13-Demethyl-13-substituted-13,14-dihydroretinols as potential affinity labels of retinol-binding proteins: Syntheses and stability studies

Article abstract of DOI:10.1016/j.bioorg.2003.09.002

13-Demethyl-13-substituted-13,14-dihydroretinols were synthesized and their stability under various conditions was measured in order to evaluate whether they would be useful as affinity labels of retinol binding proteins and retinol metabolizing enzymes.

Full text of DOI:10.1016/j.bioorg.2003.09.002

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 32 (2004) 38–50
www.elsevier.com/locate/bioorg
13-Demethyl-13-substituted-13,14-
dihydroretinols as potential affinity labels
of retinol-binding proteins: syntheses and
stability studies
Revital Yefidoff, Limor Weiss, David Avisar,
and Amnon Albeck*
Department of Chemistry, The Julius Spokojny Bioorganic Chemistry Laboratory,
Bar Ilan University, Ramat Gan 52900, Israel
Received 21 July 2003
Abstract
13-Demethyl-13-substituted-13,14-dihydroretinols were synthesized and their stability un-
der various conditions was measured in order to evaluate whether they would be useful as af-
finity labels of retinol binding proteins and retinol metabolizing enzymes. The 13-chloro
analog could not be isolated because it eliminated HCl under the Wittig reaction conditions
of its preparation. The trans- and cis-13,14-epoxy analogs are stable in non-protic organic sol-
vents, but undergo an elimination reaction under various chromatographic conditions and in
mixtures of organic solvents with water or alcohol. The 13-hydroxy and 13-methoxy analogs
are stable in aqueous solutions and are therefore suitable for biological studies.
Ó 2003 Elsevier Inc. All rights reserved.
Keywords: Retinoids; Affinity label; Stability; Epoxide
1. Introduction
Vitamin A and its metabolites have many biological functions. Retinal, the alde-
hyde derived from vitamin A, is a central player in vision. Retinol and retinyl esters
* Corresponding author. Fax: 972-3-5351250.
E-mail address: albecka@mail.biu.ac.il (A. Albeck).
0045-2068/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2003.09.002

R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
39
also participate in the visual cycle. The latter is also used for storage of retinoids.
Retinoic acids are important for cellular differentiation and morphogenesis. 14-Hy-
droxy-retro-retinol and anhydroretinol play a role in cell growth, proliferation and
death. 13,14-Dihydroxyretinol, 4-oxo-retinol, and 4-oxo-retinal probably also have
cellular functions. Retinal is also essential for the proton pumping activity of the
bacterial pigment bacteriorhodopsin [1].
Many enzymes and proteins interact with vitamin A and its derivatives.
Among these are the various opsins, which combine with retinal to form the vi-
sual pigments, dehydrogenases, ester synthases, transferases and hydrolases, trans
retinyl ester isomerohydrolase, specific retinoid binding proteins and receptors
and bacterioopsin. Some of these proteins have been isolated and (partially) struc-
turally characterized, while others are only poorly characterized. One useful tool
in the study of the interaction between these proteins and their retinoid ligands is
affinity labeling by retinol/al analogs. Diazo ketones and esters, azides, diazirines,
allyl bromides, halomethyl ketones and a-bromo esters are examples of affinity
labels that have been used in the study of visual pigments [2], bacteriorhodop-
sin [2e,3], lecithin:retinol acyltransferase (LRAT) [4] and retinoid binding proteins
[5].
In the present paper we describe the design of a new family of selective affinity
labels for enzymes and proteins that interact with retinol. It is based on 13-substitut-
ed-13,14-dihydroretinol, which can undergo a nucleophilic displacement at at least
two possible carbons (Scheme 1). This paper describes their synthesis, as well as
structure–stability correlation studies, which were carried out in order to assess their
suitability for affinity labeling experiments.
2. Results and discussion
The design of the potential affinity labels is based on the introduction of a
leaving group at the C13 sp3 carbon of a retinol skeleton (Scheme 1). This leav-
ing group can be displaced by nucleophilic residues of the target enzyme/protein.
The structure of these compounds confers high versatility since they can interact
with nucleophiles that are spatially close to C13 (SN2 reaction) and C11 (SN20 re-
action) and even to C9 and C7 of the retinol skeleton (though with higher loss
Scheme 1.

