Synthesis of apocarotenoids by acyclic cross metathesis and characterization as substrates for human retinaldehyde dehydrogenases

Article abstract of DOI:10.1016/j.tet.2018.03.050

A new synthesis of three apocarotenoids, namely 14′-apo-β-carotenal, 12′-apo-β-carotenal and 10′-apo-β-carotenal, has been achieved that is based on the acyclic cross-metathesis of the hexaene derived from retinal and the corresponding partners. These compounds can be enzymatically converted to their carboxylic acids by the human aldehyde dehydrogenases involved in retinaldehyde oxidation. Their kinetic parameters suggest that these enzymes might play a role in the physiological metabolism of apocarotenoids.

Full text of DOI:10.1016/j.tet.2018.03.050

Accepted Manuscript
Synthesis of apocarotenoids by acyclic cross metathesis and characterization as
substrates for human retinaldehyde dehydrogenases
Marta Domínguez, Raquel Pequerul, Rosana Alvarez, Joan Giménez-Dejoz, Eszter
Birta, Sergio Porté, Ralph Rühl, Xavier Parés, Jaume Farrés, Angel R. de Lera
PII:
S0040-4020(18)30323-5
10.1016/j.tet.2018.03.050
TET 29390
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 26 February 2018
Accepted Date: 23 March 2018
Please cite this article as: Domínguez M, Pequerul R, Alvarez R, Giménez-Dejoz J, Birta E, Porté S,
Rühl R, Parés X, Farrés J, de Lera AR, Synthesis of apocarotenoids by acyclic cross metathesis and
characterization as substrates for human retinaldehyde dehydrogenases, Tetrahedron (2018), doi:
10.1016/j.tet.2018.03.050.
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Synthesis of apocarotenoids by acyclic cross
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for human retinaldehyde dehydrogenases
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Marta Domínguez,a Raquel Pequerul,b Rosana Alvarez,a Joan Giménez-Dejoz,b Eszter Birta,c Sergio Porté,b
Ralph Rühl,c,d Xavier Parés,b Jaume Farrésb,* and Angel R. de Leraa,*

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Synthesis of apocarotenoids by acyclic cross metathesis and characterization as
substrates for human retinaldehyde dehydrogenases
Marta Domínguez,a Raquel Pequerul,b Rosana Alvarez,a Joan Giménez-Dejoz,b Eszter Birta,c Sergio Porté,b
Ralph Rühl,c,d Xavier Parés,b Jaume Farrésb,* and Angel R. de Leraa,*
a Departamento de Química Orgánica, Facultade de Química, Universidade de Vigo, CINBIO and IBIV, Campus As Lagoas-Marcosende, 36310 Vigo, Spain.
Fax: +34 986 811940; Tel: +34 986 812316; E-mail:qolera@uvigo.es
b Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain. E-mail: jaume.farres@uab.cat
c Department of Biochemistry and Molecular Biology, University of Debrecen, Debrecen, H-4028 Kassai út 26, PO Box 400, Hungary.
d Paprika Bioanalytics BT, Debrecen, H-4032.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A new synthesis of three apocarotenoids, namely 14’-apo-β-carotenal, 12’-apo-β-carotenal and
10’-apo-β-carotenal, has been achieved that is based on the acyclic cross-metathesis of the
hexaene derived from retinal and the corresponding partners. These compounds can be
enzymatically converted to their carboxylic acids by the human aldehyde dehydrogenases
involved in retinaldehyde oxidation. Their kinetic parameters suggest that these enzymes might
play a role in the physiological metabolism of apocarotenoids.
Available online
2009 Elsevier Ltd. All rights reserved
.
Keywords:
Apocarotenoids; Metabolism; Metathesis
1. Introduction
BCO2-mediated cleavage is the preferred pathway for
xanthophylls.9
Carotenoids are a family of natural compounds synthesized by
plants, microorganisms and some animals but not by humans.1
Carotenoids are partly responsible for the colour in nature and
play a key role both in the photosynthesis process and the
photoprotection of the producing organisms. They can be broadly
divided into two classes of chemical compounds: carotenes (e.g.,
β,β-carotene and lycopene) and their oxygenated derivatives
termed xanthophylls (e.g., lutein, zeaxanthin and cryptoxanthin).
Both carotenes and xanthophylls exhibit relevant physiological
functions, serving as antioxidants in lipophilic environments,2 a
property that might contribute to the prevention of certain human
diseases such as cardiovascular, ocular diseases and cancer.3, 4
The metabolism of β,β-carotene 1 to all-trans-retinal 2 (Figure
1) promoted by BCO1 is the first step in the production of the
natural retinoids (including, but not restricted to, vitamin A, 11-
cis-retinal and all-trans-retinoic acid 3) required for eliciting the
various functions of these compounds in the human body. Thus,
enzymes responsible for isomerization and changes in the
oxidation state provide the cognate ligands for receptors
implicated in vision, cell proliferation, cell differentiation,
immunity and development.4 In particular, the oxidation of all-
trans-retinal 2 promoted by aldehyde dehydrogenases (ALDHs)12
generate all-trans-retinoic acid 3, the natural hormone that binds
to and activates the retinoic acid receptors (RARs),13 members of
the nuclear receptor superfamily of ligand-inducible transcription
factors.14
Within carotenoids, the term apocarotenoid is used to
designate those with a backbone of less than 40 carbon atoms.5
Apocarotenoids are formed by the oxidative degradation of one
or both termini of carotenoids, a process that is catalyzed by
carotenoid cleavage enzymes. The oxidation products derived
from dietary β,β-carotene 1 (Figure 1) can be C20 all-trans-
retinal 2 resulting from the central C15-C15’ cleavage catalyzed
7
by BCO16, or non-symmetrical β-apocarotenoids obtained by
eccentric cleavage catalyzed by BCO2.8, 9 Carotenoid metabolism
appears to be cell compartmentalized,10 as BCO1 is a cytosolic
enzyme,11 while BCO2 has been associated with mitochondria.9

2
Tetrahedron
ACCEPTED MANUSCRIPT
Figure 1. β-apocarotenals 2,4-7 potentially formed by (enzyme-mediated) oxidative cleavage of β,β-carotene 1, and the corresponding
carboxylic acids 3, 8-11. The shorter fragments of putative BCO2 cleavage are not shown.
Apocarotenoids (such as 4-11, Figure 1) have been detected in
food and in the blood of animals,15 and the concentration of some
of them are similar to that of all-trans-retinoic acid 3. Their
application on the synthesis of retinoids27 and apocarotenoids
starting from carotenoids was described by Wojtkielewicz and
coworkers, although the products were obtained in very low
yields.28, 29 We30, 31 and others32 have also contributed to this field
with the synthesis of symmetrical and non-symmetrical
carotenoids by dimerization and cross-metathesis processes.33
17
biological functions, however, remain unclear.16, Recently, it
has been proposed that the activity of BCO2-derived metabolites
can protect against the damage induced by β,β-carotene 1. This
compound is deemed partially responsible for oxidative stress in
the mitochondria, a process that can trigger signaling pathways
related to cell survival and proliferation.17 Moreover, some
apocarotenoids, namely 10’-apo-β-carotenoic acid 10, 12’-apo-β-
carotenoic acid 9,17 14’-apo-β-carotenoic acid 8, 14’-apo-β-
carotenal 4, and 13’-apo-β-carotenone (not shown), have been
described as low-affinity agonists potentially acting as
We considered that β-apocarotenoids could also be prepared
by cross metathesis of appropriate precursors.33 We envisioned
the synthesis of β-apocarotenoids with different lengths of the
polyene chain by acyclic cross-metathesis33 starting from a
common precursor, the already known hexaene 1230 and the
complementary component functionalized as ester.30 More
specifically, we undertook the synthesis of the three β-
apocarotenoids (10’-apo-β-carotenal 4, 12’-apo-β-carotenal 5 and
14’-apo-β-carotenal 6; 8’-apo-β-carotenal 7 is a commercial
compound) that could potentially by formed by eccentric
cleavage of β,β-carotene 1 at the C9’=C10’, C11’=C12’ and
C13’=C14’ double bonds.4 Only the products of cleavage at the
C9’=C10’ bond have been characterized in mice.7 However,
since β-apocarotenoids can potentially be formed from β,β-
carotene 1 by non-enzymatic autoxidation processes, their
availability by synthesis can provide useful tools for biochemical
research. A general method for oxidation of β,β-carotene 1 with a
mixture of KMnO4 and H2O2, that provided a mixture of three
apo-carotenoids (8’-, 10’- and 12’-apo), was reported half a
century ago.34
17
endogenous antagonists of RARs,16, and also were found to
regulate other functions, such as the placental lipoprotein
biosynthesis.18
It is believed that β-apocarotenals can be a potential source of
β-apocarotenoic acids, which can be considered as vinylogues of
all-trans-retinoic acid 3, through the oxidation of the functional
group. However the enzymes involved in these transformations
have not been described. Logical candidates are aldehyde
dehydrogenases (ALDHs),12 enzymes that transform aldehydes
into carboxylic acids. Within the ALDH superfamily,
ALDH1A1, ALDH1A2 and ALDH1A3 are closely related
enzymatic forms that catalyze the oxidation of retinaldehydes.19
In order to shed some light on the metabolism and biological
functions of β-apocarotenoids, here we present an efficient
synthetic strategy for the preparation of these molecules and their
biological characterization as substrates for human ALDHs.
