Towards carotenoid dendrimers: Carotenoid diesters and triesters with aromatic cores

Article abstract of DOI:10.1002/hlca.200900338

For the evaluation of the synthesis of dendritic esters from carotenoids, various carotenols or their succinates were reacted with bi- and trifunctional aromatic acids and alcohols. Several methods were tested, from which the Steglich esterification was f

Full text of DOI:10.1002/hlca.200900338

Helvetica Chimica Acta – Vol. 93 (2010)
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Towards Carotenoid Dendrimers: Carotenoid Diesters and Triesters with
Aromatic Cores
by Magdolna Ha´da, Veronika Nagy, Gergely Gulya´s-Fekete, Jo´zsef Deli, and Attila Ago´cs*
´
´
Department of Biochemistry and Medical Chemistry, University of Pecs, Medical School, Szigeti ut 12,
´
H-7624 Pecs (phone: þ 36-72-536001/ext. 1864; fax: þ 36-72-536225; e-mail: attila.agocs@aok.pte.hu)
For the evaluation of the synthesis of dendritic esters from carotenoids, various carotenols or their
succinates were reacted with bi- and trifunctional aromatic acids and alcohols. Several methods were
tested, from which the Steglich esterification was found to give the best yields. Apocarotenoids showed
higher reactivity in most of the reactions and served as model compounds for C40 carotenoids and their
derivatives. The synthesized triesters can be regarded as first generation dendrimers.
Introduction. – Carotenoids are naturally occuring antioxidants having various
biological effects including anti-cancer and cardioprotective, and have been in the focus
of food biochemists recently [1]. Trimers or first generation dendrimer-like structures
of these compounds have not been synthesized until now, although they can have
interesting physicochemical properties, especially with respect to their carotenoid
nature. These compounds may have enhanced biological activity [2] and could be used
for supramolecular aggregation studies [3]. Synthesis of carotenoid esters is not
unknown in the literature, there are examples including enzymatic reactions with
carotenoid acids [4], reactions between carotenoid acids and carotenols [5], and in our
previous article, the syntheses of some carotenoid homo- and heterodimers were
described [6]. The fine tuning of this kind of reactions were of a great help for the
reactions shown in our present article.
Results. – Herein, we describe the synthesis of some carotenoid di- and triesters
with aromatic core molecules. The apocarotenoids retinol and 8’-b-apocarotenol
(Fig. 1), being not too expensive and easily accessible, were chosen as model
compounds for other carotenoids. The simplest method for esterification would be
the reaction of commercially available aromatic di- or triacyl chlorides with the
corresponding carotenol (Fig. 1) in excess. Many experiments have been performed
with different bases and solvents, but none of them gave the desired product in
acceptable yields, most of the pigments were decomposed under the reaction
conditions.
Activated carboxylic acid derivatives (e.g., di- or triimidazoles) proved to be
unreactive, which led us to investigate the use of the N,N’-dicyclohexylcarbodiimide
(DCC)/4-(dimethylamino)pyridine (¼ N,N-dimethylpyridin-4-amine; DMAP) cou-
pling used before for diesters [6][7]. Interestingly, aromatic di- or tricarboxylic acids
proved to be rather unreactive, whereas non-aromatic acids like benzenediyl- or
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Fig. 1. Structures of the carotenoid starting materials
triylacetic acids gave promising results (Scheme 1). Most probably, both stereo-
electronic and steric factors impede the reactions with aromatic carboxylic acids.
Unfortunately, C40 carotenoids bearing secondary OH groups (i.e., b-crytoxanthin, 4’-
hydroxyechinenone, zea xanthin, and lutein) gave poor yields or none of the desired
products, which may be a consequence of steric hindrance. The utilization of some kind
of spacer for the secondary OH groups (e.g., zeaxanthin) could bring the required
result. If the alcohol to be esterified is cheap, it can be used in large excess to complete
the reaction. Unfortunately, the carotenoid alcohols are much more valuable than the
core molecules and could be applied only in a 20 – 30% molar excess. This also
contributed to the relatively low yields of the reactions.
