Synthesis of enantiopure C3- and C4-hydroxyretinals and their enzymatic reduction by ADH8 from Xenopus laevis

Article abstract of DOI:10.1039/b514273c

(R)-all-trans-3-hydroxyretinal 1, (S)-all-trans-4-hydroxyretinal 3 and (R)-all-trans-4-hydroxyretinal 5 have been synthesized stereoselectively by Horner-Wadsworth-Emmons and Stille cross-coupling as bond-forming reactions. The CBS method of ketone reduct

Full text of DOI:10.1039/b514273c

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of enantiopure C3- and C4-hydroxyretinals and their enzymatic
reduction by ADH8 from Xenopus laevis
a
a
b
b
b
a
´
Marta Dom´ınguez, Rosana Alvarez, Emma Borra`s, Jaume Farre´s, Xavier Pare´s and Angel R. de Lera*
Received 10th October 2005, Accepted 8th November 2005
First published as an Advance Article on the web 30th November 2005
DOI: 10.1039/b514273c
(R)-all-trans-3-hydroxyretinal 1, (S)-all-trans-4-hydroxyretinal 3 and (R)-all-trans-4-hydroxyretinal
5 have been synthesized stereoselectively by Horner–Wadsworth–Emmons and Stille cross-coupling as
bond-forming reactions. The CBS method of ketone reduction was used in the enantioface-
differentiation step to provide the precursors for the synthesis of the 4-hydroxyretinal enantiomers.
The kinetic constants of Xenopus laevis ADH8 with these retinoids have been determined.
for the reversible conversion of retinol to retinal: alcohol dehydro-
genases (ADH) of the medium-chain dehydrogenase/reductase
(MDR), retinol dehydrogenases of the short-chain dehydroge-
nase/reductase (SDR)13 and several members of the aldo-keto
reductases (AKR).14
Introduction
Vitamin A (retinol) and its metabolites (retinoids) are essential
to the proper function of a number of biological processes. The
visual cycle1 is perhaps the best characterized2 with 11-cis-retinal
acting as the inverse agonist of the apoprotein opsin in verte-
brates. Reproduction,3 cell growth and differentiation, embryonic
development (e.g. limbs, nervous system, heart, kidney), immune
response4 and intermediary metabolism are regulated by all-trans-
retinoic acid and 9-cis-retinoic acid. These gene transcription
regulators are the ligands for two classes of nuclear receptors,
acting as ligand-dependent transcription factors: the retinoic acid
receptors (RARa, b and c) and the retinoid X receptors (RXRa,
b and c).5
An increasing number of studies attribute a physiological role
as signalling molecules to ring-oxidized derivatives at position C4,
which are major metabolites of retinoids.6 In fact, C4-oxidized
retinoids are natural ligands of several RAR receptors.6a, 6b, 7
Studies in Xenopus embryos revealed that 4-hydroxyretinol and
4-hydroxyretinoic acid can transactivate RAR receptors.7 Retinol
metabolism to 4-hydroxy and 4-oxoretinol is detected in murine
stem cells8 and a wide variety of cancer cell lines.7a, 9 In addition,
4-hydroxyretinol endogenously occurs in serum and liver of
normal neonatal rats.10 A specific role in the onset of neuronal
differentiation is postulated for 4-hydroxyretinoic acid,11 and
both 4-hydroxyretinoic and 4-oxoretinoic acid exhibit biological
activity in skin retinoid responsive systems in vivo.12 In Xenopus
eggs and early embryos, the major bioactive retinoids are 4-
oxoretinol and, particularly, 4-oxoretinal, which are critical for
proper cell differentiation,6b whereas 4-oxoretinoic acid is a potent
modulator of positional specification.6a
In vertebrates, the ADH family comprises eight different classes,
involved in ethanol metabolism and also in the transformation of
a variety of alcohols and aldehydes of physiological relevance,
such as retinoids, steroids and cytotoxic aldehydes.15 ADH1 and
ADH4 have been the best studied in mammals because of their
wide expression in tissues and their activity towards ethanol and
retinol. While ADH1 is present in all vertebrate groups, ADH4
has only been reported in mammals. In amphibians, an enzyme
showing kinetic properties similar to those of ADH4 has been
described, but it exhibits specificity towards NADP(H) instead
of NAD(H), the common cofactor for these enzymes. ADH8,
isolated from the stomach of Rana perezi, displayed high activity
towards retinal.16 This property and the coenzyme requirement
suggested that this enzyme might have a significant role in retinal
metabolism, probably as a retinal reductase.
We are interested in deciphering the substrate specificity of
ADHs towards retinoids in order to understand more deeply the
biochemical pathways for the formation of vitamin A metabo-
lites through oxidation/reduction reactions. Previous reports
have dealt with the kinetic constants of ADH1 and ADH4
from humans and mice towards some ring-oxidized derivatives
(rac-4-hydroxyretinol, 4-oxoretinal, 3,4-didehydroretinol and 3,4-
didehydroretinal). All these compounds are good substrates for
ADHs, judging from the low and similar Km values measured in
all cases. The 4-hydroxyretinol derivative appeared to be the most
active, showing a catalytic efficiency ca. 30 times higher than parent
compound retinol. The data supported the involvement of the
enzymes in the red-ox metabolism of the C4-oxidized retinoids.17
The availability of recombinant ADH8 from Xenopus laevis, the
interest of this species to developmental studies, the high activity of
ADH8 with retinal, and the physiological presence of several ring-
oxidized retinoids in Xenopus prompted us to study the specificity
and catalytic efficiency of this enzyme with chemically-modified
retinals of biological significance.
Retinoic acid is synthesized from retinol by a metabolic
sequence involving two consecutive oxidation reactions, in which
the reversible oxidation of retinol to retinal is considered to be
the rate-limiting step. Further oxidation of retinal to retinoic
acid appears to be irreversible.13 Three different enzyme types,
grouped as superfamilies, have been suggested to be responsible
aDepartamento de Qu´ımica Orga´nica, Universidade de Vigo, 36310, Vigo,
Spain. E-mail: qolera@uvigo.es; Fax: +34-986-811940; Tel: +34-986-
812316
Whereas in previous reports we determined the kinetic
characteristics of racemic 4-hydroxyretinoids17, we consider the
preparation of the corresponding enantiopure substrates to be
bDepartment of Biochemistry and Molecular Biology, Universitat Auto`noma
de Barcelona, E-08193 Bellaterra, Barcelona, Spain
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of interest in order to determine whether enantiomeric discrim-
ination by the enzyme can occur. In addition to the C4-hydroxyl
derivatives, the study was extended to C3-hydroxyretinal, easily
obtained from enantiopure starting material. The C4 and C3-ring-
oxidized retinoids of the R configuration are components of the
visual cycle in the animal kingdom, namely in the bioluminescent
squid Watasenia scintillans18 and in most insects,19 respectively.
We report herein the stereoselective synthesis of (R)-all-trans-
3-hydroxyretinal 1, (S)-all-trans-4-hydroxyretinal 3, (R)-all-trans-
4-hydroxyretinal 5, and the kinetic constants measured upon
incubation of these retinoids with Xenopus ADH8. Due to the
pharmacological interest of 13-cis-retinoids, we also isolated the
13-cis isomers (2, 4 and 6, respectively) of the above compounds,
which allowed a comparison of the catalytic efficiencies of C13–
C14-double-bond stereoisomers (Fig. 1).
The pentaene ester 13 was obtained by the Horner–Wadsworth–
Emmons reaction between aldehyde 11 and phosphonate 12, using
n-BuLi as a base in THF and DMPU as the cosolvent, in excellent
yield after purification (99%).24 Deprotection of 13 with TBAF
◦
in THF at 0 C afforded 14 in 58% yield (Scheme 2), which was
uneventfully reduced to the corresponding alcohol with Dibal-
H in THF at −78 ◦C and the latter oxidized using MnO2 (52%
combined yield) to afford a 3 : 1 mixture of 125 and 2, which was
separated by HPLC. No improvement could be achieved when
the order of reactions (reduction–deprotection and oxidation)
in the last functional group interconversion steps w◦as reversed.
Reduction of ester 13 with Dibal-H in THF at −78 C afforded
alcohol 15 in 89% yield, deprotection of which (68%), followed by
oxidation with MnO2 under basic conditions at 25 ◦C afforded a
mixture of 1 and 2 in lower yield (42%) and stereoselectivity (2 : 1
ratio) (Scheme 2).
Fig.
1
All-trans and 13-cis-isomers of enantiopure C3- and C4-
hydroxyretinals.
For the construction of the polyene side chain of these analogs,
we took advantage of palladium-catalyzed cross-coupling reac-
tions, a group of processes that have been found in general to
be chemo- and stereoselective and to take place with retention of
configuration of the coupling partners. We selected the Stille cross-
coupling reaction for the construction of the C6–C7 bond [(R)-3-
hydroxyretinal] and C8–C9 bond [(S)- and (R)-4-hydroxyretinal]
guided by our comprehensive study on the scope and limitations
of this process as applied to the synthesis of retinoids.20
Scheme 2 (a) THF, n-BuLi, DMPU, phosphonate 12, −78 − > 25 ◦C, 14 h
(99%); (b) n-Bu4NF, THF, 25 ◦C (58%); (c) Dibal-H, THF, −78 ◦C (66%);
(d) MnO2, Na2CO3, CH2Cl2, 25 ◦C (52%); (e) Dibal-H, THF, −78 ◦C
(89%); (f) n-Bu4NF, THF, 25 ◦C (68%); (g) MnO2, Na2CO3, CH2Cl2, 25 ◦C
(42%).