2. Results and discussion
Carotenoids have been traditionally synthesized following two
different strategies.4, 20 The first comprises carbonyl condensation
reactions with heteroatom-stabilized carbanions, such as Wittig,
Horner–Wadsworth–Emmons and Julia reactions, which form
Csp2=Csp2 bonds.21 The second is based on the formation of Csp2–
Csp2 bonds by palladium-catalyzed cross-coupling reactions22
(primarily Negishi, Stille, and Suzuki reactions).23 In both cases,
appropriate functionalization of the intermediates for the key
reaction is required. Recently, the olefin metathesis reaction24, 25,
2.1. Synthesis of 14’-apo-β-carotenal 4
The synthesis of 14’-apo-β-carotenal 4 started with the cross
metathesis33 of previously described hexaene 1230 and
commercial butyl (E)-but-2-enoate 13. Four different ruthenium
catalysts were tested with toluene as solvent based on the results
described by Wojtkielewicz and coworkers29 and special attention
was paid to the reaction time in order to avoid degradation of the
formed polyenes (Scheme 1). The use of Neolyst as catalyst35
was discouraging as the starting material was fully recovered
after 48 h (entry 1, Table 1). The use of Nitro-Grela catalyst36
allowed to obtain, after 7.5 h, the desired product in 29% yield
together with 15% of β,β-carotene 1, resulting from the
competing dimerization of 1230 (entry 2, Table 1).
26
has been established as one of the most general and widely
applicable synthetic methods for Csp2=Csp2 bond formation.
Despite the numerous applications of olefin metathesis reactions
in the synthesis of natural products,24 its use in the preparation of
conjugated polyene chains has been somehow limited because of
concerns about the control of site-selectivity, stereoselectivity,
and the stability of polyenes to the reaction conditions. The first

3
The 2nd generation Grubbs catalyst26, 37 proved more effective
ACCEPTED MANUSCRIPT
in the metathesis reaction (39% yield for 15), although β,β-
carotene 1 was also obtained as secondary product (entry 3, Table
1). Finally, the use of 2nd generation Hoveyda-Grubbs (HG)
catalyst38 afforded the best results, with a 62% yield of desired 15
and 16% of homodimer 1 obtained after 4.5 h (entry 4, Table 1).
The increase of the reaction time turned out to be detrimental
since it favored the cross metathesis corresponding formally to
the C11=C12 bond, with full consumption of the initially formed
apo-β-carotenoid 15 (entry 5, Table 1). Compound 15 was
readily transformed into the desired 14’-apo-β-carotenal 4 by
Dibal-H reduction followed by oxidation of 16 with MnO2 under
basic conditions (Scheme 1). In addition, saponification of ester
15 with KOH/MeOH at 70 ºC afforded 14’-apo-β-carotenoic acid
8 in 62% yield.
Scheme 2. Reagents and conditions. a) 2nd generation HG, toluene, 25 °C, 7h
(10% β,β-carotene 1, 11% starting 12, 40% methyl 12’-apo-β-carotenoate
18). b) Dibal-H, THF, -78 °C, 4h, 75%. c) MnO2, Na2CO3, CH2Cl2, 25 °C, 4h,
38%. d) KOH, MeOH, 70 ºC, 1h, 53%.
2.3. Synthesis of 10’-apo-β-carotenal 6
The anticipated component for the synthesis of 10’-apo-β-
carotenal 6, namely methyl (2E,4E)-octan-2,4,6-enoate 24, was
prepared by a six step sequence starting from methyl (2E,4E)-3-
methyl-6-oxohexa-2,4-dienoate 20. Protection of the aldehyde of
20 as dioxolane, followed by the consecutive ester reduction with
Dibal-H and MnO2 oxidation, led to aldehyde 21 in good yields.
Wittig olefination with commercially available ethyl
triphenylphosphonium iodide 22 provided triene 23 in 80% yield
as an inconsequential 1:1 mixture of double bond isomers.
Subsequent deprotection of the acetal followed by oxidation of
the trienal with MnO2 and KCN in MeOH provided ester 24 as a
mixture of isomers (Scheme 3). An excess of trienyl ester 24 was
reacted with hexaene 12 under the conditions already described,
which produced the desired methyl 10’-apo-β-carotenoate 25 in
24% yield along with 58% yield of β,β-carotene 1. A two-step
sequence of Dibal-H reduction and MnO2 oxidation of ester 25
afforded 10’-apo-β-carotenal 6 in a combined 59% yield.
Scheme 1. Reagents and conditions. a) Table 1. b) Dibal-H, THF, -78 ºC, 5h,
71%. c) MnO2, Na2CO3, CH2Cl2, 25 ºC, 5h, 88%. d) KOH, MeOH, 70 ºC, 1h,
62%.
Table 1. Olefin cross-metathesis of hexaene 12 and enoate 13.
Reagents and conditions
Yield (%)
1
14
15
1
2
3
4
5
13 (6 equiv.), Neolyst (0.15 equiv.),
toluene, 25 °C, 48 h
0
0
0
0
13 (6 equiv.), Nitro-Grela (0.15 equiv.),
toluene, 25 °C, 7.5 h
15
15
16
0
29
39
62
0
13 (6 equiv.), 2nd gen. Grubbs, toluene,
25 °C, 4.5 h
0
13 (6 equiv.), 2nd gen. Hoveyda-Grubbs
(0.15 equiv.), toluene, 25 °C, 4.5 h
13 (6 equiv.), 2nd gen. Hoveyda-Grubbs
(0.15 equiv.), toluene, 25 °C, 24 h
1
Scheme 3. Reagents and conditions: a) ethylene glycol, pTsOH, benzene, 96
ºC, 16h, 99%. b) Dibal-H, THF -78 ºC, 2h, 99%. c) MnO2, Na2CO3, THF, 25
ºC, 5h, 75%. d) ethyl triphenylphosphonium iodide 22, NaHMDS, THF, -78
ºC, 2.5h, 80%. e) pTsOH, acetone, 25 ºC, 17h, 98%. f) MnO2, KCN, MeOH,
25 ºC, 3.5h, 79%. g) 2nd generation HG, 12, toluene, 5h (58% β,β-carotene 1,
49% starting trienoate 24, 24% methyl 10’-apo-β-carotenoate 25). h) Dibal-H,
THF, -78 ºC. i) MnO2, Na2CO3, CH2Cl2, 59% (2 steps).
