Synthesis of biotinylated retinoids for cross-linking and isolation of retinol binding proteins

Article abstract of DOI:10.1016/S0040-4020(02)00667-1

The synthesis of (3R)-3-[Boc-Lys(biotinyl)-O]-11-cis-retinol bromoacetate and 3-[Boc-Lys(biotinyl)-O]-all trans-retinol chloroacetate is described. These biotinylated retinoids are instrumental in labeling the retinol binding proteins (RBPs) via a nucleophilic displacement of the haloacetate by a residue in the binding site of the protein. The covalently linked biotin will allow for a facile isolation and purification of the protein on a streptavidin column thus rendering the protein ready for a tryptic digest followed by mass spectrometric analysis. The 11-cis retinoid was synthesized via metal reduction of an alkyne intermediate generated from a Horner-Wadsworth-Emmons (HWE) reaction whereas the all-trans was synthesized via two consecutive HWE couplings.

Full text of DOI:10.1016/S0040-4020(02)00667-1

Tetrahedron 58 (2002) 6577–6584
Synthesis of biotinylated retinoids for cross-linking and isolation
of retinol binding proteins
a
Nasri Nesnas,a Robert R. Randob and Koji Nakanishi
,
,
*
*
aDepartment of Chemistry, Columbia University, New York, NY 10027, USA
bDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
Received 11 June 2002; accepted 21 June 2002
This article is dedicated to Professor Yoshito Kishi on his receipt of the Tetrahedron Prize
Abstract—The synthesis of (3R )-3-[Boc-Lys(biotinyl)-O]-11-cis-retinol bromoacetate and 3-[Boc-Lys(biotinyl)-O]-all trans-retinol
chloroacetate is described. These biotinylated retinoids are instrumental in labeling the retinol binding proteins (RBPs) via a nucleophilic
displacement of the haloacetate by a residue in the binding site of the protein. The covalently linked biotin will allow for a facile isolation and
purification of the protein on a streptavidin column thus rendering the protein ready for a tryptic digest followed by mass spectrometric
analysis. The 11-cis retinoid was synthesized via metal reduction of an alkyne intermediate generated from a Horner–Wadsworth–Emmons
(HWE) reaction whereas the all-trans was synthesized via two consecutive HWE couplings. q 2002 Elsevier Science Ltd. All rights
reserved.
1. Introduction
some are involved in catalyzing esterification, hydrolysis,
and isomerization of retinols at various stages in the visual
cycle. A summary of the mammalian visual cycle is
presented in Fig. 2. The visual protein, rhodopsin, is
comprised of the protein opsin (ca. 348 amino acids in
bovine) and 11-cis-retinal covalently linked via a schiff base
with Lys296 residue. Vision begins with the absorption of
light by rhodopsin causing the isomerization of 11-cis-
retinylidene to all trans-retinylidene. This occurs over
several states of rhodopsin leading to a meta II state
(unprotonated schiff base) which is responsible for
G-protein activation ultimately leading to neural signaling.2
Retinoids are derivatives of vitamin A (all trans-retinol)
which can generally be synthesized for studies directed to a
better understanding of human and non-human vision. We
hereby report the synthesis of (3R)-3-[Boc-Lys(biotinyl)-
O ]-11-cis-retinol bromoacetate (1) and 3-[Boc-Lys(bio-
tinyl)-O ]-all trans-retinol chloroacetate (2) (Fig. 1). These
retinoids can act as ligands for proteins known as retinol
binding proteins (RBPs) present in the retinal pigment
epithelial (RPE) cells.1 These RBPs are believed to be
responsible for the transport of retinols in RPE cells and
Figure 1. The target retinoid biotinylated structures.
Keywords: biotinylated retinoids; biotin; retinol binding proteins; RBP.
*
Corresponding author. Tel.: þ1-212-854-2169; fax: þ1-212-932-8273; e-mail: kn5@columbia.edu
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00667-1

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N. Nesnas et al. / Tetrahedron 58 (2002) 6577–6584
Figure 2. The mammalian visual cycle.
This isomerization is accompanied by various confor-
mational changes of the protein which in turn trigger the
binding of G-protein. These photo intermediates have been
well studied through photoaffinity labeling experiments,
protein digestion and mass spectrometric characterization.3
The meta II state is then protonated generating meta III (see
Fig. 2) which undergoes hydrolysis regenerating the protein
opsin and all trans-retinal. To complete the visual cycle, the
all trans-retinal needs to be re-isomerized back to 11-cis-
retinal. This is achieved through a cascade of several events
involving proteins such as dehydrogenase which initially
reduces all trans-retinal to all trans-retinol (vitamin A). The
latter is then isomerized to 11-cis-retinol through the action
of numerous RBPs which we are interested in further
investigating.1 The actions of these proteins include the
esterification of vitamin A with a palmitoyl group followed
by an isomerization of the resulting palimitate through a
possible hydrolytic mechanism. One such protein that has
been already under investigation is known as lecithin retinol
acyl transferase (LRAT).4,5 To regenerate the visual
pigment, 11-cis-retinol undergoes oxidation via a dehydro-
genase rendering 11-cis-retinal ready for incorporation into
opsin forming the Schiff base rhodopsin.
the transformation while the retinoid 1 may prove helpful
for isolating RBPs that act in the later stages of pigment
regeneration. The features of the target molecules are
outlined in Fig. 3 as exemplified through retinoid 2. The all
trans retinol portion of the structures acts as a ligand for the
binding site of the RBP. The chloroacetyl moiety acts as a
potent electrophile for a nearby residue to carry out a
nucleophilic displacement of the halogen forming a
covalent bond with a residue in the active site of the
protein. This nucleophilic displacement resulting in a
covalent bond with the protein is referred to as the labeling
step of the protein. The ultimate result is that the RBPs
which inherently bind retinol (hence the name) will be
specifically labeled with these structures bearing an
extended tether (Boc-Lys) to a biotin. The latter will
allow for the facile isolation of the protein via affinity
chromatography through an avidin column. This technique
proved to be successful in the isolation and identification of
mammalian LRAT using biotinylated affinity labeling
agents.6 The biotin affinity labeling approach was important
here because membrane bound LRAT was not susceptible to
purification due to its instability when solubilized in
detergent.