Results and discussion
Synthesis of (S) and (R)-4-hydroxyretinal
Synthesis of (R)-3-hydroxyretinal
The C8–C9 Stille disconnection for the preparation of (S)-4-
hydroxyretinal 3 and (R)-4-hydroxyretinal 5 required the con-
densation of known stannylated trienol 1620 (Scheme 4) and
dienyliodides (S)-17 or (R)-17, both having trans geometries. The
ketone 1817b already described was envisaged as the precursor
of the electrophilic component of the Stille cross-coupling. For
the enantioselective reduction of enynone 18, we selected the
CBS chiral oxazaborolidine catalysts.26 Treatment of enynone 18
with (R)- or (S)-2-methyl-CBS-oxazaborolidine and BH3·SMe2 in
THF at −30 ◦C afforded alcohols (S)-19 or (R)-19 in excellent
yields (97% and 94%, respectively). Enantiomeric ratios [95% ee
for (S)-19 and 91% ee for (R)-19] were determined by chiral HPLC
(Chiralcel OD-H; 0,46 cm φ × 15 cm, cellulose on 5 lm silica
gel and 95 : 5 hexane/MeOH as eluent) (Scheme 3). The CBS
model for enantioface differentiation27 was adopted to assign
the absolute configuration of the major enantiomer in each
case.
The Stille cross-coupling reaction of the already described triflate
721 and tributylstannyldienol 8,22 using Pd2dba3/AsPh3 as a
catalyst and a LiCl additive, provided, after stirring for 14 h at
80 ◦C, alcohol 10 in 68% yield. Oxidation of 10 with catalytic
quantities of tetra-n-propylammonium perruthenate (TPAP) and
N-methylmorpholine N-oxide (NMO) as co-oxidant,23 afforded
aldehyde 11 in good yield (Scheme 1).
Scheme 1 (a) Pd2dba3, AsPh3, NMP, tributylstannyldiene 8, LiCl, 80 ◦C,
14 h (68%); (b) TPAP, NMO, CH2Cl2, 25 ◦C (83%).
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the most significant. In order to circumvent this problem, the
alkenyl boronate was chosen instead of the alkenylstannane.
Hydroboration of alkynes (S)-21 and (R)-21 using pinacol borane
regio- and stereoselective provided the boronic esters (S)-22 and
(R)-22 (54% and 57%, respectively, based on recovered starting
material). In large-scale reactions, variable amounts of the minor
regioisomer are obtained. Boron–halogen exchange with retention
of co◦nfiguration was effected by treatment of 22 with MeONa at
−78 C followed by addition of ICl. Alkenyl iodides (S)-17 and
(R)-17 were isolated in very high yields, and their E geometry was
confirmed by the value of the coupling constant (J = 14.8 Hz) for
the alkenyl protons in their 1H-NMR spectrum (Scheme 4).
Coupling of iodides 17 with stannanyltrienol 16 under Farina’s
conditions [Pd2(dba)3, AsPh3, NMP] required 7 h at 40 ◦C and
afforded (S)-23 and (R)-23 in 70 and 65% yield, respectively.
Oxidation of diols (S)-23 and (R)-23 was carried out with the
Dess–Martin reagent in CH2Cl2-pyridine to minimize isomer-
ization at the terminal double bond. Despite these precautions,
8.5 : 1 and 6.5 : 1 mixtures of the trans:13-cis-isomers for the
(S) and (R) enantiomers were obtained in 71 and 63% yield,
respectively. Although they could be separated at this stage by
column chromatography if desired, additional isomerization of
the C13–C14 bond took place upon deprotection of the acetates
with K2CO3 in MeOH, affording 3 : 1 mixtures of all-trans:13-cis-
isomers of both (S)-4-hydroxyretinal and (R)-4-hydroxyretinal.
Scheme 3 (a) (R) or (S)-2-methyl-CBS-oxazaborolidine, BH3·SMe2,
THF, 21 h, −30 ◦C [97% for (S)-19 and 94% for (R)-19]; (b) K2CO3,
MeOH, 25 ◦C, 3.5 h [91% for (S)-20 and 98% for (R)-20]; (c) Ac2O, DMAP,
Et3N, CH2Cl2 [96% for (S)-21 and 94% for (R)-21].
R
These isomers were separated by HPLC (Preparative Nova Pakꢀ
Scheme 4 (a) i. pinacol, BH3·SMe2, CH2Cl2, 0–>25 ◦C, ii. Enyne (S)-20
or (R)-20, CH2Cl2, 0–>50 ◦C [54% for (S)-22 and 57% for (R)-22]; (b) i.
MeONa, MeOH, THF, −78 ◦C, ii. ICl, CH2Cl2, −78 ◦C [81% for (S)-17
and 86% for (R)-17]; (c) trienylstannane 16, Pd2(dba)3, AsPh3, NMP, 40 ◦C,
7 h [70% for (S)-23 and 65% for (R)-23]; (d) Dess–Martin Periodinane,
CH2Cl2, pyridine, 25 ◦C, 6 h, [71% for (S) and 63% for (R)]; e) K2CO3,
MeOH, 25 ◦C, 5 h [73% for 3 and 67% for 5].
˚
HR silica, 60 A, 19 × 300 mm and 95:5 hexane/ethyl acetate as
eluent) (Scheme 4). The ee measured for the final compounds (89%
ee for 3 and 93% ee for 4)29 indicates that enantiopurity has been
mostly preserved along the sequence.
Kinetics of ADH8 with retinoids
Deprotection of (S)-19 and (R)-19 with K2CO3 in MeOH
provided efficiently unstable alkynes (91% and 98%, respectively),
which were protected as acetates using Ac2O, DMAP and Et3N in
CH2Cl2 in excellent yield (Scheme 3).
We attempted first to prepare the alkenyliodides by tin-iodide
exchange of the corresponding alkenylstannanes, themselves ob-
tained from the terminal alkynes by radical addition of tributyltin
hydride,28 a methodology already used in the synthesis of the
racemic material,17b but we encountered a number of difficulties,
obtaining mixtures of alkenylstannanes with E and Z geometry
as a result of the quality of the tributyltinhydride used being
The kinetics of Xenopus ADH8 with these C3- and C4-oxidized
derivatives of all-trans-retinal and 13-cis-retinal are shown in
Table 1. All the hydroxyretinals assayed were very active, suggest-
ing that they can be physiological substrates for ADH8. When
compared with the corresponding parent compounds, the kcat
values for the ring-oxidized retinals showed a 10-fold increase,
while Km values remained essentially constant. This effect was
independent of the position of the hydroxyl group since no
substantial differences were found between the C3- and the C4-
hydroxyl derivatives of either all-trans-retinal or 13-cis-retinal.
Furthermore, no differences were observed between R and S
Table 1 Kinetic constants of Xenopus laevis ADH8 with retinoids 1–6a
Substrate
Km/lM
k
cat/min−1
k
cat/Km/mM−1·min−1
All-trans-retinal
21.5 3.5
13.2 1.8
12.6 1.3
10.1 0.9
15
270 13
12560 2100
157600 23500
187300 20000
243600 23800
4200
(R)-all-trans-4-hydroxy-retinal
(S)-all-trans-4-hydroxyretinal
(R)-all-trans-3-hydroxyretinal
13-cis-retinalb
2080 90
2360 70
2460 70
63
(R)-13-cis-4-hydroxyretinal
(S)-13-cis-4-hydroxyretinal
(R)-13-cis-3-hydroxyretinal
Hexanal
23.7 2.3
20.3 2.3
21.9 2.5
79.4 11.8
860 30
860 30
760 30
18600 900
36300 3700
42400 5000
34700 4200
234000 37000
a Activities were determined in 0.1 M sodium phosphate, pH 7.5, 0.02% Tween 80, using 0.6 mM NADPH, at 25 ◦C. b Constants measured for the ADH8
enzyme of Rana perezi.16
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enantiomers of the C4-hydroxyl derivatives of both all-trans-retinal
and 13-cis-retinal, thus concluding that ADH8 does not exhibit
chiral discrimination, at least at position C4. On the other hand,
the trans-ring-oxidized retinal stereoisomers showed 2.4- to 3.2-
fold higher kcat values with respect to the derivatives of 13-cis-
retinal, known to be a poor substrate for several ADH enzymes
studied.17a, 30
even using compounds, such as 13-cis-retinoids, with poor activity
with ADHs.
Experimental
General
Solvents were dried according to published methods and distilled
before use. HPLC grade solvents were used for the HPLC purifica-
tion. All other reagents were commercial compounds of the highest
purity available. All reactions were carried out under an argon
atmosphere, and those not involving aqueous reagents were carried
out in oven-dried glassware. Analytical thin layer chromatog-
raphy (TLC) was performed on aluminium plates with Merck
Kieselgel 60F254 and visualised by UV irradiation (254 nm)
or by staining with solution of phosphomolibdic acid. Flash
column chromatography was carried out using Merck Kieselgel
60 (230–400 mesh) under pressure. High performance liquid
chromatography was performed using a Waters instrument using
a dualwave detector (254 and 300 nm) with a Preparative Nova
The kcat values of ADH8 toward C3- and C4-hydroxyretinals are
the highest among all retinoid substrates assayed for this enzyme.
Similarly, C4-oxo- and C4-hydroxyretinoids were the most active
retinoids for ADH1 and ADH4.17b,31 In general, ADH shows low
turnover numbers (kcat) with retinol isomers, in comparison to
those for aliphatic substrates.17a,32 For retinoids the release of
product may be a significant rate-limiting step, in contrast to
the kinetics with aliphatic substrates, where the limiting step is,
in general, the cofactor release.33 For ADH8 the kcat for ring-
oxidized retinals (about 2000 min−1, Table 1) is still lower than that
for the best aliphatic aldehydes (kcat for hexanal = 18600 min−1
and 14000 min−1 for Xenopus laevis (Table 1) and Rana perezi
enzymes, respectively). Thus, it can be suggested that the kcat
for the ring-oxidized-retinals is higher than that for the parent
compounds because the extra polarity facilitates the delivery to the
aqueous environment of the reduced product, but still this product
release is rate limiting in the reduction of hydroxy-retinals by
ADH8.