36
2.2. Synthesis of 12’-apo-β-carotenal 5
The previously optimized reaction conditions were next
applied to the synthesis of 12’-apo-β-carotenal 5. The reaction of
an excess of 17 with hexaene 12 afforded methyl 12’-apo-β-
carotenoate 18 in 40% yield along with the product of
homodimerization 1 (10%). Some reactant hexaene 12 was also
recovered (11%). The geometry of the newly formed double
bond was confirmed by NOE experiments. Dibal-H reduction of
ester 18 and subsequent oxidation with MnO2 under basic
conditions furnished 12’-apo-β-carotenal 5 (Scheme 2). Ester
hydrolysis as described led to 12’-apo-β-carotenoic acid 9 in 53%
yield.
2.4. ALDH enzymatic assay
The activity of ALDH1A enzymes with apo-β-carotenals 4
and 5 (the study with 6 was not completed due to the low overall
yield of the synthetic sequence) was monitored using an
improved HPLC-based method for the simultaneous detection of
substrates and reaction products. In all cases, concomitant
consumption of apo-β-carotenals 4 and 5, and production of the
corresponding apo-β-carotenoic acids 8 and 9, respectively, were
observed (see Figure 2 and Supporting Information). Thus,
ALDH1A enzymes turned out to be active with 14’- and 12’-apo-
β-carotenals (4 and 5).

4
Tetrahedron
ACCEPTED MANUSCRIPT
Table 2. Kinetic constants of human ALDH1A enzymes with apo-β-carotenals.
ALDH1A1
ALDH1A2
ALDH1A3
Km
(ꢀM)
kcat
kcat/Km
Km
(ꢀM)
kcat
kcat/Km
Km
(ꢀM)
kcat
kcat/Km
(min−1)
(mM−1—min−1)
(min−1)
(mM−1—min−1)
(min−1)
(mM−1—min−1)
Substrate
12’ꢁapoꢁβꢁcarotenal 5
14’ꢁapoꢁβꢁcarotenal 4
NS
−
44
1.8 ± 0.2
5.0 ± 0.5
8.5 ± 0.2
4.7 ± 0.2
4800 ± 600
930 ± 93
3.0 ± 0.4
NS
1.3 ± 0.1
−
450 ± 62
303
12 ± 2
3.1 ± 0.2
257 ± 47
aEnzymatic activity was measured in 50 mM HEPES, pH 8.0, 0.5 mM NAD+, including 0.5 mM EDTA and 0.5 mM DTT for ALDH1A1
and ALDH1A2, or 30 mM MgCl2 and 5 mM DTT for ALDH1A3, at 37 °C. Apocarotenoic acid production was quantified by using an HPLC-
based method. NS, no saturation (60 ꢀM was the highest substrate concentration tested in the assay), where the kcat/Km value was calculated
from the slope of V/[E] vs [S] plot.
mobile phase, at a flow rate of 2 mL/min using a Waters Alliance 2695
Table 2 compares the kinetic constants of the ALDH1A
HPLC. Elution of 12’-apo-β-carotenal 5 and 12’-apo-β-carotenoic acid 9 was
monitored with a Waters 2996 photodiode array detector at 415 and 400 nm,
respectively. The retention times for 12’-apo-β-carotenal 5 and 12’-apo-β-
enzymes with 12’- and 14’-apo-β-carotenals (5 and 4), showing
some differential substrate specificity. In general, Km values were
in the micromolar range, except for ALDH1A1 and ALDH1A3,
which were not saturated with 12’-apo-β-carotenal 5 and 14’-
apo-β-carotenal 4, respectively (Figure 3). While ALDH1A1
showed higher specificity for 14’-apo-β-carotenal 4, ALDH1A2
exhibited higher catalytic efficiency for 12’-apo-β-carotenal 5.
This catalytic efficiency is about ten-fold higher than those of
ALDH1A1 and ALDH1A3, and similar to that observed for these
enzymes with all-trans-retinaldehyde 2. The kcat values are in the
same range in all the experiments performed, but lower than
those for all-trans-retinaldehyde 2 (data not shown; to be
reported elsewhere).
carotenoic acid 9 were 2.85 and 10.60 min, respectively. Insets: UV-vis
absorption spectra of the two compounds.
To date, the enzymatic activity of ALDH with these
compounds had only been reported in microorganisms.39 This is
the first report on the apocarotenal dehydrogenase activity of
human ALDH1A enzymes.
0.60
Figure 3. Representative Michaelis-Menten kinetics of ALDH1A2 with
12’-apo-β-carotenal 5. The reaction was carried out in 50 mM HEPES, 0.5
mM EDTA, 0.5 mM DTT, pH 8.0, at 37 °C. The initial rates were measured
with at least six different substrate concentrations. Experimental values were
adjusted to the Michaelis-Menten equation using the non-linear regression
program GraFit 5.0 (Eritacus software).
12’-apo-β-
0.50
0.40
0.30
0.20
0.10
0.00
A
3. Conclusion
We have developed a new stereoselective synthesis of three
apocarotenoids (14’-apo-β-carotenal 4, 12’-apo-β-carotenal 5 and
10’-apo-β-carotenal 6) and demonstrated that the olefin
metathesis protocol is a valid synthetic method for accessing
these conjugated polyenes. Whereas the acyclic cross-metathesis
of the common required hexaene was efficiently performed with
the shorter enoate and dienoate partners, the chemoselectivity
with the longer trienoates is poor due to the presence of several
trans-disubstituted olefins that can compete. Previous syntheses
of these apo-β-carotenoids have in general featured carbonyl
condensation reactions, such as Wittig and aldol condensations
(439, 539, 40 2-417) or enol ether reactions with acetals followed by
elimination and hydrolysis in the case of 441.
2.00
4.00
6.00
8.00
10.00
12.00 14.00 16.00 18.00 20.00
Minutes
12’-apo-β-
0.10
0.08
0.06
0.04
0.02
0.00
B
396.9
0.10
0.08
0.06
0.04
0.02
226.3
285.2
240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00
nm
2.00
4.00
6.00
8.00
10.00 12.00 14.00 16.00 18.00 20.00
Minutes
We have shown that human retinaldehyde dehydrogenases are
capable of using apo-β-carotenals as substrates with good
catalytic efficiency. ALDH1A1 and ALDH1A2 exhibit higher
substrate specificity for 4 and 5, respectively, while ALDH1A3
shows similar specificity for the two compounds. Overall, the
substrate specificity of ALDH1 enzymes with apo-β-carotenals is
comparable to that with all-trans-retinaldehyde 2, indicating that
Figure 2. HPLC elution profiles of 12’-apo-β-carotenal 5 (A) and 12’-
apo-β-carotenoic acid 9 (B). Stock solutions of apocarotenoids were
prepared in ethanol and 10 ꢀM of either compound was injected. Each
compound was separated by liquid chromatography on a NovaPak® silica gel
column (4 ꢀm, 3.9 x 150 mm) in hexane/tert-butyl methyl ether (96:4, v/v)

5
these enzymes might play a physiological role in the metabolism
of apocarotenoids.
for C21H3 O2 ([M+H]+), 317.2475; found, 317.2472. IR (NaCl):
3
ACCEPTED MANUSCRIPT
υ 2958 (m, C-H), 2929 (m, C-H), 1711 (s, C=O), 1598 (m), 1144
(m), 977 (m) cm-1. UV (MeOH): λmax 329 nm.
4. Experimental Section
4.2. (2E,4E,6E,8E,10E)-5,9-Dimethyl-11-(2,6,6-
trimethylcyclohex-1-en-1-yl) undeca-2,4,6,8,10-pentaenal (14’-
apo-β-carotenal) (4).
General experimental procedures. See E.S.I.
