Synthesis and characterization of all-E-(4,4'-13C2)-astaxanthin strategies for labelling the C15-end groups of carotenoids

Article abstract of DOI:10.1002/(SICI)1099-0690(200003)2000:5<829::AID-EJOC829>3.0.CO;2-Z

The all-E isomer of (4,4'-¹³C₂)astaxanthin (1a) has been prepared by total synthesis starting from commercially available 99% ¹³C enriched acetonitrile. The labelled astaxanthin was obtained in high purity and with high isotope incorporation. For this synthesis, the C₁₅ + C₁₀ + C₁₅ strategy was used. The central C₁₀-synthon, 2,7-dimethylocta-2,4,6-triene- 1,8-dial (3), was coupled with ¹³C-enriched C₁₅-phosphonium salt 2a. The new synthetic scheme for the preparation of the C₁₅-phosphonium salt is discussed in this paper; the same scheme can be used to label all positions and combinations of positions of the C₁₅-phosphonium salt.

Full text of DOI:10.1002/(SICI)1099-0690(200003)2000:5<829::AID-EJOC829>3.0.CO;2-Z

FULL PAPER
Synthesis and Characterization of all-E-(4,4Ј-13C2)-Astaxanthin
Strategies for Labelling the C15-End Groups of Carotenoids
Frans Jos H. M. Jansen[a] and Johan Lugtenburg*[b]
Keywords: Astaxanthin / Carotenoids / Isotopic labelling / 13C label / Total synthesis
The all-E isomer of (4,4Ј-13C2)astaxanthin (1a) has been pre- (3), was coupled with 13C-enriched C15-phosphonium salt 2a.
pared by total synthesis starting from commercially available The new synthetic scheme for the preparation of the C15
-
99% 13C enriched acetonitrile. The labelled astaxanthin was phosphonium salt is discussed in this paper; the same
obtained in high purity and with high isotope incorporation. scheme can be used to label all positions and combinations
For this synthesis, the C15 + C10 + C15 strategy was used. The of positions of the C15-phosphonium salt.
central C10-synthon, 2,7-dimethylocta-2,4,6-triene-1,8-dial
Introduction
oxo-groups are protonated.[5,6] Although a charge at C-4 is
not directly reflected in the chemical shift, modern NMR
techniques allow for the determination of the charge density
at this location.[5,6] We have therefore set out to label these
positions with 13C isotopes in order to be able to study
these important positions in the molecule.
In this paper we wish to report the synthesis and charac-
terisation of [4,4Ј-13C2]astaxanthin. The presented scheme
allows for the labelling of all positions and combinations in
the six-membered ring and, with minor modifications, of
all carbons of the C15-end groups. Moreover, this synthetic
scheme can be used to label a great number of other carot-
enoids. The results from the resonance Raman and solid
state NMR spectra of the α-crustacyanin reconstituted with
the (4,4Ј) labelled astaxanthin, will be the subject of separ-
ate articles.[5,6,9]
α-Crustacyanin is the carotenoprotein complex of the
lobster, which is responsible for the deep blue colour of the
lobsterЈs carapace (λmax ϭ 632 nm).[1] The 320 kDa protein
complex is an octamer of dimers with each of the dimers
containing two noncovalently bound astaxanthin molecules
as chromophores.[1,2]
Upon boiling the lobster, the protein complex denatures
and the astaxanthin chromophore is released, giving the
lobster the red colour of free astaxanthin (λmax ϭ 469 nm).
The binding of the astaxanthin in α-crustacyanin results in
a large bathochromic shift[1] in the absorption spectrum of
5500 cm–1. For explaining this large shift, detailed informa-
tion on the structure of the chromophore in the active site
and information on the noncovalent interactions between
the chromophore and the protein is necessary. The method
of choice to obtain structural information at the atomic
level of the chromophore in the active site in chromoprote- Synthesis
ins is to prepare specific isotopically enriched chromoph-
ores, introduce them in the active site of the protein[3] and
For the preparation of the enriched astaxanthin, we use
study these isotopically (13C, 2H) labelled systems with non- the convergent C15 ϩ C10 ϩ C15 scheme to construct the
invasive physical techniques such as solid state 13C NMR C40-carotenoid. The central C10-unit of this Scheme is 2,7-
and laser resonance Raman spectroscopy. Astaxanthins dimethylocta-2,4,6-triene-1,8-dial. By the means of
a
specifically labelled with 13C in the central C10-part have double Wittig reaction with two identical C15-phosphonium
previously been synthesised by us.[4] These astaxanthins salts the C10-synthon is converted in one step into the la-
have been reconstituted into α-crustacyanin and have been belled carotenoid (Scheme 2).
studied with solid state 13C NMR,[5–7] resonance Raman
and Fourier Transform Raman spectroscopy.[8]
We have developed a scheme for specifically labelling the
end groups of astaxanthin (1). For this, the C15-phosphon-
It is known that chemical modification of the end groups ium salt 2 is prepared in an isotopically enriched form (see
of astaxanthin prevents reconstitution with the apoprotein Scheme 1). The procedure is based on the schemes that have
to form the coloured protein complex: both oxo-groups at been used to prepare vitamin A and vitamin A derivatives
the positions 4 and 4Ј need to be present.[1] It is thought isotopically labelled in the six-membered rings. The C10-
that the oxo-groups undergo an interaction with the protein compound 3,7-dimethyl-2,6-octadienenitrile (7) is prepared
complex: the working hypothesis is that in the protein both by a C5 ϩ C2 ϩ C1 ϩ C2 scheme, starting from 3,3-di-
methylallyl bromide (4), acetonitrile and methyllithium, by
the method of Courtin et al.[10,11] The open-chain com-
[a]
Unilever Research Vlaardingen,
P. O. Box 114, 3130 AC Vlaardingen, The Netherlands
pound 7 is subsequently cyclized to the desired six-mem-
[b]
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden
bered ring, 2,6,6-trimethyl-2-cyclohexenecarbonitrile (8), by
University,
P. O. Box 9502, 2300 RA, Leiden, The Netherlands
the method described by Oh et al.,[12] with some modifica-
Eur. J. Org. Chem. 2000, 829Ϫ836
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0305Ϫ0829 $ 17.50ϩ.50/0
829

F. J. H. M. Jansen, J. Lugtenburg
FULL PAPER
Scheme 1. Synthesis of the C15-phosphonium salt 2; when (2-13C)acetonitrile is used as starting material, (4-13C)phosphonium salt 2a
is obtained
tions in the workup procedure; this cyclization reaction has workup procedure, it proved to be important to keep the
also been used by Groesbeek et al.[13] to prepare vitamin A conditions acidic to avoid a double bond shift to the β-
isotopically labelled in the end groups. After these steps, the position. After purification, the cyclonitrile is obtained as
scheme diverges from the scheme for the synthesis of vit- a mixture of α-cyclonitrile (90–95%) and β-cyclonitrile (5–
amin A, with the introduction of the carbonyl and hydroxyl 10%) in an overall yield of 86%. The α/β mixture cannot be
groups and the chain elongation step to give the C15-com- separated by column chromatography and so the mixture is
pound.