The fact that ADH8 does not discriminate between the hydroxyl
groups at the C3 or C4 positions of the cyclohexene ring, or between
the C4-hydroxyretinal enantiomers, is consistent with docking
studies using several retinoids as ligands and the crystallographic
ADH8 structure from Rana perezi34 and other ADH structures.35
While the functional group of the retinoid interacts with the active
site Zn, buried in the inner part of the molecule, the cyclohexene
ring localizes in the wide entrance of the substrate-binding pocket,
accessible to the solvent, and presumably with little structural
constraints.
R
ꢀ
˚
Pak HR silica, 60 A, 19 × 300 mm and 95 : 5 hexane/ethyl acetate
as eluent. Enantiomeric excess was calculated by chiral HPLC
with a WatersTM 996 (Photodiode Array) detector with a Chiralcel
OD–H Column 0,46 cm × 15 cm cellulose on 5 lm silica gel and
95 : 5 hexane/MeOH as eluent. UV–Vis spectra were recorded on a
Cary 100 Bio spectrophotometer using MeOH as solvent. Infrared
spectra were obtained on JASCO FT-IR 4200 spectrophotometer,
from a thin film deposited onto a NaCl glass. Specific rotation
was obtained on JASCO P-1020. Mass spectra were obtained
on a Hewlett-Packard HP59970 instrument operating at 70 eV
by electron ionisation. High resolution mass spectra were taken
1
on a VG Autospec instrument. H NMR spectra were recorded
in CDCl3, C6D6 and (CD3)2CO at ambient temperature on a
Bruker AMX-400 spectrometer at 400 MHz with residual protic
solvent as the internal reference (CDCl3, dH = 7.26 ppm; C6D6,
dH = 7.16 ppm; (CD3)2CO, dH = 2.05 ppm); chemical shifts (d)
are given in parts per million (ppm), and coupling constants
(J) are given in Hertz (Hz). The proton spectra are reported as
follows: d (multiplicity, coupling constant J, number of protons,
assignment). 13C NMR spectra were recorded in CDCl3, C6D6
and (CD3)2CO at ambient temperature on the same spectrometer
at 100 MHz, with the central peak of CDCl3 (dC = 77.0 ppm), C6D6
(dC = 128.0 ppm) or (CD3)2CO (dC = 30.8 ppm) as the internal
reference. DEPT135 are used to aid in the assignment of signals
in the 13C NMR spectra.
Finally, this is the first time that ring-oxidized derivatives of
the 13-cis-retinal isomer have been assayed for ADH activity, and
they also show a great increase in their kcat values when compared
with 13-cis-retinal. The rate increase is about 10-fold, similar
to that observed for the hydroxyl derivatives of all-trans-retinal.
The 13-cis-isomers are among the worst retinoid substrates for
ADHs because of their low kcat values, being virtually inactive
with ADH1.17a In fact, ADH8 is the most active known ADH
with 13-cis-retinoids.16
(−)-(2E,4E)-5-[(R)-4-(tert-Butyldimethylsilyloxy)-2,6,6-tri-
methylcyclohex-1-en-1-yl]-3-methylpenta-2,4-dien-1-ol 10. General
procedure for Stille cross coupling. A solution of (R)-5-(tert-
butyldimethylsilyloxy)-2-[(trifluoromethanesulfonyl)oxy]-1,3,3-tri-
methylcyclohex-1-ene 7 (0.70 g, 1.75 mmol) in NMP (15.6 mL)
was added to a solution of Pd2(dba)3 (0.04 g, 0.042 mmol), AsPh3
(0.11 g, 0.35 mmol) in NMP (3.2 mL). After stirring for 10 min,
a solution of (2E,4E)-3-methyl-5-(tri-n-butylstannyl)penta-2,4-
dien-1-ol 8 (0.82 g, 2.10 mmol) in NMP (3.2 mL) and LiCl
(0.22 g, 5.26 mmol) was added. The mixture was stirred for
14 h at 80 ◦C. An aqueous solution of KF (10 mL) was added
and the mixture was stirred for 10 min and then extracted with
t-BuOMe (3x). The combined organic layers were washed with
H2O (3x), dried (Na2SO4) and the solvent was evaporated. The
Conclusions
The catalytic constant (kcat) values of Xenopus laevis ADH8
with enantiopure C3- and C4-hydroxyretinals were about 10-fold
those for the parent all-trans-retinal. The kinetic studies revealed
neither enantiomeric discrimination (4R vs 4S) nor positional
discrimination (3R vs 4R) by the enzyme, consistent with an
external position of the cyclohexene ring in the ADH-retinoid
complex structure. The corresponding 13-cis isomers of the ring
oxidized derivatives, obtained as isomerization by-products in the
synthetic sequence, showed from 2.4- to 3.2-fold lower kcat than
the trans-isomers.
The OH substitution at the cyclohexene ring would facilitate
the study of the kinetic effect of the double bond isomerization,
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residue was purified by column chromatography (silica gel, 90 :
10 hexane/ethyl acetate) to afford 0.42 g (68%) of a yellow
acetate/Et3N) to afford 81 mg (99%) of a yellow oil identified
25
1
as 13. [a]D −; 94.0 (c 0.034, MeOH). H-NMR (400.16 MHz,
(CD3)2CO): d 7.13 (dd, J = 15.1, 11.4 Hz, 1H, H11), 6.42 (d, J =
15.1 Hz, 1H, H12), 6.25 (d, J = 11.4 Hz, 1H, H10), 6.3–6.2 (m, 2H,
H7 + H8), 5.82 (s, 1H, H14), 4.13 (q, J = 7.1 Hz, OCH2–CH3), 4.1–
4.0 (m, 1H, H3), 2.36 (s, 3H, C13–CH3), 2.4–2.3 (m, 1H, H4), 2.1 (d,
J = 11.1 Hz, 1H, H4), 2.05 (s, 3H, C9–CH3), 1.75 (s, 3H, C5–CH3),
1.7–1.6 (m, 1H, H2), 1.48 (t, J = 11.9 Hz, 1H, H2), 1.25 (t, J =
7.1 Hz, 3H, OCH2–CH3), 1.11 (s, 3H, C1–CH3), 1.09 (s, 3H, C1–
CH3), 0.93 (s, 9H, SiC(CH3)3), 0.11 (s, 6H, Si(CH3)2). 13C-NMR
(100.62 MHz, (CD3)2CO): d 167.6 (s), 153.7 (s), 140.4 (s), 139.3 (d),
138.6 (s), 136.8 (d), 132.3 (d), 131.4 (d), 128.8 (d), 128.4 (s), 119.9
(d), 66.6 (d), 60.4 (t), 50.1 (t), 44.3 (t), 38.0 (s), 31.1 (q), 29.4 (q),
26.7 (q, 3x), 22.3 (q), 19.1 (s), 15.1 (q), 14.2 (q), 13.3 (q), −3.9 (q,
2x). MS (EI+): m/z (%) 459 (M+ + 1, 37), 458 (M+, 100), 326 (23),
285 (91), 133 (16), 131 (15), 121 (27), 105 (19), 91(13), 74 (31),
73 (35). HRMS (EI+): Calcd. for C28H46O3Si, 458.3216; found,
oil identified as 10. [a]D − 64.9 (c 0.02, MeOH). 1H-NMR
18
(400.16 MHz, CDCl3): d 6.1–6.0 (m, 2H, H4 + H5), 5.61 (t, J =
6.8 Hz, 1H, H2), 4.30 (d, J = 6.8 Hz, 2H, 2H1), 4.0–3.8 (m, 1H,
ꢀ
ꢀ
ꢀ
H4 ), 2.22 (dd, J = 17.0, 5.2 Hz, 1H, H3 ), 2.1–2.0 (m, 1H, H3 ),
ꢀ
ꢀ
1.85 (s, 3H, C3–CH3), 1.68 (s, 3H, C2 –CH3), 1.6–1.5 (m, 1H, H5 ),
ꢀ
ꢀ
1.45 (t, J = 12.0 Hz, 1H, H5 ), 1.03 (s, 3H, C6 –CH3), 1.02 (s,
ꢀ
3H, C6 –CH3), 0.90 (s, 9H, SiC(CH3)3), 0.08 (s, 6H, Si(CH3)2).
13C-NMR (100.62 MHz, CDCl3): d 137.9 (d), 137.5 (s), 137.1 (s),
129.2 (d), 127.0 (s), 126.7 (d), 66.1 (d), 59.8 (t), 49.1 (t), 43.3 (t),
37.4 (s), 30.5 (q), 28.9 (q), 26.4 (q, 3x), 21.9 (q), 18.6 (s), 12.8 (q),
−4.2 (q, 2x). MS (EI+): m/z (%) 332 (M+–H2O, 10), 219 (100), 185
(12), 174 (35), 159 (50), 145 (11), 75 (39). HRMS (EI+): Calcd. for
C21H36OSi (M+–H2O)+, 332.2535; found, 332.2529. IR (NaCl): m
3500–3100 (br, OH), 2955 (s, C–H), 2928 (s, C–H), 2856 (s, C–H),
1084 cm−1.
458.3216. IR (NaCl): m 2955 (s, C–H), 2927 (s, C–H), 2856 (s, C–H),
(−)-(2E,4E)-5-[(R)-4-(tert-Butyldimethylsilyloxy)-2,6,6-trimethyl-
cyclohex-1-en-1-yl]-3-methylpenta-2,4-dienal 11. To a cooled
(0 ◦C) stirred solution of NMO (0.11 g, 0.94 mmol) in CH2Cl2
−1
=
1708 (s, C O), 1149 cm . UV (MeOH): kmax 348 nm (e = 26800).
Ethyl (−)-(R)-all-trans-3-hydroxyretinoate 14. A cooled (0 ◦C)
solution of 13 (0.078 g, 0.17 mmol) in THF (4 mL) was treated
with n-Bu4NF (0.21 mL, 1 M in THF, 0.21 mmol) and stirred
for 7 h. The mixture was diluted with Et2O (2 mL) and washed
with an aqueous solution of NaHCO3 (1x). The aqueous layer
was extracted with AcOEt (3x) and the combinated organic layers
were washed with brine (3x), dried (Na2SO4) and concentrated.
