Synthesis of an 11-cis-locked biotinylated retinoid for sequestering 11-cis-retinoid binding proteins

Article abstract of DOI:10.1139/V06-092

The synthesis of a seven-membered ring locked analogue of 11-cis-retinol, tethered to a cross-linking moiety on C-15 and a lysine-extended biotin on the C-3, was accomplished for its utilization as a probe to fish out retinol binding proteins that may be involved in the reisomerization of retinal from all-trans to 11-cis in the visual cycle.

Full text of DOI:10.1139/V06-092

1363
Synthesis of an 11-cis-locked biotinylated retinoid
for sequestering 11-cis-retinoid binding proteins1
Hiroko Matsuda, Shenglong Zhang, Andrea E. Holmes, Sonja Krane,
Yasuhiro Itagaki, Koji Nakanishi, and Nasri Nesnas
Abstract: The synthesis of a seven-membered ring locked analogue of 11-cis-retinol, tethered to a cross-linking moiety
on C-15 and a lysine-extended biotin on the C-3, was accomplished for its utilization as a probe to fish out retinol
binding proteins that may be involved in the reisomerization of retinal from all-trans to 11-cis in the visual cycle.
Key words: retinoids, biotin, 11-cis-locked, cross-linking, retinol binding proteins.
Résumé : La synthèse d’un analogue de 11-cis-rétinol sous la forme d’un anneau verrouillé, comportant sept membres,
attachés en partie, au C-15, avec un groupe ayant la possibilité de se liér aux protéines a été réalisée. En plus,
l’analogue est attaché de l’autre côté sur le C-3, avec une lysine biotine étendue. Cette synthèse a été réalisée afin
d’utilizer le composé comme agent détecteur des protéines qui portent des rétinols et qui pourraient être impliquées
dans la réisomerization du all-trans retinal au 11-cis dans le cycle de vision.
Mots clés : rétinoides, biotine, 11-cis-verrouillé, liés, protéines qui portent des rétinols.
Matsuda et al. 1370
Introduction
able their isolation and purification for further studies of
their detailed function.
We have previously synthesized (6) a biotin-linked 11-cis-
retinol analogue (structure 1 without the trimethylene bridge
linking C-10 and C-13), as well as the all-trans-retinol ana-
logue, 1a. The latter derivative, bearing a biotin moiety and
a chloroacetate cross-linking unit, enabled us to successfully
isolate and characterize certain retinol binding proteins (7).
We attempted to use the biotin-linked 11-cis analogue in the
isolation of RBPs involved in the isomerization of all-trans-
retinal to 11-cis-retinal. Despite the synthetic accomplish-
ment (6), the retinoid was prone to undergoing an inevitable
isomerization from 11-cis to all-trans upon incubation in the
retinal pigment epithelial (RPE) cells, thus losing its affinity
toward the RBPs of interest. To circumvent this problem, we
resorted to ret7 (Fig. 1), the first of the 11-cis-locked
analogues, which is comprised of a seven-membered ring
locking the double bond at the C-11 position into a cis-con-
formation (8). This retinoid was particularly useful because
it retains the full chromophore properties of native 11-cis-
retinal and readily forms, with bovine opsin, the
nonbleachable pigment, rhodopsin 7 or rh7. Both the UV–
vis λmax of 490 nm and the CD maxima at 330 and 488 nm
The basic process of vision, first elucidated by Wald (1),
involves the light-triggered isomerization of retinal (or vita-
min A aldehyde) from its 11-cis conformation to the all-
trans conformation. Retinal, in its 11-cis geometry, is bound
by the apoprotein opsin, a G-protein coupled receptor, form-
ing a protonated Schiff base with Lys296, present in Helix G
of the seven-membered transmembrane protein,which gener-
ates rhodopsin, the active photoreceptor unit in the cones
and rods of eyes (2, 3). Upon absorption of a photon by 11-
cis-retinal, the retinoid undergoes an isomerization to the all-
trans geometry, resulting in a conformational change in the
protein that leads to the activation of the G protein, which
ultimately results in vision (4). The all-trans-retinal is subse-
quently hydrolyzed off the lysine residue, freeing the protein
for a fresh 11-cis-retinal molecule. The completion of the
visual cycle is ultimately dependent on the less well-
understood proteins that are capable of binding all trans-
retinoids and converting them to the 11-cis-retinal geometry
(5). The latter proteins are collectively termed retinol bind-
ing proteins (RBPs). Their crucial involvement in the visual
cycle warrants the need for the development of tools that en-
Received 31 October 2005. Accepted 24 January 2006. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on
27 September 2006.
This article is part of a Special Issue dedicated to Dr. Alfred Bader in celebration of his 80th birthday.
H. Matsuda, S. Zhang, A.E. Holmes, S. Krane, Y. Itagaki, K. Nakanishi,2 and N. Nesnas.3,4 Department of Chemistry,
Columbia University, New York, NY 10027, USA.
1This article is part of a Special Issue dedicated to Dr. Alfred Bader.
2Corresponding author (e-mail: kn5@columbia.edu).
3Corresponding author (e-mail: nesnas@fit.edu).
4Present address: Department of Chemistry, Florida Institute of Technology, Melbourne, FL 32901, USA.
Can. J. Chem. 84: 1363–1370 (2006)
doi:10.1139/V06-092
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Can. J. Chem. Vol. 84, 2006
Scheme 1. Retrosynthetic analysis for the biotinlylated retinoid 1.
O
NH
HN
S
O
NH
Wittig reaction
O
11
11
9
BocHN
O
15
Cl
O
HWE reaction
9
10
13
13
10
14
14
3
O
O
1a
1
Br
15
O
O
P
11
9
OEt
OEt
PPh3Br
OHC
14
3
RO
OTBS
CN
15
4
2
3
ence of the seven-membered ring would not prevent the
retinoid from binding into the protein (8, 12, 13).
Fig. 1. The structures of ret7 and the native 11-cis-retinal.