In the interest of isolating and investigating the RBPs
involved in the transport and transformation of all trans-
retinol to the 11-cis-retinol the target molecules 1 and 2
were synthesized. The all trans biotinylated retinoid 2 is
needed as a ligand for RBPs involved in the earlier stages of
One important advantage that is featured in the target
structures needs to be emphasized. The biotin allows for the
successful isolation of the protein due to its remarkable
binding to streptavidin with association constants often
exceeding 1015 M21 6,7
However, this often presents a
.
challenge for the scientist who is interested in isolating the
protein via breaking this association. The above structure
features a labile ester of the primary alcohol at C-15 of the
retinol hydroxyl which can be easily cleaved in basic media
(pH,11).
1.1. Retrosynthetic analysis of 1 and 2
Scheme 1 represents the two approaches for the installation
of a biotin on one end of the retinoid and a haloester on the
other end. Structure 7 represents the general formula for
retinoids 1 and 2. Structure 3 represents the retinoid
backbone with some orthogonal protection on the hydroxy
groups at C-3 and C-15. Route A will entail installing the
Boc-Lys(biotinyl)-OH (B-OH) first directly after deprotec-
tion of the C-3 alcohol (unless prior steps involve releasing
the 3-hydroxy) and then attachment of the haloacid via an
EDC coupling reaction. Route B, however, will require the
release of the primary alcohol at C-15, attachment of the
haloester, and finally deprotection of the 3-hydroxy
Figure 3. A schematic diagram of structure 2 in the binding site of a
representative RBP featuring key elements of the structure and their role in
protein labeling and isolation.

N. Nesnas et al. / Tetrahedron 58 (2002) 6577–6584
6579
Scheme 1. Retrosynthetic analysis using two poteintal route A and B.
biological assays since the biotin linker is not directly
involved in the binding of the protein. We have in fact
generated 3-hydroxy-b-ionone 9 from two independent
routes and have arbitrarily proceeded with (R)-9 for the
synthesis of 1 and the racemic 9 for the synthesis of 2. The
stereoselective route for the generation of (R)-9 was needed
for another purpose that will be published elsewhere.
Scheme 4 presents that two routes for the generation of 9.
The non-stereoselective route will involve the introduction
of a double at the C-3,412 followed by hydroboration
treatment13 to afford (^)-9.14 The stereoselective route will
involve the stereoselective reduction of 18 followed by a
second reduction of the unhindered carbonyl resulting in
17.15 The latter can be transformed into (R )-9 via triflation16
followed by a Heck reaction.17
Scheme 2.
followed by esterification with B-OH. The first route is
favored as the labile haloester which is attached last will not
need to endure the steps presented in Route B. However,
both routes were used as will become apparent in due course
owing to the nature of the retinoid precursor 3 for each of
the two target molecules, the cis and the trans.
The retrosynthesis of 4a is outlined in Scheme 2. The 11-cis
double bond can be generated via a zinc metal reduction of
the alkyne intermediate 8.8 The latter can be constructed via
a Horner–Wadsworth–Emmons (HWE) coupling of the
3-hydroxy-b-ionone (R)-9 and phosphonate 10 followed by
a Sonogashira coupling8 with vinyl iodide 11, which can be
obtained from a reduction of 12 followed by iodination with
I2.9
Scheme 4.
2. Results and discussion
The synthesis of the racemic 3-hydroxy-b-ionone (^)-9
from b-ionone 16 was carried out according to Scheme 5.14
Treatment of the latter with N-bromosuccinimide (NBS)
under basic and refluxing conditions yielded a 4-bromo
derivative which eliminated when heated in pyridine for 2 h
affording 3,4-dehydro-b-ionone 19 in a 50% overall yield.12
Protection of the ketone occurred smoothly with ethylene
glycol in benzene catalyzed by p-toluenesulfonic acid in the
presence of triethylorthoformate.13 The latter acted as a
water trap and its presence resulted in enhancement of the
yield from 50% (with a Dean–Stark distillation) to 80%.
The 3-hydroxy-b-ionone can also be used in the synthesis of
4b as shown in the retrosynthetic analysis in Scheme 3. Two
consecutive HWE couplings: the first between (^)-9 and 14
followed by DIBAH reduction to generate an aldehyde
ready for the second coupling with phosphonate 15 thus
generating 13. Reduction of the latter with LiAlH4 will
afford the desired 3-siloxylated-all trans-retinol 4b.10,11
Retrosynthetic Schemes 2 and 3 indicate the key role of
the compound 3-hydro-b-ionone 9. The stereochemistry at
the 3-hydroxy position does not need to be defined for the
Scheme 5. Reagents: (a) (i) CaO, NaHCO3, NBS/CCl4 reflux, 2 h;
(ii) PhN(CH3)2, 908C, 1 h; (iii) pydrine, 908C, 2 h; 50%; (b) HOCH2CH2-
OH, TsOH, HC(OEt)3, PhH, reflux, 2 h; 80%; (c) (i) 9-BBN/THF 558C 4 h;
(ii) MeOH; (iii) NaOH, H2O2, 08C, 55%; (d) 5% HCl/acetone, 5 min, 99%;
(e) TESCl, DMAP, Et3N/CH2Cl2; 97%. TES, triethylsilyl.
Scheme 3.

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N. Nesnas et al. / Tetrahedron 58 (2002) 6577–6584
Hydroboration of 20 with 9-BBN in THF followed by
oxidative methanolic work up afforded a mixture of
hydroxylated compound enriched in the desired 21
(racemic) with a yield of 55%.13 Deprotection of the latter
in 5% HCl in acetone for 5 min afforded (^)-9 in
quantitative yield. Silylation of 3-hydroxy-b-ionone 9 was
carried out, as necessary for further transformation, using
triethylsilyl as a protecting group with a yield up to 97%
resulting in 22. The overall yield of transformation from 16
to 22 was 21%.