The residue was purified by column chromatography (silica gel,
60 : 40 hexane/ethyl acetate) to afford 53 mg (58%) of a yellow
˚
(3.6 mL) containing 4 A molecular sieves was added a solution
of 10 (0.22 g, 0.63 mmol) in CH2Cl2 (1.8 mL). After stirring for
10 min, TPAP (0.012 g, 0.032 mmol) was added and the mixture
was stirred at 25 ◦C for 3 h. The mixture was diluted with CH2Cl2
(4 mL) and washed with aqueous Na2SO3 (3x). The organic layer
was dried (Na2SO4) and the solvent was evaporated. The residue
was purified by column chromatography (silica gel, 93 : 4 : 3
hexane/ethyl acetate/Et3N) to afford 0.17 g (83%) of a yellow
26
1
26
1
oil identified as 11. [a]D − 130.6 (c 0.026, MeOH). H-NMR
(400.16 MHz, C6D6): d 10.01 (d, J = 7.8 Hz, 1H, H1), 6.38 (d,
J = 16.1 Hz, 1H, H5), 5.96 (d, J = 16.1 Hz, 1H, H4), 5.92 (d, J =
oil identified as 14. [a]D − 114.1 (c 0.028, MeOH). H-NMR
(400.16 MHz, (CD3)2CO): d 7.15 (dd, J = 15.1, 11.2 Hz, 1H, H11),
6.43 (d, J = 15.1 Hz, 1H, H12), 6.26 (d, J = 11.2 Hz, 1H, H10),
6.3–6.2 (m, 2H, H7 + H8), 5.82 (s, 1H, H14), 4.13 (q, J = 7.1 Hz,
OCH2–CH3), 4.0–3.9 (m, 1H, H3), 2.36 (s, 3H, C13–CH3), 2.3–2.2
(m, 1H, H4), 2.07 (s, 3H, C9–CH3), 2.1–2.0 (m, 1H, H4), 1.8–1.7
(m, 1H, H2), 1.74 (s, 3H, C5–CH3), 1.43 (t, J = 11.9 Hz, 1H, H2),
1.26 (t, J = 7.1 Hz, 3H, OCH2–CH3), 1.10 (s, 3H, C1–CH3), 1.07
(s, 3H, C1–CH3). 13C-NMR (100.62 MHz, (CD3)2CO): d 166.2 (s),
152.4 (s), 139.0 (s), 137.7 (d), 137.2 (s), 135.3 (d), 130.9 (d), 129.9
(d), 127.6 (d), 127.1 (s), 118.4 (d), 63.3 (d), 58.9 (t), 48.5 (t), 42.5 (t),
36.5 (s), 29.7 (q), 27.9 (q), 20.8 (q), 13.6 (q), 12.8 (q), 11.9 (q). MS
(EI+): m/z (%) 345 (M+ + 1, 24), 344 (M+, 100), 285 (19), 271 (35),
197 (37), 192 (22), 191 (25), 173 (33), 171 (45), 159 (25), 157 (30),
131 (30), 119 (39), 107 (38), 105 (39), 91 (42). HRMS (EI+): Calcd.
ꢀ
7.8 Hz, 1H, H2), 4.1–4.0 (m, 1H, H4 ), 2.26 (dd, J = 17.0, 5.7 Hz,
ꢀ
ꢀ
1H, H3 ), 2.15 (dd, J = 17.0, 8.9 Hz, 1H, H3 ), 1.8–1.7 (m, 1H,
ꢀ
ꢀ
H5 ), 1.71 (s, 3H, C3–CH3), 1.7–1.6 (m, 1H, H5 ), 1.54 (s, 3H,
ꢀ
ꢀ
C2 –CH3), 1.05 (s, 9H, SiC(CH3)3), 1.01 (s, 3H, C6 –CH3), 0.97 (s,
3H, C6 –CH3), 0.15 (s, 6H, Si(CH3)2). 13C-NMR (100.62 MHz,
ꢀ
(CD3)2CO): d 190.4 (d), 153.8 (s), 136.6 (s), 136.4 (d), 134.0 (d),
129.2 (d), 128.9 (s), 65.1 (d), 48.6 (t), 42.9 (t), 36.6 (s), 29.5 (q),
27.9 (q), 25.3 (q, 3x), 20.8 (q), 17.7 (s), 11.9 (q), −5.3 (q, 2x). MS
(EI+): m/z (%) 291 (M+-t-Bu, 28), 235 (90), 216 (21), 205 (23),
199 (25), 197 (21), 190 (48), 175 (59), 173 (29), 171 (33), 157 (36),
147 (38), 145 (26), 143 (38), 133 (78), 121 (29), 119 (49), 105 (41),
95 (42), 91 (25), 75 (100). HRMS (EI+): Calcd. for C21H36O2Si,
348.2485; found, 348.2487. IR (NaCl): m 2956 (s, C–H), 2928 (s,
for C22H32O3, 344.2351; found, 344.2350. IR (NaCl): m 3500–3150
−1
−1
=
=
C–H), 2856 (s, C–H), 1666 (s, C O), 1083 cm . UV (MeOH):
(br, OH), 2957 (s, C-H), 2921 (s, C–H), 1706 (s, C O), 1151 cm .
kmax 280 nm.
UV (MeOH): kmax 353 nm (e = 47000).
Ethyl (−)-(R)-all-trans-3-(tert-butyldimethylsilyloxy)-retinoate
13. A cooled (0 ◦C) solution of diethyl (E)-3-(ethoxycarbonyl)-
2-methylprop-2-en-1-ylphosphonate 12 (0.09 g, 0.35 mmol) and
DMPU (0.064 mL, 0.53 mmol) in THF (0.2 mL) was treated with
n-BuLi (0.25 mL, 1.32 M in hexane, 0.33 mmol) and stirred for
30 min. The mixture was cooled down to −78 ◦C, and a solution of
11 (0.07 g, 0.19 mmol) in THF (0.2 mL) was added. The resulting
mixture was allowed to warm to 25 ◦C for 14 h, and H2O (1 mL)
was added. The reaction was extracted with Et2O (3x) and the
organic layers were washed with H2O (3x) and brine (3x), dried
(Na2SO4) and the solvent was evaporated. The residue was purified
by column chromatography (silica gel, 91 : 6 : 3 hexane/ethyl
(−)-(R)-All-trans-3-hydroxyretinal 1. Dibal-H (0.85 mL, 1 M
in hexane, 0.85 mmol) was added to a solution of 14 (0.074 g,
0.21 mmol) in THF (2 mL), at −78 ◦C, and the resulting suspension
was stirred for 1 h. After careful addition of H2O, the mixture
was extracted with Et2O (3x) and the organic layers were dried
(Na2SO4) and concentrated. The residue was purified by column
chromatography (silica gel, 97:3 hexane/Et3N) to afford 43 mg
(66%) of a yellow oil that was oxidized immediately.
To a solution of this compound (0.04 g, 0.14 mmol) in CH2Cl2
(2.6 mL) was added MnO2 (0.22 g, 2.54 mmol) and Na2CO3 (0.27 g,
2.54 mmol), and the suspension was stirred for 5 h. The mixture
was filtered throught Celite and the solvents were removed. The
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residue was purified by column chromatography (silica gel, 97 :
3 hexane/ethyl acetate) to afford 0.022 g (52%) as a yellow oil
identified as a mixture of 1 and 2 in a 3 : 1 ratio, which were
separated by HPLC.
(−)-(R)-All-trans-3-hydroxyretinal 1. Following the gen-
eral procedure for TBAF deprotection, the reaction of
15 (0.036 g, 0.087 mmol) with n-Bu4NF (0.35 mL,
1 M in THF, 0.35 mmol) in THF (2 mL) afforded, after purification
by column chromatography (silica gel, 60 : 40 hexane/ethyl
acetate) 18 mg (68%) of a yellow oil that was used immediately.
Following the general procedure for MnO2 oxidation, the reaction
of the above alcohol (0.018 g, 0.14 mmol) with MnO2 (0.093 g, 1.06
mmol) and anhydrous Na2CO3 (0.11 g, 1.06 mmol) in CH2Cl2 (1.2
mL) afforded, after purification by column chromatography (silica
gel, 80 : 20 hexane/ethyl acetate), 7 mg (42%) of a yellow mixture
containing 1 and 2 in a 2 : 1 ratio.
20
Data for (−)-(R)-all-trans-3-hydroxyretinal 1: [a]D − 68.6
(c 0.022, MeOH). 1H-NMR (400.16 MHz, (CD3)2CO): d 9.81
(d, J = 8.0 Hz, 1H, H15), 6.98 (dd, J = 15.1, 11.4 Hz, 1H, H11), 6.17
(d, J = 15.1 Hz, 1H, H12), 6.03 (d, J = 16.1 Hz, 1H, H7), 5.98 (d, J =
11.5 Hz, 1H, H10), 5.90 (d, J = 16.1 Hz, 1H, H8), 5.59 (d, J = 8.0Hz,
1H, H14), 3.7–3.5 (m, 1H, H3), 2.49 (s, 3H, C13–CH3), 2.1–2.0 (m,
1H, H4), 1.74 (s, 3H, C9–CH3), 1.7–1.6 (m, 1H, H4), 1.4–1.3 (m,
1H, H2), 1.41 (s, 3H, C5–CH3), 1.09 (t, J = 11.9 Hz, 1H, H2), 0.75
(s, 3H, C1–CH3), 0.74 (s, 3H, C1–CH3). 13C-NMR (100.62 MHz,
(CD3)2CO): d 190.3 (d), 154.2 (s), 140.3 (s), 137.7 (d), 137.1 (s),
135.0 (d), 132.2 (d), 129.9 (d), 128.9 (d), 128.3 (d), 127.4 (s), 63.3
(d), 48.5 (t), 42.5 (t), 36.5 (s), 29.7 (q), 27.9 (q), 20.8 (q), 12.0
(q), 11.9 (q). MS (EI+): m/z (%) 300 (M+, 72), 171 (63), 159 (25),
157 (26), 147 (25), 145 (27), 133 (25), 131 (26), 119 (43), 105
(39), 95 (36), 91 (40), 69 (100). HRMS (EI+): Calcd. for C20H28O2,
(−)-(S)-2,4,4-Trimethyl-3-(trimethylsilylethynyl)cyclohex-2-en-
1-ol (S)-19. General procedure for enantioselective reduction of
ketones. To a cooled (−78 ◦C) solution of 2,4,4-trimethyl-
3-(trimethylsilylethynyl)cyclohex-2-en-1-one 18 (1.0 g, 4.26
mmol) in THF (50 mL) were sequentially added a solution of
(R)-2-methyl-CBS-oxazaborolidine (0.43 mL, 1 M in toluene,
0.43 mmol) and BH3·SMe2 (0.43 mL, 4.26 mmol). After stirring
for 21 h at −30 ◦C, H2O (30 mL) was added and the mixture
was extracted with t-BuOMe (4x). The combined organic layers
were dried (Na2SO4) and the solvent was evaporated. The
residue was purified by column chromatography (silica gel, 93 :
7 hexane/ethyl acetate) to afford 0.98 g (97%) of a white solid
300.2089; found, 300.2086. IR (NaCl): m 3550–3150 (br, OH), 2957
−1
=
(s, C–H), 2918 (s, C–H), 2850 (s, C–H), 1658 (s, C O), 1574 cm .