4.1. Butyl (2E,4E,6E,8E,10E)-5,9-Dimethyl-11-(2,6,6-
trimethylcyclohex-1-en-1-yl)undeca-2,4,6,8,10-pentaenoate
(butyl 14’-β-apocarotenoate) (15).
General procedure for Dibal-H reduction of esters. To a
cooled (-78 °C) solution of (2E,4E,6E,8E,10E)-5,9-dimethyl-11-
(2,6,6-trimethylcyclohex-1-en-1-yl)undeca-2,4,6,8,10-
General procedure for the olefin metathesis reaction. To a
pentaenoate 15 (13.2 mg, 34.5 mmol) in THF (0.175 mL) was
added Dibal-H (0.103 mL, 1M in hexanes, 0.103 mmol). After
stirring for 5 h at -78 °C, H2O was added and the mixture was
extracted with EtOAc (3x). The combined organic layers were
washed with brine (3x), dried (Na2SO4) and the solvent was
evaporated. The residue was purified by column chromatography
(silica gel, 93:4:3 hexane/EtOAc/Et3N) to afford 7.7 mg (71%) of
a yellow oil identified as (2E,4E,6E,8E,10E)-5,9-dimethyl-11-
(2,6,6-trimethylcyclohex-1-en-1-yl)undeca-2,4,6,8,10-pentaene-
1-ol 16, which was used without further purification.
solution
of
(1E,3E,5E,7E)-2-(3,7-dimethyldeca-1,3,5,7,9-
pentaenyl)-1,3,3-trimethylcyclohex-1-ene 12 (14 mg, 0.049
mmol) and butyl (E)-but-2-enoate 13 (46.4 ꢀL, 0.294 mmol) in
toluene (0.25 mL), 2nd generation Hoveyda-Grubbs catalyst (0.46
mg, 0.0073 mmol) was added and the reaction mixture was
thoroughly degassed. After stirring at 25 °C for 4.5 h, the mixture
was filtered through a Celite™ pad, which was washed with Et2O
and the solvent was evaporated. The residue was purified by
column chromatography (silica gel, 99:1 hexane/EtOAc) to
afford, in order of elution, 2.2 mg (16%) of a red solid identified
as β,β-carotene 1, 11.8 mg (62%) of a red oil identified as butyl
(2E,4E,6E,8E,10E)-5,9-dimethyl-11-(2,6,6-trimethylcyclohex-1-
en-1-yl)undeca-2,4,6,8,10-pentaenoate 15 and 0.2 mg (1%) of a
yellow oil identified as butyl (2E,4E,6E)-5-methyl-7-(2,6,6-
trimethylcyclohex-1-en-1-yl) hepta-2,4,6-trienoate 14.
General procedure for MnO2 oxidation of alcohols. To a
cooled (0 ºC) solution of (2E,4E,6E,8E,10E)-5,9-dimethyl-11-
(2,6,6-trimethylcyclohex-1-en-1-yl)undeca-2,4,6,8,10-pentaene-
1-ol 16 (28.5 mg, 0.0912 mmol) in CH2Cl2 (1.7 mL) MnO2
(0.143 g, 1.64 mmol) and Na2CO3 (0.174 g, 1.64 mmol) were
added. After stirring for 5h at 25 ºC, the reaction mixture was
filtered through a CeliteTM pad and the solvent was removed. The
residue was purified by column chromatography (silica gel,
95:2:3 hexane/EtOAc/Et3N) to afford 24.8 mg (88%) of a yellow
oil identified as (2E,4E,6E,8E,10E)-5,9-dimethyl-11-(2,6,6-
trimethylcyclohex-1-en-1-yl)undeca-2,4,6,8,10-pentaenal 4. 1H-
NMR (400 MHz, (CD3)2CO): δ 9.65 (d, J = 8.0 Hz, 1H, H14’),
7.76 (dd, J = 14.9, 12.0 Hz, 1H, H15), 7.07 (dd, J = 15.0, 11.5 Hz,
1H, H11), 6.50 (d, J = 15.0 Hz, 1H, H12), 6.48 (d, J = 11.8 Hz, 1H,
H14), 6.38 - 6.18 (m, 3H, H7 + H8 + H10), 6.16 (dd, J = 14.9, 8.0
Hz, 1H, H15’), 2.17 (s, 3H, CH3), 2.08 - 2.04 (m, 2H, CH2), 2.05
(s, 3H, CH3), 1.73 (s, 3H, CH3), 1.68 - 1.60 (m, 2H, CH2), 1.52 -
1.47 (m, 2H, CH2), 1.05 (s, 6H, 2 x CH3) ppm. 13C-NMR (100
MHz, (CD3)2CO): δ 193.7 (d), 148.4 (d), 147.2 (s), 139.5 (s),
138.5 (s), 138.4 (d), 136.8 (d), 131.9 (d), 131.4 (d), 130.5 (d),
130.2 (s), 129.7 (d), 128.8 (d), 40.3 (t), 34.8 (s), 33.6 (t), 29.3 (q,
2x), 22.0 (q), 19.9 (t), 13.2 (q), 12.9 (q) ppm. HRMS (ESI+):
Calcd. for C22H30O ([M+H]+), 311.2369; found, 311.2376. IR
(NaCl): υ 2926 (m, C-H), 2863 (w, C-H), 1670 (s, C=O), 1559
(m), 1124 (m), 967 (m) cm-1. UV (MeOH): λmax 402 nm (ε = 45
950 mol-1 L cm-1).
1
Data for β,β-carotene (1): H-NMR (400 MHz, C6D6): δ 6.79
(dd, J = 14.8, 11.4 Hz, 2H, 2H11), 6.70-6.67 (m, 2H, H15), 6,49 (d,
J = 14.8 Hz, 2H, H12), 6.42 - 6.31 (m, 8H, 2H7 + 2H8+ 2H10
2H14), 1.99 (t, J = 5.6 Hz, 4H, 2 x CH2), 1.94 (s, 6H, 2 x CH3),
1.88 (s, 6H, 2 x CH3), 1.82 (s, 6H, 2 x CH3), 1.64 - 1.56 (m, 4H,
2 x CH2), 1.52 - 1.49 (m, 4H, 2 x CH2), 1.16 (s, 12H, 4 x CH3)
ppm. HRMS (ESI+): Calcd. for C40H56 ([M+H]+), 536.4376;
found, 536.4376. UV (MeOH): λmax 449, 241 nm.
+
Data for butyl (2E,4E,6E,8E,10E)-5,9-dimethyl-11-(2,6,6-
trimethylcyclohex-1-en-1-yl)undeca-2,4,6,8,10-pentaenoate (15):
1H-NMR (400 MHz, CO(CD3)2): δ 7.71 (dd, J = 15.0, 12.0 Hz,
1H, H15), 7.02 (dd, J = 15.0, 11.4 Hz, 1H, H11), 6.48 (d, J = 15.0
Hz, 1H, H12), 6.34 (d, J = 11.8 Hz, 1H, H14), 6.42 - 6.18 (m, 3H,
H7 + H8 + H10), 5.97 (d, J = 15.0 Hz, 1H, H15’), 4.16 (t, J = 6.6
Hz, 2H, O-CH2-), 2.13 (s, 3H, CH3), 2.10 - 2.06 (m, 2H, CH2),
2.06 (s, 3H, CH3), 1.75 (s, 3H, CH3), 1.72 - 1.61 (m, 4H, 2 x
CH2), 1.54 - 1.48 (m, 2H, CH2), 1.47 - 1.39 (m, 2H, CH2), 1.07
(s, 6H, 2 x CH3), 0.97 (t, J = 7.4 Hz, 3H, CH2CH3) ppm. 13C-
NMR (100 MHz, (CD3)2CO): 167.4 (s), 145.2 (s), 140.8 (d),
138.8 (s), 138.7 (s), 138.6 (d), 137.0 (d), 131.4 (d), 130.1 (s),
129.5 (d), 129.4 (d), 128.4 (d), 121.4 (d), 64.4 (t), 40.4 (t), 34.9
(s), 33.6 (t), 31.6 (t), 29.3 (q, 2x), 21.9 (q), 19.9 (t), 19.8 (t), 14.0
(q), 13.1 (q), 12.9 (q) ppm. HRMS (ESI+): Calcd. for C26H39O2
([M+H]+), 383.2945; found, 383.2946. IR (NaCl): υ 2957 (m, C-
H), 2928 (m, C-H), 2866 (w, C-H), 1708 (s, C=O), 1615 (m),
1563 (m), 1135 (s), 970 (m) cm-1. UV (MeOH): λmax 394 nm.