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R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
Scheme 2.
of conjugation). On the other hand, these affinity labels will exhibit selectivity
only towards retinol binding proteins and enzymes bearing a nucleophilic residue
at the appropriate position. In contrast, diazo- or azido-based affinity labels
produce reactive intermediates that interact non-selectively with neighbouring
residues.
The leaving groups tested were 13,14-epoxy- (trans and cis), 13-chloro-, 13-hy-
droxy- and 13-methoxy- of 13-demethyl-13,14-dihydroretinol (compounds 1–5,
respectively. Scheme 2). The chlororetinol analog was expected to be the most
reactive and the hydroxy- and methoxy analogs to be the least reactive affinity labels.
The substituents replace the 13-methyl group of the native retinol, thus avoiding
unexpected steric problems from additional substitution.
2.1. Synthesis
A convergent strategy for the synthesis of the 13-substituted retinol analogues
1–3 was employed, based on the assembly of two fragments, the all-trans isomer
of the C15 phosphorane 6 and an appropriate four-carbon chain aldehyde, via
the Wittig–Horner condensation (Scheme 3). The C4 aldehydes 7–9, which carry
the electrophilic functionalities, were synthesized via a simple functionalization–
oxidation or oxidation–functionalization sequence (Scheme 4). An efficient linear
approach, based on a key Reformatsky reaction, was employed in the synthesis of
the hydroxy- and methoxyretinol analogs 4 and 5 (Scheme 6).

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41
β
Scheme 3.
Scheme 4.
2.1.1. Epoxyretinol
The synthesis of 13-demethyl-13,14-trans-epoxyretinol 1 and 13-demethyl-13,14-
cis-epoxyretinol 2 ({3-[4-methyl-6-(2,6,6-trimethylcyclohex-1-enyl)hexa-1,3,5- trienyl]-
oxyranyl}methanol) is outlined in Scheme 3 [6]. trans and cis C4 epoxy aldehydes
(7 and 8, respectively) were prepared from monosilylated trans and cis 2-butene-
1,4-diol by a short epoxidation and oxidation sequence (Scheme 4) [7]. The readily
available C15 phosphorane 6 [8] was condensed with the appropriate C4 epoxy alde-
hyde (either 7 or 8), yielding a mixture of all-trans and 11-cis O-protected epoxyret-
inol analogs 10 and 11, respectively. Removal of the silyl protecting group furnished
the epoxyretinol analogs 1 and 2 (as an all-trans/11-cis mixture).

42
R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
Scheme 5.
β
Scheme 6.
It should be noted that initially we attempted to synthesize 13,14-epoxyretinol by
direct mCPBA epoxidation of retinol. We assumed that the directing effect of the al-
lylic alcohol [9] would lead to preferred epoxidation of the C13@C14 double bond
over the other more substituted double bonds. Indeed, such a directing effect was
observed, but, to our surprise, the major product was 11,14-epoxyretinol 12 (Scheme
5) [10]. Small amounts of a bisepoxy product 13 were also isolated, as determined
by MS analysis. Attempts to carry out Sharpless chiral epoxidation yielded starting
material only. The same was also true for H2O2/OHꢀ epoxidation of retinal.
2.1.2. Chlororetinol
The same approach was applied to the synthesis of 13-chloro-13,14-dihydro-
retinol 3 (3-chloro-7-methyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-4,6,8-trien-1-ol)
(Scheme 3). The C4 a-chloro aldehyde unit 9 was prepared from butane-1,4-diol
by mono silylation, Swern oxidation and chlorination (Scheme 4) [11]. It was then
reacted with the same C15 phosphorane 6 used for the synthesis of the epoxyretinols.

R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
43
No traces of the desired O-protected-13-chlororetinol analog 14 were detected. In-
stead, the major crude product of the reaction was identified as the 4,13-elimination
product 15 (Scheme 3). It exhibited a 328, 344, 360 nm triplet in its UV absorption
1
spectrum (in ether) and its H- NMR and MS supported the suggested structure.
We did not apply any other synthetic strategy for the preparation of the chloro-
retinol analog 3 since it became clear from our stability studies that the putative
chlororetinol would be unstable under the normal aqueous conditions of biological
manipulations (Section 2.2).
2.1.3. Hydroxy- and methoxyretinol
Reformatsky reaction [12] on the readily available C17 aldehyde 16 [13] yielded
ethyl 13-hydroxy-13,14-dihydroretinoate 17 (Scheme 6). The latter could be selec-
tively alkylated on the 13-hydroxy substituent (MeI, Ag2O, and K2CO3 [14]) to
the corresponding methoxy ester 18. Reduction of either hydroxy ester 17 or
methoxy ester 18 afforded the corresponding retinol analogs 4 (7-methyl-9-(2,6,
6-trimethylcyclohex-1-enyl)nona-4,6,8-triene-1,3-diol) and 5 (3-methoxy-7-methyl-
9-(2,6,6-trimethylcyclohex-1-enyl)nona-4,6,8-trien-1-ol), respectively (Scheme 6).
2.2. Stability
The five 13-substituted-13,14-dihydroretinol analogs (Scheme 2) span a wide
range of chemical stability. The 13-chloro-analog 14 is so unstable that it could
not be isolated even as a crude product. In fact, only the elimination product, the
retro-retinol analog 15 (Scheme 3), was identified in the crude reaction mixture
and isolated in good yield. The conditions of the Wittig reaction are probably basic
enough to promote the observed 4,13 HCl elimination. The hydroxy- and methoxy-
retinol analogs (4 and 5, respectively; Scheme 6) are stable in organic solvents and in
neutral aqueous solution. No elimination product could be detected upon incubation
in a water: acetonitrile (1:1) solution for four days. The epoxyretinol analogs 1 and 2
(Scheme 2) exhibited marginal stability. They were obtained in good yield (and spec-
trally characterized) as crude products, but underwent an elimination reaction under
a variety of chromatographic purification protocols. They were stable in organic sol-
vents (CH2Cl2, CH3CN, DMF, and DMSO), but extremely unstable in organic sol-
vent mixtures with protic solvents such as water or alcohols. Thus, the half-life of the
13,14-trans-epoxyretinol in wet dichloromethane or ethanol: dichloromethane solu-
tion is about 40 min (Fig. 1). It was previously demonstrated that the half-life of a
similar compound (the corresponding retinoate) in methanol is only 7 min [6b]. In
more polar solvents such as 4:1 acetonitrile: water solution, the half-life of the epoxy-
retinol analog 1 is much shorter—just a couple of minutes. The cis epoxyretinol an-
alog 2, as well as the two corresponding silyl ether protected analogs 10 and 11
(Scheme 3), exhibited similar elimination kinetics.
It is interesting to note that two different products were obtained under different
conditions. Silica gel chromatography of the epoxyretinol analogs, or their incuba-
tion in wet dichloromethane or in methanol, afforded the expected 4,13 elimination
products that exhibited a 334, 349, 368 nm triplet in the UV absorption spectrum (in