UV (MeOH): kmax 378 nm (e = 28000).
20
Data for (−)-(R)-13-cis-3-hydroxyretinal 2: [a]D − 64.8 (c
1
0.012, MeOH). H-NMR (400.16 MHz, (CD3)2CO): d 10.25 (d,
J = 7.9 Hz, 1H, H15), 7.50 (d, J = 15.0 Hz, 1H, H12), 7.20 (dd,
J = 15.0, 11.5 Hz, 1H, H11), 6.37 (d, J = 15.9 Hz, 1H, H7), 6.35 (d,
J = 11.1 Hz, 1H, H10), 6.23 (d, J = 15.9 Hz, 1H, H8), 5.80 (d, J =
7.9 Hz, 1H, H14), 3.9–3.8 (m, 1H, H3), 2.4–2.3 (m, 1H, H2), 2.17 (s,
3H, C13–CH3), 2.04 (s, 3H, C9–CH3), 2.0–1.9 (m, 1H, H2), 1.8–1.7
(m, 1H, H4), 1.74 (s, 3H, C5–CH3), 1.42 (t, J = 12.0 Hz, 1H, H4),
1.08 (s, 3H, C1–CH3), 1.07 (s, 3H, C1–CH3). MS (EI+): m/z (%)
300 (M+, 100), 147 (22), 121 (24), 119 (40), 105 (25), 95 (24), 91
(29), 77 (19). HRMS (EI+): Calcd. for C20H28O2, 300.2089; found,
identified as (S)-19. MP: 57–59 ◦C (t-BuOMe). [a]D − 38.4
22
(c 0.026, MeOH).1H-NMR (400.13 MHz, CDCl3): d 4.01 (t,
J = 5.0 Hz, 1H, H1), 2.00 (s, 3H, C2–CH3), 1.9–1.8 (m, 1H, H6),
1.7–1.6 (m, 1H, H6), 1.6–1.5 (m, 1H, H5), 1.4–1.3 (m, 1H, H5),
1.14 (s, 3H, C4–CH3), 1.08 (s, 3H, C4–CH3), 0.2 (s, 9H, Si–(CH3)3)
ppm. 13C-NMR (100.62 MHz, CDCl3): d 141.7 (s), 127.7 (s),
103.3 (s), 99.4 (s), 68.8 (d), 34.1 (s), 33.0 (t), 28.8 (q), 28.1 (t),
27.6 (q), 19.3 (q), 0.0 (q, 3x) ppm. MS (FAB+): m/z (%) 237
(M+ + 1, 10), 236 (M+, 22), 219 (M+–OH, 100), 180 (33), 165 (13).
HRMS (FAB+): Calcd. for C14H25OSi (M + 1)+, 237.1675; found,
237.1671. IR (NaCl):m 3600–3100 (br, OH), 2961 (m, C–H), 2938
(m, C–H), 2866 (m, C–H), 2136 (w, C≡C), 1249 cm−1. Elemental
analysis: calcd. for C14H25OSi: C, 71.12; H, 10.23; found: C, 71.12;
H, 10.27.
300.2086. IR (NaCl): m 3580–3150 (br, OH), 2956 (s, C–H), 2927
−1
=
(s, C–H), 2855 (s, C–H), 1658 (s, C O), 1575 cm . UV (MeOH):
kmax 374 nm (e = 22000).
(−)-(R)-All-trans-3-(tert-butyldimethylsilyloxy)retinol 15.
Following the general procedure for Dibal-H reduction, the
reaction of 13 (0.08 g, 0.17 mmol) with Dibal-H (0.7 mL, 1 M in
hexane, 0.7 mmol) in THF (1.6 mL) at −78 ◦C for 1.5 h, afforded,
after purification by column chromatography (silica gel, 85 : 15
hexane/ethyl acetate) 0.064 g (89%) of a yellow oil identified
(+)-(R)-2,4,4-Trimethyl-3-(trimethylsilylethynyl)cyclohex-2-en-
1-ol (R)-19. Following the general prodedure for enantioselective
reduction of ketones, the reaction of 18 (1.0 g, 4.26 mmol) in
THF (50 mL) with (S)-2-methyl-CBS-oxazaborolidine (0.43 mL,
1 M in toluene, 0.43 mmol) and BH3·SMe2 (0.43 mL, 4.26 mmol)
afforded, after purification by column chromatography (silica
gel, 93 : 7 hexane/ethyl acetate), 0.95 g (94%) of a white solid
25
1
as 15. [a]D − 89.9 (c 0.03, MeOH). H-NMR (400.16 MHz,
(CD3)2CO): d 6.65 (dd, J = 15.1, 11.3 Hz, 1H, H11), 6.34 (d,
J = 15.1 Hz, 1H, H12), 6.3–6.2 (m, 3H, H7 + H8 + H10), 5.70 (t,
J = 5.8 Hz, 1H, H14), 4.25 (t, J = 5.8 Hz, 2H, 2H15), 4.1–4.0 (m,
1H, H3), 2.8–2.7 (m, 1H, H2), 2.4–2.3 (m, 1H, H2), 1.97 (s, 3H,
C13–CH3), 1.83 (s, 3H, C9–CH3), 1.73 (s, 3H, C5–CH3), 1.8–1.7
(m, 1H, H4), 1.5–1.4 (m, 1H, H4), 1.10 (s, 3H, C1–CH3), 1.07 (s,
3H, C1–CH3), 0.92 (s, 9H, SiC(CH3)3), 0.11 (s, 6H, Si(CH3)2).
13C-NMR (100.62 MHz, (CD3)2CO): d 139.7 (d), 138.7 (s), 138.6
(d), 136.2 (s), 135.9 (s), 134.2 (d), 132.3 (d), 127.6 (s), 126.6 (d),
125.4 (d), 66.7 (d), 59.7 (t), 50.1 (t), 44.2 (t), 38.0 (s), 31.0 (q),
29.4 (q), 26.7 (q, 3x), 22.2 (q), 19.1 (s), 13.1 (q), 13.0 (q), −3.9
(q, 2x). MS (EI+): m/z (%) 253 (44), 209 (13), 201 (20), 143 (24),
123 (24), 121 (25), 75 (100). HRMS (EI+): Calcd. for C26H44O2Si,
416.3126; found, 416.3111. IR (NaCl): m 3550–3150 (br, OH),
2929 (s, C–H) cm−1. UV (MeOH): kmax 323 nm (e = 19000).
26
identified as (R)-19. [a]D + 37.6 (c 0.027, MeOH). Elemental
analysis: calcd. for C14H25OSi: C, 71.12; H, 10.23; found: C, 71.09;
H, 10.26.
(−)-(S)-3-Ethynyl-2,4,4-trimethylcyclohex-2-en-1-ol (S)-20.
General procedure for deprotection with K2CO3. To a cooled
(0 ◦C) solution of (S)-19 (0.34 g, 1.45 mmol) in MeOH (6.5 mL)
was added K2CO3 (0.38 g, 2.91 mmol) and the mixture was
stirred at 25 ◦C for 3.5 h. H2O was added (4 mL) and the
mixture was extracted with Et2O (4x). The combined organic
layers were dried (Na2SO4) and the solvent was evaporated. The
residue was purified by column chromatography (silica gel, 90:10
hexane/ethyl acetate) to afford 0.22 g (91%) of a white solid
1 6 0 | Org. Biomol. Chem., 2006, 4, 155–164
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identified as (S)-20. MP : 47–49 ◦C (t-BuOMe). [a]D − 77.6
mixture was stirred at 25 ◦C for◦1 h and at 50 ◦C for an additional
5 h. After cooling down to 25 C, Et2O (1 mL) and a saturated
aqueous NH4Cl (1 mL) were added. The organic layer was
washed with saturated aqueous NH4Cl (3x), dried (Na2SO4)
and the solvent was evaporated. The residue was purified by
column chromatography (silica gel, 95 : 5 hexane/ethyl acetate)
to afford 0.086 g (54% based on recovered starting alkyne (S)-21)
22
(c 0.036, MeOH).1H-NMR (400.13 MHz, CDCl3): d 4.02 (t,
J = 4.8 Hz, 1H, H1), 3.13 (s, 1H, H2 ), 2.00 (s, 3H, C2–CH3),
ꢀ
1.7–1.5 (m, 2H, 2H6), 1.5–1.4 (m, 2H, 2H5), 1.15 (s, 3H, C4–CH3),
1.08 (s, 3H, C4–CH3) ppm. 13C-NMR (100.62 MHz, CDCl3): d
142.3 (s), 126.9 (s), 82.2 (d), 81.6 (s), 69.0 (d), 34.2 (s), 33.2 (t),
28.8 (q), 28.2 (t), 27.6 (q), 19.2 (q) ppm. MS (EI+): m/z (%) 164
(M+, 47), 149 (28), 109 (10), 108 (100), 107 (46), 91 (18), 90 (13), 79
(18), 77 (12). HRMS (EI+): Calcd. for C11H16O, 164.1201; found,
164.1206. IR (NaCl): m 3600–3100 (br, OH), 2961 (s, C–H), 2937
(s, C–H), 2866 (m, C–H), 2087 (w, C≡C) cm−1.