4.3. (2E,4E,6E,8E,10E)-5,9-Dimethyl-11-(2,6,6-
trimethylcyclohex-1-en-1-yl)undeca-2,4,6,8,10-pentaenoic Acid
(14’-β-apocarotenoic acid) (8)
General procedure for hydrolysis of esters with KOH. To a
solution
of
(2E,4E,6E,8E,10E)-5,9-dimethyl-11-(2,6,6-
Data
for
butyl
(2E,4E,6E)-5-methyl-7-(2,6,6-
1
trimethylcyclohex-1-en-1-yl)undeca-2,4,6,8,10-pentaenoate 15
(19.6 mg, 51.2 mmol) in MeOH (3.53 mL) was added KOH (0.82
mL, 2M in H2O, 1.64 mmol). After stirring at 70 °C for 1 h, the
reaction mixture was cooled down to 25 °C, CH2Cl2 and brine
were added and the layers were separated. The organic layer was
washed with H2O (3x). The combined aqueous layers were
acidified until acidic pH and extracted with CH2Cl2 (3x). The
combined organic layers were dried (Na2SO4) and the solvent
was evaporated. The residue was purified by column
chromatography (silica gel, 70:30 hexane/EtOAc) to afford 10.4
mg (62%) of a orange solid identified as (2E,4E,6E,8E,10E)-5,9-
trimethylcyclohex-1-en-1-yl)hepta-2,4,6-trienoate (14): H-NMR
(400 MHz, CDCl3): δ 7.71 (dd, J = 15.0, 11.8 Hz, 1H, H3), 6.38
(d, J = 16.1 Hz, 1H, H6 or H7), 6.18 - 6.10 (m, 2H, H6 or H7 +
H4), 5.87 (d, J = 15.5 Hz, 1H, H2), 4.16 (t, J = 6.7 Hz, 2H, O-
CH2), 2.04 (s, 3H, CH3), 2.05 - 1.95 (m, 2H, CH2), 1.71 (s, 3H,
CH3), 1.68 - 1.58 (m, 4H, 2 x CH2), 1.50 - 1.37 (m, 4H, 2 x CH2),
1.03 (s, 6H, 2 x CH3), 0.95 (t, J = 7.4 Hz, 3H, CH2CH3) ppm.
13C-NMR (100 MHz, CDCl3): δ 167.8 (s), 144.4 (s), 140.7 (d),
137.6 (s), 136.9 (d), 130.9 (s), 130.8 (d), 127.4 (d), 120.2 (d),
64.3 (t), 39.7 (t), 34.4 (s), 33.3 (t), 30.9 (t), 29.1 (q, 2x), 21.9 (q),
19.4 (t), 19.3 (t), 13.9 (q), 13.2 (q) ppm. HRMS (ESI+): Calcd.

6
Tetrahedron
dimethyl-11-(2,6,6-trimethylcyclohex-1-en-1-yl)undeca-
Following the general procedure for MnO2 oxidation of
ACCEPTED MANUSCRIPT
1
2,4,6,8,10-pentaenoic acid 8. H-NMR (400 MHz, (CD3)2CO): δ
7.68 (dd, J = 14.9, 12.0 Hz, 1H, H15), 6.98 (dd, J = 15.0, 11.5 Hz,
1H, H11), 6.45 (d, J = 15.0 Hz, 1H, H12), 6.35 (d, J = 12.1 Hz, 1H,
H14), 6.38 - 6.16 (m, 3H, H7 + H8 + H10), 5.92 (d, J = 15.0 Hz,
1H, H15’), 2.09 (s, 3H, CH3), 2.07 - 1.97 (m, 2H, CH2), 2.02 (s,
3H, CH3), 1.71 (s, 3H, CH3), 1.68 - 1.57 (m, 2H, CH2), 1.51 -
1.46 (m, 2H, CH2), 1.04 (s, 6H, 2 x CH3) ppm. 13C-NMR (100
MHz, (CD3)2CO): 168.0 (s), 145.0 (s), 141.2 (d), 138.8 (s), 138.6
(s), 138.5 (d), 137.1 (d), 131.4 (d), 130.1 (s), 129.4 (d), 129.3 (d),
128.4 (d), 121.6 (d), 40.4 (t), 34.9 (s), 33.6 (t), 29.3 (q, 2x), 21.9
(q), 19.9 (t), 13.1 (q), 12.9 (q) ppm. HRMS (ESI+): Calcd. for
C22H31O2 ([M+H]+), 327.2319; found, 327.2316. IR (NaCl): υ
2924 (m, C-H), 2856 (w, C-H), 1680 (s, C=O), 1560 (m), 1162
(s), 969 (m) cm-1. UV (MeOH): λmax 375 (ε = 46 100 mol-1 L cm-
1).
alcohols, the reaction of (2E,4E,6E,8E,10E,12E)-2,7,11-
trimethyl-13-(2,6,6-trimethylcyclohex-1-en-1-yl)trideca-
2,4,6,8,10,12-hexaene-1-ol 19 (8.4 mg, 0.0024 mmol) with MnO2
(37.3 mg, 0.429 mmol) and Na2CO3 (45.4 mg, 0.429 mmol) in
CH2Cl2 (0.443 mL) at 25 ºC for 4h, afforded, after purification by
column chromatography (silica gel, 95:2:3 hexane/EtOAc/Et3N)
3.2 mg (38%) of
(2E,4E,6E,8E,10E,12E)-2,7,11-trimethyl-13-(2,6,6-
a
orange-reddish oil identified as
trimethylcyclohex-1-en-1-yl)trideca-2,4,6,8,10,12-hexaenal
5.
1H-NMR (400 MHz, (CD3)2CO): δ 9.46 (s, 1H, H12’), 7.24 (dd, J
= 14.1, 12.1 Hz, 1H, H15), 7.13 (d, J = 11.8 Hz, 1H, H14’), 6.92
(dd, J = 14.9, 11.5 Hz, 1H, H11), 6.84 (dd, J = 14.1, 11.9 Hz, 1H,
H15’), 6.46 (d, J = 15.2 Hz, 1H, H12), 6.42 (d, J = 12.2 Hz, 1H,
H14), 6.32 – 6.15 (m, 3H, H7 + H8 + H10), 2.08 (s, 3H, CH3), 2.09
- 2.01 (m, 2H, CH2), 2.01 (s, 3H, CH3), 1.83 (s, 3H, CH3), 1.72
(s, 3H, CH3), 1.71 - 1.55 (m, 2H, CH2), 1.56 - 1.44 (m, 2H, CH2),
1.04 (s, 6H, 2 x CH3) ppm. 13C-NMR (100 MHz, (CD3)2CO): δ
194.4 (d), 149.3 (d), 142.3 (s), 138.7 (d), 138.6 (s), 138.5 (d),
138.2 (s), 137.6 (d), 137.5 (s), 132.1 (d), 131.7 (d), 130.0 (s),
128.6 (d), 128.4 (d), 128.1 (d), 40.3 (t), 34.9 (s), 33.6 (t), 29.3 (q,
2x), 22.0 (q), 19.9 (t), 13.0 (q), 12.8 (q), 9.5 (q) ppm. HRMS
(ESI+): Calcd. for C25H35O 351.2682; found, 351.2695. IR
(NaCl): υ 2924 (m, C-H), 2861 (w, C-H), 1667 (s, C=O), 1544
(m), 1185 (m) cm-1. UV (MeOH): λmax 424 nm (ε = 69 600 mol-1
L cm-1).
