New Synthesis of Natural Carotene Isorenieratene (φ,φ-Carotene) and its 3,3′-Dimethoxy Analogue

Article abstract of DOI:10.1002/hlca.200390273

The main pigments of Brevibacterium linens are the aromatic carotenoids 3,3′-dihydroxyisorenieratene (ψ,ψ-carotene-3,3′-diol), the corresponding monohydroxy compound, and the hydrocarbon isorenieratene. We report herein new syntheses of isorenieratene (φ,

Full text of DOI:10.1002/hlca.200390273

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Helvetica Chimica Acta Vol. 86 (2003)
New Synthesis of Natural Carotene Isorenieratene (f,f-Carotene) and its
3,3'-Dimethoxy Analogue
by Alain R. Valla*
¬
¡
VA R & D Pepiniere d×Entreprises 140, boulevard de Creac×hGwen, F-29561 Quimper Cedex
(phone: ‡33-2-98-64-19-22; fax: ‡33-2-98-64-19-48; e-mail: valla@univ-brest.fr)
and Dominique L. Cartier, Benoist G. Valla, Re¬gis Y. Le Guillou, Zo R. Andriamialisoa, and Roger Labia
¬
Chimie et Biologie des Substances Naturelles, 6, rue de l×Universite, F-29000 Quimper
and Dietmar E. Breithaupt
Universit‰t Hohenheim, Institut f¸r Lebensmittelchemie (170), Garbenstra˚e 28, D-70599 Stuttgart
and Ste¬phanie M. Savy, Adrien Binet, and Laurent H. Dufosse¬
¬
√
Laboratoire de Microbiologie Appliquee, EA 2651, UBO, Pole de Creac×hGwen, F-29000 Quimper
The main pigments of Brevibacterium linens are the aromatic carotenoids 3,3'-dihydroxyisorenieratene
(f,f-carotene-3,3'-diol), the corresponding monohydroxy compound, and the hydrocarbon isorenieratene. We
report herein new syntheses of isorenieratene (f,f-carotene) and its xanthophyll analogue, 3,3'-dimethoxy-f,f-
carotene, which may be considered a protected form of the natural xanthophyll, 3,3'-dihydroxy-f,f-carotene.
Introduction. The biosynthesis of the aromatic f,f-carotene (isorenieratene; 1a)
is restricted to genera of anoxygenic photosynthetic bacteria (Chlorobium, Pelochro-
matium, and Phaeobium) [1] and a few actinomycetes. This compound and
xanthophylls with 3-hydroxy or 3,3'-dihydroxy end group(s) have been previously
extracted from Brevibacterium linens [2], a member of the commercially important
group of coryneform bacteria used in cheese ripening, Mycobacterium aurum A ‡ [3],
a nonpathogenic, rapidly growing mycobacterium, and Streptomyces mediolani [4], a
bacterium isolated from soil samples [4]. Isorenieratene and derivatives are respon-
sible, in part, for the color of the rind of red-smear ripened soft cheeses (e.g. Livarot,
Epoisses, Munster...). The food and cosmetic industries also take advantage of the
antioxidant properties of natural carotenoids, often including carotenoids in their
products. To test some of their biological properties, we began a project dedicated to
the synthetic and biological production of these molecules. We report herein new
syntheses of 1a and its 3,3'-dimethoxy analogue 1b (3,3'-dimethoxy-f,f-carotene) via a
C20 ‡ C20 route. This latter compound could be considered a protected form of the
natural xanthophyll 3,3-dihydroxy-f,f-carotene. Carotenoids withtertiary MeO
groups are common in phototropic bacteria. A sponge carotenoid possesses a MeO
group at C(3) [5].