24
of a colorless oil identified as (S)-22. [a]D − 87.4 (c 0.024,
MeOH).1H-NMR (600.13 MHz, CDCl3): d 6.94 (d, J = 18.6 Hz,
ꢀ
ꢀ
1H, H1 ), 5.46 (d, J = 18.6 Hz, 1H, H2 ), 5.21 (t, J = 4.6 Hz, 1H,
H1), 2.07 (s, 3H, COOCH3), 1.9–1.8 (m, 1H, H6), 1.8–1.7 (m, 1H,
H6), 1.67 (s, 3H, C2–CH3), 1.6–1.5 (m, 1H, H5), 1.5–1.4 (m, 1H,
H5), 1.29 (s, 12H, -OC(CH3)2C(CH3)2O-), 1.07 (s, 3H, C4–CH3),
1.02 (s, 3H, C4–CH3) ppm. 13C-NMR (100.62 MHz, CDCl3): d
171.4 (s), 148.9 (d, 2x), 145.8 (s), 127.0 (s), 83.6 (s, 2x), 72.8 (d),
35.2 (t), 34.6 (s), 29.2 (q), 27.6 (q), 25.6 (t), 25.2 (q, 4x), 21.8 (q),
18.5 (q) ppm. MS (EI+): m/z (%) 274 (M+–OAc, 45), 259 (82),
172 (25), 159 (100), 158 (18), 146 (29), 145 (15), 133 (22), 131 (67),
101 (76), 84 (15), 83 (40). HRMS (EI+): Calcd. for C17H28BO2,
(+)-(R)-3-Ethynyl-2,4,4-trimethylcyclohex-2-en-1-ol (R)-20.
Following the general procedure for deprotection with K2CO3,
the reaction of (R)-19 (0.16 g, 0.66 mmol) in MeOH (3 mL) with
K2CO3 (0.17 g, 1.32 mmol) afforded, after purification by column
chromatography (silica gel, 90 : 10 hexane/ethyl acetate), 0.11 g
22
(98%) of a white solid identified as (R)-20. [a]D + 79.87 (c 0.03,
MeOH).
(−)-(S)-3-Ethynyl-2,4,4-trimethylcyclohex-2-en-1-yl acetate
(S)-21. General procedure for protection of alcohols as acetates.
To a solution of (S)-20 (0.04 g, 0.24 mmol) in CH2Cl2 (2.5 mL) were
sequentially added Et3N (0.11 mL, 0.86 mmol), DMAP (1.5 mg,
0.12 mmol) and Ac2O (37.4 mg, 0.37 mmol). After stirring for
4 h, a saturated aqueous NaHCO3 solution (3 mL) was added.
The mixture was extracted with AcOEt (3x) and the organic layers
were washed with brine (3x), dried (Na2SO4) and the solvent was
evaporated. The residue was purified by column chromatography
(silica gel, 95 : 5 hexane/ethyl acetate) to afford 0.048 g (96%)
274.2219; found, 274.2230. IR (NaCl): m 2975 (s, C–H), 2864 (m,
−1
=
C–H), 1736 (s, C O), 1619, 1349, 1242, 1142 cm .
(+)-(R)-3-[2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-
ethen-1-yl]-2,4,4-trimethylcyclohex-2-en-1-yl
acetate
(R)-22.
Following the general procedure for the preparation of pinacol
boronates, the reaction of (R)-21 (0.17 g, 0.81 mmol) with
BH3·SMe2 (0.45 mL, 4.8 mmol) and pinacol (0.57 g, 4.80 mmol)
in CH2Cl2 (0.6 mL) afforded, after purification by column
chromatography (silica gel, 95 : 5 hexane/ethyl acetate), 0.074 g
(57% based on recovered starting alkyne (R)-21) of a colorless oil
24
of a colorless oil identified as (S)-21. [a]D − 104.4 (c 0.022,
24
identified as (R)-22. [a]D + 74.9 (c 0.012, MeOH).
MeOH).1H-NMR (600.13 MHz, CDCl3): d 5.24 (t, J = 4.1 Hz,
ꢀ
1H, H1), 3.16 (s, 1H, H2 ), 2.05 (s, 3H, C2–CH3), 1.9–1.8 (m, 1H,
(R)-3-[1-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-ethen-
1-yl]-2,4,4-trimethylcyclohex-2-en-1-yl Acetate. 1H-NMR
H6), 1.86 (s, 3H, COOCH3), 1.7–1.6 (m, 1H, H6), 1.6–1.5 (m, 1H,
H5), 1.5–1.4 (m, 1H, H5), 1.16 (s, 3H, C4–CH3), 1.08 (s, 3H, C4–
CH3) ppm. 13C-NMR (100.62 MHz, CDCl3): d 170.8 (s), 138.6 (s),
129.1 (s), 82.9 (d), 81.2 (s), 70.8 (d), 34.1 (s), 33.2 (t), 28.7 (q), 27.4
(q), 25.0 (t), 21.2 (q), 19.1 (q) ppm. IR (NaCl): m 3287 (m, C–H),
2961 (s, C–H), 2938 (s, C–H), 2861 (m, C–H), 2087 (w, C≡C), 1735
ꢀ
(600.13 MHz, CDCl3): d 6.05 (d, J = 3.9 Hz, 1H, H2 ), 5.45 (d,
ꢀ
J = 3.9 Hz, 1H, H2 ), 5.28 (t, J = 5.3 Hz, 1H, H1), 2.10 (s, 3H,
COOCH3), 1.9–1.8 (m, 1H, H6), 1.8–1.7 (m, 1H, H6), 1.6–1.5 (m,
1H, H5), 1.5–1.4 (m, 1H, H5), 1.46 (s, 3H, C2–CH3), 1.27 (s, 12H,
OC(CH3)2), 1.00 (s, 3H, C4–CH3), 0.94 (s, 3H, C4–CH3) ppm.
13C-NMR (100.62 MHz, CDCl3): d 171.6 (s, COOCH3), 147.2 (s,
−1
+
+
=
(s, C O), 1236 cm . MS (EI ): m/z (%) 206 (M , 8), 147 (31),
146 (56), 132 (43), 131 (100), 129 (59), 128 (53), 116 (75), 115 (75),
108 (44), 91 (76), 77 (41), 65 (12), 63 (12). HRMS (EI+): Calcd. for
C13H18O2, 206.1307; found, 206.1310.
ꢀ
ꢀ
C3), 132.5 (t, C2 ), 132.5 (s, C1 ), 123.7 (s, C2), 83.8 (s, C(CH3)2),
73.0 (d, C1), 35.4 (t, C5), 34.8 (s, C4), 28.4 (q, C4–CH3), 27.6 (q,
C4–CH3), 25.9 (t, C6), 25.0 (q, 4x, C(CH3)2), 21.8 (q, COOCH3),
17.7 (q, C2–CH3) ppm. MS (EI+): m/z (%) 334 (M+, 2), 292 (41),
274 (93), 259 (M+–OAc, 100), 258 (25), 236 (42), 218 (28), 191
(22), 159 (35), 136 (26), 101 (60), 91 (26), 83 (33). HRMS (EI+):
Calcd. for C19H31BO4, 334.2315; found, 334.2308.
(+)-(R)-3-Ethynyl-2,4,4-trimethylcyclohex-2-en-1-yl acetate
(R)-21. Following the general procedure for protection of al-
cohols as acetates, the reaction of (R)-20 (0.86 g, 5.24 mmol) in
CH2Cl2 (52.5 mL) with Et3N (2.38 mL, 18.34 mmol), DMAP
(32 mg, 0.26 mmol) and Ac2O (0.74 mL, 7.86 mmol) afforded,
after purification by column chromatography (silica gel, 95 : 5
hexane/ethyl acetate) 1.02 g (94%) of a colorless oil identified as
(−)-(S)-3-(2-Iodoethen-1-yl)-2,4,4-trimethylcyclohex-2-en-1-yl
acetate (S)-17. General procedure for the boron/iodine exchange
reaction. A solution of (−)-(S)-3-[2-(4,4,5,5-tetramethyl-[1,3,2]-
dioxaborolan-2-yl)-ethen-1-yl]-2,4,4-trimethylcyclohex-2-en-1-yl
acetate (S)-22 (0.32 g, 0.97 mmol) in THF (15 mL) was cooled
down to −78 ◦C and treated with a suspension of MeONa
(0.11 g, 2.12 mmol) in MeOH (1 mL). After stirring for 30 min,
ICl (1.02 mL, 1 M in CH2Cl2, 1.02 mmol) was slowly added
and the mixture was stirred at −78 ◦C for an additional 2 h.
Et2O (15 mL) was added, the organic layer was separated and
washed with a saturated aqueous Na2S2O3 (2x), H2O (2x) and
brine (2x), dried (Na2SO4) and the solvent was evaporated. The
24
(R)-21. [a]D + 99.8 (c 0.016, MeOH).
