Carotenoids with two chromophores: Carotenoid retinoates

Article abstract of DOI:10.1080/14786419.2010.488629

In this study, carotenoid retinoates are described for the first time. The preparation was achieved by the azolide method. Various sec carotenols reacted with N-retinoylimidazol in the presence of catalytic amounts of sodium hydride. Mono- and diretinoates of (3R,3′R)-zeaxanthin and its (3S,3′S)- enantiomer, (9Z,9′Z; 3R,3′R)-alloxanthin, (3R,3′R)-7,8, 7′,8′-tetrahydro-3,3′-dihydroxy-β,β-carotene-8, 8′-dione and (3R,6R,3′R,6′R)-ε,ε-carotene-3, 3′-diol (lactucaxanthin), as well as monoretinoates of (3R,3′RS,6′R)-3′-methoxy-β,ε-caroten-3-ol, (3R,3′RS,6′R)-3-methoxy-β,ε-caroten-3′-ol, (2R,6′RS)-β,ε-caroten-2-ol, (3R,3′S; meso)-astaxanthin and (2′R)-aleuriaxanthin are reported in this study. Spectroscopic properties (1H-NMR mass spectrometry, visible and circular dichroism spectra) are discussed. Studies on other carotenoid derivatives with two chromophores are referred to here.

Full text of DOI:10.1080/14786419.2010.488629

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Carotenoids with two chromophores:
carotenoid retinoates
Sissel Hertzberg a , Bjart Frode Lutnaes a & Synnøve Liaaen-
Jensen a
a Department of Chemistry , Norwegian University of Science and
Technology (NTNU) , NO-7491, Trondheim, Norway
Published online: 07 Mar 2011.
To cite this article: Sissel Hertzberg , Bjart Frode Lutnaes & Synnøve Liaaen-Jensen (2011)
Carotenoids with two chromophores: carotenoid retinoates, Natural Product Research: Formerly
Natural Product Letters, 25:5, 511-525, DOI: 10.1080/14786419.2010.488629
To link to this article: http://dx.doi.org/10.1080/14786419.2010.488629
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Natural Product Research
Vol. 25, No. 5, March 2011, 511–525
Carotenoids with two chromophores: carotenoid retinoates
*
Sissel Hertzberg, Bjart Frode Lutnaes and Synnøve Liaaen-Jensen
Department of Chemistry, Norwegian University of Science and Technology
(NTNU), NO-7491, Trondheim, Norway
(Received 13 December 2009; final version received 18 April 2010)
In this study, carotenoid retinoates are described for the first time. The
preparation was achieved by the azolide method. Various sec carotenols
reacted with N-retinoylimidazol in the presence of catalytic amounts of
sodium hydride. Mono- and diretinoates of (3R,30R)-zeaxanthin and its
(3S,30S)-enantiomer, (9Z,90Z; 3R,30R)-alloxanthin, (3R,30R)-7,8,70,80-tetra-
hydro-3,30-dihydroxy-ꢀ,ꢀ-carotene-8,80-dione and (3R,6R,30R,60R)-","-car-
otene-3,30-diol (lactucaxanthin), as well as monoretinoates of (3R,30
RS,60R)-30-methoxy-ꢀ,"-caroten-3-ol, (3R,30RS,60R)-3-methoxy-ꢀ,"-caro-
ten-30-ol, (2R,60RS)-ꢀ,"-caroten-2-ol, (3R,30S; meso)-astaxanthin and
(20R)-aleuriaxanthin are reported in this study. Spectroscopic properties
(1H-NMR mass spectrometry, visible and circular dichroism spectra) are
discussed. Studies on other carotenoid derivatives with two chromophores
are referred to here.
Keywords: carotenoids; retinol; retinoates; chromophores; chirality
1. Introduction
The syntheses of carotenoids with two chromophores and their properties have
recently evoked interest. Thus, ꢀ-apo-80-carotenoic acid has been esterified with
ꢁ-tocopherol, providing 1 or the water soluble vitamin E analogue Trolox for light
exposure studies (Larsen et al., 1998) (Scheme 1). Moreover, ꢀ-apo-80-carotenol was
esterified with Trolox or a benzoic acid derivative (Karagiannidou et al., 2003)
providing 2, and its antioxidant properties were studied. Carotenylflavanoids (e.g. 3)
have recently been prepared and investigated as a novel group of dual-function
antioxidants (Beutner et al., 2007). ꢀ-Apo-120-carotenal was submitted to Wittig–
Horner–Emmons reactions with appropriate reagents to provide carotenylflavanoids
with a single, extended chromophore. (100S, 100S) and (100R,100R) dicarbamates of
geometrical isomers of (3R,30R)-zeaxanthin (4) and of the diastereomer (3R,30S;
meso)-zeaxanthin have been studied (Lutnaes, Gautun, & Liaaen-Jensen, 2001).
In the present work, a series of mono- and diretinoates of chiral, secondary,
non-allylic and allylic carotenols have been prepared by esterification with retinoic
acid (5) and characterised. Carotenoid retinoates with two separate chromophores
have so far not been available.
*Corresponding author. Email: slje@chem.ntnu.no
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2011 Taylor & Francis
DOI: 10.1080/14786419.2010.488629
http://www.informaworld.com