used in the following steps. The double bond of nitrile 8 is
In the first step (see Scheme 1), acetonitrile is coupled epoxidised with m-chloroperbenzoic acid (MCPBA) to give
to the commercially available 3,3-dimethylallyl bromide (1- the epoxide 9. The epoxide 9 is easily opened with LDA: the
bromo-3-methylbut-2-ene; 4) giving 5-methyl-4-hexenenitr- relatively acidic proton adjacent to the nitrile is abstracted,
ile (5) in 71% yield. The nitrile 5 is converted into 6-methyl- followed by the opening of the epoxide, giving a double
5-hepten-2-one (6) by treatment with methyllithium to give bond in the β-position and a deprotonated hydroxyl group
ketone 6 in 70% yield. A chain-elongation reaction with the at the 4-position. The alcohol 10 is obtained by protonation
anion of diethyl cyanomethylphosphonate gives the C10-ni- during workup and is oxidised with PCC in dichlorome-
trile 7 in 89% yield. The open-chain nitrile 7 is then cyclised thane, without purification, to give 2,6,6-trimethyl-3-oxo-1-
to the cyclic C10-nitrile 8. The ring closure mainly gives the cyclohexenecarbonitrile (11) in 61% yield based on the α-
nitrile with the double bond in the α-position. In the cyclonitrile 8 (3 steps). At this stage the side-product of the
830
Eur. J. Org. Chem. 2000, 829Ϫ836

Synthesis and Characterization of all-E-(4,4Ј-13C2)Astaxanthin
FULL PAPER
cyclization reaction, the β-cyclonitrile, which has remained of pure all-E (4,4Ј-13C2)astaxanthin (1a) (46 mg) was ob-
unchanged during the MCPBA oxidation, epoxide opening tained (yield: 28%, based on the phosphonium salt 2a).2
and PCC oxidation, is separated from the product 11.
The ketone 11 is protected as the acetal of ethylene glycol Characterization
12. The mixed ortho formate ester of ethylene glycol and
Chromatography and UV/Vis Spectroscopy
methanol formed in this reaction is removed by stirring the
product mixture for five minutes in acidified acetone. Under
these conditions the acetal 12 is stable, while the orthoester
is quickly hydrolyzed. C10-Acetalnitrile 12 is obtained in
84% yield. The nitrile 12 is reduced with Dibal-H, giving
the aldehyde 13 in 72% yield. The C10-aldehyde 13 is sub-
sequently elongated by reacting it in an HWE reaction with
the anion of the C5-phosphonate 14 (phosphonate 14 is pre-
pared by coupling the anion of commercially available trie-
thyl phosphonoacetate with chloroacetone, which is also
commercially available; the resulting C5-chloroester is con-
verted into 14 by refluxing it with triethyl phosphite in an
Arbuzov reaction, yielding the C5-phosphonate).[14] The
C15-ester 15 is obtained in 80% yield. The ester 15 is re-
duced to the alcohol by Dibal-H giving the C15-alcohol 16
in 52% yield: this reaction is carried out at low temperature
since the acetal group is highly sensitive to reduction to
the corresponding ether.[15,16] The 4-acetal group is easily
removed by stirring for a few minutes in acidified acetone
(yield: 97%). The 3-hydroxyl group is introduced by oxida-
tion of the enolate of 17. Two equivalents of LDA are used
to deprotonate the primary alcohol and to form the enolate
of 17. The enolate is reacted with the commercially available
(ϩ)-(dichlorocamphorylsulforyl)oxaziridine; no chiral re-
duction is observed due to the highly basic conditions and
the relatively high temperature needed to convert anion
17.[17] The racemic α-hydroxy ketone 18 is obtained in
69% yield.
The purity of the astaxanthins was checked by thin layer
chromatography. The synthetic astaxanthins showed one
spot (detection with visible and UV light) with the same Rf
value as astaxanthin from natural sources. Both unlabelled
1 and the labelled astaxanthin 1a have a single, solvent-
dependent absorption maximum in the UV/Vis spectra:
λmax (n-hexane) ϭ 469 nm and λmax (chloroform) ϭ
490 nm. These values are in excellent agreement with literat-
ure values.[19] They indicate that the synthetic astaxanthin
is in the all-E form: the presence of Z-isomers would lower
the absorption maximum.[19]
Mass Spectrometry
The electron impact mass spectrum of naturally occur-
ring astaxanthin has its molecular ion peak at 596 mass
units. The molecular ion peak of the 13C2 enriched astaxan-
thin (1a) is shifted two mass units to 598 mass units because
of the presence of the two isotopes. The double-focus mass
spectra were recorded for the 13C2 enriched astaxanthin
(1a). The mass found by peak matching of the molecular
ion peak is in excellent agreement with the calculated value
12
of 598.3927 for 13C2 C38H52O4; the value found for [4,4Ј-
13C2]astaxanthin (1a) is 598.3942.
The determination of the isotope enrichment of the la-
belled astaxanthin (1a) was only possible with low accuracy
(isotope enrichment Ͼ95%) from the [Mϩ – 2] peak (15%)
of the fragment from which hydrogen is eliminated. The
high isotopic enrichment of Ͼ95% was confirmed by NMR
spectroscopy (see below).
The final conversion into the phosphonium salt is ac-
complished in two steps: first, the primary alcohol of 18
is selectively converted into the allylic bromide by HBr in
dichloromethane. After workup with brine and extraction
with ethyl acetate, the excess of HBr is removed with 1,2-
epoxybutane. The reaction of the bromide with triphenyl-
1H NMR Spectroscopy
The 1H NMR spectrum (at 300 MHz) was used to deter-
phosphane in ethyl acetate is carried out in the presence of mine the purity of the astaxanthins and the positions of the
1
1,2-epoxybutane to prevent the degradation of the un- label in the 13C2 astaxanthin (1a). The H NMR spectrum
changed bromide.[18] After an overnight reaction, the C15- of unlabelled astaxanthin (1) confirms the high purity and
phosphonium salt 2 is formed as a white solid in 62% yield the all-E structure. Within experimental error, no Z-isomers
based on 18.
could be detected. The spectral values determined are ident-
The reaction scheme was first optimised using unlabelled ical with the published data of Englert et al.[19] The signal
material. The scheme was applied in the synthesis of the (4- of H-14/H-14Ј overlaps with the doublet H-10/H-10Ј and
13C)C15-phosphonium salt 2a: starting from (2-13C)aceto- the signal of H-15/H-15Ј overlaps with the double doublet
nitrile (2 g) and 3,3-dimethylallyl bromide (4), (4-13C)C15- of H-11/H-11Ј: due to this overlap the coupling constants
phosphonium salt 2a (0.32 g) was obtained. The overall with these protons could not be determined from spectral
yield of phosphonium salt relative to acetonitrile 2a is 1.2% simulation of the AAЈBBЈ spectra. The synthetic astaxan-
(16 steps).
thins consist of a mixture of 25% (3R,3ЈR), 50% (3R,3ЈS)
The 4-13C labelled phosphonium salt 2a was used in the and 25% (3S,3SЈ); as expected, no differences between the
synthesis of (4,4Ј-13C2)astaxanthin (1a) (see Scheme 2). Two different stereoisomers were observed.
equivalents are coupled with the C10-dialdehyde 3 in a
The olefinic part of the spectrum is identical to unla-
double Wittig reaction.[18] The (4,4Ј-13C2)astaxanthin (1a) belled astaxanthin (1) since no coupling between the 4- and
was purified by a combination of column chromatography 4Ј-carbonyl carbons with the olefinic protons are observed.