11
11
Results and discussion
The retrosynthetic logic for 1 is shown in Scheme 1. It
stems from our earlier work conducted on the seven-
membered ring unit 3, synthesized from 2-cyclohepten-1-
one in five steps (14). The hydroxylated β-ionone ring
structure can be introduced by employing a Wittig reaction
between aldehyde 3 and synthon 2, the latter being prepared
from dehydro-β-cyclocitral (also known as safranal) in seven
steps as outlined in Scheme 2. The Horner–Wadsworth–
Emmons (HWE) reaction can be used to install a nitrile
group at the C-14 position with reagent 4, followed by its re-
duction to the corresponding aldehyde, which would be sub-
jected to further reduction to the corresponding primary
alcohol onto which a bromoester can be esterified in the fi-
nal stages.
15
15
O
O
ret7
11-cis-retinal
(digitonin–phosphate buffer, pH 7) are similar to those of
the native chromophore (UV–vis λmax at 500 nm and CD
maxima at 340 and 490 nm), indicating that the binding sites
are the same. These aspects have made this pigment a popu-
lar substrate for various spectroscopic measurements (9–11).
We hereby report the synthesis of 11-cis-locked retinoid 1
bearing an appropriate functionality for isolation through
cross-linking and which possesses the additional advantage
of resisting isomerization under light conditions as well as in
the media of RPE cells.
Synthon 3 has been prepared from the commercially avail-
able 2-cyclohepten-1-one, according to our earlier report
(14). The synthesis featured an allylic oxidation of 2-cyclo-
hepten-1-one, a three-carbon homologation by HWE reac-
tion with the reagent bearing the nitrile functionality, and
lastly, a partial reduction of the nitrile to the corresponding
aldehyde.
The synthesis of 10 (the synthon 2 unit) is outlined in
Scheme 2. Dehydro-β-cyclocitrol (5), prepared from
dehydro-β-cyclocitral (15), was protected as the tetrahy-
dropyranyl (THP) ether (→6, 81%) and subsequently treated
The target structure 1 has significant features that merit
highlighting. It has the basic 11-cis-retinoid structure that
can bind selectively to certain RBPs involved in the reiso-
merization of all-trans- to 11-cis-retinal. It also harbors an
active cross-linking unit, the bromoacetate functionality,
which can form a stable covalent bond with the RBPs and it
also has the (Boc)Lys-biotinyl linker anchored at C-3, which
will facilitate isolation of the covalently linked protein via
an avidin affinity chromatography. Moreover, the ret7 moi-
ety in the structure ensures its stability against light and (or)
the RPE cell medium. Based on our earlier studies, the pres-
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Matsuda et al.
1365
Scheme 2. Reagents and conditions: (a) DHP, p-TsOH, CH2Cl2,
0 °C (81%); (b) 9-BBN, THF, ∆, then MeOH, NaOH, H2O2,
0 °C → 50 °C (55%); (c) (i) Ac2O, pyridine, CH2Cl2, rt, (ii) p-
TsOH, MeOH, rt (60%, two steps); (d) PPh3, CBr4, THF (94%);
(e) PPh3, benzene, ∆ (quant.).]
as a TBS ether to afford 15. Basic cleavage of the 3-acetyl
group gave the corresponding 3-OH followed by
esterification with biotinyl-(Boc)Lys-OH to give 16 (78%
yield, two steps). The TBS group was deprotected, releasing
the primary alcohol, which was eventually subjected to
esterification with bromoacetic acid to afford the target
structure 1 in 66% yield.
Preliminary studies directed towards isolation and charac-
terization of RBPs employing 1 performed by Rando’s group
(Harvard Medical School, Boston, Massachusetts) have
shown that some RBPs have been sequestered and identified.
The results of the isolation and characterization of the RBPs
using 1 will be reported in a subsequent manuscript.
OTHP
6
Conclusion
The synthesis of biotinylated retinoid 1 was accomplished
via a series of Wittig reactions, esterification of a (Boc)Lys-
biotin on the 3-hydroxy position of the β-ionone ring, and
the final attachment of the active bromoester cross-linking
unit. The significance of the seven-membered ring lock is
evident as the preliminary biological studies using this mole-
cule have already proven the robust nature of the 11-cis-ole-
fin unit. The target structure will be able to cross-link to
RBPs that are involved in the crucial isomerization process
that regenerates 11-cis-retinal. Avidin affinity chromatogra-
phy will enable facile purification of the sequestered protein,
and basic hydrolysis will release the isolated protein from
the affinity column by cleaving the ester at the C-15 posi-
tion. The purified protein can be further studied and charac-
terized for the elucidation of its detailed function.
9
10
with 9-borabicyclo[3.3.1]nonane (9-BBN) to undergo a
hydroboration reaction affording the 3-hydroxy 7 in 55%
yield. As in the case of the biotinylated all-trans analogue 1a
(6, 7), no attempts were made to resolve the C-3 enantio-
mers. The 3-hydroxy group was protected as the acetate by
treatment with acetic anhydride (Ac2O) – pyridine. The al-
lylic hydroxy group was then liberated by acidic cleavage of
the THP group to give alcohol 8, which was transformed to
the corresponding bromide 9 using triphenylphosphine
(PPh3) and carbon tetrabromide (94% yield). The phos-
phonium salt 10 was synthesized from 9 by treatment with
PPh3 under reflux conditions.
Scheme 3 shows the critical steps that assemble the
retinoid from synthons 3 and 10. A Wittig reaction between
aldehyde 3 and phosphonium salt 10 afforded the desired
tetraene 11a (33%), along with a considerable amount of
11b (40%). The trans geometry of the olefin thus formed
was confirmed from the coupling constant (J = 16 Hz) ob-
served for the H-7 and H-8 protons. The TBS group of 11a
was removed using tetrabutylammonium fluoride (TBAF) to
give the free allylic alcohol, which, upon treatment with
MnO2, underwent smooth oxidation to afford the conjugated
ketone 12 in 84% yield. A two-carbon extension through
HWE reaction was carried out on ketone 12 with cyano-
methylphoshonate (4) to afford the (E)-isomer 13 in 27%
yield (the (Z)-isomer, 53%). The E/Z geometry was deter-
mined by NOE experiments for the H-12 and H-14 protons.