Another approach for the synthesis of such kind of trimers is reverse functional-
ization, i.e., reacting carotenoid succinates with polyphenols. Once more, these
attempts were unsuccessful, but reactions with the corresponding benzene-1,4-
diyldimethanol (5) provided the products in acceptable yields, although only with
retinol and 8’-b-apocarotenol succinates (Scheme 2). Surprisingly, the reaction of triol 7

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Scheme 1. Synthesis of Di- and Tricarotenoid Esters of Benzenediyl- and Benzenetriylacetic Acid
(which was synthesized from benzene-1,3,5-tricarboxylic acid in two steps [8]) with
carotenoid succinates gave low yields under identical conditions. In the case of retinol
succinate, the yield was acceptable (8a), but with 8’-b-apocarotenol succinate, only
trace amounts of the trimer (based on MS and NMR) could be detected even after
several trials (8b).
Scheme 2. Synthesis of Carotenoid Di- and Triesters with Succinic Acid as a Spacer
In both approaches, Mitsunobu-type esterification was also tested, but the results
confirmed that DCC coupling is superior in yields and reactivity. The structures of the
carotenoid di- and triesters were proven on the basis of NMR, MALDI-TOF, and UV/
VIS data. The purity of the compounds (> 95%) was determined by HPLC.
Conclusions. – Our results confirm that even the simplest reaction such as
esterification can have unusual outcome among carotenoids, and carotenoids with close
structural resemblance can react rather differently under the same conditions. The

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synthesized tricarotenoid esters can be regarded as first generation dendrimers of
carotenoids, although their size exceeds to a great extent the usual size of such kind of
molecules (Fig. 2). The lack of reactivity is still problematic in the cases of bifunctional
carotenoids (e.g., zeaxanthin), most probably for steric reasons, although succinates
bearing a relatively large spacer on the molecule proved also to be unreactive.
Fig. 2. Fan-like structure of carotenoid triester 4b

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Preliminary test results with liver cell lines showed excellent antioxidant activity for
the new compounds in H2O2-induced oxidative stress assays.
We thank Katalin Bçddi for MALDI and Erika Turcsi for HPLC measurements. We also thank Mrs.
´
Andrea Bognar and Mr. Norbert Gçtz for their assistance. This study was supported by OTKA K 60121
and PD 77467 (Hungarian National Research Foundation). We are grateful to CaroteNature for providing
us with a sample of canthaxanthin.
Experimental Part
General. All solvents were of analytical grade, dry solvents were stored over 4 ꢂ molecular sieves.
TLC and prep. TLC: DC-Kieselgel 60 F254 (Merck). HPLC: Dionex P580 gradient pump, Dionex PDA-
100 detector; eluents: 12% H2O/MeOH (A), MeOH (B), 50% acetone/MeOH (C), flow rate: 1.250 ml/
min; column: Chromsyl RP-18 endcapped 6 mm, detection at 450 nm; gradient program: 100% A 2 min,
80% A and 20% B 6 min, 50% A and 50% B 10 min, 100% B 7 min, 100% C 8 min, 100% B 7 min, 100%
A 5 min. UV-VIS: Jasco V-530 spectrophotometer; lmax [nm] (emol values); ref. values: retinol, 326
(48324); 8’-b-apocarotenol, 425 (143540); b-cryptoxanthin, 449 (131900). NMR: Varian Unity Inova (400/
1
100 MHz H/13C), solvent: CDCl3; d [ppm] and J [Hz]1). MS: Bruker Daltonics Autoflex II MALDI,
positive-ion mode (337 nm; N2 laser, max. freq.: 50 Hz; laser intensity 20 – 30%), external calibration.
Source of Carotenoids. Carotenoids were isolated from natural sources or purchased from Sigma-
Aldrich Co. 8’-b-Apocarotenol was synthesized from 8’-b-apocarotenal and 4’-hydroxyechinenone from
canthaxanthin by NaBH4 reduction.