Scheme 8. Reagents: (a) NaH/THF; 84%; (b) DIBAH, CH2Cl2, 2608C;
74%; (c) BuLi, THF; 88%.
The stereoselective route for the synthesis of (3R )-3-
hydroxy-b-ionone (R )-9 shown in Scheme 6 involved an
enzymatic reduction of 2,6,6-trimethyl-2-cyclohexen-1,4-
dione (4-oxoisophorone) 18 with baker’s yeast to generate
23 after which a Raney Nickel reduction resulted in 17.15 A
generous amount of 17 was provided to us by Hoffmann
LaRoche from which the rest of the synthesis was actually
carried out. Silylation of the latter under standard conditions
of DMAP and triethylamine resulted in a quantitative yield
of 24.16 Triflation was carried out on 24 with 1.1 equiv. of
LDA and Tf2NPh to afford 25 in a modest 68% yield.16 This
was followed by a Heck reaction with methyl vinyl ketone
(MVK) catalyzed by Pd(0), which forms in situ from Pd(II),
to give 26 in 67% yield.17 The overall yield of transform-
ations from compound 17 to the desired (3R)-3-siloxylated-
b-ionone 26 was 44%.
nate18 10 and BuLi to afford the protected alkyne 27 in a
quantitative yield with 4:1 of the desired E/Z ratio. The
isomeric mixture inseparable at that stage was subjected to a
global deprotection of both silyl groups with TBAF
resulting in the terminal alkyne 28 ready for a Sonogashira
coupling with vinyl iodide 11 generating 8 in quantitative
yield. The 9E and 9Z isomers were then easily separated at
that stage due to the presence of the bulky silyl group via
column chromatography. Vinyl iodide 11 was prepared via
reduction of 2-butyn-1-ol 12 with titanocene dichloride9
followed by quenching with I2 and protection of the primary
alcohol with tert-butyldiphenylsilyl chloride (TBDPSCl).
The key step of generating the 11-cis from alkyne 8 utilized
a copper/silver pretreated zinc reduction8 of the latter in a
1:1 MeOH/H2O mixture over a period of ca. 24 h resulting
in a nearly quantitative yield of exclusively the 11-cis
isomer 4a.
It was completely arbitrary and a result of synthetic timing
that resulted in the usage of 26 in the synthesis of 11-cis
retinoid 1 and the usage of the racemic precursor 22 in the
synthesis of the all trans retinoid 2. Scheme 7 presents the
synthesis of the 11-cis precursor monoprotected retinoid 4a.
Synthesis of the all trans retinoid 13 involved a series of two
HWE couplings outlined in Scheme 8.10,11 The siloxylated
b-ionone 22 was treated with the NaH generated anion of
phosphonate 14 to afford 29 in a mixture of 3.5:1 of 9E/9Z
which were separated after DIBAH reduction resulting in
the desired 9E 30 in an overall yield of 61%. The second
HWE of the latter with 15 pretreated with BuLi resulted in a
4:1 of 13E/13Z mixture separable by column chromato-
graphy to afford 13.
The silyloxylated b-ionone 26 was treated with phospho-
Due to the nature of the structures of the retinoid precursors
4a and 13, both routes A and B (see Scheme 1) were utilized
in installing the biotin group. For instance, in the case of 13
the only means of introducing the primary alcohol at C-15 is
through an LAH reduction. Therefore the Boc-Lys-biotin
(B) had to be introduced last (according to route B) as it
would not have survived the latter reduction. Thus 13 must
be first reduced with LAH to the primary alcohol, esterified
with the appropiate a-haloacid, then desilylated without
disturbing the labile haloester, and finally biotinylated with
Boc-Lys(biotinyl)-OH. The 11-cis was fortunately carried
through as the protected primary alcohol and a free C-3
hydroxyl allowing for the transformations depicted by route
A (the more desirable of the two routes).
Scheme 6. Reagents: (a) Baker’s yeast, sucrose; 24%; (b) Raney Ni; 66%;
(c) TBSCl, DMAP, Et3N/CH2Cl2; 97%; (d) (i) LDA (ii) Tf2NPh; 68%;
(e) PdCl2(PPh3)2, DMF, 808C, 30 h; 67%. TBS, tert-butyldimethylsilyl.
Scheme 9 describes the transformations required for the
generation of the target biotinylated retinoid 1 according to
route A. The monoprotected 3-hydroxy retinoid 4a was
subjected to EDC coupling conditions with Boc-Lys(bio-
tinyl)-OH affording 5a in 78% yield. This was followed by
TBAF deprotection of the primary alcohol resulting in 6a
with a yield of 79%. The unstable primary alcohol was
immediately treated with bromoacetic acid under EDC
Scheme 7. Reagents: (a) BuLiþ10 at 08C then 26/THF; 99%; (b) TBAF,
4 h; 99%; (c) CuI, Pd(PPh3)4, i PrNH2; 99%; (d) Zn(Cu/Ag), MeOH/H2O,
21 h; 99%. TMS, trimethylsilyl; TBDPS, tert-butyldiphenylsilyl.

N. Nesnas et al. / Tetrahedron 58 (2002) 6577–6584
6581
3. Conclusion
The synthesis of biotinylated retinoids 1 and 2 was
accomplished using two different routes. Installing the
biotin first, route A, was preferred due to the labile nature of
a-haloesters (in particular chloroesters) which were
preferably attached last. Route A was used in the synthesis
of 1, however, to avoid multiple protection and deprotection
procedures, route B (attaching the chloroester first) was
employed in the synthesis of 2. The overall yield for the
synthesis of 1 and 2 were 20% (from 17) and 0.7%,
respectively. The biotinylated targets are currently in use for
labeling RBPs in the retinal pigment epithelial (RPE) cells
allowing for facile isolation via avidin affinity chromatog-
raphy, hydrolysis off the column, and final characterization
by mass spectrometry.
Scheme 9. Reagents: (a) B-OH/DMSO, EDC, DMAP, CH2Cl2; 78%;
(b) 2 equiv. TBAF, 08C–rt, 2 h; 79%; (c) BrCH2CO2H, EDC, DMAP,
CH2Cl2, 2108C, 15 min; 99%. B, Boc-Lys(biotinyl)-.