Nitrile 13 was subjected to partial reduction with diisobutyl-
aluminum hydride (DIBAH) to the corresponding aldehyde
accompanied by an inevitable reductive cleavage of the ace-
tate group at the C-3 position. After reprotection of the free
alcohol with an acetyl group, resulting in 14, the aldehyde
was further reduced to the corresponding primary alcohol by
treatment with sodium borohydride and was then protected
Experimental section
General considerations
1H NMR spectra were measured on a Bruker DMX 400 or
Bruker DPX 300 spectrometer (400 or 300 MHz). The
chemical shifts are expressed in ppm downfield from the
signal of tetramethylsilane, used as an internal standard in
the solvent CDCl3. Splitting patterns are designated as s
(singlet), d (doublet), t (triplet), q (quartet), qn (quintet),
m (multiplet), and br (broad). High resolution mass spec-
trometry (HRMS) were measured on a JEOL (Tokyo) JMS-
HX110/110 tandem mass spectrometer equipped with a Xe
beam FAB gun (6 kV), using 3-nitrobenzyl alcohol as a ma-
trix. Analytical and preparative thin layer chromatography
(TLC) was carried out using precoated silica gel plates
(Merck silica gel 60F254). The silica gel used for column
chromatography was 230–400 mesh (Merck). All reactions
were carried out under an Ar atmosphere using predried sol-
vents. Anhydrous CH2Cl2, Et2O, and THF were obtained
from the solvent purification system via passage through an
activated alumina cartridge.
Tetrahydropyranyl safranol (6)
To a solution of safranol 5 (7.67 g, 50.4 mmol) in CH2Cl2
(100 mL) was added 3,4-dihydro-2H-pyran (DHP, 8.48 g,
101 mmol). The mixture was stirred and cooled to –10 °C
and p-toluenesulfonic acid monohydrate (p-TsOH, 190 mg,
1.00 mmol) was then added. After 10 min of stirring,
triethylamine (1.0 mL) was added to quench the reaction.
© 2006 NRC Canada

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Can. J. Chem. Vol. 84, 2006
Scheme 3. Reagents and conditions: (a) n-BuLi, THF, –78 °C → rt, (for 11a, 33%; for 11b, 40%); (b) Ac2O, pyridine, DMAP, rt for 11b
(63%); (c) (i) TBAF, THF, 0 °C → rt (99%), (ii) MnO2, CH2Cl2, 0 °C → rt (84%); (d) 4, NaH, THF, 0 °C → rt (E:Z, 27%:53%);
(e) (i) DIBAH, Et2O, –78 °C → 0 °C (82%), (ii) Ac2O, pyridine, CH2Cl2, 0 °C → rt (50%); (f) (i) NaBH4, MeOH–CHCl3 (1:10), 0 °C,
(ii) TBSCl, imidazole, CH2Cl2, 0 °C (70%, two steps); (g) (i) K2CO3, MeOH, 0 °C → rt, (ii) biotinyl (Boc)lys-OH (B-OH), EDC, DMAP,
CH2Cl2, 0 °C → rt (78%, two steps); (h) (i) TBAF, THF, 0 °C → rt (60%), (ii) BrCH2COOH, EDC, DMAP, 0 °C → rt (66%).
a
c
10 +
3
OR'
11a: R = Ac, R' = TBS
11b: R = H, R' = TBS
RO
b
e
d
O
AcO
AcO
CN
12
13
f
AcO
AcO
CHO
OTBS
14
15
g
h
1
BO
OTBS
16
H
N
O
NHBoc
O
B =
N
H
NH
S
O
The mixture was poured into water (150 mL) and extracted
with Et2O (3 × 150 mL). The organic layers were washed
with brine, combined, dried over MgSO4, and concentrated
under reduced pressure. Purification of the residue by silica
gel column chromatography (EtOAc–hexanes, 10:90) af-
forded the THP ether 6 (9.60 g, 40.8 mmol, 81%) as a color-
less oil. 1H NMR (CDCl3, ppm) δ: 5.75 (2H), 4.61 (1H, brs),
4.31 (1H, d, J = 11.2 Hz), 4.07 (1H, d, J = 11.2 Hz), 3.91
(1H, m), 3.52 (1H, m), 2.05 (2H, s), 1.81 (3H, s), 1.81–1.69
(6H), 1.04, 1.01 (each 3H, s). 13C NMR (CDCl3, ppm) δ:
134, 129.9, 129.1, 126, 97.7, 63.4, 62.4, 40.3, 33.5, 31.0,
26.7 (2C), 25.9, 19.9, 18.4. FAB-HRMS calcd. for C15H24O2
m/z: 236.1776 [M+]; found: 236.1769.
Tetrahydropyranyl 3-hydroxysafranol (7)
To a solution of THP ether 6 (6.66 g, 28.2 mmol) in THF
(40.0 mL), 9-BBN (0.5 mol/L solution in THF, 113 mL,
56.4 mmol) was added slowly over 20 min. The reaction
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Matsuda et al.
1367
mixture was heated up to 60 °C and was stirred for 4 h. Af-
ter the reaction mixture was cooled to room temperature,
methanol (68.0 mL) and an aq. NaOH solution (3 mol/L,
9.4 mL) were added to it. Subsequently, H2O2 (30% solu-
tion, 8.0 mL) was added slowly to the stirring reaction mix-
ture using an ice bath. The reaction mixture was heated for
2 h at 55 °C. The mixture was poured into water (100 mL)
and extracted with EtOAc (3 × 100 mL). The organic layers
were washed with brine, combined, dried over MgSO4, and
concentrated in vacuo. Purification of the residue by silica
gel column chromatography (EtOAc–hexanes, 30:70) af-
forded a diastereomeric mixture of 7 (3.94 g, 15.5 mmol,
overlapping with 1.75 (3H, s), 1.58 (1H, t, J = 9.5 Hz), 1.20,
1.12 (each 3H, s). 13C NMR (CDCl3, ppm) δ: 169, 133.4,
144.2, 67.2, 43.5, 38.4, 36.7, 29.0, 28.6, 28.3, 21.1, 19.6.