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R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
Fig. 1. Stability of epoxyretinol 1. Time course (10 min intervals) of elimination from the epoxyretinol
(kmax 302 nm) to the corresponding retro-retinol (kmax 348 nm) in dichloromethane:ethanol solution.
CH2Cl2, see Fig. 1). NMR analysis of one of these products, 19, derived from com-
pound 10, confirmed its structure. On the other hand, incubation in acetonitrile:
water yielded a product that exhibited a 302, 316, 332 nm triplet in its UV absorption
spectrum. This product was not further analyzed.
3. Conclusion
This study describes a structure–stability relationship for 13-substituted-13,14-di-
hydroretinols. The most reactive analog, 13-chloro-13,14-dihydroretinol, is so unsta-
ble it eliminates HCl spontaneously upon its formation via the Wittig reaction. The
13,14-epoxides are stable in non-protic organic solvents, but undergo a facile 4,13
elimination reaction in alcohols or aqueous solutions. In contrast, the 13-hydroxy-
and methoxyretinols are stable also in aqueous solutions. This trend correlates well
with the chemical reactivity of allylic halides, epoxides, alcohols and ethers.
From a practical standpoint, it is clear that only the hydroxy- and methoxyretinol
analogs 4 and 5 could be used for affinity labeling studies. Although they are the least
reactive, they are the most stable of the tested 13-substituted-13,14-dihydro-retinol
analogs. They are currently under investigation as affinity labels of some retinol
metabolizing enzymes.
4. Experimental
4.1. General
1H, and 13C NMR spectra were recorded at 200, 300 or 600, and 50, 75 or
150 MHz, respectively, in CDCl3 (TMS as an internal standard) or, when specified,