and repeated crystallisation from dichloromethaneme-
thanol and dichloromethane/n-hexane. In this way, a sample tons in the ring confirm the position and high incorpora-
Additional couplings between the 13C labels and the pro-
Eur. J. Org. Chem. 2000, 829Ϫ836
831

F. J. H. M. Jansen, J. Lugtenburg
FULL PAPER
Scheme 2. Synthesis of (4,4Ј-13C2)astaxanthin (1a) from (4-13C)phosphonium salt (2a); labelled positions are marked with an asterisk
tion of the labels. A three-bond coupling (3J ϭ 2.9 Hz) of
Discussion
the 13C label at the 4- and 4Ј-position splits the triplet of
the axial H-2 and H-2Ј. The triplet of the equatorial H-2
In this article the synthesis of racemic astaxanthin has
been described. In contrast to both the oxo groups, the hy-
droxyl groups at the positions 3 and 3Ј, which induce chiral-
ity in the molecule, are not essential for binding and colour
and H-2Ј is split by a three-bond coupling (3J ϭ 8.2 Hz);
the coupling of the equatorial proton, which lies in a plane
with C-4 and C-3, is larger than the coupling with the axial
proton, which is rotated out of the plane with a smaller
shift, nor does the stereoconfiguration: all three isomers
dihedral angle between C-3, C-4 and H-2. The signal of the
(RR, SS and RS) bind in the protein complex.[1] Because
of the great distance between the two stereocentres, no dif-
ferences in the spectra of these stereoisomers are ob-
5- and 5Ј-methyl groups (18 and 18Ј) is split by a three-
bond 13C-1H coupling of 3.4 Hz. A three-bond 13C-1H
coupling of 3.8 Hz is observed between the proton of the 3-
served.[20–22] For these reasons, in first instance a racemic
and 3Ј-hydroxyl group resulting in a double doublet. The
mixture of astaxanthin has been prepared. If chirality will
prove to be important from the spectroscopy experiments,
proton at the positions 3 and 3Ј shows four couplings: three
1H-1H couplings (with H-2eq, H-2ax and 3-OH) and one
the isomers can be separated according to a literature
13C-1H coupling of 2.1 Hz. Table 1 summarises these JCH
method.[10]
couplings.
The synthetic scheme, described here has been used to
prepare all-E (4,4Ј-13C2)astaxanthin (1a) starting from com-
mercially available 99% 13C enriched (2-13C)acetonitrile.
Table 1. JCH coupling constants from the 13C labelled astaxanthin
1
The same synthetic scheme, with some modifications, can
also be used to label the C15-phosphonium salt (2) at any
position or combination of positions. The positions 4, 5, 6
and 7 can be 13C enriched using labelled acetonitrile. The
starting material 3,3-dimethylallyl bromide (4) can be pre-
pared in labelled form starting from acetone and ethyl bro-
moacetate.[23] When labelled 4 is used in this scheme
(Scheme 1) the positions 1, 2, 3, 16, 17 and any combination
of positions in the ring can be labelled with 13C. In
Scheme 1, the 5-methyl carbon is introduced using methylli-
(1a) as determined by H NMR spectroscopy
Coupled protons
H-3/H-3Ј
H-2eq/H-2Јeq
H-2ax/H-2Јax
3-OH/ 3Ј-OH
H-18/H-18Ј
J (Hz)
(4,4Ј-13C2)astaxanthin (1a)
2J ϭ 2.1
3J ϭ 8.2
3J ϭ 2.9
3J ϭ 3.8
3J ϭ 3.4
13C NMR Spectroscopy
The 13C{1H} NMR spectra (at 50 MHz) of unlabelled thium. Methyllithium is not available in labelled form; to
astaxanthin (1) and the 13C2 enriched astaxanthin (1a) have introduce a 13C label at the 5-Me position, methylmagnes-
been recorded. In the spectrum of naturally occurring as- ium iodide, prepared in situ from commercially available
taxanthin 20 peaks are observed. The chemical shift values 99% (13C)methyl iodide and magnesium, can be used. For
found are in exact agreement with the values found by En- labelling the positions 8, 9, 10, 11 and 19 (9-CH3), the reac-
glert et al. for all-E astaxanthin.[19] 13C NMR spectroscopy tions that have been developed for the preparation of iso-
is the method of choice to determine the position of the topically enriched vitamin A and vitamin A derivatives can
label and to check that no scrambling of the label has taken be used.[23]
place. Furthermore, the isomeric purity of the sample can
The same scheme can be used to prepare β-carotene and
be easily determined, since the signals of the Z-isomers res- canthaxanthin labelled in the C15-end groups. For this, the
onate at higher field. No differences in the spectra of the corresponding C15-phosphonium salts are prepared in their
diastereoisomers are observed. In the spectrum of the la- labelled form. The synthesis of the phosphonium salt of
belled astaxanthin (1a) a single peak with high intensity is canthaxanthin, the C15-hydroxy ketone 17 is not oxidized
observed at δ ϭ 200.4.
with oxaziridine but directly converted into the desired 4-
832
Eur. J. Org. Chem. 2000, 829Ϫ836

Synthesis and Characterization of all-E-(4,4Ј-13C2)Astaxanthin
FULL PAPER
leum ether; detection KMnO4) showed complete conversion of the
starting material. At 0 °C, a cooled solution of conc. HCl (60 mL)
in 140 mL water was carefully added and the mixture was stirred
at 0 °C for 2 h. Solid NaHCO3 was carefully added until the mix-
ture was neutral. The mixture was extracted with diethyl ether. The
combined organic layers were washed with saturated NaCl and
dried with MgSO4. After concentration in vacuo, flash chromato-
graphy (silica gel, 25% diethyl ether/petroleum ether) yielded 6
(4.58 g, 36.4 mmol, 70%). – 1H NMR (200 MHz, CDCl3): δ ϭ 1.62
(s, 3 H, 6Z-CH3), 1.68 (s, 3 H, 6E-CH3), 2.14 (s, 3 H, H-1), 2.25
(q, J ϭ 7 Hz, 2 H, H-4), 2.45 (t, J ϭ 7 Hz, 2 H, H-3), 5.06 (t, J ϭ
7 Hz, 1 H, H-5).[10,11]
oxo-C15-phosphonium salt. For the synthesis of the C15-
phosphonium salt for the synthesis of β-carotene labelled
in the C15-end groups, all three oxidation steps (epoxid-
ation, PCC and oxaziridine oxidation) are omitted from the
synthetic scheme. In this way, all the positions and all com-
binations of positions of the C15-phosphonium salt can be
labelled. In combination with the synthetic schemes for la-
belling the central C10-dialdehyde 3,[4,23] astaxanthin (1), β-
carotene and canthaxanthin can now be labelled at any po-
sition or combination of positions.
(3-13C)6 (6a): Similarly 5a (4.31 g, 39.2 mmol) yielded 6a (3.18 g,
25.1 mmol, 82%). – 1H NMR (200 MHz, CDCl3), as for 6; in addi-
Experimental Section
1
tion: δ ϭ 2.45 (dt, JCH ϭ 126 Hz, JHH ϭ 7 Hz, 2 H, H-3). – 13C
All experiments were carried out in a dry nitrogen atmosphere. Re-
action vessels were flame-dried under nitrogen prior to use. The
astaxanthins were handled in dim red light. The following solvents
were distilled prior to use: THF was freshly distilled from LiAlH4;
n-pentane was distilled from P2O5 and stored over sodium wire.
Petroleum ether refers to low-boiling petroleum ether 40–60 °C. –
For column chromatography, Merck silica gel 60, (230–400 Mesh)
was used. – TLC analyses were performed on Schleicher Schuell
TLC-plates F1500/LS 254. Evaporation of solvents was carried out
in vacuo (1.4 ϫ 103 Pa). – 1H NMR spectra were recorded on a
Jeol FX-200, a Bruker WM-300 spectrometer or a Bruker AM-600
NMR (50.1 MHz, CDCl3): δ ϭ 43.7 (C-3).