FAB-HRMS calcd. for C12H18 79BrO2 m/z: 273.0490 [M –
H]+; found: 273.0497.
3-O-Acetoxysafranyl triphenylphosphonium bromide
(10)
To a solution of allyl bromide 9 (710 mg, 2.55 mmol) in
benzene (20.0 mL) at room temperature, PPh3 (670 mg,
2.55 mmol) was added. The resultant mixture was heated up
to the reflux temperature (~85 °C bath) for 12 h. After the
mixture was cooled to room temperature, the volatiles were
evaporated in vacuo. The residue was washed with benzene
(×3) and dried under the reduced pressure for 1 day to give
10 (1.37 g, 2.55 mmol, quantitative yield). 1H NMR (CDCl3,
ppm) δ: 7.9–7.6 (15H), 4.97 (1H, m), 4.58, 4.42 (each 1H, t,
J = 14.0 Hz), 2.26 (1H, m), 2.04 (3H, s), 1.95 (1H, m), 1.71
(1H, dd, J = 2.0, 12.0 Hz), 1.51 (1H, t, J = 12.0 Hz), 1.07
(3H, d, J = 4.0 Hz), 0.88, 0.84 (each 3H, s). FAB-HRMS
calcd. for C30H34O2P m/z: 457.2291 [M – Br]+; found:
457.2285.
1
55%). The H NMR spectra indicated that the sample con-
sisted of a mixture of diastereomers (50:50). 1H NMR
(CDCl3, ppm) δ: 4.55 (2 × 0.5H, dt, J = 3.2, 8.5 Hz), 4.26,
4.17, 3.80, 3.74 (each 0.5H, d, J = 10.7 Hz), 3.93–3.81
(0.5H × 2 × 2), 3.50 (0.5H × 2, m), 2.37 (0.5H × 2, brs),
2.27 (0.5H × 2, brdt), 2.01 (0.5H × 2, brdd), 1.78 (0.5H ×
2, m), 1.69, 1.68 (each 1.5H, s), 1.70–1.62 (0.5H × 2 × 2),
1.56–1.40 (0.5H × 2 × 5), 1.06, 1.02, 1.00, 0.993 (each
1.5H, s). 13C NMR (CDCl3, ppm) δ: 134, 133, 131.5, 131.4,
98.9, 98.6, 64.9 (0.5C × 2), 63.5, 63.4, 62.4, 62.3, 48.2
(0.5C × 2), 42.3 (0.5C × 2), 36.8 (0.5C × 2), 30.7 (0.5C ×
2), 29.4, 29.2, 28.7, 28.6, 25.5 (0.5C × 2), 19.7 (0.5C × 2),
19.69, 19.63. FAB-HRMS calcd. for C15H26O3 m/z:
254.1882 [M+]; found: 254.1891.
3-O-Acetyl-13-O-TBS (11a)
To a solution of the triphenylphosphonium bromide 10
(657 mg, 1.22 mmol) in THF (7.0 mL) at –78 °C, n-BuLi
(2.5 mol/L solution in hexanes, 1.4 mL, 3.50 mmol) was
added, and the resultant mixture was allowed to warm to
room temperature and was stirred at this temperature for 1 h
to generate the corresponding ylide. To a solution of alde-
hyde 3 (460 mg, 1.64 mmol) in THF (10.0 mL), the ylide
solution was added. After 14 h of stirring at the same tem-
perature, the mixture was poured into water and extracted
with Et2O (3 × 30 mL). The organic layers were washed
with brine, combined, dried over MgSO4, and concentrated
in vacuo. Purification of the residue by silica gel column
chromatography (EtOAc–hexanes, 5:95 and 20:80) afforded
the acetyl 11a (245 mg, 534 µmol, 33%) and alcohol 11b
(276 mg, 663 µmol, 40%) as a colorless oil. The ratio of
diastereomers was not determined and this sample was used
as a mixture.
3-O-Acetoxysafranol (8)
To a solution of hydroxy 7 (1.00 g, 3.93 mmol) in
pyridine (7.0 mL), acetic anhydride (3.5 mL) was added at
0 °C. The resultant mixture was stirred at room temperature.
After 12 h of stirring, the mixture was azeotropically con-
centrated with toluene (×3) in vacuo. The crude sample was
subjected to the next step without purification. The acetate
was treated with a catalytic amount of p-TsOH (10.0 mg) in
methanol (60.0 mL). The resultant mixture was allowed to
stir for 12 h at room temperature. The mixture was neutral-
ized with Et3N (1.0 mL) and concentrated under reduced
pressure. Silica gel purification of the residue (EtOAc–hex-
anes, 25:75) gave the 3-O-acetyl safranol 8 (576 mg,
1
2.72 mmol, 70%, in two steps). H NMR (CDCl3, ppm) δ:
4.97 (1H, m), 4.13, 4.07 (each 1H, d, J = 11.5 Hz), 2.35
(1H, dd, J = 5.7, 16.9 Hz), 2.04 (1H, dd, J = 9.4, 16.9 Hz),
2.00 (3H, s), 1.71 (1H, m) overlapping with 1.72 (3H, s),
1.52 (1H, t, J = 11.9 Hz), 1.45 (1H, br, CH2OH), 1.09, 1.05
(each 3H, s). 13C NMR (CDCl3, ppm) δ: 170, 137, 127, 68.2,
58.2, 43.8, 38.2, 36.4, 29.2, 28.5, 21.5, 19.5.