4. Experimental
4.1. General
All chemicals were obtained from Aldrich except for Boc-
Lys(biotinyl)-OH which was purchased from BAChem. The
solvents THF, Et2O, and CH2Cl2 were obtained dry from the
solvent purification system via passage through an activated
alumina cartridge. Column chromatography was performed
Scheme 10. Reagents: (a) LiAlH4, Et2O, 2108C, 10 min; (b) ClCH2CO2H,
EDC, DMAP, CH2Cl2, 2108C, 5 min; (c) 5% HF/pyridine, THF, 08C,
5 min; 27% over last three steps; (d) B-OH/DMSO, EDC, DMAP, CH2Cl2,
2108C, 5 min; 40%. B, Boc-Lys(biotinyl)-.
1
using ICN silica gel (32–63 mesh). H NMR spectra were
obtained on Bruker DMX 400 or 500 MHz spectrometers
and are reported in parts per million (ppm) relative to TMS
(d), with coupling constants (J ) in Hertz (Hz). The residual
protic sovlents (in CDCl3 or C6D6) were used as an internal
reference. Low and high resolution FAB mass spectra were
measured on a JEOL JMS-DX303 HF mass spectrometers
using a glycerol matrix and Xe ionizing gas. ESI and APCI
spectra were measured on a JEOL LC mate mass
spectrometer. The all trans retinoids were handled under
dim light conditions especially when containing more than 3
conjugated double bonds. The 11-cis compounds were
handled strictly in the darkroom under dim red lights.
Retinoids are best stored in benzene solutions frozen at
2788C, unless they are not soluble.
coupling conditions to generate the desired final product 1 in
quantitative yield.
The transformations that brought about the final stitches in
the construction of 2 in Scheme 10 were certainly a lot more
cumbersome due to the undesirable route B that was
pursued. Needless to say, several attempts at completing this
route proved unsuccessful due to the labile nature of the
chloroester (which indeed is much more unstable than its
bromo analog). The latter information regarding the
stability of the bromoester versus the chloroester was not
available to us until after the completion of this work and
hence the reason for the synthesis of the chloroester analog
2. Moreover, preliminary unpublished biological assay
results, which were not available during the time of the
synthesis of the chloroester, have shown no preference
toward having one halogen over the other, and therefore
future targets will employ the exclusive use of
a-bromoesters.
4.2. Synthesis of biotinylated retinoid 1
4.2.1. (R )-5-(tert-Butyldimethylsiloxy)-1,3,3-trimethyl-2-
[3-methyl-6-(trimethylsilyl)-hexa-1,3-dien-5-ynyl]-cyclo-
hexene (27). Dimethyl (3-trimethylsilyl-2-propynyl)phos-
phonate 10 (0.550 g, 2.5 mmol) in 10 mL of THF at 08C was
treated with nBuLi (2.5 mmol) to give a red solution. The
reaction mixture was then stirred at room temperature for
15 min, after which time 26 (0.400 g, 1.24 mmol) in 1 mL
of THF was added. The mixture was stirred for an additional
2 h, and was then quenched with aqueous NH4Cl, extracted
with Et2O, washed with brine, and dried over Na2SO4. The
product was purified by column chromatography using 2%
Et2O in hexanes as an eluent to afford 27 as an oil (0.511 g,
99% yield; 4:1 9E/9Z ). 1H NMR (500 MHz, CDCl3):
d¼6.90 (d, 1H of 9Z, J¼16.4 Hz), 6.31 (d, 1H of 9Z,
J¼16.4 Hz), 6.21 (d, 1H of 9E, J¼16.4 Hz), 6.07 (d, 1H of
9E, J¼16.4 Hz), 5.44 (s, 1H of 9E), 5.37 (s, 1H of 9Z ), 3.93
(m, 1H), 2.22 (m, 1H), 2.07 (s, 3H of 9E), 2.1–2.0 (m, 1H),
1.92 (s, 3H of 9Z), 1.78 (s, 3H of 9Z), 1.68 (s, 3H of 9E ),
The silylated ester 13 was reduced with LAH resulting in 4b
which was subsequently used without purification in an
EDC coupling reaction with chloroacetic acid affording 5b
which was purified rapidly by flash chromatography to
avoid major decomposition. The latter was mildly desilyl-
ated by treatment with 5% HF/pyridine in THF for 5 min
affording 6b in a modest overall yield of 27% from the
starting ester 13. An EDC coupling of 6b with Boc-
Lys(biotinyl)-OH resulted in the formation of an apparent
quantitative yield of 2 by TLC; however a maximum of 40%
recovery was achieved after workup and purification of the
desired compound 2.

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N. Nesnas et al. / Tetrahedron 58 (2002) 6577–6584
1.49 (t, 1H, J¼12.2 Hz), 1.11 and 1.08 (s, 2£3H), 0.91 (s,
6H), 0.20 (s, 9H). [C25H44OSi2þHþ]: calcd 417.30; found
417.2 (APCIþ).
EtOAc and the combined organic layers were washed with
brine and dried over Na2SO4. The solvent was removed and
the residue was suspended in 50% EtOAc/hexanes and
filtered through a silica plug to afford purified 4a as an oil
with an exclusive 11-cis geometry (194 mg, 99% yield). 1H
NMR (500 MHz, C6D6): d¼7.81 (m, 4H), 7.71 (m, 6H),
6.86 (d, 1H, J¼12.0 Hz), 6.36 (t, 1H, J¼11.8 Hz), 6.31 (d,
1H, J¼16.1 Hz), 6.17 (d, 1H, J¼16.1 Hz), 5.99 (t, 1H,
J¼6.2 Hz), 5.88 (d, 1H, J¼11.6 Hz), 4.37 (d, 2H,
J¼6.3 Hz), 3.79 (m, 1H), 2.21 (dd, 1H, J¼16.8, 5.3 Hz),
1.95 (dd, 1H, J¼16.8, 9.4 Hz), 1.82 (s, 3H), 1.68 (s, 3H),
ovrlp with 1.6–1.5 (ddd, 1H, J¼12.1, 3.3, 2.1 Hz), 1.53 (s,
3H), 1.42 (t, 1H, J¼12.0 Hz), 1.17 (s, 9H), 1.05 and 1.04
(2s, 2£3H). [C36H48O2SiþHþ]: calcd 541.35; found 541.3
(APCIþ).