512
S. Hertzberg et al.
Scheme 1. Structures of the carotenoids discussed in Section 1.
According to the studies by Nakanishi’s group (Harada & Nakanishi, 1983;
Nakanishi et al., 1984), a chiral through-space interaction between the two
chromophores may be expected to give rise to split or coupled curves (Davydov
splitting). If such effects were observed, the result could possibly be used as an
alternative approach for the determination of chirality of carotenoids by circular
dichroism (CD). Non-covalent chromophore interaction is observed in certain
carotenoid-protein complexes. The CD spectrum of the blue carotenoprotein
crustacyanin (6) is independant of the chirality of the bound astaxanthin
(Renstrøm, Rønneberg, Borch, & Liaaen-Jensen, 1982). From recent X-ray studies,
the large colour shift between orange free astaxanthin and the blue carotenoprotein
is mainly ascribed to the exciton interaction between two astaxanthin neighbours
(Weesie et al., 1997). Further interaction with a chiral protein may serve to
rationalise the Cotton effect.
Non-covalent interaction between salinixanthin (7) and retinal (8) is also evident
from recent X-ray structures of the light driven proton pump of xanthorhodopsin,
causing energy transfer, as demonstrated by fluorescence studies (Balashov,
Imasheva, & Lanyi, 2006; Luecke et al., 2008).
Carotenoid retinoates may possibly serve as dual antioxidants. With light
absorption in the visible as well as the ultraviolet (UV), a potential application as, for

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513
example, sunscreen is considered. Hydrolysis of carotenoid retinoates in biological
systems to the native carotenol, and retinoic acid (5) with possible metabolism to
retinal (8) and its hydroxy analogue retinol is another aspect. It is well known that
retinoic acid (5) is an essential regulatory factor in growth and development
(Palozza, 2004). Moreover, there is considerable evidence for natural and synthetic
retinoids being effective in the prevention and treatment of various chronic diseases,
including cancer (Altucci & Gronemeyer, 2001), through their ability to regulate gene
expression (Duester, 2008; Fields, Soprano, & Soprano, 2007).
2. Results and discussion
2.1. Preparative method
The esterification of zeaxanthin (4) with retinoic acid (5) in the presence of DCC,
which was attempted, yielded no retinoates. However, retinoic acid (5) was converted
to the isourea ester 9 and retinoic anhydride (10) (Scheme 2). The intermediate
O-acylisourea 9 is known to be an activated carboxylic acid derivative (Streitweiser &
Heathcock, 1985). Nevertheless, attempts to react the sec, non-allylic carotenol
zeaxanthin (4) or the primary allylic alcohol ꢀ-apo-40-carotenol (11) in separate
experiments either with the ester 9 or with retinoic anhydride (10) failed to provide
carotenoid retinoates.
Treatment of zeaxanthin (4) with retinoic acid (5) in chloroform in the presence
of catalytic amounts of p-toluene sulphonic acid was unsuccessful, probably due
to carotenoid cation formation (Lutnaes, Kildahl-Andersen, Krane, & Liaaen-
Jensen, 2004).
It is reported that the esterification of carboxylic acids by the azolide method
proceeds under mild conditions and with high yields (Staab, 1962). The method has
previously been employed in the carotenoid field for the preparation of glycosyl
esters of the conjugated dicarboxylic acid crocetin (Pfander & Wittwer, 1979).
Scheme 2. Attempted esterification reactions using dicyclohexylcarbodiimide (DCC) as a
catalyst.

514
S. Hertzberg et al.
Scheme 3. Carotenoid retinoid formation by the imidazole method.
Methyl and ethyl esters of retinoic acid (5) were reported already in 1961
(Staab, Rohn, & Mannschreck, 1961). Several steroid retinoates have been prepared
via N-acyl imidazole derivatives using p-toluenesulphonic acid as a catalyst (Carlen
& Draper, 1984).
In this study, retinoic acid (5) was reacted with 1,10-carbonyldiimidazole (CDI)
(Staab & Braunling, 1962) providing the imidazolide 12 (Staab, 1962), Scheme 3.
1
N-retinoylimidazol (12) exhibited H-NMR and mass spectral data consistent with
the structure assigned and was used for the preparation of the desired carotenoid
retinoates. The reaction was carried out with various carotenols dissolved in pyridine
in the presence of catalytic amounts of sodium hydride (Pfander & Wittwer, 1979).
The catalyst presumably acts by converting the carotenol to its alcoholate,
which serves as a better nucleophile in the ester synthesis. The imidazol anion
formed during this process serves to generate more alcoholate from the carotenol
(Staab, 1962) (Scheme 3).
2.2. Retinoates of sec non-allylic carotenols
The reaction of (3R,30R)-zeaxanthin (4) with N-retinoylimidazol (12) provided
the monoretinoate 4a and the diretinoate 4b, Scheme 4. The enantiomeric
(3S,30S)-zeaxanthin (13) afforded its monoretinoate (13a) and diretinoate (13b) in
an analogous reaction. With three times molar excess of reagent, pigment recoveries
after 3 h at room temperature were around 70%. The monoretinoates (4a and 13a)
comprised ca 50%, diretinoates (4b and 13b) 26–40% and unreacted zeaxanthin
(4 and 13) 10–23% of the recovered carotenoid. These reaction conditions were
generally employed in the esterifications treated below.
(3R,30RS,60R)-Lutein 30-methyl ether (14) (Liaaen-Jensen & Hertzberg, 1966)
with the same type of 3-hydroxy-ꢀ-end group afforded the monoretinoate (14a, 40%
of recovered carotenoid) besides unreacted 14.
The stable (9Z,90Z)-isomer of the diacetylenic diol (3R,30R)-alloxanthin (15)
(Fiksdahl, Tauber, Liaaen-Jensen, Saucy, & Weber, 1979) provided the correspond-
ing monoretinoate (15a) and diretinoate (15b) with similar yields as for the
zeaxanthin (4 and 13) above.
The reaction with the synthetic diol (3R,30R)-7,8,70,80-tetrahydro-3,30-dihydroxy-
ꢀ,ꢀ-carotene-8,80-dione (16) (G. Saucy, unpublished) proceeded with lower pigment