R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
45
1
in CD3OD. H NMR assignments were supported by COSY experiments, while 13C
NMR assignments were supported by DEPT and hetero COSY experiments. J val-
ues are given in Hz. Mass spectra were recorded in DCI mode with methane or am-
monia as the reagent gas, unless otherwise stated. TLC was performed on E. Merck
0.2 mm precoated silica gel F-254 plates, and viewed by UV and vanillin [15]. Chro-
matography refers to flash column chromatography [16], carried out on silica gel 60
(230–400 mesh ASTM, E. Merck). Anhydrous solvents were dried and freshly dis-
tilled (THF and ether from sodium/benzophenone, pyridine and triethylamine from
ꢀ
CaH2, CH2Cl2 from CaCl2 and DMF from 4 A molecular sieves).
4.2. Synthesis
Compound 6 was prepared according to a literature procedure [8]. Compound 7
and compound 8 [17] were prepared according to [18], except a Swern oxidation was
used instead of the SO3-pyridine oxidation of the primary alcohol in the last step
[17b]. Compound 16 was prepared as described elsewhere [13].
4.2.1. 4-(t-Butyldimethylsilyloxy)-2-chlorobutanal (9)
A solution of sulfuryl chloride (distilled, 65 mg, 0.5 mmol) in CH2Cl2 (2 ml) was
added slowly to a solution of 4-(t-butyldimethylsilyloxy)butanal [19] (100 mg,
0.5 mmol) in CH2Cl2 (5 ml). After stirring for 1 h, the reaction mixture was extracted
twice with saturated NaHCO3 solution and then with saturated NaCl solution until
the aqueous layer became neutral. The organic phase was dried (MgSO4) and the sol-
vent was removed to provide 76 mg (64% yield) of 9. 1H NMR d 9.53 (1 H, d, J 1.9),
4.41 (1H, ddd, J 7.3, 5.0, 1.9), 3.81 (2H, m), 2.23 (1H, ddt, J 14.5, 7.2, 5.0), 2.05 (1H,
dddd, J 14.5, 7.3, 5.9, 4.2), 0.88 (9H, s), 0.06 (3H, s); 13C NMR d 195.2, 61.5, 58.2,
35.6, 25.8, 18.2, )5.5, )5.5; MS m=z 239, 237 (MHþ), 238, 236.
4.2.2. O-t-butyldimethylsilyl-13-demethyl-13,14-trans-epoxy-13, 14-dihydroretinol (10)
n-BuLi (0.6 ml, 1 M in hexane) was injected to a solution of the phosphonium salt
of 6 (350 mg, 0.64 mmol) in dry THF (7 ml) under Ar atmosphere at )78 °C. The re-
action mixture became red. After stirring for 20 min at )78 °C, a solution of aldehyde
7 (70 mg, 0.32 mmol) in dry THF (3 ml) was added and the reaction mixture was stir-
red for additional 1 h. Water was added and the mixture was extracted twice with
hexane. The organic phase was dried (MgSO4) and the solvents were removed to give
the product as a mixture of 1.3:1 11-cis:11-trans isomers. The crude product could
1
not be chromatographed due to its instability. trans Isomer: H NMR d 6.75 (1H,
dd, J 15.2, 11.4, H-11), 6.14 (1H, d, J 16.1, H-7), 6.02 (1H, d, J 16.1, H-8), 5.99
(1H, d, J 11.4, H-10), 5.37 (1H, dd, J 15.2, 8.3, H-12), 3.77 (1H, dd, J 12.0, 3.2,
H-15), 3.74 (1H, m, H-15), 3.32 (1H, dd, J 8.3, 2.1, H-13), 2.99 (1H, m, H-14),
1.97 (2H, br t, J 5.9, CH2-4), 1.88 (3H, s, Me-19), 1.64 (3H, s, Me-18), 1.61 (2H,
m, CH2-3), 1.46 (2H, m, CH2-2), 1.00 (6H, s, Me-16,17), 0.87 (9H, s, t-Bu), 0.06
1
(6H, s, SiMe2). cis Isomer: H NMR d 6.57 (1H, dd, J 12.0, 10.2, H-11), 6.37 (1H,
d, J 12.0, H-10), 6.19 (1H, d, J 15.8, H-7), 6.08 (1H, d, J 15.8, H-8), 5.07 (1H,
dd, J 10.2, 8.0, H-12), 3.85 (1H, dd, J 12.8, 3.1, H-15), 3.68 (2H, m, H-13, H-15),