3,7-Dimethyl-2,6-octadienenitrile (7): The mineral oil of NaH
(1.9 g, 60% in mineral oil, 47 mmol) was removed by washing three
times with petroleum ether: in the reaction flask, 20 mL dry petro-
leum ether was added and the suspension was stirred for a few
seconds. After the NaH had settled, the petroleum ether was care-
fully removed with a pipette. After removing the remaining petro-
leum ether under a stream of nitrogen, 150 mL THF was added.
The suspension was cooled to 0 °C and diethyl cyanomethylphos-
phonate (9.0 g, 51 mmol) in 35 mL THF was added dropwise. The
reaction mixture was stirred at room temperature until all the NaH
had disappeared. At 0 °C 6 (4.58 g, 36.4 mmol) in 20 mL THF was
slowly added. The mixture was stirred for an additional 2 h at room
temperature. TLC analysis (25% diethyl ether/petroleum ether; de-
tection KMnO4) showed complete conversion of the starting mat-
erial. Workup was accomplished by adding saturated NH4Cl and
extracting the mixture twice with diethyl ether. The combined or-
ganic layers were washed with saturated NaCl and dried with
MgSO4. Flash chromatography (silica gel, 25% diethyl ether/petro-
leum ether) yielded 7 (4.84 g, 32.5 mmol, 89.4%) as a mixture of 2-
E/2-Z isomers. – 1H NMR (200 MHz, CDCl3) of E-isomer: δ ϭ
1.60 (s, 3 H, 7Z-CH3), 1.69 (s, 3 H, 7E-CH3), 2.05 (s, 3 H, 3-CH3),
2.19 (br s, 4 H, H-4/H-5), 5.03 (m, 1 H, H-6), 5.10 (s, 1 H, H-
2).[10,11]
1
using tetramethylsilane (TMS; 0 ppm) as internal standard. – H-
noise decoupled 13C NMR spectra were recorded on a Jeol FX-
200 at 50.1 MHz. NMR data are reported for purified all-E iso-
mers. – UV/Vis spectra were recorded with a Varian DMS 200 spec-
trophotometer. – Mass spectra were recorded on a V.G. Micromass
ZAB-2HF mass spectrometer. The experimental conditions and
spectral assignments are given for the unlabelled compounds. For
labelled compounds, only the changes relative to the corresponding
unlabelled compounds are given. [2-13C]Acetonitrile was purchased
from Cambridge Isotope Laboratories U.S.A. All other reagents
were purchased from Aldrich or Acros.
5-Methyl-4-hexenenitrile (5): To a solution of acetonitrile (3.0 g,
73.2 mmol) in 75 mL THF at –70 °C was added nBuLi (46 mL, 1.6
, 75.2 mmol) dropwise with a syringe. After 15 min 3,3-dimethyl-
allyl bromide (1-bromo-3-methyl-but-2-ene; 4) (12.6 g, 80 mmol,
95%) in 50 mL THF was added dropwise at the same temperature.
After addition, the reaction mixture was allowed to warm to room
temperature. Workup was accomplished by adding saturated
NH4Cl and extraction with diethyl ether. The combined organic
layers were washed with saturated NaCl and dried with MgSO4.
After concentration in vacuo, the crude product was purified by
column chromatography (silica gel; 20% diethyl ether/petroleum
ether; TLC-detection KMnO4) yielding 5 (4.31 g, 39.2 mmol, 82%)
(4-13C)7 (7a): Similarly, 6a (3.18 g, 25.1 mmol) yielded 7a (1.90 g,
12.7 mmol, 51%). – 1H NMR (200 MHz, CDCl3) of E-isomer, as
3
for 7; in addition: δ ϭ 2.05 (d, JCH ϭ 3.8 Hz, 3 H, 3-CH3), 2.18
1
(q, JHH ϭ 7 Hz, 2 H, H-5), 2.20 (dt, JCH ϭ 129 Hz, JHH ϭ 7 Hz,
3
2 H, H-4), 5.10 (d, JCH ϭ 6.2 Hz, 1 H, H-2). – 13C NMR
(50.1 MHz, CDCl3); δ ϭ 36.3 (C-4; Z-isomer), 38.6 (C-4; E-iso-
mer).
1
as a colourless oil. – H NMR (200 MHz, CDCl3): δ ϭ 1.66 (s, 3
2,6,6-Trimethyl-2-cyclohexenecarbonitrile (8):
A solution of 7
H, 5E-CH3), 1.73 (s, 3 H, 5Z-CH3), 2.34 (m, 4 H, H-2/H-3), 5.13
(4.84 g, 32.5 mmol) in 20 mL nitromethane was slowly added to a
solution of 5 mL concentrated sulfuric acid in 50 mL nitromethane
at 0 °C. After stirring for 30 min at 0 °C, TLC analysis (10% diethyl
ether/petroleum ether; detection KMnO4) showed complete conver-
sion of the starting material. Workup was accomplished by adding
100 mL water and extracting the reaction mixture three times with
diethyl ether. The combined organic layers were washed twice with
saturated NaCl and once with a saturated solution of sodium acet-
ate in water. After drying the organic layers with MgSO4, the solu-
(m, 1 H, H-4).[10,11]
(2-13C)5 (5a): Similarly (2-13C)acetonitrile (2 g, 48.8 mmol) yielded
5a (4.31 g, 39.2 mmol, 82%). – H NMR (200 MHz, CDCl3): δ ϭ
1
1.66 (s, 3 H, 5E-CH3), 1.73 (s, 3 H, 5Z-CH3), 2.34 (quintet, J ϭ
1
7 Hz, 2 H, H-3), 2.34 (dt, JCH ϭ 136.3 Hz, JHH ϭ 7.0 Hz, 2 H,
H-2), 5.13 (m, 1 H, H-4). – 13C NMR (50.1 MHz, CDCl3): δ ϭ
17.7 (C-2).
6-Methyl-5-hepten-2-one (6): At 0 °C, a solution of 5 (5.66 g, tion was concentrated in vacuo. Flash chromatography (silica gel,
52.0 mmol) in 80 mL diethyl ether was added dropwise to 80 mL
MeLi (1.6  solution in diethyl ether, 130 mmol). The reaction mix-
ture was stirred for 1 h at 0 °C. TLC analysis (50% CH2Cl2/petro-
10% diethyl ether/petroleum ether) yielded 4.17 g (28.0 mmol) of a
mixture of α-cyclonitrile 8 and β-cyclonitrile (86%). – 1H NMR
(200 MHz, CDCl3) of 8 (α-cyclonitrile): δ ϭ 1.04 (s, 3 H, 1-CH3),
Eur. J. Org. Chem. 2000, 829Ϫ836
833

F. J. H. M. Jansen, J. Lugtenburg
FULL PAPER
1.14 (s, 3 H, 1-CH3), 1.5–1.6 (m, 2 H, H-2), 1.83 (s, 3 H, 5-CH3),
2.05 (m, 2 H, H-3), 2.77 (br s, 1 H, H-6), 5.59 (br s, 1 H, H-4).[13]
(t, J 6.8 Hz, 2 H, H-2), 2.06 (s, 3 H, 5-CH3), 2.56 (t, J 6.8 Hz, 2
H, H-3).