General Procedure for the Synthesis of Di- and Tricarotenoid Esters. To a dry CH2Cl2 soln. (20 mg/ml)
of carotenol or carotenoid succinate (1 equiv.), the corresponding di- or triacid (0.4 equiv./0.25 equiv.,
resp.) or di- or triol (0.4 equiv./0.25 equiv., resp.) was added under N2. To the mixture, 1.5 equiv. of
DMAP and 1.5 equiv. of DCC were added, and the mixture was stirred overnight at r.t. After completion
of the reaction (TLC), the mixture was diluted with Et2O and washed with 5% citric acid soln. and sat.
NaCl soln., and finally dried with MgSO4, and evaporated at 358. The crude product was purified by prep.
TLC (hexane/acetone 8 :2 or 7:3). The di- and tricarotenoid esters were found in the apolar region of the
chromatogram. All products are red solids. The synthesized compounds have > 95% purity according to
HPLC, and the UV/VIS spectra confirm the presence of the starting carotenoids.
Bis(retinyl)benzene-1,4-diacetate (¼ Bis[(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-
en-1-yl)nona-2,4,6,8-tetraen-1-yl] 2,2’-Benzene-1,4-diyldiacetate; 2a). UV/VIS (hexane): (225), 327
(92521). 1H-NMR: 1.02 (s, 2 Me(16), 2 Me(17)); 1.25 – 2.01 (m, 2 CH2(2), 2 CH2(3), 2 CH2(4),
2 Me(18), 2 Me(19), 2 Me(20)); 3.60 (s, 2 CH2); 4.74 (d, J(14,15) ¼ 7.2, 2 CH2(15)); 5.59 (t, J(14,15) ¼
7.2, 2 HꢀC(14)); 6.07 – 6.67 (m, 10 olefin. H); 7.23 (s, 4 arom. H). 13C-NMR: 12.7 (C(19), C(20)); 19.2
(C(3)); 21.7 (C(18)); 28.9, 30.1 (C(16), C(17)); 33.0, 34.9 (C(4), C(1)); 39.6, 40.9 (C(2), CH2); 61.7
(C(15)); 124.2, 125.8, 129.9, 128.3, 129.3, 129.4, 129.9, 132.8, 135.7, 136.6, 137.5, 137.8, 139.2 (polyene
chain); 171.4 (2 C¼O). MALDI-MS: 730 (Mþ).
Bis(8’-b-apocarotenyl)benzene-1,4-diacetate (¼ Bis[(2E,4E,6E,8E,10E,12E,14E,16E)-2,6,11,15-tet-
ramethyl-17-(2,6,6-trimethylcyclohex-1-en-1-yl)heptadeca-2,4,6,8,10,12,14,16-octaen-1-yl] 2,2’-Benzene-
1,4-diyldiacetate; 2b). UV/VIS (hexane): (224), 402, 425 (252340), 450. 1H-NMR: 1.03 – 1.97 (m,
2 CH2(2), 2 CH2(3), 2 CH2(4), 2 Me(17), 2 Me(18), 2 Me(16), 2 Me(19), 2 Me(19’), 2 Me(20),
2 Me(20’)); 3.60 – 3.68 (m, 2 CH2); 4.57 (s, 2 CH2(8’)); 6.15 – 6.62 (m, 24 olefin. H); 7.23 (s, 4 arom.
H). 13C-NMR: 12.6 – 12.8 (C(19), C(19’), C(20), C(20’)); 19.2 (C(3)); 21.6 (C(18)); 29.1, 30.2 (C(16),
C(17)); 33.1, 34.3 (C(1), C(4)); 39.6, 41.0 (CH2, C(2)); 68.1 (C(8’)); 123.0 – 138.2 (polyene chain); 171.2
(2 C¼O). MALDI-MS: 994 (Mþ).
Bis(b-cryptoxanthinyl)benzene-1,4-diacetate (¼ Di-(3S)-b,b-caroten-3-yl 2,2’-Benzene-1,4-diyldiace-
tate; 2c). UV/VIS (hexane): (226), 425, 450 (225200), 476. 1H-NMR: 0.85 – 2.11 (m, 78 H); 2.30 – 2.36 (m,
CH2(4)); 3.60 – 3.65 (m, 2 CH2); 5.06 – 5.10 (m, 2 HꢀC(3’)); 6.13 – 6.84 (m, 28 olefin. H); 7.21 (s, 4 arom.