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R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
2.99 (1H, m, H-14), 1.97 (2H, br t, J 5.9, CH2-4), 1.88 (3H, s, Me-19), 1.67 (3H, s,
Me-18), 1.61 (2H, m, CH2-3), 1.46 (2H, m, CH2-2), 1.02 (6H, s, Me-16,17), 0.87 (9H,
s, t-Bu), 0.04 (6H, s, SiMe2); 13C NMR (mixture of cis and trans isomers) d 137.3,
137.1, 132.0, 131.8, 130.1, 129.0, 128.1, 127.8, 126.5, 123.6, 63.0 (C-15, cis), 63.0
(C-15, trans), 60.9 (C-14, trans), 60.4 (C-14, cis), 56.1 (C-13, trans), 51.9 (C-13,
cis), 39.5, 34.1, 32.9, 28.8, 25.8, 21.6, 19.1, 18.2, 12.5, 12.2, )5.4; kmax(ether)
298 nm; HRMS m=z 402.2950 (Mþ. C25H42SiO2 requires 402.2954).
4.2.3. 13-Demethyl-13,14-trans-epoxy-13,14-dihydroretinol (1)
A solution of tetrabutylamonium fluoride (0.4 ml, 1 M in THF) was added drop-
wise to a solution of 10 (160 mg, 0.4 mmol) in dry THF (10 ml). After 30 min the mix-
ture was diluted with hexane and extracted twice with water. The organic phase was
dried (MgSO4) and the solvents were evaporated to give a 1.6:1 mixture of 11-cis:11-
trans isomers of the product. The crude product could not be chromatographed due
to its instability. cis Isomer: 1H NMR d 6.65 (1H, dd, J 12.0, 11.8, H-11), 6.42 (1H, d,
J 11.8, H-10), 6.24 (1H, d, J 16.0, H-7), 6.13 (1H, d, J 16.0, H-8), 5.10 (1H, dd, J 12.0,
9.1, H-12), 3.93 (1H, dd, J 12.5, 2.3, H-15), 3.86 (1H, dd, J 9.1, 2.1, H-13), 3.72 (1H,
dd, J 12.5, 3.4, H-15), 3.11 (1H, m, H-14), 2.01 (2H, br t, J 6.7, CH2-4), 1.93 (3H, s,
Me-19), 1.71 (3H, s, Me-18), 1.64 (2H, m, CH2-3), 1.45 (2H, m, CH2-2), 1.02 (6H, s,
Me-16,17); trans isomer: 1H NMR d 6.80 (1H, dd, J 15.2, 11.4, H-11), 6.20 (1H, d, J
16.1, H-7), 6.06 (1H, d, J 16.1, H-8), 6.03 (1H, d, J 11.4, H-10), 5.40 (1H, dd, J 15.2,
8.1, H-12), 3.90 (1H, dd, J 10.9, 2.2, H-15), 3.68 (1H, dd, J 10.9, 3.8, H-15), 3.48 (1H,
dd, J 8.1, 2.4, H-13), 3.11 (1H, m, H-14), 2.01 (2H, br t, J 6.7, CH2-4), 1.93 (3H, s,
Me-19), 1.69 (3H, s, Me-18), 1.64 (2H, m, CH2-3), 1.45 (2H, m, CH2-2), 1.01 (6H,
s, Me-16,17); cis isomer: 13C NMR d 137.3 (C-8), 130.7 (C-11), 128.2 (C-7), 126.0
(C-12), 123.5 (C-10), 61.1 (C-15), 60.4 (C-14), 51.7 (C-13), 39.6 (C-2), 34.2 (C-1),
33.0 (C-4), 28.9 (Me-16,17), 21.7 (Me-18), 19.2 (C-3), 12.3 (Me-19); kmax(ether)
300 nm; HRMS m=z 288.2096 (Mþ. C19H28O2 requires 288.2089).
4.2.4. O-t-butyldimethylsilyl-13-demethyl-13,14-cis-epoxy-13, 14-dihydroretinol (11)
Compound 11 was prepared as described for 10, yielding a mixture of 1.5:1 11-
cis:11-trans isomers. The crude product could not be chromatographed due to its in-
stability. cis Isomer: 1H NMR d 6.64 (1H, ddd, J 12.0, 10.9, 1.1, H-11), 6.41 (1H, d, J
12.0, H-10), 6.24 (1H, d, J 16.3, H-7), 6.13 (1H, d, J 16.3, H-8), 5.23 (1H, dd, J 10.9,
7.9, H-12), 3.87 (1H, ddd, J 7.9, 4.5, 1.1, H-13), 3.79 (1H, dd, J 11.7, 4.5, H-15), 3.71
(1H, dd, J 11.7, 6.0, H-15), 3.29 (1H, br q, J 4.5, H-14), 2.01 (2H, br t, J 6.0, CH2-4),
1.93 (3H, s, Me-19), 1.71 (3H, s, Me-18), 1.61 (2H, m, CH2-3), 1.46 (2H, m, CH2-2),
1.03 (6H, s, Me-16,17), 0.90 (9H, s, t-Bu), 0.10 (6H, s, SiMe2); trans isomer: 1H NMR
d 6.80 (1H, dd, J 15.0, 11.3, H-11), 6.21 (1H, d, J 16.2, H-7), 6.08 (1H, d, J 16.2, H-
8), 6.05 (1H, d, J 11.3, H-10), 5.51 (1H, dd, J 15.0, 7.7, H-12), 3.80 (1H, dd, J 11.8,
4.6, H-15), 3.72 (1H, dd, J 11.8, 6.0, H-15), 3.55 (1H, dd, J 7.7, 4.4, H-13), 3.28 (1H,
m, H-14), 2.01 (2H, br t, J 6.0, CH2-4), 1.93 (3H, s, Me-19), 1.71 (3H, s, Me-18), 1.61
(2H, m, CH2-3), 1.46 (2H, m, CH2-2), 1.02 (6H, s, Me-16,17), 0.91 (9H, s, t-Bu), 0.08
(6H, s, SiMe2); 13C NMR (mixture of cis and trans isomers) d 137.3, 137.1, 130.6,
128.5, 128.3, 127.9, 127.6, 125.6, 123.7, 123.2, 61.9 (cis), 61.6 (trans), 59.3 (trans),