4.4. Methyl (2E,4E,6E,8E,10E,12E)-2,7,11-Trimethyl-13-(2,6,6-
trimethylcyclohex-1-en-1-yl)trideca-2,4,6,8,10,12-hexaenoate
(methyl 12’-apo-β-carotenoate) (18).
Following the general procedure for the olefin metathesis, the
reaction of (1,3,3-trimethyl-2-((1E,3E,5E,7E)-3,7-dimethyldeca-
1,3,5,7,9-pentaenyl)cyclohex-1-ene 12 (21 mg, 0.074 mmol) with
methyl (E)-2-methylpenta-2,4-dienoate 17 (37.5 mg, 0.298
mmol) and 2nd generation Hoveyda-Grubbs catalyst (7 mg, 0.011
mmol) in toluene (0.8 mL) at 25 °C for 7 h, afforded, after
purification by column chromatography (silica gel, gradient from
100:0 to 99:1 hexane/EtOAc), in order of elution, 2.4 mg (11%)
of a yellow oil identified as 1,3,3-trimethyl-2-((1E,3E,5E,7E)-
3,7-dimethyldeca-1,3,5,7,9-pentaenyl)cyclohex-1-ene 12, 1.9 mg
(10%) of a red solid identified as β-β-carotene 1 and 10.3 mg
4.6. (2E,4E,6E,8E,10E,12E)-2,7,11-Trimethyl-13-(2,6,6-
trimethylcyclohex-1-en-1-yl)trideca-2,4,6,8,10,12-hexaenoic Acid
(12’-apo-β-carotenoic acid) (9).
(40%)
of
a
red
solid
identified
as
methyl
Following the general procedure for hydrolysis of esters, the
reaction of (2E,4E,6E,8E,10E,12E)-2,7,11-trimethyl-13-(2,6,6-
trimethylcyclohex-1-en-1-yl)trideca-2,4,6,8,10,12-hexaenoate 18
(10.1 mg, 0.026 mmol) with KOH (0.425 mL, 2M in H2O, 0.85
mmol) in MeOH (1.83 mL) at 70 °C for 1 h, afforded, after
purification by column chromatography (silica gel, 70:30
hexane/EtOAc) 5.1 mg (53%) of a red solid identified as
(2E,4E,6E,8E,10E,12E)-2,7,11-trimethyl-13-(2,6,6-
trimethylcyclohex-1-en-1-yl)trideca-2,4,6,8,10,12-hexaenoic acid
9. 1H-NMR (400 MHz, CDCl3): δ 7.42 (d, J = 11.8 Hz, 1H, H14’),
6.95 (dd, J = 14.0, 12.1 Hz, 1H, H15), 6.76 (dd, J = 14.9, 11.5 Hz,
1H, H11), 6.54 (dd, J = 13.8, 12.1 Hz, 1H, H15’), 6.36 (d, J = 15.0
Hz, 1H, H12), 6.27 (d, J = 12.4 Hz, 1H, H14), 6.23 – 6.10 (m, 3H,
H7 + H8 + H10), 2.02 (s, 3H, CH3), 1.99 (s, 3H, CH3), 2.10 – 1.99
(m, 2H, CH2), 1.72 (s, 3H, CH3), 1.66 - 1.57 (m, 2H, CH2), 1.51 -
1.47 (m, 2H, CH2), 1.03 (s, 6H, 2 x CH3) ppm. 13C-NMR (100
MHz, CDCl3): δ 173.1 (s), 141.0 (d), 140.8 (s), 138.0 (s), 137.7
(d), 137.6 (s), 137.1 (d), 136.6 (d), 131.1 (d), 130.6 (d), 129.8 (s),
127.8 (d), 127.7 (d), 127.2 (d), 125.2 (s), 39.8 (t), 34.4 (s), 33.3
(t), 29.2 (q, 2x), 21.9 (q), 19.4 (t), 13.1 (q), 13.0 (q), 12.6 (q)
ppm. HRMS (ESI+): Calcd. for C25H35O2 ([M+H]+), 367.2632;
found, 367.2629. IR (NaCl): υ 2924 (m, C-H), 2858 (w, C-H),
1670 (s, C=O), 1419 (m), 1248 (m), 968 (m) cm-1. UV (MeOH):
λmax 402 (ε = 61 450 mol-1 L cm-1).
(2E,4E,6E,8E,10E,12E)-2,7,11-trimethyl-13-(2,6,6-
trimethylcyclohex-1-en-1-yl)trideca-2,4,6,8,10,12-hexaenoate 18.
1H-NMR (400 MHz, (CD3)2CO): δ 7.34 (d, J = 11.8 Hz, 1H,
H14’), 7.10 (dd, J = 14.3, 12.0 Hz, 1H, H15), 6.89 (dd, J = 14.9,
11.5 Hz, 1H, H11), 6.69 (dd, J = 14.2, 12.1 Hz, 1H, H15’), 6.46 (d,
J = 15.0 Hz, 1H, H12), 6.39 (d, J = 11.8 Hz, 1H, H14), 6.34 – 6.16
(m, 3H, H7 + H8 + H10), 3.74 (s, 3H, OCH3), 2.09 (s, 3H, CH3),
2.10 - 2.02 (m, 2H, CH2), 2.03 (s, 3H, CH3), 1.99 (s, 3H, CH3),
1.75 (s, 3H, CH3), 1.70 - 1.62 (m, 2H, CH2), 1.54 - 1.48 (m, 2H,
CH2), 1.07 (s, 6H, 2 x CH3) ppm. 13C-NMR (100 MHz,
(CD3)2CO): δ 168.8 (s), 140.8 (s), 139.4 (d), 138.8 (d), 138.7 (s),
137.8 (d), 137.6 (s), 137.0 (d), 132.3 (d), 131.8 (d), 129.9 (s),
128.9 (d), 127.8 (d), 127.7 (d), 126.9 (s), 51.9 (q), 40.4 (t), 34.9
(s), 33.6 (t), 29.3 (q, 2x), 22.0 (q), 19.9 (t), 13.0 (q), 12.9 (q),
12.8 (q) ppm. HRMS (ESI+): Calcd. for C26H37O2 ([M+H]+),
381.2788; found, 381.2786. IR (NaCl): υ 2926 (m, C-H), 2862
(w, C-H), 1703 (m, C=O), 1235 (m) cm-1. UV (MeOH): λmax 410
nm.
4.5. (2E,4E,6E,8E,10E,12E)-2,7,11-trimethyl-13-(2,6,6-
trimethylcyclohex-1-en-1-yl)trideca-2,4,6,8,10,12-hexaenal (12’-
apo-β-carotenal) (5).
Following the general procedure for Dibal-H reduction of
esters, the reaction of methyl (2E,4E,6E,8E,10E,12E)-2,7,11-
trimethyl-13-(2,6,6-trimethylcyclohex-1-en-1-yl)trideca-
2,4,6,8,10,12-hexaenoate 18 (15.7 mg, 0.041 mmol) with Dibal-
H (0.124 mL, 0.124 mmol, 1M in hexanes) in THF (0.209 mL) at
-78 ºC for 4h, afforded, after purification by column
chromatography (silica gel, 90:7:3 hexane/EtOAc/Et3N) 10.9 mg
(75%) of a reddish oil identified as (2E,4E,6E,8E,10E,12E)-
2,7,11-trimethyl-13-(2,6,6-trimethylcyclohex-1-en-1-yl)trideca-
2,4,6,8,10,12-hexaene-1-ol 19, which was used without further
purification.
4.7. Ethyl (2E,4E)-5-(1,3-Dioxolan-2-yl)-3methylpenta-2,4-
dienoate (20a).