(3-13C)8 (8a): Similarly, 7a (1.90 g, 12.7 mmol) yielded 8a (1.73 g,
11.5 mol, 91%). – 1H NMR (200 MHz, CDCl3) of 8a (α-cycloni-
(3-13C)11 (11a): Similarly, crude 10a (1.92 g) yielded 11a (0.91 g,
5.5 mmol, 60%) and β-cyclonitrile (0.18 g, 1.2 mmol, 13%). –
1H NMR (200 MHz, CDCl3) of 11a, as for 11; in addition: δ ϭ
3
trile), as for 8; in addition: δ ϭ 1.83 (d, JCH ϭ 5.5 Hz, 3 H, 5-
1
CH3), 5.59 (d br s, JCH ϭ 155.5 Hz, 1 H, H-4). – 13C NMR
1.95 (q, JCH ϭ 6.8 Hz, JHH ϭ 6.8 Hz, 2 H, H-2), 2.06 (d, JCH ϭ
(50.1 MHz, CDCl3); δ ϭ 124.5 (C-4).
3.4 Hz, 3 H, 5-CH3), 2.56 (dt, JCH ϭ 5.8 Hz, JHH ϭ 6.8 Hz, 2 H,
H-3). – 13C NMR (50.1 MHz, CDCl3); δ ϭ 196.4 (C-4).
2,3-Epoxy-2,6,6-trimethylcyclohexanecarbonitrile (9): A solution of
8 (4.17 g, 28.0 mmol) in 40 mL ethyl acetate, was added to a solu-
tion of m-chloroperbenzoic acid (12.4 g, 50%, 36 mmol) in 40 mL
ethyl acetate at 0 °C. The reaction mixture was stirred overnight at
room temperature. The solution was concentrated in vacuo and
200 mL petroleum ether was added. The reaction mixture was
stirred at 0 °C and triethylamine (2.7 g, 45 mmol) was slowly ad-
ded. After stirring for 10 min, the reaction mixture was filtered over
silica gel. The silica gel was washed with 25% diethyl ether/3% trie-
2,10,10-Trimethyl-4,7-dioxaspiro[4,5]dec-1-enecarbonitrile (12): To
a mixture of 11 (2.77 g, 17.0 mmol) were added trimethyl orthofor-
mate (5.0 g, 34 mmol), ethylene glycol (3.2 g, 51 mmol) and a cata-
lytic amount of p-toluenesulfonic acid. After fifteen minutes, TLC
analysis (25% diethyl ether/petroleum ether) showed complete con-
version of the starting material. The reaction mixture was poured
into a saturated NaHCO3 solution. The mixture was extracted
three times with diethyl ether. The combined organic layers were
thylamine/petroleum ether and the filtrate was concentrated in va- washed with a saturated NaCl solution, dried with MgSO4 and
cuo. In this way, 4.39 g crude product (a mixture of 9 and β-cycloni- concentrated in vacuo. The crude product was dissolved in 75 mL
trile) was obtained (95%). – Compound 9: 1H NMR (200 MHz, acetone and one drop 1  HCl was added. After 5 min at room
CDCl3): δ ϭ 1.05 (s, 3 H, 1-CH3), 1.09 (s, 3 H, 1-CH3), 1.2–1.3 (m,
2 H, H-2), 1.55 (s, 3 H, 5-CH3), 2.0 (m, 2 H, H-3), 2.75 (s, 1 H, H-
6), 3.05 (s, 1 H, H-4).
temperature, solid K2CO3 and MgSO4 were added. The solids were
filtered off and washed with diethyl ether. After removal of the
solvents in vacuo and purification by column chromatography (2%
triethylamine/25% diethyl ether/petroleum ether) 12 (3.13 g,
15.1 mmol, 89%) was obtained. – 1H NMR (200 MHz, CDCl3):
δ ϭ 1.19 (s, 6 H, 1-CH3), 1.62–1.83 (m, 4 H, H-2/H-3), 1.97 (s, 3
H, 5-CH3), 4.03 (s, 4 H, OCH2CH2O).
(3-13C)9 (9a): Similarly, 8a (1.73 g, 11.5 mol) yielded 1.53 g product
(mixture of 9a and β-cyclonitrile) (80%). – 1H NMR (200 MHz,
3
CDCl3) of 9a, as for 9; in addition: δ ϭ 1.55 (d, JCH ϭ 4.1 Hz, 3
1
H, 5-CH3), 3.05 (d, JCH ϭ 174 Hz, 1 H, H-4). – 13C NMR
(50.1 MHz, CDCl3); δ ϭ 58.6 (C-4).
(3-13C)12 (12a): Similarly, 11a (0.91 g, 5.5 mmol) yielded 12a
(1.06 g, 5.1 mmol, 92%). – 1H NMR (200 MHz, CDCl3), as for 12,
in addition: δ ϭ 1.97 (d, JCH ϭ 3.4 Hz, 3 H, 5-CH3), 4.03 (d, JCH ϭ
3.4 Hz, 4 H, OCH2CH2O). – 13C NMR (50.1 MHz, CDCl3): δ ϭ
105.5 (C-4).
3-Hydroxy-2,6,6-trimethyl-1-cyclohexenecarbonitrile (10): At –40
°C, a solution of 9 (4.39 g, 26.6 mmol) in 10 mL THF was added
dropwise to a solution of 30 mmol LDA in 50 mL THF [prepared
in situ from nBuLi (18.8 mL, 1.6  solution, 30 mmol) and diiso-
propylamine (3.2 g, 32 mmol)]. The reaction mixture was allowed to
warm to room temperature. Workup was accomplished by adding
saturated NH4Cl and extraction of the mixture with diethyl ether.
The combined organic layers were washed with saturated NaCl and
dried with MgSO4. After concentration in vacuo, 4.24 g crude
product was obtained (96.5%). – 1H NMR (200 MHz, CDCl3) of
2,10,10-Trimethyl-4,7-dioxaspiro[4,5]dec-1-enecarbaldehyde (13): A
solution of 12 (3.13 g, 15.1 mmol) in 60 mL dry petroleum ether
was cooled to –60 °C and 19.6 mL 1  (19.6 mmol) Dibal-H was
added with a syringe. The reaction mixture was allowed to warm
to room temperature during one hour. After half an hour at room
temperature, the reaction mixture was cooled to –20 °C and a
10: δ ϭ 1.15 (s, 3 H, 1-CH3), 1.20 (s, 3 H, 1-CH3), 1.3–2.0 (m, 4 homogeneous mixture of 26 g silica gel and 10 mL H2O was added.
H, H-2/H-3), 2.10 (s, 3 H, 5-CH3), 4.06 (t, J 6 Hz, 1 H, H-4).
The mixture was stirred for an additional hour at 0 °C. Solid
K2CO3 and MgSO4 were then added. Subsequently, the solids were
filtered off and thoroughly washed with diethyl ether. After concen-
tration in vacuo, the product was purified by flash chromatography
(50 % diethyl ether in petroleum ether) to yield 13 (2.67 g,
10.8 mmol, 72%). – 1H NMR (200 MHz, CDCl3): δ ϭ 1.21 (s, 6 H,
1-CH3), 1.54 –1.64 (m, 2 H, H-2), 1.75–1.82 (m, 4 H, H-3), 2.01 (s,
3 H, 5-CH3), 4.07 (s, 4 H, OCH2CH2O), 10.16 (s, 1 H, CHO).
(3-13C)10 (10a): Similarly, 9a (1.53 g, 0.92 mmol) yielded 1.92 g
1
crude product. – H NMR (200 MHz, CDCl3) of 10a, as for 10; in
addition: δ ϭ 2.10 (d, 3JCH ϭ 3.4 Hz, 3 H, 5-CH3), 4.06 (dt, 1JCH ϭ
172 Hz, JHH ϭ 6 Hz, 1 H, H-4). – 13C NMR (50.1 MHz, CDCl3):
δ ؍ 68.1 (C-4).