R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
47
58.9 (cis), 56.7 (trans), 52.7 (cis), 41.5, 39.5, 34.1, 32.9, 28.8, 25.8, 21.6, 19.1, 18.2,
12.5 (trans), 12.1 (cis), )5.3, )5.4; kmax(ether) 298 nm; HRMS m=z 402.2940 (Mþ.
C25H42SiO2 requires 402.2954).
4.2.5. 13-Demethyl-13,14-cis-epoxy-13,14-dihydroretinol (2)
Compound 2 was prepared as described for 1, yielding a 3.5:1 mixture of 11-
1
cis:11-trans isomers of the product. trans Isomer: H NMR d 6.81 (1H, dd, J 15.2,
11.6, H-11), 6.06 (1H, d, J 16.0, H-8), 6.04 (1H, d, J 11.6, H-10), 5.53 (1H, dd, J
15.2, 8.2, H-12), 3.59 (1H, dd, J 8.2, 4.4, H-13), 3.36 (1H, obscured, H-14), 2.01
(2H, br t, J 6.4, CH2-4), 1.93 (3H, s, Me-19), 1.70 (3H, s, Me-18), 1.61 (2H, m,
1
CH2-3), 1.45 (2H, m, CH2-2), 1.02 (6H, s, Me-16,17); cis Isomer: H NMR d 6.65
(1H, dd, J 12.1, 10.9, H-11), 6.38 (1H, d, J 12.1, H-10), 6.25 (1H, d, J 16.1, H-7),
6.13 (1H, d, J 16.1, H-8), 5.26 (1H, dd, J 10.9, 8.2, H-12), 3.91 (1H, dd, J 8.2,
4.4, H-13), 3.81 (1H, dd, J 12.4, 4.4, H-15), 3.68 (1H, dd, J 12.4, 6.6, H-15), 3.36
(1H, dt, J 6.6, 4.4, H-14), 2.01 (2H, br t, J 6.4, CH2-4), 1.93 (3H, s, Me-19), 1.71
(3H, s, Me-18), 1.61 (2H, m, CH2-3), 1.45 (2H, m, CH2-2), 1.02 (6H, s, Me-16,17);
cis isomer: 13C NMR d 137.3 (C-8), 130.7 (C-11), 128.1 (C-7), 126.0 (C-12), 123.5
(C-10), 61.1 (C-15), 58.7 (C-14), 51.7 (C-13), 39.5 (C-2), 34.2 (C-1), 33.0 (C-4),
28.9 (C-16,17), 21.7 (C-18), 19.2 (C-3), 12.2 (C-19); kmax(ether) 300 nm; HRMS
m=z 288.2090 (Mþ. C19H28O2 requires 288.2089).
4.2.6. 11,14-Epoxyretinol (12)
mCPBA (27 mg, 0.09 mmol) was added to a mixture of trans retinol (30 mg,
0.1 mmol), K2HPO4 (27 mg, 0.15 mmol) and water (40 ll) in CH2Cl2 (4 ml) at r.t. After
30 min, saturated NaHCO3 solution was added and the mixture was extracted twice
with CH2Cl2. The organic phase was dried (MgSO4) and evaporated to dryness. Chro-
matography afforded 9 mg (30% yield) of 12. A bisepoxy product 13 was also isolated
(about 10% yield), as determined by its mass spectrum. 1H NMR d 6.15 (1H, d, J 16.0,
H-7), 5.99 (1H, d, J 16.0, H-8), 5.47 (1H, m, H-10), 5.42 (1H, d, J 11.1, H-12), 4.95 (1H,
br d, J 8.1, H-11), 4.03 (1H, dd, J 11.5, 3.7, H-15), 3.90 (1H, m, H-14), 3.62 (1H, dd, J
11.5, 4.6, H-15), 2.00 (2H, br t, J 6.4, CH2-4), 1.89 (3H, d, J 1.2, CH3-19), 1.85 (3H, m,
CH3-20), 1.67 (3H, d, J 0.7, CH3-18), 1.59 (2H, m, CH2-3), 1.46 (2H, m, CH2-2), 1.00
(6H, s, CH3-16,17); 13C NMR d 137.5 (C-6), 136.9 (C-8), 129.0 (C-5), 127.3 (CH), 127.2
(C-7), 126.0 (CH), 70.5 (C-11), 67.7 (C-15), 66.1 (C-14), 39.6 (C2), 34.2 (C-1), 32.9 (C-
4), 28.9 (C-16,17), 21.6 (C-18), 19.5 (C-20), 19.3 (C-3), 12.8 (C-19); MS m=z 320
(MNHþ4 ), 303 (MHþ).
4.2.7. Bisepoxy retinol (13)
MS m=z 336 (MNHþ4 ), 319 (MHþ), 303, 301.
4.2.8. O-t-butyldimethylsilyl-13-demethyl-retroretinol (15)
Aldehyde 9 (70 mg, 0.3 mmol) was coupled to phosphorane 6 (330 mg, 0.6 mmol)
as described for 10. After drying the organic phase and evaporation of the solvent,
1
the elimination product 15 was obtained. H NMR d 6.75 (1H, d, J 12, H-8), 6.58
(1H, dd, J 15.5, 8.5, H-11), 6.36 (1H, d, J 12, H-7), 6.2 (2H, m, H-10, H-12), 5.76