H). MALDI-MS: 1263 (Mþ).
1
)
Carotenoid numbering is used in all cases, see Fig. 1.

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Tris(retinyl)benzene-1,3,5-triacetate (¼ Tris[(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-
1-en-1-yl)nona-2,4,6,8-tetraen-1-yl] 2,2’,2’’-Benzene-1,3,5-triyltriacetate; 4a). UV/VIS (hexane): (225),
1
326 (151610). H-NMR: 1.02 – 2.15 (m, 3 CH2(2), 3 CH2(3), 3 CH2(4), 3 Me(16), 3 Me(17), 3 Me(18),
3 Me(19), 3 Me(20)); 3.58 (s, 3 CH2); 4.73 (d, J(14,15) ¼ 6.4, 3 CH2(15)); 5.59 (t, J(14,15) ¼ 6.4,
3 HꢀC(14)); 6.06 – 6.65 (2m, 18 olefin. H); 7.05 (s, 3 arom. H). 13C-NMR: 12.7 – 12.8 (C(19), C(20)); 19.3
(C(3)); 21.7 (C(18)); 29.0, 30.1 (C(16), C(17)); 33.0, 34.3 (C(4), C(1)); 39.6, 41.0 (C(2), CH2); 61.8
(C(15)); 124.3, 125.8, 126.9, 129.0, 129.3, 129.8, 129.9, 128.3, 129.3, 129.4, 129.9, 134.5, 135.8, 136.6, 137.6,
137.8, 139.2 (polyene chain); 171.4 (3 C¼O). MALDI-MS: 1057 (Mþ).
Tris(8’-b-apocarotenyl)benzene-1,3,5-triacetate (¼ Tris[(2E,4E,6E,8E,10E,12E,14E,16E)-2,6,11,15-
tetramethyl-17-(2,6,6-trimethylcyclohex-1-en-1-yl)heptadeca-2,4,6,8,10,12,14,16-octaen-1-yl] 2,2’,2’’-Ben-
zene-1,3,5-triyltriacetate; 4b). UV/VIS (hexane): (227), 402, 426 (382218), 451. 1H-NMR: 1.03 – 1.97
(m, 3 CH2(2), 3 CH2(3), 3 CH2(4), 3 Me(17), 3 Me(18), 3 Me(16), 3 Me(19), 3 Me(19’), 3 Me(20),
3 Me(20’)); 3.64 (s, 3 CH2); 4.56 (s, 3 CH2(8’)); 6.15 – 6.62 (m, 36 olefin. H); 7.16 (s, 3 arom. H).
13C-NMR: 12.8 (C(19), C(19’), C(20), C(20’)); 19.3 (C(3)); 21.7 (C(18)); 29.1, 30.3 (C(16), C(17)); 33.9
(C(4)); 34.3 (C(1)); 39.6, 41.2 (CH2, C(2)); 70.3 (C(8’)); 123.3 – 130.8; 131.6, 132.2, 132.9, 134.6, 135.7 –
138.4 (polyene chain); 171.0 (3 C¼O). MALDI-MS: 1454 (Mþ).
1,4-Di-({3-[(retinyloxy)carbonyl]propionyloxy}methyl)benzene (¼1,1’-(Benzene-1,4-diyldimethane-
diyl) 4,4’-Bis[(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraen-1-yl]
Dibutanedioate; 6a). UV/VIS (hexane): (227), 325 (88143). 1H-NMR: 1.02 – 2.02 (m, 2 CH2(2),
2 CH2(3), 2 CH2(4), 2 Me(16), 2 Me(17), 2 Me(18), 2 Me(19), 2 Me(20)); 2.62 – 2.70 (m,
2 ꢀCH2CH2ꢀ); 4.74 (d, J(14,15) ¼ 7.6, 2 CH2(15)); 5.13 (s, 2 CH2); 5.58 (t, J(14,15) ¼ 7.6, 2 HꢀC(14));
6.10 – 6.65 (m, 12 olefin. H); 7.35 (s, 4 arom. H). 13C-NMR: 12.9 (C(19), C(20)); 19.3 (C(3)); 21.8
(C(18)); 25.6, 28.9 – 29.1, 29.8 (C(16), C(17), ꢀCH2CH2ꢀ); 33.1, 34.3 (C(4), C(1)); 39.6 (C(2)); 61.6
(C(15)); 66.2 (CH2); 124.2, 125.9, 129.9, 127.0, 128.3, 128.4 – 128.6, 129.4, 129.9, 135.7, 135.9, 136.7, 137.6,
137.8, 139.3 (polyene chain); 172.2 (2 C¼O). MALDI-MS: 875 (Mþ).
