from natural sources and structurally characterized,nd each year a few new examples are discovered. They areiosynthesized de novo by various plants, algae, and pho-osynthetic bacteria.3,4 Carotenoids perform critically im-ortant functions within photosynthetic systems. They func-ion as accessory pigments, harvesting the light energy ofavelengths, which are only weakly absorbed by chloro-hyll, thus, increasing the efficiency of photosynthesis. Inddition, carotenoids serve a protective role by effectively

aCardax Pharmaceuticals, Aiea, Hawaii, USA; and bBASF AG,-67056 Ludwigshafen, Germany.

Statement of author disclosure: Please see the Author Disclosuresection at the end of this article.

*Address for reprints: Hansgeorg Ernst, PhD, Fine Chemicals andiocatalysis Research, GVF/A-B009, BASF AG D-67056, Ludwigshafen,ermany.

bE-mail address: [email protected].

002-9149/08/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.amjcard.2008.02.008

issipating excess energy, preventing the formation of re-ctive oxygen species, and by deactivating singlet oxygenenerated during the photosynthetic process.5

Animals, including humans, cannot synthesize carote-oids, therefore, these compounds must be assimilated fromheir diets.6 The typical human diet contains about 40 caro-enoids, most of which are obtained from fruits, vegetables,nd seafood.7 For example, the major dietary source ofitamin A for mammals, including humans, is derived fromarotenoids. Vitamin A is an essential micronutrient for cellrowth, embryonic development, vision, and immune sys-em function.8 On the basis of structure, carotenoids thatossess an unsubstituted �-ionone ring (eg, �-carotene) areotential vitamin A precursors. Astaxanthin acts as a pro-itamin A carotenoid in salmon and is used as a feed supple-ent for pigmentation.9,10 More importantly, humans have had

significant history of multiple exposures to astaxanthinhrough dietary intake (eg, salmon, lobster, shrimp) and USood and Drug Administration (FDA)–approved food addi-

ives. Numerous reports in the literature have suggested aotential medical therapeutic role for astaxanthin based on itshemistry, mechanism of action, and antioxidant properties in
oth in vitro and in vivo models.11–13
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51DJackson et al/The Chemistry of Novel Xanthophyll Carotenoids

In addition to being potent quenchers of singlet oxygen,arotenoids are also excellent direct free radical scaven-ers.14 Numerous epidemiologic studies suggest that dietsigh in fruits and vegetables are associated with a reducedisk for certain cancers, cardiovascular disease, and otheriseases, such as age-related macular degeneration, Alzhei-er disease, and arthritis.15,16 Oxidative stress and the re-

ultant free radical damage to biologic macromolecules,uch as DNA, proteins, and lipids, are believed to be majoractors in the pathogenesis of such diseases. Dietary anti-xidants, including carotenoids, may be among the possibleources of the protective effects observed in these studies.

In addition to their antioxidant activity, carotenoids mayxert biologic effects through other mechanisms. A numberf cell and tissue culture experiments and in vivo animaltudies suggest that carotenoids exert beneficial biologicffects through modulation of both transcription17 and im-une response.18 Several biologic effects attributed to caro-

Figure 1. Carotenoid

enoids may, in fact, be caused by the actions of carotenoid t

etabolites (eg, apocarotenoids). The oxidative metabolitesf various carotenoids have been shown in vitro to induceap junctional communication,19,20 inhibit the growth ofeukemias21 and other cancers,22,23 induce apoptosis in can-er cells,24 and promote gene activation.25 Research is on-oing to determine the potential role of these carotenoidxidation products as biologic mediators. To date, only aew carotenoid oxidative metabolites have been detected inumans.26

Carotenoids are absorbed along with dietary lipidshrough passive diffusion into intestinal epithelial cellsenterocytes).27 For dietary carotenoids to be absorbed ef-ectively, they must first be released from the food matrix,nd their bioavailability varies from a small percentage foraw whole foods to �50% for some cooked and processedoods.28 The initial step in carotenoid absorption involvesncorporation into mixed micelles composed of bile salts,holesterol, and other lipids. The mixed micelles are then

re found in nature.2

ransferred into the enterocytes, where they are incorporated

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52D The American Journal of Cardiology (www.AJConline.org) Vol 101 (10A) May 22, 2008

nto triglyceride-rich lipoproteins called chylomicrons andeleased into the lymphatic system. From the lymphaticystem, chylomicrons enter systemic circulation thoughhe subclavian vein. Through the action of the endothelialnzyme lipoprotein lipase, triglycerides are removedrom chylomicrons and distributed to extrahepatic tis-ues. The resulting chylomicron remnants are taken up byhe liver, where the remaining carotenoids are incorpo-ated into lipoproteins and secreted back into the circu-ation for delivery to the tissues.