Helvetica Chimica Acta Vol. 86 (2003)
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Isorenieratene (1a)
Results and Discussion. The usual process for preparation of carotenoids is a
C15 ‡ C10 ‡ C15 strategy (for books related to syntheses, see [6]) but, when C20
intermediates are readily available, the synthesis of symmetric carotenoids via the
coupling of two C20 units by Wittig olefination [7] (C20 phosphonium salt ‡ C20
aldehyde) or Julia coupling [8] (C20 sulfone ‡ C20 aldehyde) is also viable. An
important contribution to the synthesis of symmetric carotenoids was the discovery by
McMurry [9] that low-valent titanium succeeds in the reductive coupling of aldehydes
or ketones to give high yields of the corresponding alkenes.
The synthesis of isorenieratene and 3,3'-dimethoxyisorenieratene has been
previously reported. Apart from Akiyama et al. [10], who used a McMurry coupling
of two C20 aldehydes, other reported methods have not made use of the above-outlined
strategies. Yamaguchi [11] used a C16 ‡ C8 ‡ C16 method, Weedon [12] a C10 ‡ C20 ‡ C10
approach, and Okukado et al. [13], for the synthesis of dimethoxyisorenieratene, used a
C11 ‡ C20 ‡ C11 strategy.
We use here C15 ‡ C5 synthons to generate C20 units from methyl isopropylidene
cyanoacetate, an easily available C5 unit, and the C15 and C16 −b-methylene aldehyde×
synthons 5 [14], thereby avoiding the problems linked to the configuration of the
double bond that occur with the usual procedures [15][16]. The C15 units were obtained
by formylation of ketones 2a and 2b ((3E)-4-(2,3,6-trimethylphenyl)but-3-en-2-one
and (3E)-4-(4-methoxy-2,3,6-trimethylphenyl)but-3-en-2-one, resp. [17][18]) with
MeONa and HCOOMe, and acetalization of the Na salts of the hydroxymethylene
intermediates by H2SO4 in MeOH furnished the b-ketoacetals 3 (Scheme 1). A Wittig
reaction with (triphenyl)methylenephosphorane led to the corresponding methylene
derivatives, which, after heterogeneous acidic hydrolysis in pentane and HCOOH of
the obtained b-methylidene acetals 4, gave the b-methylidene aldehydes 5 (60% yield
based on 2) [19]. Catalytic conjugation by triethylamine provided regioselectively the
desired (E,E)-a,b-unsaturated aldehydes 6 ((E,E)/(E,Z) 97:3), as previously de-
scribed [14].
Scheme 1
a R ˆ H; b R ˆ OMe.

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Helvetica Chimica Acta Vol. 86 (2003)
A Stobbe-like condensation with methyl 2-cyano-3-methylbut-2-enoate (Scheme 2)
followed by decarboxylation of the crude cyano acids in pyridine led to the nitriles 7a
and 7b in 90% yield (7a: (13E)/(13Z) 80 :20; 7b: (13E)/(13Z) 72 :28). The latter
compounds were further reduced with diisobutylaluminium hydride (DIBAL-H) to the
aldehydes 8, and the isomers were separated by column chromatography (SiO2,
CH2Cl2). A McMurry (low-valent titanium) coupling of the (all-E) isomers gave
isorenieratene 1a and its 3,3'-dimethoxy analogue 1b in 80% yield. These products were
purified first by rapid column chromatography (neutral Al2O3, pentane/CH2Cl2 50 :50)
and then by HPLC (Lichro 5 mm CART RP 18 Merck, MeOH/hexane 80 :20).
Scheme 2
a: R, R' ˆ H; b: R, R' ˆ OMe.
Liquid-chromatography/atmospheric-pressure-chemical-ionization mass spectrom-
etry (LC/(APcI)MS) was performed on the products [20]. The MS spectra of both
‡
carotenoids clearly showed the respective quasi-molecular ions ([M ‡ H] ) at m/z 529
(isorenieratene) and m/z 589 (3,3'-dimethoxyisorenieratene). The mass of isorenier-
atene was in accordance withthat obtained by electron-impact mass spectrometry
[10][21]. The fragmentation pattern (Fig. 1 and 2) observed was dominated in both
‡
cases by loss of one complete f-endring (120 Da and 150 Da) from [M ‡ H] , resulting
in the formation of daughter ions at m/z 409 and m/z 439, respectively. The formation of
M À 92, M À 106, and M À 158 ions, which is typical for carotenoids under ionization by
electron bombardment (e.g., at 70 eV) and direct inlet systems, was not observed
[22][23]. As far as we know, aromatic carotenoids have not been studied before by LC/
MS analyses, and cleavage of the a-bond has not yet been described. However, the
fragmentation unequivocally substantiates the structures of isorenieratene and its 3,3'-
dimethoxy analog.