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515
Scheme 4. Sec. non-allylic carotenols employed for the preparation of retinoates.
recovery (45%) affording the monoretinoate (16a, 35% of recovered) and diretinoate
(16b, 20% of recovered carotenoid).
Reactions with the alkali labile (Liaaen & Sørensen, 1956; Kjøsen et al., 1976)
fucoxanthin (17) and peridinin (18) were unsuccessful. Experiments with
(3S,30S)-astaxanthin, its (3R,30R)-enantiomer and the meso form (19) were
hampered by the facile conversion to astacene (20) in the presence of base (NaH)
and traces of oxygen (Kuhn & Sørensen, 1938). Pigment recoveries were 60–82%.
A minor product obtained from (3R,30S; meso)-astaxanthin (19) under attempted
anaerobic conditions had spectroscopic properties compatible with astaxanthin
monoretinoate (19a). No retinoates of astaxanthin were formed in the absence
of sodium hydride or in the presence of p-toluene sulphonic acid (Carlen &
Draper, 1984).
(2R)-ꢀ,"-Caroten-2-ol (21) (Kjøsen, Arpin, & Liaaen-Jensen, 1972) with a
sterically hindered sec hydroxyl group reacted slowly with 41% pigment recovery
to the monoretinoate (21a), yielding 10% recovered carotenoid.
2.3. Retinoates of sec allylic carotenols
The bisallylic diol (3R,6R,30R,60R)-ꢀ,"-carotene-3,30-diol (22, lactucaxanthin)
(Siefermann-Harms, Hertzberg, Borch, & Liaaen-Jensen, 1981) reacted with
N-retinoylimidazol (12) with 66% pigment recovery, providing the monoretinoate
(22a, 45% of recovered) and the diretinoate (22b, 15%), besides unreacted 22.
The monoretinoate (22a) gave the acetate 22c upon acetylation.

516
S. Hertzberg et al.
Scheme 5. Sec. allylic carotenoids employed for the preparation of retinoates.
Also the allylic mono-ol (3R,30RS,60R)-lutein 3-methyl ether (23) (Fiksdahl et al.,
1979) revealed no enhanced reactivity versus the non-allylic carotenols and afforded
the monoretinoate (23a, 30% of recovered carotenoid). (20R)-Aleuriaxanthin (24)
showed similar reactivity; 60% pigment recovery and formation of the mono-
retinoate (24a, 20% of recovered).
In conclusion, the formation of several mono- and diretinoates of sec, non-allylic
or allylic carotenols was achieved by the azolide method. The reaction proceeded
more slowly and with lower yields than other esterifications such as acetylation
(Liaaen-Jensen & Jensen, 1965), but yields were not optimised.
2.4. Spectroscopic properties of carotenoid retinoates
The carotenoid retinoates prepared here possess two chromophores, the monocyclic
pentaenoate chromophore of the retinoyl moiety and the polyene chromophore of
the parent carotenols. Retinoic acid (5) has its ꢂmax at 350 nm (acetone) with
" ¼ 49,200. For most of the carotenols employed, " ¼ ca 150,000 at ꢂmax. The
absorption spectra recorded were consistent with the additive effect of one
(monoretinoates) and two (diretinoates) retinoyl chromophores and the carotenoid
chromophore, here conveniently expressed by the % DX/DII value. By analogy with
the previous % DB/DII definition (Ke, Imsgard, Kjøsen & Liaaen-Jensen, 1970), the
term DX/DII expresses the intensity ratio for ꢂmax at ca 350 nm, mainly due to the
retinoyl chromophore, and ꢂmax in the visible region. This general result is illustrated
by the absorption spectra of (3R,30R)-zeaxanthin (4) (Britton, Liaaen-Jensen, &
Pfander, 2004) and its mono (4a) and diretinoate (4b) given in Figure 1.
Whereas molecular ions could not be observed upon electron impact MS for the
diretinoates (M 4 m/z 1100), all the monoretinoates (4a, 13a, 14a, 15a, 16a, 22a, 23a
and 24a) provided molecular ions and exhibited the common fragmentations of the
polyene chain and the underivatised end group (Enzell & Wahlberg, 1980).
Characteristic fragment ions for the monoretinoates were medium to strong ions
due to the loss of 300 mass units (retinoic acid, 5) and m/z 300. Such fragment
ions were also observed for zeaxanthin diretinoate (4b) and for lactucaxanthin
diretinoate (22b).