ierarchy of Antioxidants

n early 2001, Beutner and coworkers29 published the re-ults of a series of studies in which they used in vitro assayso quantify the antioxidant capacity of several classes ofhytochemicals, including carotenoids. The antioxidant ca-acity was characterized by: (1) the ability to quench singletxygen, (2) the inhibition of peroxide formation, and (3) theorrelation of antioxidant dependency with oxygen partialressures.29 As a class, carotenoids proved to be excellentuenchers of singlet oxygen, with rate constants approach-ng the diffusion-control limit.29 The inhibition of radical-

igure 2. (A) Peroxide formation of pure methyl linoleate in the presence ool/L is lowered by 50%). (B) Normalized (with respect to actinioerythro

re extracted from the experimental 200-mbar graphs by taking the interserom J Sci Food Agric.29)

nduced formation of the hydroperoxides of methyl linoleate b

as used to assess the free radical scavenging function ofarotenoids in the presence of excess oxygen. As shown inigure 2, xanthophyll carotenoids bearing terminal carbonylroups conjugated to the polyene backbone (eg, astaxanthinnd canthaxanthin) were better free radical scavengers andntioxidants.29 Additional experiments measured the radi-al-induced oxidation of cumene under variable oxygenartial pressures and carotenoid concentrations. At a con-tant oxygen partial pressure (150 torr) and carotenoid con-entrations ranging up to 7.7 � 10�4 mol/L, the resultsupported the conclusions of the peroxide inhibition assay.eto-carotenoids, such as astaxanthin and canthaxanthin,ere better radical scavengers than carotene carotenoids,hich did not contain conjugated terminal carbonyl func-

ions (Figure 2). These findings suggest that the keto func-ion in conjugation with the polyene backbone is able totabilize carbon-centered radicals more effectively than theolyene backbone alone. In subsequent experiments, theate of oxygen consumption was determined at varyingarotenoid concentrations at constant oxygen pressures. Theesults helped to further differentiate the carotenoids into 2eneral classes. The first group contained carotene carote-oids (eg, �-carotene, lycopene), which displayed goodntioxidative properties at lower carotenoid concentrations

idants (50% astaxanthin means that the standard concentration 7.7 � 10�4

ates of oxidation at p(O2) � 200 mbar (150 Torr). The rates of oxidationoint with a vertical line at 7.7 � 10�4 mol/L. (Reprinted with permission

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ut prooxidative properties at higher concentrations. The

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econd group contained xanthophyll carotenoids (eg, can-haxanthin, astaxanthin), which displayed strong antioxidantroperties throughout the concentration range studied with-ut displaying any prooxidative properties. Surprisingly,nder conditions where the carotenoids behaved as prooxi-ants, no oxidation product of the substrate itself (cumener methyl linoleate) was detected. The oxygen consumptionas affected solely by the degradation of the carotenoids.

hemical Composition of Carotenoids

ll C40 carotenoids can be formally derived from the

Figure 3. Biosyn

igure 4. Possible geometric isomeric forms of carotenoids attributable to cSci Food Agric.29

cyclic C40 isoprenoid (tetraterpene) lycopene. Lyco- c

ene is biosynthesized from a total of 8 C5 isoprenenits. Initially, 4 C5 units combine to produce the C20ntermediate geranylgeranyl diphosphate; 2 C20 precur-ors combine in a head-to-head fashion to form the C40ntermediate phytoene, a more saturated precursor ofycopene. From phytoene, the other double bonds arentroduced by stepwise enzymatic dehydrations. Otherarotenoids are synthesized from lycopene by modifica-ions, such as cyclizations, oxidative functionalizations,earrangements, and oxidative degradations (Figure 3).ased on their chemical composition, carotenoids areivided into 2 general classes: (1) carotenes, which are

f carotenoids.2

ed double bonds of the polyene backbone. Reprinted with permission from
onjugat
omposed of only carbon and hydrogen; and (2) xantho-

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hylls, which contain �1 oxygen functionality at theyclic end groups.