Helvetica Chimica Acta Vol. 86 (2003)
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Fig. 1. Mass spectrum (LC-(APcI)/MS, positive-ion mode) of 1a (m/z 529.4 (100%); m/z 409.2 (17%))
Fig. 2. Mass spectrum (LC-(APcI)/MS, positive-ion mode) of 1b (m/z 589.5 (100%); m/z 439.4 (26%))
Experimental Part
General. All reactions were carried out under Ar. IR Spectra (film): Bruker IF-55 spectrometer; n in cmÀ1
1H-NMR Spectra (CDCl3, 400 MHz): Bruker Avance DPX-400; d in ppm rel. to SiMe4. LC/MS: HP-1100
modular HPLC system coupled to a Micromass VG platform II quadrupole MS (scan range: m/z 400 1000;
UV/VIS (DAD): 450 nm; injection vol. 20 ml), carried out in the APcI ‡ mode; data processed with
MassLynx 3.2 sofware (for further details, see [20]); separation on a YMC column (5 mm-RP-C30 (250 Â 4.6 mm
i.d.) withprecolumn (10 Â 4 mm i.d.), 358; mobile phase (flow rate: 1 ml/min.): mixtures of MeOH/t-butyl
methyl ether/H2O (A ˆ 81 :15 :4 and B ˆ 6 :90 :4), gradient program (0/99, 39/44, 45/0, 50/99, 55/99 (min/%A).
(all-E)-3,7-Dimethyl-9-(2,3,6-trimethylphenyl)nona-2,4,6,8-tetraenenitrile (7a) and (all-E)-9-(4-Methoxy-
2,3,6-trimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenenitrile (7b). General procedure: at 08, MeOK (2.1 g,
3 mmol) in 10 ml of MeOH was added to a mixture of 1 mmol of 6 [14] and 1 mmol of methyl 2-cyano-3-
methylbut-2-enoate. After 2 d at r.t., the crude mixture was poured into 100 ml H2O and extracted withether.
.