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517
Figure 1. UV–vis spectra of zeaxanthin mono- and diretinoate (4a and 4b, respectively) in
acetone, and of zeaxanthin (4) in ethanol (Britton et al., 2004).
1H-NMR spectra (500 MHz) were complex due to the partial overlap of several
methyl signals and olefinic protons from the two chromophores, but compatible with
the expected data based on the esterified carotenol (Englert, 1995) and the retinoyl
moiety.
CD is used for the determination of relative configuration of carotenoids (Liaaen-
Jensen, 1980; Noack, 1987). CD spectra of the carotenoid retinoates were offered
particular attention, since according to the studies by Nakanishi et al. (1984) and by
Harada and Nakanishi (1983), a chiral through-space interaction between the two
chromophores could be expected to give rise to CD curves exhibiting Davydov
splitting. If such effects were observed the result could possibly be used for an
alternative approach to the determination of chirality of carotenoids by CD.
From the CD spectra of TLC (kieselgel – calcium carbonate) purified samples
of all-trans (3R,30R)-zeaxanthin monoretinoate (4a) and diretinoate (4b),
(3S,30S)-zeaxanthin monoretinoate (13a) and diretinoate (13b), all-trans
(3R,30RS,60R)-lutein-30-methyl ether-3-retinoate (14a), (9Z,90Z;3R,30R)-alloxanthin
monoretinoate (15a) and diretinoate (15b) and all-trans (3S,6R,30S,60R)-","-carotene-
3,30-diol monoretinoate (21a), some generalisations may be drawn.
Carotenoids with a (3R)-retinoate function in a carotenoid ꢀ-ring exhibited
an extra medium-strength negative Cotton effect around 350 nm, near the position
of the retinoate absorption in the UV–vis spectrum. This band was enhanced in
diretinoates and was apparently additive to the corresponding CD spectrum of
the carotenol. However, split CD curves (Harada & Nakanishi, 1983) were not
obtained.
This may be rationalised by lack of exciton coupling due to the absence of
overlap between the two chromophores for stereochemical reasons. An alternative
approach using the less available in-chain prim carotenols (Britton et al., 2004) as
substrates for the esterification, with higher probability of chromophore interaction,
is pointed out.
3. Experimental
General precautions for work with carotenoids were taken (Schiedt & Liaaen-
Jensen, 1995). Visible (vis) spectra were recorded on a Perkin Elmer 552

518
S. Hertzberg et al.
UV–vis spectrophotometer. Spectral fine structure of vis absorption spectra is
expressed as % III/II (Ke et al., 1970). The term % DX/DII is defined above.
Mass spectra were obtained on an AEI MS902 instrument with direct inlet
system, ionisation energy 70 eV, with ionisation temperature as indicated for each
compound. Only prominent or diagnostically significant peaks are cited.
NMR spectra were recorded on a 500 MHz Bruker DRX instrument in CDCl3
with 1% TMS as the internal reference. For signal assignments, double dash
numbers refer to the retinoyl moiety of the carotenoid monoretinoates, and triple
dash to the second retinoid moiety of diretinoates.
CD spectra were recorded on a Jobin Yvon Auto Dicrograf Mark IV at room
temperature in EPA (diethyl ether : isopentane : ethanol, 5 : 5 : 2) solution containing
ca 20 mg (unesterified basis) carotenoid mLꢀ1, as estimated from vis spectra using
1%
1cm
1%
1cm
E
¼ 2340 for zeaxanthin derivatives and E ¼ 2500 for the other carotenoids.
The reported D"-values serve to redraw the spectra, but they should not be
considered as absolute values.
Thin-layer chromatography (TLC) was carried out on silica plates, using acetone
in hexane (A/H) or mixtures of methanol in ethyl acetate (M/E) as eluents, or on
kieselgel : CaCO3 2 : 1 plates with 5–20% acetone in hexane as eluent.
3.1. Esterification using dicyclohexylcarbodiimide
For further information about the use of this procedure, see Neelakantan,
Padmasani and Seshadri (1965) and L. Fieser and M. Fieser (1967).
(1) Dicyclohexylcarbodiimide (DCC) (7.5 mg, 36 mmol) and zeaxanthin (4,
10.5 mg, 18 mmol) were added to retinoic acid (5, tretinoin Roche,
1%
1cm
E
¼ 1640, " ¼ 49200 at ꢂmax 350 nm in acetone, 10.7 mg, 36 mmol) dissolved
in dry pyridine (2 mL). The reaction mixture was stirred overnight at room
temperature. Transfer of products to ether followed by TLC (20% A/H)
revealed the formation of retinoic anhydride (10, 0.5 mg) and O-retinoyl-N-
cyclohexyl-N0-cyclohexyl isourea (9, 0.4 mg) besides unreacted 4 and 5.
Products 9 and 10 were also prepared in two subsequent experiments from
retinoic acid (5, 30 mg, 10 mmol) and DDC (21 mg, 10 mmol) in dry tetrahydrofurane
(2 mL) overnight, recovery 53%, providing 9 (54% of recovered), 10 (7%) and
unreacted 5 (39%).
O-Retinoyl-N-cyclohexyl-N0-cyclohexyl-isourea (9). Available 10 mg, RF ¼ 0.51 (10%
A/H); ꢂmax (acetone)/nm 350; m/z (EI–MS 210ꢁC) 506 (M, 18%), 491 (3, M-15), 396
(10), 381 (100), 366 (18), 316 (29), 299 (10), 283 (18), 255 (42) and 243 (69).
Retinoic anhydride (10). Available 4 mg, RF ¼ 0.61 (10% A/H); ꢂmax (acetone)/
nm 388; m/z (EI–MS 210ꢁC) 582 (M, 100%), 567 (4, M-15), 510 (4, M-72), 444
(44, M-138), 404 (M-72-106), 300 (18), 291 (5, M2þ), 283 (27), 243 (90); ꢃH (500 MHz;
CDCl3; Me4Si) 1.03s (4 Me, Me-16, 17, 160, 170), 1.72s (2 Me, Me-18, 180), 2.02s (2
Me, Me-19, 190), 2.41s (2 Me, Me-20, 200), 5.78s (2 H, H-14, 140), 6.10d (2 H, H-8, 80),
6.21d (2 H, H-10, 100), 6.27d (2 H, H-7, 70), 6.38d (2 H, H-12, 120) and 7.09dd (2 H, H-
11, 110). Recorded chemical shifts for retinoic acid (5), consistent with reported data
(Englert, 1995), are given for comparison: ꢃH (500 MHz; CDCl3; Me4Si) 1.03s (2 Me,
Me-16, 17), 1.76s (Me, Me-18), 2.01s (Me, Me-19), 2.37s (Me, Me-20), 5.80s (H, H-