4.2.2. (R )-5-Hydroxy-1,3,3-trimethyl-2-[3-methyl-6-
hexa-1,3-dien-5-ynyl]-cyclohexene (28). Acetylene 27
(0.511, 1.23 mmol) in 5 mL THF at 08C was treated with
TBAF (5 mL, 1 M in THF, 5 mmol) and the stirred at room
temperature for 2 h. The mixture was then quenched with
aqueous NH4Cl, extracted with Et2O, washed with brine,
and dried over Na2SO4. The product was purified by column
chromatography using 30% EtOAc in hexanes as an eluent
to afford 28 as an oil (0.282 g, 99% yield). 1H NMR
(400 MHz, CDCl3): d¼6.83 (d, 1H of 9Z, J¼16.4 Hz), 6.31
(d, 1H of 9Z, J¼16.4 Hz), 6.26 (d, 1H of 9E, J¼16.4 Hz),
6.11 (d, 1H of 9E, J¼16.4 Hz), 5.44 (s, 1H of 9E), 5.37 (s,
1H of 9Z), 4.02 (m, 1H), 3.32 (s, 1H of 9E ), 3.20 (s, 1H of
9Z ), 2.4 (dd, 1H, J¼17.2, 5.3 Hz), 2.10 (s, 3H), 2.15–2.00
(m, 1H), 1.96 (s, 3H of 9Z ), 1.8 (m, 1H) ovrlp with 1.8 (s,
3H of 9Z), 1.74 (s, 3H of 9E ), 1.51 (t, 1H, J¼11.9 Hz), 1.1–
1.08 (2s, 2£3H). [C25H22OþHþ]: calcd 231.17; found 231.1
(APCIþ).
4.2.5. 3-[Boc-Lys(biotinyl)-O ]-11-cis-retin-O-tert-butyl-
diphenylsilyl (5a). To a 1 mL solution of retinoid 4a
(16 mg, 0.03 mmol) in CH2Cl2 was added a mixture of Boc-
Lys(biotinyl)-OH (28 mg, 0.06 mmol) in 100 mL DMSO
and EDC (18 mg, 0.094 mmol) in 1 mL of CH2Cl2. After
5 min of stirring, DMAP (1 mg, 0.008 mmol) was added and
the reaction mixture was stirred for 20 h after which it was
washed with NaHCO3, NH4Cl, extracted with CH2Cl2 and
chromatographed with 5–12% MeOH/CH2Cl2 gradient
affording 5a as an oil (23 mg, 78% yield). 1H NMR
(500 MHz, CDCl3): d¼7.7 (m, 4H), 7.4 (m, 6H), 6.56 (d,
1H, J¼12.0 Hz), 6.32 (t, 1H, 11.9 Hz), 6.09 (‘s’, 2H), 5.95
(br t, 1H), 5.87 (d, 1H, J¼11.7 Hz), 5.7 (t, 1H) ovrlp with
(br, 1H), 5.16 (d, 1H, J¼8.4 Hz), 5.10 (m, 1H), 5.00 (br,
1H), 4.52 (dd, 1H, J¼7.2, 5.3 Hz), 4.33 (d, 1H, J¼6.6 Hz)
ovrlp with (t, 2H), 4.23 (br t, 1H), 3.24 (dd, 2H, J¼6, 5 Hz),
3.17 (dd, 1H, J¼7.0, 4.6 Hz), 2.93 (dd, 1H, J¼12.8, 4.9 Hz),
2.74 (d, 1H, J¼12.8 Hz), 2.41 (dd, 1H, J¼17.5, 5.3 Hz),
2.20 (m, 2H), 2.11 (dd, 1H, J¼17.2, 9.6 Hz), 1.93 (s, 3H),
1.9–1.5 (m, 8H), 1.67 (s, 3H), 1.64 (s, 3H), 1.5–1.4 (m,
6H), 1.45 (s, 9H), 1.10 and 1.07 (2s, 2£3H), 1.05 (s, 9H).
[C57H82N4O7SSiþHþ]: calcd 995.5752; found 995.5745
(HRMS).
4.2.3. (R )-3-Hydroxy-retin-11-yne-O-tert-butyldiphenyl-
silyl (8). Vinyl iodide 11 (872 mg, 1.6 mmol) was dissolved
in i PrNH2 (4 mL), and tetrakis(triphenylphosphine)palla-
dium (13.8 mg, 0.0124 mmol) was then added. The solution
was stirred at room temperature for 5 min prior to the
addition of CuI (2.4 mg, 0.0124 mmol). After 5 min of
stirring, aceylene 28 (282 mg, 1.23 mmol) was introduced,
and the mixture was stirred at room temperature for 2 h. The
reaction was then quenched by the removal of solvent
followed by the addition of Et2O and then NH4Cl. The
resultant mixture was extracted with Et2O, washed with
brine, and dried over Na2SO4. The product was purified by
column chromatography using 12–20% EtOAc in hexanes
as an eluent to afford 8 as an oil (432 mg of 9E, and 660 mg
total weight, 99% yield). 1H NMR (500 MHz, CDCl3):
d¼7.69 (m, 4H), 7.42 (m, 6H), 6.19 (d, 1H, J¼16.0 Hz),
6.10 (d, 1H, J¼16.0 Hz), 6.02 (t, 1H, J¼5.1 Hz), 5.54 (s,
1H), 4.29 (d, 2H, J¼6.28 Hz), 4.00 (m, 1H), 2.38 (dd, 1H,
J¼16.9, 5.2 Hz), 2.1–2.0 (m, 1H) ovrlp with 2.06 (s, 3H),
1.77 (ddd, 1H, J¼12.1, 3.3, 2.1 Hz), 1.72 (s, 3H), 1.67 (s,
3H), 1.47 (t, 1H, J¼11.9 Hz), 1.35 (d, 1H, J¼4.9 Hz), 1.06
and 1.05 (2s, 2£3H) ovrlp with 1.05 (s, 9H).