To a solution of 3-hydroxy 11b (294 mg, 706 µmol) in
pyridine (3.0 mL) at room temperature, Ac2O (1.5 mL) and
N,N-dimethylaminopyridine (DMAP, 5.0 mg) were added,
and the resultant mixture was stirred at the same temperature
for 12 h. The mixture was poured into a satd. aq. solution of
NaHCO3 (30 mL) and extracted with Et2O (3 × 30 mL). The
organic layers were washed with brine, combined, dried over
MgSO4, and concentrated in vacuo. Purification of the resi-
due by silica gel column chromatography (EtOAc–hexanes,
2:98) provided the acetyl 11a (205 mg, 446 µmol, 63%) as a
colorless oil. The ratio of diastereomers was not determined,
3-O-Acetoxysafranyl bromide (9)
To a solution of alcohol 8 (576 mg, 2.72 mmol) in THF
(20.0 mL) at 0 °C, PPh3 (1.07 g, 4.08 mmol) and CBr4
(1.35 g, 4.08 mmol) were added. The resultant mixture was
stirred at room temperature for 1 h. The mixture was poured
into water (50 mL) and extracted with Et2O (3 × 50 mL).
The organic layers were washed with brine, combined, dried
over MgSO4, and concentrated in vacuo. The residue was
purified with silica gel chromatography (EtOAc–hexanes,
2:98) to afford the allyl bromide 9 (710 mg, 2.55 mmol,
1
and this sample was used as a diastereomeric mixture. H
NMR for 11a (CDCl3, ppm, major signals are only de-
scribed.) δ: 6.49, 6.09 (each 1H, dd, J = 1.8, 16.0 Hz), 6.45,
5.69 (each 1H, dd, J = 2.2, 11.8 Hz), 5.06 (1H, m), 4.46
(1H, m), 2.67 (1H, dt, J = 5.0, 13.7 Hz), 2.44 (1H, dd, J =
5.8, 17.0 Hz), 2.23 (1H, m), 2.10 (1H, dd, J = 9.4, 16.6 Hz),
2.04 (3H, s), 1.87 (3H, s), 1.83–1.75 (2H), 1.72 (3H, s), 1.8–
1.7 (2H), 1.58 (2H, t, J = 12.0 Hz), 1.10, 1.07 (each 3H, s),
0.906 (3H × 3, s), 0.090, 0.081 (each 3H, s). 13C NMR
1
94%). H NMR (CDCl3, ppm) δ: 4.99 (1H, m), 4.07, 3.96
(each 1H, d, J = 8.2 Hz), 2.43 (1H, dd, J = 4.5, 13.8 Hz),
2.11 (1H, dd, J = 7.4, 13.8 Hz), 2.02 (3H, s), 1.72 (1H, m)
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Can. J. Chem. Vol. 84, 2006
(CDCl3, ppm) δ: 170, 138, 137, 134, 132, 130, 129, 125.8,
125.0, 71.0, 68.4, 43.9, 38.3, 36.6, 35.6, 30.0, 29.8. 28.4,
25.8 (3C), 23.8, 21.4 (2C), 18.2, 14.3, –4.6 (2C). FAB-
HRMS calcd for C28H46O3Si m/z: 458.3216 [M+]; found:
was allowed to warm to room temperature and was stirred at
this temperature for 1 h to generate the corresponding ylide.
To a solution of ketone 12 (174 mg, 507 µmol) in THF
(25.0 mL), the generated ylide was added. After 12 h of stir-
ring at the same temperature, the mixture was poured into
water (30 mL) and extracted with Et2O (3 × 30 mL). The or-
ganic layers were washed with brine, combined, dried over
MgSO4, and concentrated in vacuo. Purification of the resi-
due by silica gel column chromatography (EtOAc–hexanes,
7:93) afforded the (E)-nitrile 13 (47.1 mg, 135 µmol, 27%)
and the (Z)-nitrile (99.3 mg, 265 µmol, 53%) as yellowish
1
458.3196. H NMR for 11b (CDCl3, ppm, major signals are
only described.) δ: 6.11, 6.49 (each 1H, d, J = 16.0 Hz),
5.69, 6.46 (each 1H, d, J = 11.9 Hz), 4.46 (1H, m), 4.01
(1H, m), 2.68 (1H, dt, J = 5.4, 8.8 Hz), 2.38 (1H, dd, J =
5.4, 16.6 Hz), 2.24 (1H, m), 2.04 (1H, m), 1.86 (3H, s),
1.71–1.85 (4H) overlapping with 1.73 (3H, s), 1.65 (1H,
brs), 1.48 (1H, t, J = 11.9 Hz), 1.07 (3H × 2, s), 0.907 (3H ×
3, s), 0.0815, 0.0914 (each 3H, s). 13C NMR (CDCl3, ppm)
δ: 138, 136, 134, 132, 130, 130, 125.67, 125.64, 71.0, 64.9,
48.2, 42.4, 37.0, 35.6, 30.2, 29.8, 28.6, 25.8 (3C), 23.8, 21.5,
18.2, 14.4, –4.6 (2C). FAB-MS m/z (%, rel. int.): 416 (52,
M+), 398 (25, [M – H2O]+). FAB-HRMS calcd. for
C26H44O2Si m/z: 416.3111 [M+]; found: 416.3106.
1
oils, respectively. H NMR for (E)-isomer 13 (CDCl3, ppm)
δ: 6.82, 6.13 (each 1H, d, J = 11.3 Hz), 6.52, 6.25 (each 1H,
d, J = 15.9 Hz), 5.15 (1H, s), 5.04 (1H, m), 2.67 (2H, t, J =
6.7 Hz), 2.52 (2H, t, J = 6.7 Hz), 2.46 (1H, dd, J = 5.9,
17.2 Hz), 2.12 (1H, dd, J = 9.5, 17.2 Hz), 2.05, 1.96 (each
3H, s), 1.87 (2H, qn, J = 6.7 Hz), 1.79 (1H, m), 1.73 (3H, s),
1.59 (1H, t, J = 12.2 Hz), 1.11, 1.08 (each 3H, s). 13C NMR
(CDCl3, ppm) δ: 170, 162, 139, 138, 134 (2C), 132, 129,
128, 126, 117, 96.3, 68.2, 43.9, 38.4, 36.6, 33.0, 31.3, 30.0,
28.5, 26.9, 21.5, 21.4, 14.9. FAB–HRMS calcd. for
13-Ketone (12)
To a solution of 11a (205 mg, 446 mmol) in THF
(10.0 mL) at 0 °C, tetrabutylammonium fluoride (TBAF,
1.0 mol/L solution in THF, 892 µL, 892 µmol) was added,
and the resultant mixture was allowed to warm to room tem-
perature and was continuously stirred at this temperature.