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519
14), 6.10d (H, H-8), 6.20d (H, H-10), 6.24d (H, H-7), 6.39d (H, H-12) and 7.04dd (H,
H-11).
(2) (3R,30R)-Zeaxanthin (4, 2.5 mg, 5 mmol) and 9 (4 mg, 8 mmol) dissolved in dry
tetrahydrofurane (2.5 mL) were heated to 50ꢁC and kept at room temper-
ature for 24 h. No new products were isolated.
(3) 4 (0.7 mg, 1.2 mmol) and 10 (1.5 mg, 2.9 mmol) in dry pyridine (2 mL) were
kept overnight at room temperature, and then at 40–50ꢁC for 5 h. No
retinoates were formed.
(4) ꢀ-Apo-40-carotenol (11) was prepared by LiAlH4 reduction of synthetic
ꢀ-apo-40-carotenal (C35). 11 (0.15 mg) in dry pyridine (1 mL) and 10 (0.5 mg)
were kept at room temperature overnight; pigment recovery was 53%. TLC
revealed unreacted 11 only.
(5) An analogous reaction with 11 (0.15 mg) in dry tetrahydrofurane (1 mL)
and 3 (0.5 mg) overnight gave 80% pigment recovery and no retinoates.
3.2. Esterification using p-toluene sulphonic acid
To (3R, 30R)-zeaxanthin (4, 4 mg) and retinoic acid (5, 2 mg) in dry CHCI3 (10 mL)
was added a spatula tip of p-toluene sulphonic acid. The reaction mixture turned
green; after few minutes the pigment recovery was 21%. TLC showed a series
of products. No retinoates were identified.
3.3. Carotenoid retinoates
3.3.1. Preparation of N-retinoylimidazol (12)
To retinoic acid (5, 160 mg, 67 mmol) dissolved in dry benzene (5 mL) was added
N,N0-carbonyl-di-imidazol (100 mg, mp. 117ꢁC). The mixture was kept at room
temperature for 1 h, recovery 83%. TLC showed 12 (60% of recovered, RF ¼ 0.65
(10% A/H)) and unreacted 5 (RF ¼ 0.60, 10% A/H; ꢂmax (acetone)/nm 385; m/z
(EI–MS 200ꢁC) 350 (M, 20%), 335 (1, M-15), 300 (20), 282 (80, M-68),
267 (21, M-83), 159 (89) and 118 (100); ꢃH (500 MHz, CDCl3, Me4Si) 1.04s (2 Me,
gem-Me), 1.74s (1 Me, Me-18), 2.05s (1 Me, Me-19), 2.49s (1 Me, Me-20) and 6–8 m
(ca 9H, olefinic).
3.3.2. Preparation of (3R,30R)-zeaxanthin (4) retinoates
(3R,30R)-ꢀ,ꢀ-Carotene-3,30-diol (4, 6 mg, 11 mmol; zeaxanthin) and N-retinoylimida-
zol (12) (6 mg, 32 mmol) were dissolved in dry pyridine (2 mL). A spatula tip of NaH
(prewashed with hexane) was added. The mixture was kept for 3 h at room
temperature, and pigments transferred to ethyl acetate upon dilution with water;
pigment recovery 70%. TLC showed the diretinoate (4b, 26% of recovered),
monoretinoate (4a, 50%) and unreacted 4 (23%, RF ¼ 0.64, 15% M/E). The
products were repeatedly purified by TLC.
Unless otherwise stated, this procedure was used for the preparation of
carotenoid diretinoates. When monoretinoates were the target compounds, the
amount of reagent 12 was 1.5 molar excess.