[C36H46O2SiþHþ]: calcd 539.33; found 539.3 (APCIþ).
4.2.6. 3-[Boc-Lys(biotinyl)-O ]-11-cis-retinol (6a). To a
500 mL solution of 5a (10 mg, 0.01 mmol) in THF (at 08C)
˚
and two beads of activated molecular sieves 4 A was added
TBAF (18 mL of 1 M/THF, 0.018 mmol) and the reaction
mixture was stirred to rt for 1 h. The mixture was then
chromatographed and eluted with 5–12% MeOH/CH2Cl2
gradient affording 6a as a film coating the vial (6 mg, 79%
yield). Due to the extreme instability of this compound,
TLC was the sole means of characterization and the product
was used directly in following step.
4.2.4. (R )-3-Hydroxy-11-cis-retin-O-tert-butyldiphenyl-
silyl (4a). Argon was bubbled through a suspension of Zn
dust (10 g) in distilled H2O (60 mL) for 15 min after which
Cu(OAc)2 (1 g) was added and the flask sealed immediately.
The mixture was stirred vigorously at room temperature for
15 min prior to the addition of AgNO3 (1 g) which resulted
in an exothermic reaction. The mixture was stirred for
30 min. The resultant activated Zn was then filtered and
washed successively with H2O, MeOH, acetone, and
Et2O. The moist activated Zn was then transferred to a
flask to which 20 mL of H2O and 20 mL of MeOH were
added. Acetylene 8 (194 mg, 0.36 mmol) dissolved in 2 mL
MeOH was then added to the activated Zn and the reaction
mixture was stirred at room temperature in the dark for 24 h.
The Zn dust was then filtered through Celite, washed with
Et2O and H2O. The aqueous layer was extracted further with
4.2.7. 3-[Boc-Lys(biotinyl)-O ]-11-cis-retinol bromo-
acetate (1). To a 1 mL solution of 6a (6 mg, 0.008 mmol)
in CH2Cl2 at 2108C, and two beads of activated molecular
˚
sieves 4 A, was added EDC (13 mg, 0.068 mmol) and
bromoacetic acid (4 mg, 0.029 mmol). The mixture was
stirred for 2 min prior to the addition of DMAP (1 mg,
0.008 mmol) after which it was stirred for an additional
15 min. The reaction mixture was then washed with
NaHCO3 and NH4Cl, dried over Na2SO4 and then
chromatographed using 5–8% MeOH/CH2Cl2 affording 1
as a film coating the inside of a vial (6.9 mg, 99% yield). 1H
NMR (500 MHz, CDCl3): d¼6.50 (d, 1H, J¼12.0 Hz), 6.39

N. Nesnas et al. / Tetrahedron 58 (2002) 6577–6584
6583
(t, 1H, J¼11.9 Hz), 6.11 (s, 2H), 5.99 (br t, 1H), 5.89 (d, 1H,
J¼11.6 Hz), 5.68 (br, 1H), 5.64 (t, 1H, J¼7.0 Hz), 5.19 (d,
1H, J¼8.3 Hz), 5.10 (m, 1H), 5.02 (br, 1H), 4.81 (d, 2H,
J¼7.2 Hz), 4.52 (dd, 1H, J¼7.5, 5.1 Hz), 4.33 (dd, 1H,
J¼7.7, 4.8 Hz), 4.24 (br t, 1H), 3.85 (s, 2H), 3.24 (dd, 2H,
J¼6, 5 Hz), 3.17 (dd, 1H, J¼12.0, 7.3 Hz), 2.93 (dd, 1H,
J¼12.8, 4.9 Hz), 2.74 (d, 1H, J¼12.8 Hz), 2.42 (dd, 1H,
J¼17.5, 5.4 Hz), 2.20 (dd, 2H, J¼12.1, 7.0 Hz), 2.12 (dd,
1H, J¼17.2, 9.6 Hz), 1.91 (2s-ovrlp, 2£3H), 1.8–1.5 (m,
8H), 1.71 (s, 3H), 1.5–1.1.4 (m, 6H), 1.45 (s, 9H), 1.11 and
1.08 (2s, 2£3H). [C43H65BrN4O8SþHþ]: calcd 877.3785;
found 877.3791 and 879.2012 (HRMS) due to Br isotope
effect.
ovrlp with 2.31 (s, 3H), 2.11 (dd, 1H, J¼16.8, 8.9 Hz), 1.71
(m, 1H) ovrlp with 1.73 (s, 3H), 1.52 (t, 1H, J¼12.1 Hz),
1.10 (2s, 2£3H), 0.98 (t, 9H, J¼8.0 Hz), 0.62 (q, 6H,
J¼8.0 Hz). [C21H36O2SiþHþ]: calcd 349.26; found 349.3
(APCIþ).
4.3.4. 3-Triethylsiloxy-all trans-retinoic acid, ethyl ester
(13). Phosphonate 15 (459 mg, 422 mL, 1.74 mmol) was
dissolved in 5 mL of THF and mixture stirred at 08C for
10 min prior to the dropwise addition of n-BuLi (1 mL of
1 M in THF, 1.5 mmol). The reaction was stirred for 30 min
and then aldehyde 30 (263 mg, 0.754 mmol) in 5 mL THF
was added to the reaction mixture at 08C (under dim light
conditions). The mixture was stirred at room temperature
for 2 h and then quenched with NH4Cl and extracted with
Et2O, washed with brine, and dried over Na2SO4. The
product was purified by column chromatography using 2%
Et2O in hexanes affording a 4:1 mixture of 13E/13Z of 13
(230 mg of 13E, total mass of products: 305 mg, 88% yield).