(−)-(S)-3-[2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-
ethen-1-yl]-2,4,4-trimethylcyclohex-2-en-1-yl acetate (S)-22. Gen-
eral procedure for the preparation of pinacol boronates. BH3·SMe2
(0.35 mL, 3.77 mmol) was slowly added to a cooled (0 ◦C) solution
of pinacol (0.45 g, 3.77 mmol) in CH2Cl2 (0.4 mL). The mixture
was stirred at this temperature for 1h and at 25 ◦C for an
additional 1h. A solution of (S)-21 (0.131 g, 0.63 mmol) in
CH2Cl2 (0.1 mL) was slowly added at 0 ◦C, and the reaction
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24
residue was purified by column chromatography (silica gel, 97 :
3 hexane/ethyl acetate) to afford 0.20 g (81%) of a colorless oil
(65%) of a yellow oil identified as (R)-23. [a]D + 42.8 (c 0.014,
MeOH).
identified as (S)-17. [a]D − 84.2 (c 0.032, MeOH).1H-NMR
24
(−)-(S)-All-trans-4-acetoxyretinal (S)-24. General procedure for
Dess–Martin oxidation. To a solution of (−)-(S)-all-trans-3-
acetoxyretinol (S)-23 (0.055 g, 0.16 mmol) in CH2Cl2 (7.5 mL)
were sequentially added pyridine (0.145 mL) and Dess–Martin
periodinane (0.09 g, 0.21 mmol). After stirring for 6 h, a saturated
aqueous NaHCO3 solution (3 mL) was added. The mixture was
extracted with CH2Cl2 (3x) and the organic layers were washed
with NaHCO3 (3x) and Na2S2O3 (3x), dried (Na2SO4) and the
solvent was evaporated. The residue was purified by column
chromatography (silica gel, 93 : 7 hexane/ethyl acetate) to afford
0.04 g (71%) of a mixture of (S)-24 and (S, 13Z)-24 in a 8.5 : 1
ꢀ
(400.16 MHz, CDCl3): d 6.95 (d, J = 14.8 Hz, 1H, H1 ), 6.07 (d,
ꢀ
J = 14.8 Hz, 1H, H2 ), 5.18 (t, J = 4.6 Hz, 1H, H1), 2.07 (s, 3H,
COOCH3), 1.9–1.8 (m, 1H, H6), 1.7–1.6 (m, 1H, H6), 1.64 (s, 3H,
C2–CH3), 1.6–1.5 (m, 1H, H5), 1.5–1.4 (m, 1H, H5), 1.04 (s, 3H,
C4–CH3), 0.99 (s, 3H, C4–CH3) ppm. 13C-NMR (100.62 MHz,
C6D6): d 170.3 (s), 144.9 (s), 143.6 (d), 128.7 (s), 80.6 (d), 72.0 (d),
35.0 (t), 34.5 (s), 28.8 (q), 27.3 (q), 25.9 (t), 21.2 (q), 18.5 (q) ppm.
MS (EI+): m/z (%) 275 (M+–OAc, 1), 274 (24), 259 (47), 147 (53),
131 (100), 117 (40), 105 (32), 91 (54). HRMS (EI+): Calcd. for
C11H16I, 275.0297; found, 275.0298. IR (NaCl): m 2929 (s, C–H),
−1
=
2926 (s, C–H), 2864 (m, C–H), 1735 (s, C O), 1241 cm .
ratio.
24
Data for (−)-(S)-all-trans-4-acetoxyretinal (S)-24. [a]D
−
(+)-(R)-3-(2-Iodoethen-1-yl)-2,4,4-trimethylcyclohex-2-en-1-
yl acetate (R)-17. Following the general procedure for the
boron/iodine exchange reaction, the reaction of (R)-22 (0.38 g,
1.15 mmol) in THF (18 mL) with MeONa (0.14 g, 2.53 mmol) in
MeOH (1.15 mL) and ICl (1.21 mL, 1 M in CH2Cl2, 1.21 mmol)
afforded, after purification by column chromatography (silica
gel, 96 : 4 hexane/ethyl acetate), 0.31 g (86%) of a colorless oil
19.4 (c 0.036, MeOH).1H-NMR (400.16 MHz, C6D6): d 10.01 (d,
J = 7.8 Hz, 1H, H15), 6.80 (dd, J = 15.0, 11.5 Hz, 1H, H11), 6.20
(s, 2H, H7 + H8), 6.04 (d, J = 15.1 Hz, 1H, H12), 6.1–5.9 (m, 2H,
H10 + H14), 5.50 (t, J = 4.6 Hz, 1H, H4), 1.83 (s, 3H, COOCH3),
1.78 (s, 3H, C–CH3), 1.73 (s, 3H, C–CH3), 1.72 (s, 3H, C–CH3),
1.7–1.6 (m, 2H, 2H3), 1.4–1.2 (m, 2H, 2H2), 1.05 (s, 3H, C1–CH3),
0.96 (s, 3H, C1–CH3) ppm. 13C-NMR (100.62 MHz, (CD3)2CO):
d 190.3 (d), 170.1 (s), 155.1 (s), 143.5 (s), 139.9 (s), 138.5 (d), 135.5
(d), 132.0 (d), 130.7 (d), 129.0 (d), 127.6 (d), 127.2 (s), 71.5 (d),
34.4 (t), 32.5 (s), 28.6 (q), 26.6 (q), 24.9 (t), 20.1 (q), 17.6 (q),
12.0 (q), 11.9 (q). MS (EI+): m/z (%) 342 (M+, 62), 300 (50), 283
(37), 282 (M+–OAc, 100), 187 (34), 119 (51), 105 (44), 95 (45), 91
(45), 77 (24). HRMS (EI+): Calcd. for C22H30O3, 342.2195; found,
24
identified as (R)-17. [a]D + 81.7 (c 0.022, MeOH).
(−)-(S)-All-trans-4-acetoxyretinol (S)-23. Following the gen-
eral procedure for Stille cross-coupling, the reaction of (−)-(S)-3-
(2-iodoethen-1-yl)-2,4,4-trimethylcyclohex-2-en-1-yl acetate (S)-
17 (0.063 g, 0.19 mmol) with (2E,4E,6E)-3,7-dimethyl-7-(tri-
n-butylstannyl)hepta-2,4,6-trien-1-ol 16 (0.09 g, 0.20 mmol),
Pd2(dba)3 (4.3 mg, 0.0047 mmol) and AsPh3 (11.9 mg, 0.038 mmol)
in NMP (3.5 mL) at 40 ◦C for 6 h, afforded, after purification by
column chromatography (silica gel, 85 : 15 hexane/ethyl acetate),
342.2193. IR (NaCl): d 2953 (s, C–H), 2924 (s, C–H), 2854 (m,
−1
=
C–H), 1735 (m, C O), 1661, 1457, 1240 cm . UV (MeOH): k
368 nm (e = 23300).
24
Data for (S)-13-cis-4-acetoxyretinal (S, 13Z)-24. 1H-NMR
(400.13 MHz, C6D6): d 10.13 (d, J = 7.3 Hz, 1H, H15), 7.10 (d,
J = 15.2 Hz, 1H, H12), 6.71 (dd, J = 15.2, 11.5 Hz, 1H, H11),
6.21 (s, 2H, H7 + H8), 6.00 (d, J = 11.5 Hz, 1H, H10), 5.75 (d,
J = 7.3 Hz, 1H, H14), 5.49 (t, J = 4.5 Hz, 1H, H4), 1.84 (s, 3H,
COOCH3), 1.77 (s, 3H, C–CH3), 1.71 (s, 3H, C–CH3), 1.58 (s, 3H,
C–CH3), 1.6–1.2 (m, 4H, 2H3 + 2H2), 1.05 (s, 3H, C1–CH3), 0.96
(s, 3H, C1–CH3) ppm.
0.046 g (70%) of a yellow oil identified as (S)-23. [a]D − 44.4
(c 0.036, MeOH).1H-NMR (400.13 MHz, (CD3)2CO): d 6.65 (dd,
J = 15.1, 11.2 Hz, 1H, H11), 6.13 (d, J = 15.1 Hz, 1H, H12), 6.3–6.2
(m, 3H, H7 + H8 + H10), 5.70 (t, J = 6.4 Hz, 1H, H14), 5.20 (t,
J = 4.6 Hz, 1H, H4), 4.32 (t, J = 6.4 Hz, 2H, 2H15), 2.05 (s, 3H,
COOCH3), 2.02 (s, 3H, C13–CH3), 1.9–1.8 (m, 1H, H3), 1.85 (s, 3H,
C9–CH3), 1.8–1.7 (m, 1H, H3), 1.69 (s, 3H, C5–CH3), 1.7–1.6 (m,
1H, H2), 1.5–1.4 (m, 1H, H2), 1.07 (s, 3H, C1–CH3), 1.04 (s, 3H,
C1–CH3) ppm. 13C-NMR (100.62 MHz, (CD3)2CO): d 171.9 (s),
145.6 (s), 141.0 (d), 139.6 (d), 136.6 (s), 136.5 (s), 135.1 (d), 133.6
(d), 127.9 (s), 126.7 (d), 125.9 (d), 73.6 (d), 60.3 (t), 36.4 (t), 36.3
(s), 30.3 (q), 28.6 (q), 27.0 (t), 22.1 (q), 19.6 (q), 13.7 (q), 13.6 (q).
MS (EI+): m/z (%) 284 (M+–OAc, 2), 278 (25), 262 (54), 247 (54),
245 (49), 232 (46), 207 (53), 195 (45), 179 (43), 171 (50), 165 (51),
157 (55), 143 (55), 141 (58), 133 (64), 131 (45), 129 (64), 128 (65),
119 (59), 117 (39), 115 (78), 105 (71), 95 (54), 91 (100), 77 (39).
HRMS (EI+): Calcd. for C20H28O, 284.2140; found, 284.2143. IR
(+)-(R)-All-trans-4-acetoxyretinal (R)-24. Following the gen-
eral procedure for Dess–Martin oxidation, the reaction of (R)-23
(0.055 g, 0.16 mmol) with piridine (0.143 mL) and Dess–Martin
periodinane (0.088 g, 0.21 mmol) in CH2Cl2 (7.3 mL) afforded,
after purification by column chromatography (silica gel, 96 : 4
hexane/ethyl acetate), 0.034 g (63%) of a yellow oil identified as a
mixture of (R)-24 and (R, 13Z)-24 in a 6.5:1 ratio.