2,6,6-Trimethyl-3-oxo-1-cyclohexenecarbonitrile (11): A solution of
10 (4.24 g, 25.7 mmol) in 20 mL dry CH2Cl2 was added dropwise
to a suspension of pyridinium chlorochromate (PCC) (7.2 g,
33.4 mmol) in 80 mL dry CH2Cl2 at 0 °C. The reaction mixture
was allowed to warm to room temperature and stirred until TLC
analysis (80% diethyl ether/petroleum ether; detection KMnO4/UV)
showed complete conversion of the starting material (after approx-
imately 1 h). Workup was accomplished by cooling the mixture to
0 °C and adding ice-cooled diethyl ether. After stirring for 15 min,
the organic layers were decanted. This procedure was repeated until
the tar-like reduced PCC was a finely dispersed black solid. The
combined organic layers were filtered twice over silica; the silica
was rinsed with diethyl ether. Column chromatography (silica gel,
gradient 20–40% diethyl ether/petroleum ether) yielded 11 (2.77 g,
17.0 mmol, 66%) and β-cyclonitrile (0.46 g, 3.0 mmol, 12%). –
1H NMR (200 MHz, CDCl3) of 11: δ ϭ 1.34 (s, 6 H, 1-CH3), 1.95
(3-13C)13 (13a): Similarly, 12a (1.06 g, 5.1 mmol) yielded 13a
(0.55 g, 2.61 mmol, 51%). – 1H NMR (200 MHz, CDCl3), identical
as for 13, in addition: δ ϭ 2.01 (d, JCH ϭ 3.8 Hz, 3 H, 5-CH3),
4.07 (d, JCH ϭ 3.5 Hz, 4 H, OCH2CH2O). – 13C NMR (50.1 MHz,
CDCl3): δ ϭ 107.2 (C-4).
Ethyl (2E,4E)-3-Methyl-5-(2,10,10-trimethyl-4,7-dioxaspiro[4,5]dec-
1-en-1-yl)-2,4-pentadienoate (15): A 50-mL three-necked round-
bottomed flask equipped with a dropping funnel, septum inlet and
a low-temperature thermometer, was charged with diisopropylam-
ine (1.5 g, 15 mmol) and 75 mL THF. The mixture was cooled to –
20 °C in an alcohol/liquid nitrogen bath, and nBuLi (8.8 mL, 1.6
, 14 mmol) was added. At –20 °C, a solution of triethyl 3-methyl-
4-phosphonocrotonate (14) (3.7 g, 14 mmol) in 10 mL THF was
added dropwise. At 0 °C, 13 (2.67 g, 10.8 mmol) in 10 mL THF
834
Eur. J. Org. Chem. 2000, 829Ϫ836

Synthesis and Characterization of all-E-(4,4Ј-13C2)Astaxanthin
FULL PAPER
was slowly added. The reaction mixture was allowed to warm to
room temperature. After one hour, TLC analysis (25% diethyl
ether/petroleum ether) showed complete conversion of the starting
material. The reaction mixture was poured into a saturated NaCl
solution. The mixture was extracted three times with diethyl ether
and the combined organic layers were washed with saturated NaCl
and dried with MgSO4. Concentration in vacuo yielded a yellow
oil. Column chromatography (silica gel, 10% diethyl ether/petro-
leum ether/2% triethylamine) gave 15 (2.75 g, 8.59 mmol, 79%). –
1H NMR (200 MHz, CDCl3): δ ϭ 1.03 (s, 6 H, 1-CH3), 1.29 (t,
J ϭ 7.2 Hz, 3 H, OCH2CH3), 1.57–1.83 (m, 4 H, H-2/H-3), 1.66
(s, 3 H, 5-CH3), 2.33 (s, 3 H, 9-CH3), 4.02 (s, 4 H, OCH2CH2O),
4.23 (q, J ϭ 7.2 Hz, 2 H, OCH2CH3), 5.76 (s, 1 H, H-10), 6.12 (d,
J ϭ 6.1 Hz, 1 H, H-8), 6.47 (d, J ϭ 6.1 Hz, 1 H, H-7).
6.9 Hz, 2 H, H-3). – 13C NMR (50.1 MHz, CDCl3): δ ϭ 199.4
(C-4).
(2E,4E)-3-Methyl-5-(4-hydroxy-2,6,6-trimethyl-3-oxo-1-cyclohexen-
1-yl)-2,4-pentadienol (18): In a 50-mL three-necked round-bot-
tomed flask equipped with a dropping funnel, septum inlet and a
low-temperature thermometer, a solution of LDA (12.9 mmol) was
prepared at –20 °C from diisopropylamine (1.4 g, 14 mmol) dis-
solved in 150 mL freshly distilled THF and nBuLi (8.0 mL, 1.6 ,
12.9 mmol). After ten minutes at –20 °C, the solution was cooled
to –80 °C and a solution of 17 (1.00 g, 4.29 mmol) in 25 mL THF
was added dropwise. After 30 minutes, (ϩ)-(8,8-dichlorocamphor-
ylsulfonyl)oxaziridine (3.8 g, 12.9 mmol) in 25 mL THF was added.
The reaction mixture was allowed to warm to room temperature in
approximately one hour. As soon as TLC analysis (100% diethyl
ether) showed complete conversion (after approximately fifteen mi-
nutes at room temperature), the reaction mixture was worked up.
Workup was accomplished by adding saturated NH4Cl and sodium
thiosulfate. The mixture was extracted three times with diethyl
ether and the combined diethyl ether layers were washed with satur-
ated NaCl and dried with MgSO4. Concentration in vacuo gave a
mixture of (ϩ)-(8,8-dichlorocamphorylsulfonyl)imine and 18. Col-
umn chromatography (gradient: 50–100% diethyl ether/petroleum
ether), gave 18 (0.74 g, 2.96 mmol, 69%). – 1H NMR (200 MHz,
CDCl3): δ ϭ 1.18 (s, 3 H, 1-CH3), 1.30 (s, 3 H, 1-CH3), 1.81 (t,
J ϭ 13.4 Hz, 1 H, H-2ax), 1.87 (s, 3 H, 9-CH3), 1.90 (s, 3 H, 5-
CH3), 2.16 (dd, J ϭ 12.7, 5.5 Hz, H-2eq), 3.72 (s, 1 H, OH), 4.32
(q, J ϭ 13.8, 5.5 Hz, H-3), 4.36 (d, J ϭ 6.5 Hz, 2 H, H-11), 5.78
(t, J ϭ 6.5 Hz, 1 H, H-10), 6.16 (d, J ϭ 16.1 Hz, 1 H, H-7), 6.29
(d, J ϭ 16.1 Hz, 1 H, H-8).
(3Ј-13C) 15 (15a): Similarly, 13a (0.55 g, 2.61 mmol) yielded 15a
(0.73 g, 2.3 mmol, 88%). – 1H NMR (200 MHz, CDCl3), as for 15,
in addition: δ ϭ 1.66 (d, JCH ϭ 3.1 Hz, 3 H, 5-CH3), 4.02 (d, JCH ϭ
3.1 Hz, 4 H, OCH2CH2O). – 13C NMR (50.1 MHz, CDCl3): δ ϭ
107.6 (C-4).