1H NMR (500 MHz, CDCl3): d¼6.98 (dd, 1H, J¼15.0,
11.5 Hz), 6.29 (d, 1H, J¼15.1 Hz), 6.22 (d, 1H, J¼16.2 Hz).
6.1 (m, 2H), 5.78 (s, 1H), 4.17 (q, 2H, J¼7.1 Hz), 3.95 (m,
1H), 2.48 (s, 3H), 2.26 (dd, 1H, J¼16.8, 5.4 Hz), 2.10 (dd,
1H, J¼16.8, 9.4 Hz), 1.99 (s, 3H), 1.71 (s, 3H), 1.67 (ddd,
1H, J¼12.1, 3.3, 2.1 Hz), 1.51 (t, 1H, J¼11.9 Hz), 1.28 (t,
1H, J¼7.1 Hz), 1.08 and 1.07 (2s, 2£3H), 0.98 (t, 9H,
J¼8.0 Hz), 0.62 (q, 6H, J¼8.0 Hz). [C28H46O3SiþHþ]:
calcd 459.33; found 459.3 (APCIþ).
4.3. Synthesis of biotinylated retinoid 2
4.3.1. 3-Triethylsiloxy-b-ionone (22). Triethylchlorosilane
(1.5 mL, 9.0 mmol) was added to a solution of 3-hydroxy-b-
ionone (^)-9 (1.55 g, 7.4 mmol), Et3N (1.2 mL,
16.2 mmol), and DMAP (1.8 g, 14.8 mmol) in 15 mL of
CH2Cl2 at 08C. The reaction mixture was stirred for 1 h at
room temperature. The mixture was then quenched with
cold H2O and extracted with CH2Cl2, washed with cold 1%
HCl, brine, and then dried over Na2SO4. The product was
purified by column chromatography using 15–20% EtOAc
in hexanes as an eluent to afford 22 as an oil (2.33 g, 97%
yield). 1H NMR (400 MHz, CDCl3): d¼7.23 (d, 1H,
J¼16.4 Hz), 6.13 (d, 1H, J¼16.4 Hz), 3.97 (m, 1H), 2.33
(m, 1H) ovrlp with 2.32 (s, 3H), 2.17 (dd, 1H, J¼16.8,
9.4 Hz), 1.79 (s, 3H), 1.71 (ddd, 1H, J¼12.1, 3.3, 2.1 Hz),
1.53 (t, 1H, J¼11.9 Hz), 1.13 and 1.11 (2s, 2£3H), 1.00 (t,
9H, J¼8.0 Hz), 0.64 (q, 6H, J¼8.0 Hz). [C19H34O2SiþHþ]:
calcd 323.24; found 323.3 (ESIþ).
4.3.5. 3-Triethylsiloxy-all trans-retinol (4b). To a 5 mL
suspension of LiAlH4 (,10 mg, ,0.3 mmol) in Et2O at 08C
a solution of 13 (74 mg, 0.16 mmol) in 2 mL of Et2O was
added. The reaction mixture was stirred for 10 min and then
quenched with NH4Cl, extracted with EtOAc, and then
dried over Na2SO4. The resultant crude oil 4b (70 mg) was
not further purified and was directly used in the following
step. 1H NMR (500 MHz, C6D6): d¼6.65 (dd, 1H, J¼15.0,
11.2 Hz), 6.30 (2d, 2£1H, J¼15.1, 5.9 Hz), 6.21 (2d,
2£1H), 5.59 (t, 1H, J¼6.6 Hz), 4.13 (m, 1H), 4.00 (t, 2H,
J¼5.6 Hz), 2.40 (dd, 1H, J¼17.2, 5.3 Hz), 2.31 (dd, 1H,
17.2, 9.6 Hz), 1.9–1.7 (2dd, 2£1H), 1.88 (s, 3H), 1.73 (s,
3H), 1.62 (s, 3H), 1.17 and 1.14 (2s, 2£3H), 1.06 (t, 9H,
J¼7.9 Hz), 0.68 (q, 6H, J¼7.9 Hz), 0.55 (t, 1H, J¼5.6 Hz).
[C26H44O2SiþHþ]: calcd 417.3189; found 417.3193
(HRMS).
4.3.2. 5-(4-Hydroxy-2,6,6-trimethyl-cyclohex-1-enyl)-3-
methyl-penta-2,4-dienenitrile (29). In a 25 mL rb flask,
NaH (124 mg, 60% dispersion in mineral oil, 3.1 mmol) was
placed and washed with hexanes prior to suspension in dry
THF (5 mL) at 08C. Then phosphonate 14 (636 mg,
3.1 mmol) was added neat to the NaH and the reaction
allowed to stir for 30 min at room temperature. The reaction
mixture was cooled again to 08C prior to the addition of
ketone 22 (500 mg, 1.55 mmol) as a 1 mL solution in THF.
The reaction mixture was stirred for 2 h at room
temperature, then quenched with cold NH4Cl, and extracted
with Et2O. The combined organic extracts were washed
with brine and dried over Na2SO4 and product was purified
by column chromatography using 20% Et2O in hexanes
affording 29 as an oil (447 mg, 84% yield, 3.5:1 9E/9Z ).
This compound was characterized after the DIBAH
reduction described in Section 4.3.3.
4.3.6. 3-Triethylsiloxy-all trans-retinol chloroacetate
(5b). To a 2 mL solution of the crude 4b (70 mg,
,0.16 mmol) in CH2Cl2 at 2108C, and two beads of
˚
activated molecular sieves 4 A, was added EDC (86 mg,
0.45 mmol) and chloroacetic acid (18 mg, 0.19 mmol). The
mixture was stirred for 5 min prior to the addition of DMAP
(7.8 mg, 0.07 mmol) after which it was stirred for an
additional 5 min. The reaction mixture was immediately
washed with NaHCO3 and NH4Cl, dried over Na2SO4 and
then chromatographed rapidly using 10% EtOAc/hexanes
affording 5b as an oil. The material was not weighed and
4.3.3. 5-(4-Hydroxy-2,6,6-trimethyl-cyclohex-1-enyl)-3-
methyl-penta-2,4-dienal (30). To
a solution of 29
(447 mg, 1.3 mmol) in CH2Cl2 (5 mL) at 2608C was
added DIBAH (2 mL of 1 M, 2.0 mmol) and the reaction
stirred for 1 h prior to quenching with cold wet SiO2/H2O. The
residue was then suspended in 10% Et2O/hexanes and
loaded on a column for purification to afford 30 as an oil
(263 mg of 9E, total mass of products: 335 mg, 74% yield).