Several geometric isomeric forms are possible becausef the many conjugated double bonds present in the polyeneackbone (Figure 4).30 The linear all-trans isomer is theost prevalent isomer found in nature, and the electron-rich

olyene backbone is responsible for many of the physico-hemical and biologic properties of these compounds.31

To understand the complexities that chirality presents inharmaceutical compound development, a brief primerbout chirality and the stereoisomers of astaxanthin will beelpful. Chirality literally means “handedness”; the termhiral is derived from the Greek word for hand, kheir. Theost common example of chirality is our hands. The left

and and right hand are mirror images of each other but areot identical. If you try to superimpose your right on youreft hand, you will see that both hands are not identical. Asllustrated in Figure 5, a carbon atom with 4 different sub-tituents is chiral because its image and mirror image areonsuperimposable. Pairs of molecules that are nonsuper-mposable mirror images are called enantiomers. A 1:1ixture of each enantiomer of a pair is a racemic mixture or

acemate. Diastereomers are molecules that contain �2

Figure 5. Exa

hiral centers and differ in configuration at �1, but not all, a

hiral centers. Astaxanthin has 2 chiral centers, which re-ults in 4 different stereoisomer configurations (Figure 6).he 3S,3=S and 3R,3=R configurational isomers constitute annantiomeric pair. The 3S,3=R, the 3R,3=S forms are super-mposable mirror images and are called the meso form. Thehemical synthesis of astaxanthin starting from racemic inter-ediates results in a 1:2 (meso form): 1 statistical mixture of

he 3 configurational isomers of astaxanthin. All 3 configura-ional isomers of astaxanthin are found in nature, and theroportions vary from species to species (Table 132–37).

he Industrial Synthesis of 3S,3=S Astaxanthin

staxanthin is 1 of only 5 C40 carotenoids that are pro-uced entirely synthetically in their natural forms on anndustrial scale.10 However, manufacturing pharmaceuticaluality astaxanthin and other carotenoids within regulatoryompliance standards has remained a barrier to drug devel-pment efforts. Astaxanthin is a symmetrical carotenoid,eaning both headgroups coupled to the polyene backbone

re identical. Initially, the most efficient and economicaltrategy for the synthesis of astaxanthin was to react an

of chirality.2

ppropriate C15 phosphonium salt with a symmetrical C10

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ialdehyde as a central building block (Figure 7). Undesiredsomers were removed by thermal and/or recrystallizationethods. Technical processes using this strategy also are

xisting for lycopene, �-carotene, and zeaxanthin.10

During the late 1970s and early 1980s, a strategy waseveloped by Hoffmann-La Roche (Basel, Switzerland) toynthesize building blocks for many xanthophylls, includingstaxanthin, starting from a C-9 unit, ketoisophorone, which isasily accessible from petroleum feedstocks. In a few simpleteps, the functionality of the final product is introduced in arotected form. Briefly, a C9 ketal building block for astaxan-hin is obtained from ketoisophorone by a series of simpleransformations (epoxidation, base-catalyzed rearrangement ofhe epoxide into a sodium enolate, and hydrogenation of theeactive carbonyl group) and is coupled with a C6 acetylide toield a C15 intermediate. The C15 phosphonium salt is gen-rated by successive reactions with hydrogen bromide andriphenylphosphine (Figure 8). In the final step, the classicouble Wittig condensation reaction leads to the formation of

Figure 6. Configuratio

Table 1

Occurrence

Crustacyanine32 (lobster)Pfaffia rhodozyma33 (yeast)Haematococcus pluvialis34 (algae)Petals of Adonis annua35

Pandalus borealis36 (shrimp)Salmo salar/Salmo37 (Atlantic/Pacific salmon)

staxanthin.10 Ideally, to yield enantiopure compounds for v

harmaceutical product development, chirality needs to bentroduced at a very early stage of the synthesis and maintainedhroughout a scalable, reproducible, and economically viableanufacturing process.

onclusion

ontrolled, reproducible, and characterizable manufactur-ng processes are an essential component in the develop-ent of any drug candidate considered for regulatory ap-

roval as a pharmaceutical. The manufacturing processust unambiguously confer the identity, purity, and stabil-

ty of the active pharmaceutical ingredient, as well as con-rol for the formation of nontoxic impurities at kilogram toetric ton production scale. Until recently, carotenoids (ie,

staxanthin) produced for the animal feed markets could noteadily comply with the pharmaceutical industry standards.