Data for (R)-24. [a]24 + 17.6 (c 0.034, MeOH)
(−)-(S)-All-trans-4-hydroxyretinal 3. Following the general
procedure for deprotection with K2CO3, the reaction of (−)-
(S)-all-trans-4-acetoxyretinal (S)-24 (0.022 g, 0.064 mmol) in
MeOH (0.6 mL) with K2CO3 (8.9 mg, 0.064 mmol) afforded,
after purification by column chromatography (silica gel, 85 : 15
hexane/ethyl acetate), 0.014 g (73%) of a yellow oil identified as a
mixture of 3 and 4 in a 3 : 1 ratio, which were separated by HPLC.
(NaCl): d 3550–3100 (br, OH), 2956 (s, C–H), 2926 (s, C–H), 1731
−1
=
(s, C O), 1241 cm . UV (MeOH): k 323 nm (e = 37000).
(+)-(R)-All-trans-4-acetoxyretinol (R)-23. Following the gen-
eral procedure for Stille cross-coupling, the reaction of (R)-
17 (0.047 g, 0.14 mmol) with (2E,4E,6E)-3,7-dimethyl-7-(tri-
n-butylstannyl)hepta-2,4,6-trien-1-ol 16 (0.064 g, 0.15 mmol),
Pd2(dba)3 (3.2 mg, 0.0035 mmol) and AsPh3 (8.7 mg, 0.028 mmol)
in NMP (3.5 mL) at 40 ◦C for 7 h afforded, after purification by
column chromatography (silica gel, 90 : 10 hexane/ethyl) 31.2 mg
24
Data for (−)-(S)-all-trans-4-hydroxyretinal 3. [a]D − 89.0
(c 0.016, MeOH).1H-NMR (400.13 MHz, (CD3)2CO): d 10.13 (d,
J = 8.0 Hz, 1H, H15), 7.31 (dd, J = 15.1, 11.5 Hz, 1H, H11),
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6.51 (d, J = 15.1 Hz, 1H, H12), 6.37 (d, J = 16.1 Hz, 1H, H7),
6.32 (d, J = 11.5 Hz, 1H, H10), 6.24 (d, J = 16.1 Hz, 1H, H8),
5.92 (d, J = 8.0 Hz, 1H, H14), 3.94 (t, J = 4.5 Hz, 1H, H4),
2.38 (s, 3H, C13–CH3), 2.07 (s, 3H, C9–CH3), 1.9–1.8 (m, 1H, H3),
1.82 (s, 3H, C5–CH3), 1.7–1.6 (m, 2H, H3 + H2), 1.4–1.3 (m, 1H,
H2), 1.04 (s, 3H, C1–CH3), 1.03 (s, 3H, C1–CH3) ppm. 13C-NMR
(100.62 MHz, (CD3)2CO): d 190.3 (d), 154.2 (s), 140.2 (s), 139.7
(s), 137.9 (d), 135.1 (d), 132.1 (d), 131.9 (s), 130.1 (d), 128.9 (d),
128.5 (d), 68.9 (d), 34.7 (t), 34.2 (s), 28.5 (t), 28.2 (q), 27.1 (q), 17.9
(q), 12.0 (q), 11.9 (q). MS (EI+): m/z (%) 300 (M+, 72), 203 (15),
190 (14), 175 (15), 161 (26), 119 (43), 105 (37), 91 (37), 86 (69).
HRMS (EI+): Calcd. for C20H28O2, 300.2089; found, 300.2086. IR
Enzyme kinetics
Enzymatic activities of purified Xenopus ADH8 were determined
in a Varian Cary 400 spectrophotometer, at 25 ◦C. Standard
activity was measured with 1.92 mM octanol (Merck) and 2.4 mM
NADP+ (Roche) in 0.1 M glycine, pH 10.0, at 340 nm, in 1-cm
pathlength cuvettes. A specific activity of 34.02 U mg−1 for octanol
was considered. One unit (U) of ADH activity is defined as the
amount of enzyme required to transform 1 lmol of substrate or
cofactor per min at 25 ◦C. Activities for retinal reduction kinetics
were determined at 400 nm, with 0.6 mM NADPH (Roche), and
0.1 M sodium phosphate, pH 7.5, 0.02% Tween 80 (assay buffer),
in 0.2-cm pathlength cuvettes. Retinoid concentration ranged from
0.1 × Km to 10 × Km and each individual rate measurement was run
in duplicate. Kinetic constants were calculated using the GraFit
5.0 program (Erithacus Software Limited), and the results were
expressed as the mean value SEM. A molecular weight of 80,000
for ADH dimer was used to calculate kcat values.
(NaCl): d 3500–3100 (br, OH), 2918 (s, C–H), 2850 (m, C–H),
−1
=
1656 (m, C O), 1575, 1164, 996 cm . UV (MeOH): kmax 375 nm
(e = 31900).
24
Data for (−)-(S)-13-cis-4-hydroxyretinal 4. [a]D − 75.8
(c 0.018, MeOH).1H-NMR (400.13 MHz, (CD3)2CO): d 10.25 (d,
J = 7.8 Hz, 1H, H15), 7.52 (d, J = 15.0 Hz, 1H, H12), 7.20 (dd,
J = 15.0, 11.3 Hz, 1H, H11), 6.38 (d, J = 16.2 Hz, 1H, H7), 6.36
(d, J = 11.3 Hz, 1H, H10), 6.25 (d, J = 16.2 Hz, 1H, H8), 5.80 (d,
J = 8.0 Hz, 1H, H14), 3.94 (t, J = 4.5 Hz, 1H, H4), 2.18 (d, J =
0.9 Hz, 3H, C13–CH3), 2.07 (s, 3H, C9–CH3), 1.9–1.8 (m, 1H, H3),
1.82 (s, 3H, C5–CH3), 1.7–1.6 (m, 2H, H3 + H2), 1.4–1.3 (m, 1H,
H2), 1.05 (s, 3H, C1–CH3), 1.03 (s, 3H, C1–CH3) ppm. MS (EI+):
m/z (%) 300 (M+, 100), 161 (25), 135 (25), 119 (38), 107 (25), 105
(33), 95 (25), 91 (33), 69 (31). HRMS (EI+): Calcd. for C20H28O2,
Molar absorption coefficients in the assay buffer, used to
calculate retinoid concentration, were e400 = 29500 M−1 cm−1 for
all-trans-retinal (Sigma) and e370 = 27000 M−1 cm−1 for 13-cis-
retinal (Sigma). Since the absorption coefficients for the ring-
oxidized retinals were not known, these values were first estimated
in methanol, and then calculated in the assay buffer: e375
=
30300 M−1 cm−1 for 4-hydroxyretinal, e378 = 26600 M−1 cm−1 for 3-
hydroxyretinal, e368 = 28500 M−1 cm−1 for 4-hydroxy-13-cis-retinal,
and e374 = 25400 M−1 cm−1 for 13-cis-3-hydroxyretinal. Substrate
solutions were prepared by diluting 1 mg retinal, dissolved in 150–
500 lL methanol, with the appropriate volume of the assay buff◦er
to have a final concentration of approximately 200 lM, at 4 C
and under dim red light. The stability of the retinoid was checked
spectrophotometrically.
300.2089; found, 300.2087. IR (NaCl): m 3580–3150 (br, OH), 2956
−1
=
(s, C–H), 2927 (s, C–H), 2855 (s, C–H), 1658 (s, C O), 1575 cm .
UV (MeOH): kmax 368 nm (e = 28500).
(+)-(R)-All-trans-4-hydroxyretinal 5. Following the general
procedure for deprotection with K2CO3, the reaction of (R)-24
(0.026 g, 0.08 mmol) in MeOH (0.7 mL) with K2CO3 (0.011 g,
0.07 mmol) afforded, after purification by column chromatogra-
phy (silica gel, 85 : 15 hexane/ethyl acetate), 0.015 g (67%) of a
yellow oil identified as a mixture of 5 and 6 in a 3 : 1 ratio, which
were separated by HPLC.
Acknowledgements
Supported by grants from Ministerio de Educacio´n y Ciencia
(FPU Fellowship to M.D., Ramo´n y Cajal Contract to R.A.), the
Spanish Direccio´n General de Investigacio´n (BMC2002-02659,
BMC2003-09606 and SAF-2004-07131) and the Generalitat de
Catalunya (2001SGR 00198). We thank Dr. Gregg Duester for
his help in the preparation of ADH8 cDNA. Graphical Abstract
graphics were kindly provided by Oriol Gallego.
24
Data for (+)-(R)-all-trans-4-hydroxyretinal 5: [a]D + 87.0
(c 0.018, MeOH).
24
Data for (+)-(R)-13-cis-4-hydroxyretinal 6: [a]D + 73.2
(c 0.012, MeOH).
References
Cloning, expression and purification of Xenopus laevis ADH8
1 (a) R. R. Rando, Chem. Rev., 2001, 101, 1881; (b) J. K. McBee, K.
Palczewski, W. Baehr and D. R. Pepperberg, Prog. Retinal Eye Res.,
2001, 20, 469.
2 J. C. Saari, in The Retinoids: Biology, Chemistry and Medicine, 2nd
Edition, ed. M. B. Sporn, A. B. Roberts and D. S. Goodman, Raven,
New York, 1993, pp. 351–385.
ADH8 cDNA sequence from Xenopus laevis was obtained by
nested-PCR, using two sets of degenerated primers based on
the sequence of the orthologous enzyme from Rana perezi,16
followed by 3ꢀ-end RACE-PCR amplification. The sequence
was deposited in the GenBank data base under the accession
no. AJ566764. The full-length cDNA was then generated by
PCR amplification, cloned in the expression vector pGEX-4T-2
(Amersham Biosciences) and used to transform E. coli BL21 cells,
as described previously.17a Expression, cell lysis and purification of
Xenopus ADH8 as a fusion protein with glutathione-S-transferase
(GST) were conducted as described for the Rana perezi enzyme.36
Finally, the homogeneity of the purified protein was assessed
by SDS-PAGE followed by Coomassie Brilliant Blue (Sigma)
staining.
3 J. T. Davis and D. E. Ong, Biol. Reprod., 1995, 52, 356.
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