1H NMR (500 MHz, CDCl3): d¼10.13 (d, 1H, J¼8.2 Hz),
6.68 (d, 1H, J¼16.2 Hz), 6.21 (d, 1H, J¼16.2 Hz), 5.94 (d,
1H, J¼8.2 Hz), 3.94 (m, 1H), 2.29 (dd, 1H, J¼16.8, 5.4 Hz)
1
used directly in the following step. H NMR (500 MHz,
C6D6): d¼6.65 (dd, 1H, J¼15.1, 11.3 Hz), 6.3–6.1 (m, 4H),
5.49 (t, 1H, J¼7.3 Hz), 4.55 (d, 2H, 7.3 Hz), 4.12 (m, 1H),
3.40 (s, 2H), 2.39 (dd, 1H, J¼16.8, 5.6 Hz), 2.30 (dd, 1H,
J¼16.8, 9.4 Hz), 1.87 (ddd, 1H, J¼13.5, 1.8, 1.7 Hz), ovrlp
with 1.84 (s, 3H), 1.76 (s, 3H), ovrlp with 1.74 (t, 1H,

6584
N. Nesnas et al. / Tetrahedron 58 (2002) 6577–6584
J¼11.9 Hz), 1.62 (s, 3H), 1.16 and 1.14 (2s, 2£3H), 1.08 (t,
9H, J¼7.9 Hz), 0.68 (q, 6H, J¼7.9 Hz).
Acknowledgments
We are greatly appreciative of the generous donation made
by Hoffmann La Roche of a sample of (4R,6R )-4-hydroxy-
2,2,6-trimethylcyclohexanone 17. We are also pleased to
acknowledge the National Institute of Health for their
financial support.
4.3.7. 3-Hydroxy-all trans-retinol chloroacetate (6b). To
a 2 mL solution of 5b (the entire sample from the above
procedure) in THF at 08C, and two beads of activated
˚
molecular sieves 4 A, was added 100 mL of HF/pyridine.
The mixture was stirred for 5 min prior to concentration and
direct loading on a rapid pre-washed column eluted with
20–35% EtOAc/hexanes affording 6b as a coating on the
inside of a flask (16 mg, 27% yield over last three steps). 1H
NMR (500 MHz, C6D6): d¼6.64 (dd, 1H, J¼15.1, 11.3 Hz),
6.3–6.1 (m, 4H), 5.49 (t, 1H, 7.3 Hz), 4.55 (d, 2H, 7.3 Hz),
3.82 (m, 1H), 3.41 (s, 2H), 2.23 (dd, 1H, J¼16.8, 5.5 Hz),
1.99 (dd, 1H, J¼16.8, 9.4 Hz), 1.82 (s, 3H), 1.71 (s, 3H),
1.64 (ddd, 1H, J¼13.5, 1.8, 1.7 Hz), ovrlp with 1.62 (s, 3H),
1.44 (t, 1H, J¼11.9 Hz), 1.07 and 1.06 (2s, 2£3H).
[C22H31ClO3þHþ]: calcd 379.2040; found 379.2038
(HRMS).
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acetate (2). To a 2 mL solution of 6b (8 mg, 0.02 mmol) in
CH2Cl2 at 2108C, and five beads of activated molecular
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sieves 4 A, was added a mixture of Boc-Lys(biotinyl)-OH
(20 mg, 0.04 mmol) in 200 mL DMSO and EDC (12 mg,
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DMAP (1 mg, 0.008 mmol) was added and the reaction
mixture was stirred for 4 h which was sufficient for a
quantitative amount of product by TLC. The reaction
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CH2Cl2, dried over Na2SO4 and chromatographed rapidly
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1
inside of a vial (6.8 mg, 40% yield). H NMR (500 MHz,
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CDCl3): d¼6.66 (dd, 1H, J¼15.1, 11.3 Hz), 6.23 (d, 1H,
J¼15.1 Hz), 6.11 (d, 1H, J¼11.3 Hz), ovrlp with 6.10 (s,
2H), 6.03 (br t, 1H), 5.89 (br, 1H), 5.62 (t, 1H, J¼7.3 Hz),
5.17 (br, 2H), 5.09 (m, 1H), 4.85 (d, 2H, J¼7.3 Hz), 4.53
(dd, 1H, J¼7.2, 4.9 Hz), 4.34 (dd, 1H, J¼7.7, 4.8 Hz), 4.23
(br, 1H), 4.07 (s, 2H), 3.24 (br, 2H), 3.17 (dd, 1H, J¼11.9,
7.3 Hz), 2.93 (dd, 1H, J¼12.8, 4.9 Hz), 2.75 (d, 1H,
J¼12.8 Hz), 2.43 (dt, 1H, J¼16.4, 5 Hz), 2.20 (br t, 2H),
2.12 (dd, 1H, J¼17.2, 9.6 Hz), 1.96 (s, 3H), 1.91 (s, 3H),
1.9–1.7 (m, 8H), ovrlp with 1.71 (s, 3H), 1.7–1.2 (m, 6H),
ovrlap with 1.45 (s, 9H), 1.10 and 1.07 (2s, 2£3H).
[C43H65ClN4O8SþHþ]: calcd 833.4290; found 833.4281
and 835.3402 (HRMS) due to Cl isotope effect.
13. Chen, R.-L.; Liu, R. S. H. Tetrahedron 1996, 52, 7809–7816.
14. Broom, S. J.; Ede, R. M.; Wilkins, A. L. Tetrahedron Lett.
1992, 33, 3197–3200.
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Helv. Chim. Acta 1976, 59, 1832–1849.
16. Yamano, Y.; Ito, M. J. Chem. Soc., Perkin Trans. 1 1993,
1599–1610.
17. Yamano, Y.; Tode, C.; Ito, M. J. Chem. Soc., Perkin Trans. 1
1998, 2569–2582.
18. Gibson, A. W.; Humphrey, G. R.; Kennedy, D. J.; Wright, S. H.
B. Synthesis 1991, 414–416.