The production of enantiopure (3S,3=S) astaxanthin pro-

mers of astaxanthin.2

Configurational Isomer (%)

3S,3=S 3R,3=R meso

33 39 28– 100 –99 – –

100 – –12–25 23–46 50–5378–85 12–17 2–6

ides a platform for drug discovery and development for

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oth astaxanthin and chemically modified, water dispersible3S,3=S) astaxanthin derivatives. These compounds can thene used for the treatment of diseases characterized by in-ammation and oxidative stress, which are key factors at-

ributable to the onset and maintenance of the underlyingathophysiologic processes. Recent reports have demon-trated that derivative selection not only maintains stereo-hemistry, but it also impacts biologic effects in rodents andonrodent species, paving the way for the development ofatural source compounds in humans.

cknowledgment

he authors acknowledge the efforts of Thomas H. Goodin,

Figure 7. Industrial synthesis of symmetrical C40-carot

Figure 8. Synthesis of C15

hD, in the preparation of this manuscript.

uthor Disclosures

he authors who contributed to this article have disclosedhe following industry relationships:

Henry Jackson, PhD, is a chemist employed by Cardaxharmaceuticals.

Cristi L. Braun, BS, is a chemist employed by Cardaxharmaceuticals.

Hansgeorg Ernst, PhD, is a scientist employed byASF AG; is a member of the Council of the Internationalarotenoid Society; and serves on the Scientific Advisoryoard of Cardax Pharmaceuticals.

1. Britton G, Liaaen-Jensen S, Pfander H, eds. Carotenoids Handbook.Basel: Birkhäuser Verlag, 2004.

(Reprinted with permission from Pure Appl Chem.10)

onium salt intermediate.2

2. Data on file. Ludwigshafen, Germany: BASF AG.

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3. Sandmann G. Carotenoid biosynthesis in microorganisms and plants.Eur J Biochem 1994;223:7–24.

4. Britton G. Carotenoids and polyterpenoids. Nat Prod Rep 1991:223–249.

5. Krinsky NI. The antioxidant and biological properties of the carote-noids. Ann N Y Acad Sci 1998;854:443–447.

6. Schweigert FJ. Metabolism of cartenoids in mammals. In: Britton G,Liaaen-Jensen S, Pfander H (eds). Carotenoids: Isolation and Analy-sis. Basel: Birkhäuser Verlag, 1998.

7. Bendich A. Biological functions of dietary carotenoids. Ann N Y AcadSci 1993;691:61–67.

8. Olson JA. Absorption, transport, and metabolism of carotenoids inhumans. Pure Appl Chem 1994;66:1011–1016.

9. Christiansen RT, Torrissen OJ. Growth and survival of Atlanticsalmon. Aquaculture Nutrition 1996;2:55–62.

0. Ernst H. Recent advances in industrial carotenoid synthesis. Pure ApplChem 2002;74:2213–2226.

1. Guerin M, Huntley ME, Olaizola M. Haematococcus astaxanthin:applications for human health and nutrition. Trends Biotechnol 2003;21:210–216.

2. Higuera-Ciapara I, Felix-Valenzuela L, Goycoolea FM. Astaxanthin: areview of its chemistry and applications. Crit Rev Food Sci Nutr2006;46:185–196.

3. Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H. Astax-anthin, a carotenoid with potential in human health and nutrition. J NatProd 2006;69:443–449.

4. Krinsky NI. Carotenoid protection against oxidation. Pure Appl Chem1979;51:649–660.

5. Kirsh VA, Mayne ST, Peters U, Chatterjee N, Leitzmann MF, DixonLB, Urban DA, Crawford ED, Hayes RB. A prospective study oflycopene and tomato product intake and risk of prostate cancer. Can-cer Epidemiol Biomarkers Prev 2006;15:92–98.

6. Ruano-Ravina A, Figueiras A, Freire-Garabal M, Barros-Dios JM.Antioxidant vitamins and risk of lung cancer. Curr Pharm Des 2006;12:599–613.

7. Sharoni Y, Agbaria R, Amir H, Ben-Dor A, Bobilev I, Doubi N, GiatY, Hirsh K, Izumchenko G, Khanin M, et al. Modulation of transcrip-tional activity by antioxidant carotenoids. Mol Aspects Med 2003;24:371–384.