(2E,4E)-3-Methyl-5-(2,10,10-trimethyl-4,7-dioxaspiro-
[4,5]dec-1-en-1-yl)-2,4-pentadienol (16): A solution of 15 (2.75 g,
8.59 mmol) in 150 mL dry petroleum ether was cooled to –80 °C
in an alcohol/liquid nitrogen bath and Dibal-H (1.9 mL, 1 ,
1.9 mmol) was slowly added with a syringe. The mixture was al-
lowed to warm to –20 °C. TLC analysis (50% diethyl ether/petro-
leum ether) showed complete conversion of the starting material.
A homogeneous mixture of 2.6 g silica gel and 1 mL H2O was
slowly added and the mixture was stirred for one hour at 0 °C.
Solid K2CO3 and MgSO4 were added and the solids were filtered
off and thoroughly washed with diethyl ether. After concentration
in vacuo, the product was purified by column chromatography (sil-
ica gel, gradient 50–98% diethyl ether/petroleum ether/2% triethyla-
mine), giving 1.23 g (4.4 mmol; 52%) 16. – 1H NMR (200 MHz,
CDCl3): δ ϭ 1.04 (s, 6 H, 1-CH3), 1.55–1.90 (m, 4 H, H-2/H-3),1.67
(s, 3 H, 5-CH3), 1.84 (s, 3 H, 9-CH3), 4.02 (s, 4 H, OCH2CH2O),
4.29 (d, J ϭ 6.5 Hz, 2 H, H-11), 5.64 (t, J ϭ 6.5 Hz, 1 H, H-10),
6.08 (s, 2 H, H-7/H-8).
(3Ј-13C)18 (18a): Similarly, 17a (0.31 g, 1.31 mmol) yielded 18a
1
(0.22 g, 0.88 mmol) (68%). – H NMR (200 MHz, CDCl3), as for
3
18, in addition: δ ϭ 1.81 (dt, JCH ϭ 2.7 Hz, JHH ϭ 13.4 Hz, 1 H,
3
3
H-2ax), 1.90 (d, JCH ϭ 3.8 Hz, 3 H, 5-CH3), 2.16 (ddd, JCH
ϭ
8.0 Hz, JHH ϭ 12.7, 5.5 Hz, H-2eq), 4.32 (m, coupling constants
not determined due to overlap with signal of H-11, H-3). – 13C
NMR (50.1 MHz, CDCl3): δ ϭ 200.4 (C-4).
[(2E,4E)-3-Methyl-5-(4-hydroxy-2,6,6-trimethyl-3-oxo-1-
cyclohexen-1-yl)-2,4-pentadien-1-yl]phosphonium Bromide (2): A so-
lution of 18 (0.74 g, 2.96 mmol) in 50 mL dichloromethane was co-
oled with an ice-bath to 0 °C and HBr (0.7 mL, 47%, 5.9 mmol)
was added in small portions. The reaction mixture was stirred until
TLC analysis (100% diethyl ether) showed complete conversion of
the starting material. If the reaction was not complete after half an
hour, an additional equivalent of HBr was added. The reaction
mixture was poured into a saturated NaCl solution and extracted
three times with ethyl acetate. The combined organic layers were
dried with MgSO4 and 1,2-epoxybutane was added until the solu-
tion was neutral. The solution was concentrated in vacuo to a vol-
ume of 20 mL and ethyl acetate (30 mL) and 1,2-epoxybutane
(0.5 mL) were added. Subsequently, the solution was again concen-
trated in vacuo to a volume of 20 mL. This solution, containing the
product (2E,4E)-1-bromo-3-methyl-5-(4-hydroxy-2,6,6-trimethyl-3-
oxo-1-cyclohexen-1-yl)-2,4-pentadiene, was slowly added to a solu-
tion of triphenylphosphane (0.78 g, 2.98 mmol) in 20 mL ethyl
acetate/0.2 mL 1,2-epoxybutane and the mixture was stirred at
room temperature for 24 h. The clear solution formed a white sus-
pension. TLC analysis (100% diethyl ether) showed complete disap-
pearance of the starting material. The solids were filtered off and
washed with diethyl ether. After drying in vacuo at 50 °C, 2 (1.06 g,
1.83 mmol, 62%) was obtained. – 1H NMR (600 MHz, CDCl3):
δ ϭ 1.11 and 1.25 (2s, 6 H, 1-CH3), 1.49 (d, JPH ϭ 3.5 Hz, 3 H, 9-
CH3), 1.77 (t, J ϭ 13.2 Hz, 1 H, H-2ax), 1.81 (s, 3 H, 5-CH3), 2.14
(3Ј-13C)16 (16a): Similarly, 15a (0.73 g, 2.3 mmol) yielded 16a
(0.37 g, 1.3 mmol, 88%). – 1H NMR (200 MHz, CDCl3), as for 16,
in addition: δ ϭ 1.67 (d, JCH ϭ 3.4 Hz, 3 H, 5-CH3), 4.02 (d, JCH ϭ
3.7 Hz, 4 H, OCH2CH2O). – 13C NMR (50.1 MHz, CDCl3): δ ϭ
108.1 (C-4).
(2E,4E)-3-Methyl-5-(2,6,6-trimethyl-3-oxo-1-cyclohexen-
1-yl)-2,4-pentadienol (17): To a solution of 16 (1.23 g, 4.4 mmol) in
125 mL acetone was added one drop of 1  HCl. TLC analysis
(50% diethyl ether/petroleum ether) showed complete conversion of
the starting material after a few minutes. Workup was accomp-
lished by adding solid K2CO3 and MgSO4. The solids were filtered
off and washed with diethyl ether. Concentration in vacuo gave the
crude product, which was purified by column chromatography (sil-
ica gel, 100% diethyl ether) to give 17 (1.00 g, 4.3 mmol, yield
97%). – 1H NMR (200 MHz, CDCl3): δ ϭ 1.17 (s, 6 H, 1-CH3),
1.83 (s, 3 H, 5-CH3), 1.86 (t, J ϭ 6.9 Hz, 2 H, H-2), 1.88 (s, 3 H,
9-CH3), 2.51 (t, J ϭ 6.9 Hz, 2 H, H-3), 4.35 (d, J ϭ 6.9 Hz, 2 H,
H-11), 5.76 (t, J ϭ 6.9 Hz, 1 H, H-10), 6.21 (s, 2 H, H-7/H-8).
(3Ј-13C)17 (17a): Similarly, 16a (0.37 g, 1.33 mmol) yielded 17a
(0.31 g, 1.31 mmol, 98%). – 1H NMR (200 MHz, CDCl3), as for
17, in addition: δ ϭ 1.86 (m, JCH not determined due to overlap),
1.83 (d, JCH ϭ 3.4 Hz, 3 H, 5-CH3), 2.51 (q, JCH ϭ 6.9 Hz, JHH ϭ
Eur. J. Org. Chem. 2000, 829Ϫ836
835

F. J. H. M. Jansen, J. Lugtenburg
FULL PAPER
(dd, J ϭ 13.2, 5.6 Hz, 1 H, H-2eq), 3.62 (d, J ϭ 1.9 Hz, 1 H, 3-
Acknowledgments
OH), 4.29 (ddd, J ϭ 13.2, 5.6, 1.9 Hz, 1 H, H-3), 5.53 (q, JHH
7.7 Hz, JPH ϭ 7.7 Hz, 1 H, H-10), 4.99 (dt, JHH ϭ 15.7, 8.3 Hz,
JPH ϭ 15.7 Hz, 1 H, H-11), 5.11 (dt, JHH ϭ 15.7, 7.7 Hz, JPH
ϭ
We would like to thank Dr. P. P. Lankhorst of Gist Brocades for
making a sample of (3R,3ЈR)-astaxanthin available to us, A. W. M.