After 3 h of stirring, the mixture was poured into water
(30 mL) and extracted with Et2O (3 × 30 mL). The organic
layers were washed with brine, combined, dried over
MgSO4, and concentrated under reduced pressure. Purifica-
tion of the residue by silica gel column chromatography
(EtOAc–hexanes, 15:85) afforded the 13-hydroxy compound
(145 mg, 425 µmol, 99%) as a colorless oil. 1H NMR
(CDCl3, ppm) δ: 6.51, 5.73 (each 1H, d, J = 11.8 Hz), 6.47,
6.10 (each 1H, d, J = 15.8 Hz), 5.05 (1H, m), 4.47 (1H, m),
2.70 (1H, m), 2.44 (1H, dd, J = 5.4, 6.8 Hz), 2.23 (1H, m),
2.12 (1H, m), 2.05 (3H, s), 2.00–1.89 (2H), 1.88 (3H, s),
1.81–1.68 (2H, m), 1.71 (3H, s), 1.58 (2H, t, J = 11.9 Hz),
1.10, 1.07 (each 3H, s).
1
C24H31NO2 m/z: 365.2355 [M+]; found: 365.2354. H NMR
for the (Z)-isomer (CDCl3, ppm) δ: 6.94, 6.57 (each 1H, d,
J = 11.4 Hz), 6.50, 6.25 (each 1H, d, J = 16.0 Hz), 5.07
(1H, s), 5.04 (1H, m), 2.51 (2H, t, J = 6.7 Hz), 2.46 (1H, m)
2.42 (2H, t, J = 6.7 Hz), 2.11 (1H, dd, J = 9.5, 17.0 Hz),
2.04, 1.97 (each 3H, s), 1.83 (2H, qn, J = 6.7 Hz), 1.72
(3H, s), 1.59 (2H, t, J = 11.9 Hz), 1.11, 1.08 (each 3H, s).
14-Aldehyde (14)
To a solution of the (E)-nitrile 13 (35.7 mg, 97.7 µmol) in
Et2O (3.0 mL) at –78 °C, DIBAH (1.0 mol/L solution in to-
luene, 900 µL, 900 µmol) was added, and the resultant mix-
ture was stirred at 0 °C. After 40 min of stirring, methanol
(0.63 mL) was carefully added, followed by a 30% aqueous
solution of Rochelle salt (potassium sodium tartrate)
(1.08 mL), the resultant mixture was then stirred for 40 min.
The organic layer was washed with a 30% Rochelle salt
aqueous solution (2 × 0.45 mL and 1 × 0.23 mL). The com-
bined aqueous layer was back-extracted with Et2O (3 ×
30 mL). The organic layers were washed with brine, com-
bined, dried over MgSO4, and concentrated under reduced
pressure. Purification of the residue by silica gel column
chromatography (Et2O–hexanes, 50:50) afforded the alde-
hyde without an acetyl group at C-3 (26.1 mg, 80.1 µmol,
To a solution of the 13-hydroxy (209 mg, 607 µmol) in
CH2Cl2 (15.0 mL) at 0 °C, MnO2 (1.60 g, 18.2 mmol) was
added, and the resultant mixture was stirred at this tempera-
ture. After 40 min of stirring at the same temperature, the
mixture was filtered and the volatiles were removed under
reduced pressure. Purification of the residue by silica gel
column chromatography (EtOAc–hexanes, 20:80) afforded
ketone 12 (174 mg, 507 µmol, 84%) as a colorless oil.
1H NMR (CDCl3, ppm) δ: 7.32, 5.92 (each 1H, d, J =
12.0 Hz), 6.55, 6.32 (each 1H, d, J = 18.0 Hz), 5.03 (1H, m),
2.63 (2H, t, J = 6.6 Hz), 2.57 (2H, t, J = 6.6 Hz), 2.45 (1H,
dd, J = 5.4, 16.9 Hz), 2.11 (1H, dd, J = 9.6, 16.9 Hz), 2.04,
2.02 (each 3H, s), 1.87 (2H, qn, J = 6.6 Hz), 1.79 (1H, brt),
1.72 (3H, s), 1.58 (1H, t, J = 12.0 Hz), 1.10, 1.07 (each
3H, s). 13C NMR (CDCl3, ppm) δ: 203, 170, 143, 137.9,
137.0, 134, 132, 130, 129, 126, 68.1, 43.8, 42.9, 38.3, 36.5,
31.2, 29.9, 28.4, 23.1, 21.4, 21.3, 15.0. FAB–HRMS calcd.
1
82%) as an oil. H NMR (CDCl3, ppm) δ: 10.0 (1H, d, J =
8.2 Hz), 6.90, 6.24 (each 1H, d, J = 11.3 Hz), 6.53, 6.27
(each 1H, d, J = 15.9 Hz), 5.93 (1H, d, J = 8.2 Hz), 3.97
(1H, m), 2.85 (2H, t, J = 6.7 Hz), 2.56 (2H, t, J = 6.7 Hz),
2.40 (1H, dd, J = 6.2, 17.0 Hz), 2.05 (1H, dd, J = 10.0,
17.0 Hz), 1.98 (3H, s), 1.89 (2H, qn, J = 6.7 Hz), 1.83–1.74
(1H, m), 1.74 (3H, s), 1.49 (1H, t, J = 11.8 Hz), 1.08 (3H ×
2, s).