8. Camara B, Bouvier F. Oxidative remodeling of plastid carotenoids.Arch Biochem Biophys 2004;430:16–21.

9. Bertram JS. Induction of connexin 43 by carotenoids: functional con-sequences. Arch Biochem Biophys 2004;430:120–126.

0. Aust O, Ale-Agha N, Zhang L, Wollersen H, Sies H, Stahl W. Lyco-pene oxidation product enhances gap junctional communication. FoodChem Toxicol 2003;41:1399–1407.

1. Nara E, Hayashi H, Kotake M, Miyashita K, Nagao A. Acycliccarotenoids and their oxidation mixtures inhibit the growth of HL-60
human promyelocytic leukemia cells. Nutr Cancer 2001;39:273–283.
2. Ben-Dor A, Nahum A, Danilenko M, Giat Y, Stahl W, Martin HD,Emmerich T, Noy N, Levy J, Sharoni Y. Effects of acyclo-retinoic acidand lycopene on activation of the retinoic acid receptor and proliferationof mammary cancer cells. Arch Biochem Biophys 2001;391:295–302.

3. Russell K, Murgatroyd PR, Batt RM. Net protein oxidation is adaptedto dietary protein intake in domestic cats (Felis silvestris catus). J Nutr2002;132:456–460.

4. Zhang H, Kotake-Nara E, Ono H, Nagao A. A novel cleavage productformed by autoxidation of lycopene induces apoptosis in HL-60 cells.Free Radic Biol Med 2003;35:1653–1663.

5. Wang Y, Jones Voy B, Urs S, Kim S, Soltani-Bejnood M, Quigley N,Heo YR, Standridge M, Andersen B, Dhar M, et al. The human fattyacid synthase gene and de novo lipogenesis are coordinately regulatedin human adipose tissue. J Nutr 2004;134:1032–1038.

6. Carail M, Caris-Veyrat C. Carotenoid oxidation products: from villainto saviour? Pure Appl Chem 2006;78:1493–1503.

7. Faulks RM, Southon S. Challenges to understanding and measuringcarotenoid bioavailability. Biochim Biophys Acta 2005;1740:95–100.

8. Kopsell DA, Kopsell DE. Accumulation and bioavailability of dietarycarotenoids in vegetable crops. Trends Plant Sci 2006;11:499–507.

9. Beutner S, Bloedorn B, Frixel S, Blanco IH, Hoffman T, Martin HD,Mayer B, Noach P, Ruck C, Schimdt M, et al. Quantitative assessmentof antioxidant properties of natural colorants and phytochemicals;carotenoids, flavonoids, phenols and indigoids: the role of �-carotenein antioxidant functions. J Sci Food Agric 2001;81:559–568.

0. Zechmeister L. Cis-Trans Isomeric Carotenoids, Vitamins A and Aryl-polyenes. New York: Academic Press; and Vienna: Springer Verlag,1962.

1. Britton G. Structure and properties of carotenoids in relation to func-tion. FASEB J 1995;15:1551–1558.

2. Renstroem B, Rønneberg H, Borch G, Liaaen-Jensen S. Animal caro-tenoids. 27. Further studies on the carotenoproteins, crustacyanin andovoverdin. Comp Biochem Physiol 1982;71B:249–252.

3. Andrewes AGS, Mortimer P. (3R,3=R)–Astaxanthin from the yeastPhaffia rhodozyma. Phytochemistry 1976;15:1009–1011.

4. Renstroem B, Borch G, Skulberg OM, Liaaen-Jensen S. Natural oc-currence of enantiomeric and meso-astaxanthin. Part 3. Optical purityof (3,S,3=S)-astaxanthin from Haematococcus pluvialis. Phytochemis-try 1981;20:2561–2564.

5. Renstroem B, Hanni R, Liaaen-Jensen S. Esterified, optically pure(3S,3=S)-astaxanthin from flowers of Adonis annua. Biochem Syst Ecol1981;9:249–250.

6. Renstroem B, Borch G, Liaaen-Jensen S. Natural occurrence of enan-tiomeric and meso-astaxanthin. 4. Ex shrimps (Pandalus borealis).Comp Biochem Physiol B Biochem Mol Biol 1981;69B:621–624.

7. Schiedt KL, Franz J, Vecchi M. Natural occurrence of enantiomericand meso-astaxanthin. 5. Ex wild salmon (Salmo salar and Oncorhyn-
chus). Helvetica Chimica Acta 1981;64:449–457.

The Chemistry of Novel Xanthophyll Carotenoids

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