To a 500 mL round-bottomed flask armed with a Dean-Stark
trap, were added ethyl (2E,4E) 3-methyl-6-oxohexa-2,4-dienoate
20 (2.5 g, 14.8 mmol), benzene (256.3 mL), p-TsOH (170 mg,
0.89 mmol) and ethyleneglycol (8.7 mL, 156 mmol), and the
resulting mixture was heated under reflux for 16 h. The cooled

7
reaction mixture was diluted with H2O, an aqueous solution of
NaHCO3 was added and the mixture was extracted with Et2O
(3x). The combined organic layers were dried (Na2SO4) and the
solvent was removed to obtain 3.12 g (99%) of a colorless oil
identified as ethyl (2E,4E)-5-(1,3-dioxolan-2-yl)-3-methylpenta-
2,4-dienoate 20a. 1H-NMR (400 MHz, C6D6): δ 6.29 (d, J = 15.8
Hz, 1H, H4), 5.96 (dd, J = 15.8, 5.4 Hz, 1H, H5), 5.82 (s, 1H, H2),
5.23 (d, J = 5.4 Hz, 1H, H6), 3.99 (q, J = 7.1 Hz, 2H, OCH2CH3),
3.53 - 3.45 (m, 2H, CH2), 3.43 - 3.36 (m, 2H, CH2), 2.23 (s, 3H,
CH3), 0.96 (t, J = 7.1 Hz, 3H, OCH2CH3) ppm. 13C-NMR (101
MHz, C6D6): δ 166.3 (s), 150.8 (s), 137.2 (d), 131.8 (d), 121.7
(d), 103.4 (d), 65.0 (t, 2x), 59.8 (t), 14.3 (q), 13.7 (q) ppm.
HRMS (ESI+): Calcd. for C11H17O4 ([M+H]+), 213.1121; found,
213.1120. IR (NaCl): υ 2979 (m, C-H), 2887 (m, C-H), 1713 (s,
C=O), 1616 (m), 1231 (m), 1154 (s), 772 (m) cm-1. UV (MeOH):
λmax 253 nm.
(100.62 MHz, (CD3)2CO): δ 167.8 (s), 149.3 (d), 140.3 (d),
ACCEPTED MANUSCRIPT
139.8 (s), 138.8 (d), 138.7 (s), 137.9 (d), 137.4 (s), 135.0 (d),
134.2 (s), 132.8 (d), 131.9 (d), 129.9 (d), 129.9 (s), 127.6 (d),
127.3 (d), 116.8 (d), 51.5 (q), 40.3 (t), 34.9 (s), 33.6 (t), 29.3 (q,
2x), 22.0 (q), 19.9 (t), 12.9 (q), 12.8 (q), 12.6 (q) ppm. HRMS
(ESI+): Calcd. for C28H39O2 ([M+H]+), 407.2945; found,
407.2954. IR (NaCl): υ 2921 (m, C-H), 2856 (m, C-H), 1714 (m,
C=O), 1701 (m), 1539 (m) cm-1. UV (MeOH): 429 nm.
4.10. (2E,4E,6E,8E,10E,12E,14E)-4,9,13-Trimethyl-15-(2,6,6-
trimethylcyclohex-1-en-1-yl)pentadeca-2,4,6,8,10,12,14-
heptaenal (10’-apo-β-carotenal) (6).
Following the general procedure for Dibal-H reduction of
esters, the reaction of methyl (2E,4E,6E,8E,10E,12E,14E)-
4,9,13-trimethyl-15-(2,6,6-trimethylcyclohex-1-en-1-
yl)pentadeca-2,4,6,8,10,12,14-heptaenoate 25 (11.2 mg, 0.028
mmol) with Dibal-H (6.9 ꢀL, 0.069 mmol, 1M in hexanes) in
THF (0.138 mL) at -78 ºC for 2h, afforded, after purification by
column chromatography (silica gel, gradient from 85:12:3 to
80:20:0 hexane/EtOAc/Et3N) 10 mg of a red solid identified as
(2E,4E,6E,8E,10E,12E,14E)-4,9,13-trimethyl-15-(2,6,6-
4.8. (2E,4E)-5-(1,3-Dioxolan-2-yl)-3-methylpenta-2,4-dien-1-ol
(20b)
Following the general procedure for Dibal-H reduction of
esters, the reaction of ethyl (2E,4E)-5-(1,3-dioxolan-2-yl)-3-
methylpenta-2,4-dienoate 20a (1.39 g, 6.59 mmol) with Dibal-H
(16.5 mL, 1M in hexanes, 16.5 mmol) in THF (33.4 mL) at -78
ºC for 2h, afforded 1.11 g (99%) of a colorless oil identified as
(2E,4E)-5-(1,3-dioxolan-2-yl)-3-methylpenta-2,4-dien-1-ol 20b.
1H-NMR (400 MHz, C6D6): δ 6.43 (d, J = 15.8 Hz, 1H, H4), 5.77
(dd, J = 15.8, 5.9 Hz, 1H, H5), 5.54 (t, J = 5.4 Hz, 1H, H2), 5.32
(d, J = 5.9 Hz, 1H, H6), 3.90 (d, J = 5.4 Hz, 2H, 2H1), 3.65 - 3.54
(m, 2H, CH2), 3.47 - 3.41 (m, 2H, CH2), 1.47 (s, 3H, CH3) ppm.
13C-NMR (101 MHz, C6D6): δ 138.8 (d), 134.1 (s), 134.0 (d),
125.3 (d), 104.4 (d), 65.0 (t, 2x), 59.2 (t), 12.4 (q) ppm. HRMS
(ESI+): Calcd. for C9H15O3 ([M+H]+), 171.1016; found,
171.1020. IR (NaCl): υ 3500-3100 (br, O-H), 2950 (m, C-H),
2883 (m, C-H), 1386 (m), 1156 (m), 1085 (m), 1014 (m), 950 (s)
cm-1. UV (MeOH): λmax 233 nm.
trimethylcyclohex-1-en-1-yl)pentadeca-2,4,6,8,10,12,14-heptaen-
1-ol 26 which was used without further purification.
Following the general procedure for MnO2 oxidation of
alcohols, the reaction of (2E,4E,6E,8E,10E,12E,14E)-4,9,13-
trimethyl-15-(2,6,6-trimethylcyclohex-1-en-1-yl)pentadeca-
2,4,6,8,10,12,14-heptaen-1-ol 26 (10.7 mg, 0.028 mmol) with
MnO2 (52.2 mg, 0.51 mmol) and Na2CO3 (54.1 mg, 0.51 mmol)
in THF (0.423 mL) at 25 ºC for 2h afforded, after purification by
column chromatography (silica gel, gradient from 98:0:2 to
97:3:0 hexane/EtOAc/Et3N) 6.1 mg (58% combined yield) of a
red solid identified as (2E,4E,6E,8E,10E,12E,14E)-4,9,13-
trimethyl-15-(2,6,6-trimethylcyclohex-1-en-1-yl)pentadeca-
2,4,6,8,10,12,14-heptaenal 6 which was purified by HPLC
(Waters SpherisorbTM 10µm CN, 10x250 mm Semipreparative
column with NovaPak CN 4µm precolumn, 95:5 hexane/acetone,
flow rate: 3mL/min, tR = 12 min). 1H-NMR (400.16 MHz,
(CD3)2CO): δ 9.59 (d, J = 7.6 Hz, 1H, H10’), 7.33 (d, J = 15.3 Hz,
1H, H12’), 7.08 - 6.96 (m, 1H, H15 or H15’), 6.86 (dd, J = 15.0,
11.4 Hz, 1H, H11), 6.81 - 6.74 (m, 2H, H14 or H14’ + H15 or H15’),
6.44 (d, J = 15.0 Hz, 1H, H12), 6.39 (d, J = 12.0 Hz, 1H, H14 or
H14’), 6.30 - 6.11 (m, 4H, H7 + H8 + H10 + H11’), 2.07 - 2.04 (m,
2H, CH2), 2.04 (s, 6H, 2 x CH3), 2.00 (s, 3H, CH3), 1.71 (s, 3H,
CH3), 1.66 - 1.59 (m, 2H, CH2), 1.50 - 1.46 (m, 2H, CH2), 1.03
(s, 6H, 2x CH3) ppm. 13C-NMR (100.62 MHz, (CD3)2CO): δ
193.7 (d), 156.9 (d), 141.7 (d), 140.7 (s), 138.7 (d), 138.7 (s),
137.9 (d), 137.6 (s), 136.1 (d), 134.7 (s), 132.6 (d), 131.9 (d),
129.9 (s), 129.9 (d), 128.1 (d), 127.8 (d), 127.6 (d), 40.3 (t), 34.9
(s), 33.6 (t), 29.3 (q, 2x), 22.0 (q), 19.9 (t), 13.0 (q), 12.8 (q),
12.70 (q) ppm. HRMS (ESI+): Calcd. for C27H37O ([M+H]+),
377.2839; found, 377.2842. IR (NaCl): υ 2920 (m, C-H), 2852
(m, C-H), 1671 (s, C=O), 1123 (s), 968 (m) cm-1. UV (MeOH):
450 nm (ε 66 400 mol-1 L cm-1).