To a solution of the aldehyde (26.1 mg, 80.0 µmol) in a
mixture of CH2Cl2 (1.0 mL) and pyridine (0.2 mL) at 0 °C,
Ac2O (0.1 mL) and DMAP (2.0 mg) were added, and the re-
sultant mixture was allowed to warm to room temperature
and was stirred at this temperature. After 1 h of stirring, the
volatiles were removed in vacuo. Purification of the residue
by silica gel column chromatography (Et2O–hexanes, 15:85)
afforded the alcohol (14.5 mg, 39.3 µmol, 50%) as an oil.
+
for C22H31O3 m/z: 343.2273 [M + H]+; found: 343.2266.
13-Nitrilemethylene (13)
To a solution of NaH (freshly prepared by washing with
hexanes and drying under reduced pressure, 140 mg,
5.84 mmol) in THF (15.0 mL) at 0 °C, (EtO)2P(O)CH2CN
(4, 944 µL, 5.84 mmol) was added, and the resultant mixture
© 2006 NRC Canada

Matsuda et al.
1369
1H NMR (CDCl3, ppm) δ: 10.0 (1H, d, J = 8.2 Hz), 6.90,
6.24 (each 1H, d, J = 11.3 Hz), 6.53, 6.25 (each 1H, d, J =
15.9 Hz), 5.93 (1H, d, J = 8.2 Hz), 5.05 (1H, m), 2.85 (2H,
t, J = 6.7 Hz), 2.56 (2H, t, J = 6.7 Hz), 2.45 (1H, dd, J = 5.5,
16.4 Hz), 2.12 (1H, dd, J = 9.4, 16.4 Hz), 2.05 (3H, s), 1.98
(3H, s), 1.90 (2H, qn, J = 6.7 Hz), 1.79 (1H, m), 1.74
(3H, s), 1.59 (1H, t, J = 12.0 Hz), 1.11, 1.08 (each 3H, s).
13C NMR (CDCl3, ppm) δ: 190, 170, 160, 139, 138, 135,
134, 132.4, 132.2, 128.8, 128.5, 126, 68.2, 43.8, 38.4, 36.6,
31.2, 29.9, 29.0, 28.4 (2C), 21.4 (2C), 14.9. FAB–HRMS
calcd. for C24H31O3: 367.2273 [M – H]+; found: 367.2271.
6.11 (each 1H, d, J = 11.4 Hz), 5.57 (1H, t, J = 6.3 Hz), 4.28
(2H, d, J = 6.3 Hz), 4.00 (1H, m), 2.52 (2H, t, J = 6.6 Hz),
2.38 (1H, dd, J = 5.0, 16.7 Hz), 2.31 (2H, t, J = 6.6 Hz),
2.04 (1H, dd, J = 9.7, 16.7 Hz), 1.91 (3H, s), 1.77 (1H, m),
1.73 (2H, qn, J = 6.7 Hz), 1.73 (3H, s), 1.47 (2H, t, J =
11.9 Hz), 1.07 (3H × 2, s), 0.906 (3H × 3, s), 0.0807 (3H ×
2, s).
To a mixture of the crude 3-OH compound, biotinyl
(Boc)Lys-OH (7.9 mg, 16.7 µmol),1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide (EDC, 6.4 mg, 33.3 µmol) at
0 °C, and CH2Cl2 (0.7 mL) were added. DMAP (2.0 mg)
was also added at this temperature. The mixture was warmed
to room temperature and stirred. After 3 h of stirring, the
mixture was poured into a satd. aq. solution of NaHCO3
(20 mL) and the resultant mixture was extracted with CHCl3
(20 mL). The organic layer was washed with a satd. aq.
NH4Cl solution, water, and brine, followed by drying over
MgSO4. Purification of the residue by silica gel column
chromatography (methanol–CHCl3, 7:93) afforded 16
15-O-TBS (15)
To a solution of the aldehyde 14 (14.5 mg, 39.3 µmol) in a
mixture of CHCl3 (0.2 mL) and methanol (1.0 mL) at
–10 °C, NaBH4 (8.9 mg, 236 µmol) was added. After 2 min
of stirring, a satd. aq. solution of NH4Cl (20 mL) was added
and the resultant mixture was extracted with EtOAc (3 ×
20 mL). The organic layers were washed with brine, com-
1
1
(7.8 mg, 8.69 µmol, 78%, in two steps) as a colorless oil. H
bined, and dried over MgSO4. H NMR (CDCl3, ppm) δ:
NMR (CDCl3, ppm) δ: 6.52, 6.11 (each 1H, d, J = 16.1 Hz),
6.44, 6.13 (each 1H, d, J = 11.3 Hz), 6.24 (1H, brs), 6.18
(1H, brs), 5.59 (1H, t, J = 6.5 Hz), 5.45–5.38 (1H, m), 5.37
(1H,brs), 5.20 (1H, d, J = 6.5 Hz), 5.10 (1H, m), 4.53 (1H,
dd, J = 4.8, 7.5 Hz), 4.37–4.28 (1H, m), 4.29 (2H, d, J =
6.5), 4.23 (1H, m), 3.23 (2H, qn, J = 5.9 Hz), 3.16 (1H, m),
2.92 (1H, dd, J = 4.8, 12.6 Hz), 2.75 (1H, d, J = 12.6 Hz),
2.52 (2H, t, J = 6.4 Hz), 2.44 (1H, m), 2.32 (2H, t, J =
7.4 Hz), 2.20 (2H, t, J = 7.4 Hz), 2.13 (1H, m), 1.91 (3H, s),
1.85–1.50 (15H), 1.73 (3H, s), 1.45 (3H × 3, s), 1.10, 1.08
(each 3H, s), 0.908 (3H × 3, s), 0.0813 (3H × 2, s). FAB-
HRMS calcd. for C49H81N4O7SSi+ m/z: 897.5590 [M + H]+;
found: 897.5574. FAB-HRMS calcd. for C49H81N4NaO7SSi:
920.5493 [M + H + Na]+; found: 920.5489.