48
R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
(1H, br t, J 4, H-4), 5.67 (1H, dt, J 16, 7, H-13), 3.66 (2H, m, CH2-15), 2.33 (2H, m,
CH2-14), 2.03 (2H, m, CH2-3), 1.88 (3H, s, Me-19), 1.4 (2H, m, CH2-2), 1.24 (3H, s,
Me-18), 1.02 (6H, s, Me-16,17), 0.90 (9H, s, t-Bu), 0.07 (6H, s, SiMe2); kmax(ether)
328, 344, 366 nm triplet; MS m=z (EI) 386 (Mþ), 255, 201, 185.
4.2.9. Ethyl 13-demethyl-13-hydroxy-13, 14-dihydroretinoate (17)
Ethyl bromoacetate (1.1 ml, 10 mmol) was added dropwise to a mixture of freshly
activated Zinc (0.67 g, 10 mmol) in triethylborate (8 ml) and dry THF (3 ml), under
Ar atmosphere. After the addition was completed, the mixture was heated to 55 °C
and after a few minutes aldehyde 16 (1 g, 4.1 mmol), dissolved in dry THF (5 ml),
was added. After 3 h the reaction mixture was cooled to r.t. and dilute aqueous NH4OH
was added. The mixture was extracted twice with ether and the combined organic phase
was washed with water, dried (MgSO4) and concentrated. Chromatography
1
(EtOAc:hexane 1:9) yielded 990 mg (73% yield) of clean 17. H NMR d 6.66 (1H,
ddd, J 15.1, 11.2, 1.2, H-11), 6.16 (1H, d, J 16.2, H-7), 6.05 (1H, d, J 16.2, H-8), 6.00
(1H, d, J 11.2, H-10), 5.70 (1H, dd, J 15.1, 6.3, H-12), 4.64 (1H, br quint, J 5.2, H-
13), 4.16 (2H, q, J 7.2, OCH2), 3.12 (1H, d, J 4.2, OH), 2.58 (1H, dd, J 16.2, 4.2,
CH2-14), 2.54 (1H, dd, J 16.2, 8.2, CH2-14), 2.01 (2H, t, J 6.0, CH2-4), 1.92 (3H, d,
J 1.2, Me-19), 1.69 (3H, s, Me-18), 1.61 (2H, m, CH2-3), 1.46 (2H, m, CH2-2), 1.28
(3H, t, J 7.2, Me), 1.01 (6H, s, Me-16,17); 13C NMR d 172.2 (C-15), 137.7 (C-9),
137.4 (C-8), 136.7 (C-6), 133.1 (C-12), 129.2 (C-5), 128.5 (C-10), 127.4 (C-11), 127.2
(C-7), 68.9 (C-13), 60.8 (OCH2), 41.7 (C-14), 39.6 (C-2), 34.2 (C-1), 33.0 (C-4), 28.9
(Me-16,17), 21.7 (C-18), 19.3 (C-3), 14.7 (Me), 12.6 (C-19); kmax(CH2Cl2) 296 nm;
HRMS m=z 332.2354 (Mþ. C21H32O3 requires 332.2351).
4.2.10. 13-Demethyl-13-hydroxy-13,14-dihydroretinol (4)
Lithium borohydride (85 mg, 3.9 mmol) was added to a solution of 17 (1.3 g,
3.9 mmol) in freshly distilled dry ether (30 ml) under N2 atmosphere at 0 °C. The re-
sulting mixture was stirred at 0 °C. After 20 min water was added and the mixture
was extracted twice with ether. The combined organic phase was dried (MgSO4)
and concentrated. Chromatography (EtOAc:hexane 1:1) afforded 645 mg (57% yield)
1
of the product 4. H NMR d 6.64 (1H, ddd, J 15.1, 11.3, 1.3, H-11), 6.17 (1H, d, J
16.5, H-7), 6.07 (1H, d, J 16.5, H-8), 6.02 (1H, d, J 11.3, H-10), 5.75 (1H, dd, J 15.1,
6.6, H-12), 4.49 (1H, td, J 6.6, 5.7, H-13), 3.89 (1H, dt, J 11.0, 5.3, H-15), 3.83 (1H,
ddd, J 11.0, 6.7, 5.0, H-15), 2.55 (1H, br s, OH), 2.48 (1H, br s, OH), 2.01 (2H, t, J
6.2, CH2-4), 1.93 (3H, d, J 1.2, Me-19), 1.82 (2H, m, CH2-14), 1.70 (3H, d, J 1.0, Me-
18), 1.61 (2H, m, CH2-3), 1.46 (2H, m, CH2-2), 1.01 (6H, s, Me-16,17); 13C NMR d
137.8 (C-9), 137.4 (C-8), 136.6 (C-6), 134.9 (C-12), 129.3 (C-5), 128.6 (C-10), 127.2
(C-7), 127.0 (C-11), 72.7 (C-13), 61.2 (C-15), 39.6 (C-2), 38.7 (C-14), 34.2 (C-1),
33.0 (C-4), 28.9 (Me-16,17), 21.7 (C-18), 19.3 (C-3), 12.6 (C-19); kmax(CH2Cl2)
294 nm; HRMS m=z 290.2253 (Mþ. C19H30O2 requires 290.2246).
4.2.11. Ethyl 13-demethyl-13-methoxy-13,14-dihydroretinoate (18)
Ag2O (1.2 g, 5.18 mmol), K2CO3 (1.45 g, 10.5 mmol) and methyliodide (1.75 ml,
28 mmol) were added to hydroxyester 17 (0.9 g, 2.7 mmol) in acetonitrile (14.5 ml).