520
S. Hertzberg et al.
3.3.2.1. (3R,30R)-Zeaxanthin monoretinoate (4a). RF ¼ 0.75 (15% M/E), 0.46 (40%
A/H); ꢂmax (acetone)/nm 360, 425sh, 453 and 480, % III/II ¼ 33, % DX/DII ¼ 38,
Figure 1; m/z (EI–MS 250ꢁC) 850 (M, 20%), 832 (1, M-18), 758 (4, M-92), 744 (2, M-
106), 568 (58, M-282), 550 (40, M-300), 538 (100, M-300-18) and 300 (70); 1H-NMR
as for 13a below; CD nm (D") 214 (0), 233 (ꢀ11.7), 236 (0), 248 (7.9), 262 (0), 284
(ꢀ12.8), 349 (ꢀ1.4), 372 (ꢀ2.7), 407 (0) and 488 (4.8).
3.3.2.2. (3R,30R)-Zeaxanthin diretinoate (4b). RF ¼ 0.86 (15% M/E), 0.76 (40%
A/H); CD nm(D") 218 (0), 226 (6.0), 233 (0), 247 (7.7), 265 (0), 286 (ꢀ5.6), 306
(ꢀ3.6), 344 (ca 8.5), 398 (0), 416 (1.6), 439 (0.6) and 488 (3.9); ꢂmax (acetone)/nm 355,
425sh, 453 and 480, % III/II ¼ 37, % DX/DII ¼ 68, Figure 1; m/z (EI–MS,
250ꢁC) 4 820 not countable m/z 1132 (M, 5%), 1040 (1, M-92), 1026 (1, M-106),
832 (25, M-300), 742 (24, M-300-92), 532 (32, M-300-300) and 300 (100); CD nm (D")
218 (0), 226 (6.0), 233 (0), 247 (7.7), 265 (0), 286 (ꢀ5.6), 306 (ꢀ3.6), 344 (ca 8.5), 398
(0), 416 (1.6), 439 (0.6) and 488 (3.9).
3.3.3. Preparation of (3S,30S)-zeaxanthin (13) retinoates
(3S,30S)-Zeaxanthin (13) (5.4 mg, 9.5 mmol) and N-retinoylimidazol (12, 10 mg,
28 mmol) were reacted as described for 4 for 2.5 h; pigment recovery 80%. TLC
showed formation of the diretinoate (13b, 40%) and monoretinoate (13a, 50%)
besides unreacted 13 (10%).
3.3.3.1. (3S,30S)-Zeaxanthin monoretinoate (13a). RF as for 4a, inseparable by
co-chromatography; ꢂmax (acetone)/nm 360, 425sh, 453 and 480, % III/II ¼ 32, %
DB/DII ¼ 44, ꢂmax (ether)/nm 277, 355, 425sh, 449 and 476, % III/II ¼ 31, %
DX/DII ¼ 44; m/z (EI–MS, 250ꢁC) as for 4a; ꢃH (500 MHz; CDCl3; Me4Si) 1.03s
(2 Me, Me-1600, 1700), 1.08s (3 Me, Me-16, 160, 170), 1.14s (1 Me, Me-17), 1.72s (3 Me,
Me-18, 180, 1800), 1.97 (5 Me, Me-19, 20, 190, 200, 1900), 2.37s (1 Me, Me-2000), 5.77s
(1H, H-1400) and olefinic signals; CD nm (D") 217 (0), 223 (3.3), 233 (0), 252 (ꢀ2.8)
269 (0), 288 (3.4), 325 (0), 363 (ꢀ1.6), 420 (ꢀ0.7), 436 (0), 482 (3.8) and 532 (0).
3.3.3.2. (3S,30S)-Zeaxanthin diretinoate (13b). RF as for 4b; inseparable by
co-chromatography; ꢂmax (acetone)/nm 356, 425sh, 453 and 481, % III/II ¼ 35, %
DX/DII: ¼ 65, ꢂmax (ether)/nm 275, 357, 425, 449 and 478, % III/II ¼ 40, %
DX/DII ¼ 68; m/z 4 800 not countable, cf 4b; ꢃH (500 MHz; CDCl3; Me4Si) 1.03s
(4 Me, Me-1600, 1700, 16000, 17000), 1.08s (2 Me, Me-18, 180, 1800 and 18000), 1.98s (6 Me,
Me-19, 20, 190, 200, 1900, 19000), 2.387s (2 Me, Me-20, 2000, 20000) and olefinic signals;
CD nm (D") 217 (0), 273 (3.3), 233 (0), 252 (ꢀ4.6), 270 (0), 285 (4.5), 328 (0), 330–450
(50), 432 (0) and 480 (4.2).
3.3.4. Preparation of the retinoate of (3R,30RS,60R)-lutein 30-methyl ether (14)
(3R,30RS,60R)-30-Methoxy-ꢀ,"-caroten-3-ol (14, 2 mg; lutein 30-methyl ether), see
Liaaen-Jensen and Hertzberg (1966), was treated as described for 4 with 13 (1.5 mg)
for 2 h. TLC revealed the formation of 14a (40% of recovered) besides unreacted 14.