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Helvetica Chimica Acta Vol. 86 (2003)
The aq. layer was acidified with a 10% HCl soln. and extracted with ether. The solvent was removed under
reduced pressure and the crude cyanoacid was dissolved in benzene (20 ml) and decarboxylated by reflux for
20 hwith5 equiv. of pyridine. Benzene and pyridine were removed under reduced pressure, and the crude
mixture was extracted with ether and washed successively with 5% aq. HCl and H2O. The solvent was
evaporated and the crude product was purified by column chromatography (CC; SiO2, CH2Cl2).
Data of 7a: IR: 2940, 2206, 1625, 1465, 1306, 1154, 1119, 974. 1H-NMR1): 6.96 7.02 (m, HÀC(3), HÀC(4),
HÀC(7)); 6.77 (d, J ˆ 16.4, HÀC(8)); 6.17 (d, J ˆ 11.3, HÀC(10)); 6.32 (d, J ˆ 15.5, HÀC(12)); 5.21
(s, HÀC(14)); 2.27 2.29 (m, MeÀC(2), MeÀC(5), MeÀC(13)); 2.24 (s, MeÀC(1)); 2.14 (s, MeÀC(9)). Anal.
calc. for C20H23N: C 86.59, H 8.36, N 5.05; found: C 86.40, H 8.47, N 6.13.
Data of 7b: IR: 2995, 2928, 2853, 2206, 1620, 1586, 1464, 1384, 1309, 1289, 1121, 972, 837, 797. 1H-NMR1):
6.99 (dd, J ˆ 15.2, 11.3, HÀC(11)); 6.74 (d, J ˆ 16.16, HÀC(8)); 6.32 (d, J ˆ 15.2, HÀC(12)); 6.25 (d, J ˆ 16.16,
HÀC(7)); 6.17 (d, J ˆ 11.3, HÀC(10)); 3.82 (s, MeO); 2.30 (s, MeÀC(13)); 2.23, 2.17 (2s, MeÀC(1),
MeÀC(5)); 2.15 (s, MeÀC(2)); 2.09 (s, MeÀC(9)). Anal. calc. for C21H25NO: C 82.04, H 8.20, N 4.56, O 5.20;
found: C 81.89, H 8.32, N 4.49, O 5.30.
(all-E)-3,7-Dimethyl-9-(2,3,6-trimethylphenyl)nona-2,4,6,8-tetraenal (8a) and (all-E)-9-(4-Methoxy-2,3,6-
trimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenal (8b). General procedure: DIBAL-H (2 mm, 2.84 g) in
toluene (as a 1m soln.) was slowly added at 08 to a stirred soln. of 7 (1 equiv.) in toluene (10 ml). After 2 hat 5 8,
the mixture was slowly hydrolyzed with 10% aq. HCl (100 ml). The salts were filtered off and washed with ether
(2 Â 25 ml). The org. layer was washed with brine and dried (Na2SO4). The solvent was evaporated and the
crude product was purified by CC (SiO2, CH2Cl2/MeOH 97:3).
Data of 8a: IR: 2954, 2864, 1660, 1583, 1445, 1389, 1154, 1112, 967, 801. 1H-NMR1): 10.12 (d, J ˆ 8.1, CHO);
6.98 7.36 (m, HÀC(3), HÀC(4), HÀC(7), HÀC(11)); 6.77 (d, J ˆ 15.2, HÀC(8)); 6.42 (d, J ˆ 15.02,
HÀC(12)); 6.27 6.29 (m, J ˆ 15.8, HÀC(10)); 5.99 (d, J ˆ 8.1, HÀC(14)); 2.33 (s, MeÀC(13)); 2.28
(s, MeÀC(2)); 2.23 (s, MeÀC(5)); 2.14 (s, MeÀC(1)); 1.55 (s, MeÀC(9)). Anal. calc. for C20H24O: C 85.67;
H 8.63; O 5.70; found: C 85.48, H 8.77, O 5.75.
Data of 8b: IR: 2995, 2943, 2829, 1659, 1577, 1460, 1399, 1310, 1289, 1262, 1214, 1193, 1152, 1111, 967, 796.
1H-NMR1): 10.12 (d, J ˆ 8.03, CHO); 7.16 (dd, J ˆ 15.7, 11.1, HÀC(11)); 6.76 (d, J ˆ 15.75, HÀC(7)); 6.60
(s, HÀC(4); 6.40 (d, J ˆ 15.75, HÀC(8)); 6.26 (m, 2 H, HÀC(10), HÀC(12)); 5.98 (d, J ˆ 8.03, HÀC(14)); 3.82
(s, MeO); 2.35 (s, MeÀC(13)); 2.30 (s, MeÀC(2)); 2.24 (s, MeÀC(1)); 2.20 (s, MeÀC(5)); 2.17 (s, MeÀC(9)).
Anal. calc. for C21H26O2: C 81.25, H 8.44, O 10.31; found: C 80.98, H 8.57, O 10.45.
Isorenieratene (ˆ f,f-Carotene; 1a) and 3,3'-Dimethoxy-f,f-carotene (1b). The reductive coupling of 8
was carried out as described by Paust and Manchand [24]. General procedure: Under Ar, 190 mg (5 mmol) of
powdered LiAlH4 was added to 1.53 g (10 mmol) of TiCl3 in 30 ml of anh. THF. After 2 h at r.t., 5 mmol of 8a or
8b in 10 ml THF was added. The mixture was stirred overnight, and 50 ml of 2m HCl was slowly added at 08. Th e
crude mixture was extracted with ether and washed with brine. The solvents were distilled off under reduced
pressure, and the residues obtained were purified first by rapid CC (neutral Al2O3, pentane/CH2Cl2 50 :50) to
give 85% of 1a and 75% of 1b and, to obtain a pure sample, further by HPLC (Lichro 5 mm CART RP 18 Merck,
1
MeOH/hexane 80 :20). H-NMR spectra were identical to those previously described [25].
Data of 1a: 1H-NMR: 6.95 (s, 4 arom. H); 6.1 6.08 (m, 14 CˆCH); 2.26 (s, 4 Me); 2.23 (s, 2 Me); 2.08
(s, 2 Me); 2.00 (s, 2 Me).
Data of 1b: 1H-NMR: 7.00 6.10 (m, 16 H, 2 arom. H, 14 CˆCH); 3.82 (s, 2 MeO); 2.47 (s, MeÀC(13,13'));
2.27 (s, MeÀC(5,5')); 2.25 (s, MeÀC(1,1')); 2.15 (s, MeÀC(2,2')); 2.07 (s, MeÀC(9,9')).
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Helvetica Chimica Acta Vol. 86 (2003)
3319
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Received April 14, 2003