R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
49
The reaction mixture was stirred at r.t. for 60 h. The mixture was filtered through
celite and concentrated under vacuum. Chromatography (EtOAc:hexane 1:9) affor-
ded 440 mg (47% yield) of the product. 1H NMR d 6.64 (1H, dd, J 15.0, 11.3, H-11),
6.19 (1H, d, J 16.0, H-7), 6.07 (1H, d, J 16.0, H-8), 6.03 (1H, d, J 11.3, H-10), 5.55
(1H, dd, J 15.0, 8.2, H-12), 4.16 (2H, q, J 7.2, CH2-O), 4.13 (1H, td, J 8.2, 5.4, H-13),
3.29 (3H, s, MeO-13), 2.62 (1H, dd, J 15.0, 8.2, CH-14), 2.47 (1H, dd, J 15.0, 5.4,
CH-14), 2.03 (2H, t, J 6.0, CH2-4), 1.93 (3H, s, Me-19), 1.70 (3H, s, Me-18), 1.61
(2H, m, CH2-3), 1.46 (2H, m, CH2-2), 1.25 (3H, t, J 7.2, Me), 1.01 (6H, s, Me-
16,17);13C NMR d 170.9 (C-15), 137.7 (C-9), 137.3 (C-8), 136.8 (C-6), 131.4 (C-
12), 129.7 (C-10), 129.3 (C-5), 128.3 (C-11), 127.5 (C-7), 78.7 (MeO), 60.5 (OCH2),
56.4 (C-13), 41.4 (C-14), 39.5 (C-2), 34.2 (C-1), 33.0 (C-4), 28.9 (Me-16,17), 21.6 (C-
18), 19.2 (C-3), 14.2 (Me), 12.6 (C-19); kmax(CH2Cl2) 296 nm; HRMS m=z 346.2517
(Mþ. C22H34O3 requires 346.2508).
4.2.12. 13-Demethyl-13-methoxy-13,14-dihydroretinol (5)
Lithium borohydride (26 mg, 1.2 mmol) was added to a solution of 18 (0.418 g,
1.2 mmol) in freshly distilled dry ether (9 ml) under Ar atmosphere at 0 °C. After
40 min water was added and the mixture was extracted twice with ether. The com-
bined organic phase was dried (MgSO4) and concentrated. Chromatography
1
(EtOAc:hexane 4:1) yielded 255 mg (70% yield) of the product. H NMR d 6.57
(1H, dd, J 15.2, 11.3, 0.6, H-11), 6.17 (1H, d, J 16.1, H-7), 6.06 (1H, d, J 16.1, H-
8), 6.02 (1H, d, J 11.3, H-10), 5.55 (1H, dd, J 15.2, 8.1, H-12), 3.89 (1H, dt, J 8.1,
4.5, H-13), 3.75 (1H, ddd, J 10.8, 6.0, 4.8, H-15), 3.72 (1H, ddd, J 10.8, 6.0, 4.8,
H-15), 3.28 (3H, s, MeO-13), 1.99 (1H, dd, J 6.0, CH2-4), 1.92 (3H, d, J 0.9, Me-
19), 1.78 (2H, m, CH2-14), 1.68 (3H, d, J 0.6, Me-18), 1.60 (2H, m, CH2-3), 1.45
(2H, m, CH2-2), 1.00 (6H, s, Me-16,17); 13C NMR d 137.6 (C-9), 137.3 (C-8),
136.4 (C-6), 132.4 (C-12), 129.3 (C-10), 129.1 (C-5), 128.4 (C-11), 127.2 (C-7), 81.8
(C-13), 60.4 (C-15), 56.1 (MeO), 39.4 (C-2), 38.0 (C-14), 34.1 (C-1), 32.9 (C-4),
28.8 (Me-16,17), 21.6 (C-18), 19.1 (C-3), 12.5 (C-19); kmax(CH2Cl2) 294 nm; HRMS
m=z 304.2395 (Mþ. C20H33O2 requires 304.2402).
4.2.13. O-t-butyldimethylsilyl-13-demethyl-14-hydroxy retroretinol (19)
Epoxyretinol 10 was dissolved in ether (5 ml). Silica (0.5 g) was added and the
mixture was stirred for 3 h at r.t. It was then filtered and evaporated to dryness.
1H NMR d 6.76 (1H, d, J 12.0, H-8), 6.50-6.25 (4H, m, H-7, H-10, H-11, H-12),
5.77 (1H, br t, J 4, H-4), 5.66 (1H, dd, J 15.5, 6.5, H-13), 4.24 (1H, m, H-14),
3.66 (1H, dd, J 10.0, 3.7, H-15), 3.46 (1H, dd, J 10.0, 8.0, H-15), 2.63 (1H, d, J
3.0, OH), 2.12 (2H, m, H-3), 1.87 (3H, s, Me-19), 1.50 (2H, m, H-2), 1.26 (3H, S,
Me-18), 0.90 (15H, s, Me-16,17, t-Bu), 0.06 (6H, s, SiMe2).
Acknowledgments
This study was supported by a United States—Israel Binational Science Founda-
tion Grant no. 2000390.

50
R. Yefidoff et al. / Bioorganic Chemistry 32 (2004) 38–50
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