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521
3.3.4.1. (3R,30R,S,60R)-30-Methoxy-ꢀ,"-caroten-3-retinoate (14a). RF ¼ 0.61 (20%
A/H, compared with 0.32 for 14); ꢂmax (ether)/nm 275, 335, 418, 442 and 470, % III/
II ¼ 75, % DX/DII ¼ 36; m/z (EI–MS 230ꢁC) 864 (M, 36%), 832 (39, M-32), 772 (4,
M-92), 740 (4, M-92-32), 726 (4, M-106-32), 564 (28, M-300), 532 (100, M-300-32),
300 (80); CD nm (D") 214 (5.2), 228 (1.5), 245 (5.1), 273 (0), 282 (ꢀ1.4), 315 (ꢀ0.3),
354 (ꢀ1.8), 388 (0), 407 (0.8), 428 (0) and 478 (3.6).
3.3.5. Preparation of (3R,30R)-alloxanthin (15) retinoates
(9Z,90Z; 3R,30R)-7,8,70,80-Tetradehydro-ꢀ,ꢀ-carotene-3,30-diol (15, 1 mg; allox-
anthin) (Saucy & Weber, unpublished) was reacted as described for 4; pigment
recovery 63%, yield monoretinoate 15a (15% of recovered), diretinoate 15b (35%)
and unreacted 15 (50%).
3.3.5.1. (9Z,90Z; 3R,30R)-Alloxanthin monoretinoate (15a). RF ¼ 0.54 (20% A/H,
compared with 0.35 for 15); ꢂmax (acetone)/nm 360, 418, 443, 472, % III/II ¼ 73, %
DX/DII ¼ 44, ꢂmax (Et2O)/nm 360, 415, 440 and 470, % III/II ¼ 72, % DX/DII ¼ 46;
m/z (EI–MS 240ꢁC) 864 (M, 75%), 828 (1, M-13), 754 (12, M-92), 740 (1, M-106),
564 (90, M-300), 546 (100, M-18-300), 300 (60); CD nm (D") 223 (ꢀ3.6), 232 (0), 252
(4.7), 291 (0.4), 346 (2.5), 420 (1.0) and 478 (5.3).
3.3.5.2. (9Z,90Z; 3R,30R)-Alloxanthin diretinoate (15b). RF ¼ 0.80 (20% A/H);
ꢂ
max (acetone)/nm 360, 418, 453 and 472, % III/II ¼ 70, % DX/DII ¼ 78; m/z (EI–MS
240ꢁC) not countable; CD nm (D") 227 (ꢀ3.7), 232 (0), 252 (2.7), 268 (0), 288
(ꢀ0.91), 354 (ꢀ2.8), 430 (0) and 476 (3.2).
3.3.6. Preparation of (3R,30R)-3,30-dihydroxy-7,8,70,80-tetrahydro-ꢀ,ꢀ-carotene-
8,80-dione (16) retinoates
(3R,30R)-3,30-Dihydroxy-7,8,70,80-tetrahydro-ꢀ,ꢀ-carotene-8,80-dione (16) (Saucy &
Weber, unpublished) (2 mg) was treated as described for 4; pigment recovery 45%.
TLC revealed the formation of the diretinoate (16b, 20% of recovered),
monoretinoate (16a, 35%) and unidentified products (10%) besides unreacted 16.
3.3.6.1. (3R,30R)-3,30-Dihydroxy-7,8,70,80-tetrahydro-ꢀ,ꢀ-carotene-8,80-dione mono-
retinoate (16a). RF ¼ 0.40 (20% A/H, compared with 0.17 for 16); ꢂmax (acetone)/
nm 363, 410sh, 435 and 462, % III/II ¼ 85, % DX/DII ¼ 57, ꢂmax (Et2O)/nm 262, 360,
405, 430 and 458, % III/II ¼ 93, % DX/DII ¼ 56; m/z (EI–MS 240ꢁC) 880 (M, 20%),
788 (15, M-92), 580 (40, M-300), 538 (24), 491 (35, 447 (25) and 300 (100).
3.3.6.2. (3R,30R)-3,30-Dihydroxy-7,8,70,80-tetrahydro-ꢀ,ꢀ-carotene-8,80-dione
dire-
tinoate (16b). RF ¼ 0.87 (20% A/H); ꢂmax (acetone)/nm 360, 410sh, 435 and 462,
% III/II ¼ 74, % DX/DII ¼ 75, (Et2O)/nm 262, 355, 406, 431 and 453, % III/II ¼ 74,
% DX/DII ¼ 75; m/z (EI–MS 240ꢁC) unsuccessful.

522
S. Hertzberg et al.
3.3.7. Reaction of 12 with fucoxanthin (17)
Natural fucoxanthin (17, 1 mg) in dry pyridine (2 mL) gave no reaction with
N-retinoylimidazol (12, 3 mg) during 30 h. Addition of a spatula tip of NaH resulted
immediately in a hypsochromic shift and products with shorter chromophore; ꢂmax
(Et2O)/420 and 448 nm.
3.3.8. Reaction of 12 with peridinin (18)
Natural peridinin (18, 5 mg) decomposed under the above reaction conditions.
3.3.9. Preparation of astaxanthin (19) retinoates
Experiments with (3S,30S)-3,30-dihydroxy-ꢀ,ꢀ-carotene-4,40-dione (6 mg; astax-
anthin), (3R,30R)-astaxanthin (3.7 mg) and (3R,30S; meso)-astaxanthin (19, 3.5 mg)
at conditions described for 4 gave 59–82% pigment recovery. TLC showed no
unreacted astaxanthin, but a product with properties compatible with astacene (20,
450% of recovered) and two less polar products, including 19a was obtained from
meso-astaxanthin (19) under attempted anaerobic conditions (ca 5% of recovered
carotenoid). In the absence of NaH, 19 (1.5 mg) failed to react with
N-retinoylimidazol (12, 2.4 mg).
(3R,30S; meso)-astaxanthin (19, 1.5 mg) was treated with 12 (5 mg) in the presence
of a spatula tip p-toluenesulphonic acid in benzene-tetrahydrofurane (2 mL)
overnight; pigment recovery 66%. Only astaxanthin (19) was recovered.
Astacene (14). RF ¼ 0.40 (30% A/H) with streaking; ꢂmax (acetone)/nm 475; m/z
(EI–MS 210ꢁC) 592 (M, 100%) 578 (M-16), 564 (M-32), 500 (M-92) and 586
(M-106).
3.3.9.1. (3R,30S)-Astaxanthin monoretinoate (19a). RF ¼ 40.40 (30% A/H); ꢂmax
(acetone)/nm 370, 472, % DB/DII ¼ 57; m/z (EI–MS 230ꢁC) 862 (M-16), 770 (M-16-
92), 756 (M-16-106), 652 (M-16-300), 300 (100%). Astaxanthin (19) had under the
same conditions as for 19a no Mþ but m/z 564 (M-16-16) and 472 (M-16-16-92).
.
3.3.10. Preparation of lactucaxanthin (22) retinoates
(3S,6R,30S,60R)-","-Carotene-3,30-diol (22, 2.3 mg; lactucaxanthin, ex Lactuca
alpinus) was treated as described for 4; pigment recovery 66%. TLC showed the
diretinoate 22b (15% of recovered), the monoretinoate 22a (45%) and unreacted
22 (40%).
3.3.10.1. (3S,6R,30S,60R)-","-Carotene-3,30-diol monoretinoate (22a). RF ¼ 0.20
(20% A/H, compared with RF ¼ 0.08 for 22); ꢂmax (acetone)/nm 360, 415, 440 and
470, % III/II ¼ 88, % DX/DII ¼ 80, ꢂmax (Et2O)/nm 265, 353, 412, 438 and 468, %
III/II ¼ 86, % DX/DII ¼ 83; m/z (EI–MS 250ꢁC) 550 (M-300, 10%), 532 (30, M-300-
18), 300 (100); CD nm (D") 212 (11.2), 228 (2.8), 236 (3.7), 242 (3.0), 269 (10.8), 312
(0.3), 337 (0.8), 362 (0.2), 402 (1.2), 424 (0.1), 472 (3.3) and 512 (0). 22a provided
upon acetylation a monoacetate 22c. RF ¼ 0.30 (20% A/H).