4.9. Methyl (2E,4E,6E,8E,10E,12E,14E)-4,9,13-Trimethyl-15-
(2,6,6-trimethylcyclohex-1-en-1-yl)pentadeca-2,4,6,8,10,12,14-
heptaenoate (methyl 10’-β-apo-carotenoate) (25).
Following the general procedure for olefin metathesis, the
reaction of (1,3,3-trimethyl-2-((1E,3E,5E,7E)-3,7-dimethyldeca-
1,3,5,7,9-pentaenyl)cyclohex-1-ene 12 (10 mg, 0.035 mmol),
methyl (2E,4E,6E) and (2E,4E,6Z)-4-methylocta-2,4,6-trienoate
24 (35.3 mg, 0.212 mmol) and 2nd generation Hoveyda-Grubbs
catalyst (4.4 mg, 0.007 mmol) in toluene (0.18 mL) at 25 ºC for 5
h, afforded, after purification by column chromatography (silica
gel, 98:2 hexane/EtOAc) in order of elution, 5.5 mg (58 %) of a
red solid identified as β,β-carotene 1, 17.2 mg (49%) of a
yellowish oil identified as starting methyl (2E,4E,6E) and
(2E,4E,6Z)-4-methylocta-2,4,6-trienoate 24 and 3.4 mg (24%) of
a red oil identified as methyl (2E,4E,6E,8E,10E,12E,14E)-4,9,13-
trimethyl-15-(2,6,6-trimethylcyclohex-1-en-1-yl)pentadeca-
2,4,6,8,10,12,14-heptaenoate 25. 1H-NMR (400.16 MHz,
(CD3)2CO): δ 7.35 (d, J = 15.5 Hz, 1H, H12’), 6.96 (dd, J = 13.9,
11.9 Hz, 1H, H15), 6.84 (dd, J = 14.9, 11.5 Hz, 1H, H11), 6.80 -
6.72 (m, 1H, H15’), 6.67 (d, J = 11.8 Hz, 1H, H14’), 6.43 (d, J =
15.0 Hz, 1H, H12), 6.37 (d, J = 11.8 Hz, 1H, H14), 6.26 (d, J =
15.8 Hz, 1H, H7), 6.23 (d, J = 11.0 Hz, 1H, H10), 6.17 (d, J = 16.1
Hz, 1H, H8), 5.90 (d, J = 15.5 Hz, 1H, H11’), 3.69 (s, 3H, OCH3),
2.07 - 2.05 (m, 2H, CH2), 2.03 (s, 3H, CH3), 2.00 (s, 3H, CH3),
1.96 (s, 3H, CH3), 1.71 (s, 3H, CH3), 1.68 - 1.57 (m, 2H, CH2),
1.51 - 1.45 (m, 2H, CH2), 1.03 (s, 6H, 2 x CH3) ppm. 13C-NMR
4.11. Purification of recombinant human ALDHs and enzymatic
assay
Human ALDH1A1, ALDH1A2 and ALDH1A3 were
recombinantly expressed from the pET-30 Xa/LIC vector and
affinity purified onto a Ni2+-NTA Chelating SepharoseTM Fast
Flow column (GE Heathcare). Activity assays with apo-β-
carotenals were carried out using detergent-free solubilization
and end-point reaction, followed by HPLC, originally devised for
retinoid analysis.42 ALDH1A1 and ALDH1A2 were assayed in
50 mM HEPES, 0.5 mM EDTA, 0.5 mM DTT, pH 8.0, while

8
Tetrahedron
5. Britton G, Liaaen-Jensen S, Pfander, H, Eds., Carotenoids. Volume 3.
ALDH1A3 was assayed in 50 mM HEPES, 30 mM MgCl2, 5
ACCEPTED MANUSCRIPT
Biosynthesis and Metabolism, Birkhäuser, Basel 1998.
mM DTT, pH 8.0. Concentration of 12’- and 14’-apo-β-carotenal
was determined based on the corresponding molar absorption
coefficient in aqueous solutions at the appropriate wavelength
(ε410 = 24,228 M−1·cm−1 and ε416 = 15,945 M−1·cm−1, for 12’-apo-
β-carotenal 5 in ALDH1A1/1A2 and ALDH1A3 reaction buffer,
respectively; and ε397 = 9,218 M−1·cm−1 and ε340 = 9,036 M−1·cm−1
for 14’-apo-β-carotenal 4 in ALDH1A1/1A2 and ALDH1A3
reaction buffer, respectively). The reaction was started by the
addition of cofactor and carried out for 15 min at 37 °C in a final
volume of 0.5 mL. With the aim to measure the steady state
enzymatic activity, the concentration of enzyme was kept from
50- to 100-fold lower than that of the substrate for all enzymatic
assays and a saturating concentration of cofactor (0.5 mM NAD+)
was used. Reaction products were extracted with
hexane/dioxane/isopropanol (50:5:1, v/v) and analyzed by an
HPLC-based method.43 The organic layer was evaporated, apo-β-
carotenoids were dissolved in hexane and injected onto a
NovaPak® silica gel column (4 ꢀm, 3.9 x 150 mm, Waters) in
hexane:tert-butyl methyl ether (96:4, v/v) mobile phase, at a flow
rate of 2 mL/min using a Waters Alliance 2695 HPLC
instrument. Elution was monitored at 415 nm for 12’-apo-β-
carotenal 5, 400 nm for 12’-apo-β-carotenoic acid 9 and 14’-apo-
β-carotenal 4, and 373 nm for 14’-apo-β-carotenoic acid 8, using
a Waters 2996 photodiode array detector. All compound
manipulations were performed under dim or red light to prevent
photoisomerization.
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5. Acknowledgments
This work work was supported by funds from the Spanish
MINECO (SAF2016-77620-R-FEDER and BIO2016-78057),
Xunta de Galicia (Consolidación GRC ED431C 29017/61 from
DXPCTSUG; ED-431G/02-FEDER "Unha maneira de facer
Europa" to CINBIO, a Galician research center 2016-2019).
INBIOMED-FEDER “Unha maneira de facer Europa”). We are
indebted to Samuel Gallego and Pablo Fernández for preliminary
results in this project and the Centro de Apoio Científico-
Tecnolóxico á Investigación (C.A.C.T.I.) for invaluable help in
structure elucidation. Raquel Pequerul is a recipient of a PIF
predoctoral fellowship from Universitat Autònoma de Barcelona.
Eszter Birta was supported by Campus Hungary, a programme
financed by the EU and the Hungarian Government in the
framework of Social Renewal Operational Program (TAMOP) of
Hungary.
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