1,4-Di-[(3-{[(8’-b-apocarotenyl)oxy]carbonyl}propionyloxy)methyl]benzene (¼1,1’-(Benzene-1,4-
diyldimethanediyl) 4,4’-Bis[(2E,4E,6E,8E,10E,12E,14E,16E)-2,6,11,15-tetramethyl-17-(2,6,6-trimethyl-
cyclohex-1-en-1-yl)heptadeca-2,4,6,8,10,12,14,16-octaen-1-yl] Dibutanedioate; 6b). UV/VIS (hexane):
1
(225), 403, 425 (226152), 450. H-NMR: 1.02 (s, 12 H, 2 Me(16), 2 Me(17)); 1.23 – 2.04 (m, 2 CH2(2),
2 CH2(3), 2 CH2(4), 2 Me(18), 2 Me(19), 2 Me(19’), 2 Me(20), 2 Me(20’)); 2.65 – 2.74 (m,
2 ꢀCH2CH2ꢀ); 4.57 (s, 2 CH2(8’)); 5.14 (s, 2 CH2); 6.14 – 6.64 (m, 24 olefin. H); 7.35 (s, 4 arom. H).
13C-NMR: 12.7, 12.8, 14.7 (C(19), C(19’), C(20), C(20’)), 19.3 (C(3)), 21.7 (C(18)), 24.9 (ꢀCH2CH2ꢀ),
28.9 – 29.1 (C(16), C(17)), 33.1 (C(4)), 34.3 (C(1)), 39.6 (C(2)), 66.2 (CH2) 70.4 (C(8’)), 123.3 – 129.6,
130.4, 130.7, 131.6, 132.2, 132.9, 135.7 – 138.5 (polyene chain), 171.9 (2 C¼O). MALDI-MS: 1139 (Mþ,
C76H98Oþ8 ).
1,3,5-Tri-({3-[(retinyloxy)carbonyl]propionyloxy}methyl)benzene (¼1,1’,1’’-(Benzene-1,3,5-triyltri-
methanediyl) 4,4’,4’’-Tris[(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-
tetraen-1-yl] Tributanedioate; 8a). UV/VIS (hexane): (225), 326 (136214). 1H-NMR: 0.95 – 2.0 (m,
3 CH2(2), 3 CH2(3), 3 CH2(4), 3 Me(16), 3 Me(17), 3 Me(18), 3 Me(19), 3 Me(20)); 2.66 (m,
3 ꢀCH2CH2ꢀ); 4.74 (d, J(14,15) ¼ 7.2, 3 CH2(15)); 5.13 (s, 3 CH2); 5.58 (t, J(14,15) ¼ 7.2, 3 HꢀC(14));
6.10 – 6.65 (m, 18 olefin. H); 7.28 (s, 4 arom. H). 13C-NMR: 12.7 (C(19), C(20)); 19.2 (C(3)); 21.6
(C(18)); 24.8, 25.5, 29.1, 29.9 (C(16), C(17), ꢀCH2CH2ꢀ); 33.0, 34.2 (C(4), C(1)); 39.5 (C(2)); 61.5
(C(15)); 65.9 (CH2); 124.1, 125.9, 129.9, 127.0, 128.3, 128.4, 129.4, 129.8, 135.7, 135.9, 136.7, 137.6, 137.8,
139.2 (polyene chain); 172.0 (2 C¼O). MALDI-MS: 1273 (Mþ).
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Received September 21, 2009