Natural Product Research
523
3.3.10.2. (3S,6R,30S,60R)-","-Carotene-3,30-diol diretinoate (22b). RF ¼ 0.52 (20%
A/H); ꢂmax (acetone)/nm 360, 398sh, 415, 440 and 470, % III/II ¼ 86, % DX/
DII ¼ 56, ꢂmax (Et2O)/nm 275, 350, 388sh, 413, 438 and 468, % III/II ¼ 76, % DX/
DII ¼ 62; m/z (EI–MS 240ꢁC) 832 (M-300, 1%), 532 (80, M-300-300), 440 (25), 426
(30) and 300 (100). 22b afforded no acetate upon acetylation.
3.3.11. Preparation of the retinoate of lutein 3-methyl ether (23)
(3R,30RS,60R)-3-Methoxy-ꢀ,"-caroten-30ol (23, 0.36 mg; lutein 3-methyl ether) was
treated with N-retinoylimidazol (12, 1.5 mg) and NaH in dry pyridine for 2 h;
pigment recovery 90%. The monoretinoate (23a) comprised 25% of the recovered
carotenoid.
3.3.11.1. (3R,30RS,60R)-3-Methoxy-ꢀ,"-caroten-30-retinoate (23a). RF ¼ 0.68 (20%
A/H); ꢂmax (Et2O)/nm 275, 350, 414, 440 and 458,% III/II ¼ 40,% DX/DII ¼ 39; m/z
(EI–MS 230ꢁC) 864 (M, 2%), 564 (100, M-300), 300 (100); CD nm (D") 214 (5.2), 228
(1.5), 245 (5.1), 273 (0), 282 (ꢀ1.4), 315 (ꢀ0.3), 354 (ꢀ1.8), 388 (0), 407 (0.8), 428 (0)
and 478 (3.6).
3.3.12. Preparation of the retinoate of aleuriaxanthin (24)
(20R)-10,160-Didehydro-10,20-dihydro-ꢀ, -carotene-20-ol (24, 0.5 mg; aleuriaxanthin)
(Bucheckert, Arpin, & Liaaen-Jensen, 1976) in dry pyridine (1 mL) was reacted with
N-retinoylimidazol (12, 1 mg) in the presence of NaH for 2 h; pigment recovery 60%.
The retinoate 24a comprised 20% of recovered carotenoid by TLC.
3.3.12.1. (20R)-Aleuriaxanthin retinoate (24a). RF 0.75 (30% A/H, compared with
RF 0.58 for 24); ꢂmax (acetone)/nm 355, 438, 462 and 493, % DX/DII ¼ 52, ꢂmax
(Et2O)/nm 278, 355, 436, 459 and 490, % DX/DII ¼ 56; m/z (EI–MS 250ꢁC) 834
(M, 2%), 742 (1, M-92), 728 (2, M-106), 634 (3, M-300) and 211 (100).
Acknowledgements
We thank Professor K. Nakanishi, Columbia University, for suggesting this study. Skilled
technical assistance including CD spectra was provided by student Einar Egeland. Vitamin A
acid and the synthetic carotenoids were mainly gifts from Dr H. Mayer, Hoffmann-La Roche,
Basel, except synthetic alloxanthin and 3,30-dihydroxy-7,8,70,80-tetrahydro-ꢀ,ꢀ-carotene-8,80-
dione, which were gifts from Dr G. Saucy, formerly of Hoffmann-La Roche, Nutley. Sissel
Hertzberg and Einar Egeland were supported by a research grant from Hoffmann-La-Roche,
Basel, to Synnøve Liaaen-Jensen.
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