Lefeber and C. Erkelens for their assistance in recording the 300-
MHz NMR spectra and R. van der Hoeven for the recording of
the mass spectra. This work was sponsored by the Netherlands
Foundation for Chemical Research (SON), with financial aid from
the Netherlands Organisation for Scientific Research (NWO). Ad-
ditional financial support was given by the European Economic
Community (grant number SC1* CT92 0813).
ϭ
15.7 Hz, 1 H, H-11), 6.06 (d, J ϭ 16.5 Hz, 1 H, H-7), 6.14 (d, J ϭ
16.5 Hz, 1 H, H-8), 7.64–7.92 (m, 15 H, Phenyl).
(3Ј-13C)2 (2a): Similarly, 18a (0.22 g, 0.88 mmol) yielded 2a (0.32 g,
0.56 mmol, 63%). – 1H NMR (200 MHz, CDCl3), as for 2, in addi-
tion: δ ϭ 1.77 (dt, JCH ϭ 3.0 Hz, JHH ϭ 13.2 Hz, 1 H, H-2ax), 1.81
(d, JCH ϭ 3.3 Hz, 3 H, 5-CH3), 2.14 (ddd, JCH ϭ 8.2 Hz, JHH
ϭ
13.2, 5.6 Hz, 1 H, H-2eq), 3.62 (dd, JCH ϭ 3.7 Hz, JHH ϭ 1.9 Hz,
1 H, 3-OH), 4.29 (dddd, JCH ϭ 1.9 Hz, JHH ϭ 13.2, 5.6, 1.9 Hz, 1
H, H-3). – 13C NMR (50.1 MHz, CDCl3): δ ϭ 200.3 (C-4).
[1]
G. Britton, G. M. Armitt, S. Y. M. Lau, A. K. Patel, C. C.
Shone, in Carotenoid Chemistry and Biochemistry (Eds.: G.
Britton, T. W. Goodwin), Pergamon Press, Oxford, 1994.
[2]
(all-E)-3,3Ј-Dihydroxy--carotene-4,4Ј-dione (Astaxanthin, 1): With
exclusion of light, dialdehyde 3 (200 mg, 1.22 mmol) and C15 Wittig
salt 2 (1.66 g, 2.89 mmol) in 1,2-epoxybutane (15 mL) were heated
under reflux (63 °C) for 20 h under a nitrogen atmosphere. TLC
analysis (50% diethyl ether/petroleum ether) showed complete con-
version of the dialdehyde 3. All subsequent handling of the astax-
anthins was performed in dim red light and with exclusion of oxy-
gen. The 1,2-epoxybutane was removed by distillation and abs. eth-
anol added. The crystallised astaxanthin was filtered off and
washed with cold ethanol. The astaxanthin was washed off the fil-
ter with dichloromethane. The dichloromethane was removed by
distillation and n-heptane added. The suspension of astaxanthin
crystals was then heated under reflux (98 °C) for 20 h under a nitro-
gen atmosphere. After cooling to 0 °C, the astaxanthin was filtered
off and washed with n-pentane. Repeated recrystallization from
CH2Cl2/MeOH and CH2Cl2/n-hexane afforded 398 mg (0.67 mmol)
pure all-E astaxanthin (55%). – M.p. 216–218 °C (ref.[3] 217–219
°C). – UV/Vis: λ ϭ 469 nm (n-hexane), 490 nm (CHCl3). – MS (EI;
70 eV); m/z (%): 488 (1.9), 489 (0.9), 490 (10.7), 491 (2.2), 492 (1.0),
502 (2.0), 503 (0.9), 504 (10.6), 505 (2.2), 506 (1.0), 594 (28.2), 595
(12.2), 596 (100; Mϩ ), 597 (43.0), 598 (16.1). – 1H NMR
(600 MHz, CDCl3; δH ϭ δHЈ): δ ϭ 1.21 ϩ 1.32 (s, 12 H, H-16/H-
17), 1.82 (t, J ϭ 13.3 Hz, 2 H, H-2ax), 1.95 (s, 6 H, H-18), 1.99 (s,
6 H, H-20), 2.00 (s, 6 H, H-19), 2.16 (dd, J ϭ 5.7, 12.6 Hz, 2 H,
H-2eq), 3.70 (d, J ϭ 1.8 Hz, 2 H, 3-OH), 4.33 (ddd, J ϭ 1.8, 5.7,
13.9 Hz, 2 H, H-3), 6.21 (d, J ϭ 16.0 Hz, 2 H, H-7), 6.30 (d, J ϭ
11.4 Hz, 2 H, H-10), 6.30–6.32 (m, 2 H, H-14), 6.43 (d, J ϭ
16.0 Hz, 2 H, H-8), 6.48 (d, J ϭ 14.8 Hz, 2 H, H-12), 6.66 (dd, J ϭ
11.4, 14.8 Hz, 2 H, H-11), 6.64–6.68 (m, 2 H, H-15). – 13C NMR
(50.1 MHz, CDCl3; δC ϭ δCЈ): δ ϭ 12.6 (C-19), 12.8 (C-20), 14.0
(C-18), 26.2 and 30.7 (C-16/C-17), 36.8 (C-1), 45.4 (C-2), 69.2 (C-
3), 123.3 (C-7), 124.6 (C-11), 126.8 (C-5), 130.6 (C-15), 133.8 (C-
14), 134.6 (C-9), 135.2 (C-10), 136.8 (C-13), 139.7 (C-12), 142.3 (C-
8), 162.2 (C-6), 200.4 (C-4).[19]
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[13]
[14]
[15]
[16]
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E. Becher, R. Albrecht, K. Bernhard, H. G. W. Leuenberger,
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[19]
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[20]
(4,4Ј-13C2)Astaxanthin (1a): Similarly, 3 (33 mg, 0.2 mmol) and 2a
(0.32 g, 0.56 mmol) yielded 46 mg (0.08 mmol) of all-E 1a (38%
based on 3; 28% based on 2a). – UV/Vis: λ ϭ 469 nm (n-hexane),
[21]
K. Bernhard, G. Englert, H. Mayer, R. K. Müller, A. Rüttim-
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[22]
490 nm (CHCl3).
– Exact mass: 598.3942 (calculated for
G. Englert, F. Kienzle, K. Noack, Helv. Chim. Acta 1977, 60,
1209–1219.
F. J. H. M. Jansen, J. Lugtenburg, in Carotenoids Volume II:
13C2 C38H52O4: 598.3927). – MS (EI; 70 eV); m/z (%): 490 (1.7),
491 (1.1), 492 (4.1), 493 (1.7), 494 (1.8), 504 (1.6), 505 (1.0), 506
(5.0), 507 (1.8), 508 (1.9), 596 (15.7), 597 (8.9), 598 (100; Mϩ ), 599
(44.8), 601 (25.6). – 1H NMR: see characterisation. – 13C NMR
(50.1 MHz, CDCl3): δ ϭ 200.4 (C-4/C-4Ј).
12
[23]
Synthesis (Eds.: G. Britton, S. Liaaen-Jensen, H.-P. Pfander),
Birkhaüser Verlag, Basel, 1996, 233–258.
Received July 6, 1999
[O99398]
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Eur. J. Org. Chem. 2000, 829Ϫ836