6.50, 6.10 (each 1H, d, J = 15.9 Hz), 6.47, 6.12 (each 1H, d,
J = 11.3 Hz), 5.66 (1H, t, J = 7.0 Hz), 5.04 (1H, m), 4.25
(2H, d, J = 7.0 Hz), 2.52 (2H, t, J = 6.7 Hz), 2.44 (1H, dd,
J = 6.0, 17.6 Hz), 2.36 (2H, t, J = 6.7 Hz), 2.11 (1H, dd, J =
8.9, 17.6 Hz), 2.05 (3H, s), 1.92 (3H, s), 1.83–1.70 (3H),
1.72 (3H, s), 1.58 (1H, t, J = 11.9 Hz), 1.10, 1.07 (each
3H, s).
To a solution of the crude alcohol in CH2Cl2 (1.0 mL) at
room temperature, imidazole (16.0 mg, 197 µmol) was
added. The mixture was cooled to 0 °C, and TBSCl
(11.8 mg, 78.6 µmol) was added at this temperature. After
2 h of stirring, the mixture was poured into water (20 mL)
and the resultant mixture was extracted with Et2O (3 ×
20 mL). The organic layers were washed with brine, com-
bined, dried over MgSO4, and concentrated in vacuo. Purifi-
cation of the residue by silica gel column chromatography
(EtOAc–hexanes, 7:93) afforded 15 (13.4 mg, 27.6 µmol,
70%, in two steps) as a colorless oil. 1H NMR (CDCl3, ppm)
δ: 6.51, 6.08 (each 1H, d, J = 16.0 Hz), 6.42, 6.11 (each 1H,
d, J = 11.4 Hz), 5.57 (1H, t, J = 6.4 Hz), 5.05 (1H, m), 4.28
(2H, d, J = 6.4 Hz), 2.52 (2H, t, J = 6.7 Hz), 2.44 (1H, dd,
J = 5.5, 17.0 Hz), 2.31 (2H, t, J = 6.7 Hz), 2.10 (1H, dd, J =
9.3, 17.0 Hz), 2.05, 1.91 (each 3H, s), 1.82–1.73 (3H), 1.72
(3H, s), 1.58 (1H, t, J = 12.0 Hz), 1.10, 1.07 (each 3H, s),
0.906 (3H × 3, s), 0.0813 (3H × 2, s). 13C NMR (CDCl3,
ppm) δ: 170, 139, 138, 135, 134, 132, 131, 130.4, 130.3,
125.7, 125.0, 68.4, 59.9, 44.0, 38.5, 36.7, 31.3, 30.4, 30.1,
28.9, 28.6, 27.9, 26.1 (3C), 21.6, 18.5, 14.7, –4.80 (2C).
FAB-HRMS calcd. for C30H48O3Si m/z: 484.3373 [M+];
found: 484.3366.
3-O-Biotinyl (Boc)Lys 15-O-bromoacetate (1)
To a solution of 16 (4.1 mg, 4.57 µmol) in THF (1.0 mL)
at 0 °C, TBAF (1.0 mol/L solution in THF, 9.2 µL,
9.2 µmol) was added. The mixture was warmed to room
temperature and stirred. After 2 h of stirring, the mixture
was poured into water (20 mL) and the resultant mixture
was extracted with CHCl3 (3 × 20 mL). The organic layer
was washed with brine, combined, dried over MgSO4, and
concentrated under reduced pressure. Purification of the resi-
due by silica gel column chromatography (methanol–CHCl3,
10:90) afforded the biotinylated alcohol (2.1 mg, 2.68 µmol,
1
60%) as a colorless oil. H NMR (CDCl3, ppm) δ: 6.52, 6.13
(each 1H, d, J = 15.3 Hz), 6.49, 6.15 (each 1H, d, J =
11.1 Hz), 5.90 (1H, brs), 5.68 (1H, t, J = 6.8 Hz), 5.56 (1H,
brs), 5.17 (1H, d, J = 7.9 Hz), 5.10 (1H, m), 4.90 (1H, brs),
4.52 (1H, m), 4.33 (1H, m), 4.26 (2H, d, J = 6.8 Hz), 4.25
(1H, brs), 3.23 (2H, brs), 3.17 (1H, q, J = 4.4 Hz), 2.93 (1H,
dd, J = 4.8, 12.9 Hz), 2.74 (1H, d, J = 12.9 Hz), 2.53 (2H),
2.44 (1H, m), 2.37 (2H, t, J = 6.6 Hz), 2.28–2.18 (2H),
2.18–2.10 (1H, m), 1.92 (3H, s), 1.85–1.48 (16H), 1.73
(3H, s), 1.45 (3H × 3, s), 1.10, 1.08 (each 3H, s).
3-O-Biotinyl (Boc)Lys-15-O-TBS (16)
To a solution of 15 (5.4 mg, 11.1 µmol) in methanol
(1.0 mL) at 0 °C, K2CO3 (2.0 mg, 14.5 µmol) was added.
The mixture was stirred and allowed to warm to room tem-
perature. After 12 h of stirring, the mixture was poured into
a satd. aq. solution of NH4Cl (20 mL) and the resultant mix-
ture was extracted with EtOAc (3 × 20 mL). The organic
layers were washed with brine, combined, dried over
To a solution of the biotinylated alcohol (2.1 mg,
2.68 µmol) in CH2Cl2 (0.7 mL) at room temperature,
BrCH2COOH (2.0 mg, 14.4 µmol) was added. The mixture
was cooled to –10 °C and EDC (3.1 mg, 16.1 µmol) and
DMAP (1.0 mg) were added. After 15 min of stirring at the
1
MgSO4, and concentrated under reduced pressure. H NMR
(CDCl3, ppm) δ: 6.51, 6.11 (each 1H, d, J = 15.9 Hz), 6.43,
© 2006 NRC Canada

1370
Can. J. Chem. Vol. 84, 2006
2. N. Fishkin, N. Berova, and K. Nakanishi. Chem. Rec. 4, 120
(2004).
3. K. Nakanishi. Chem. Pharm. Bull. 48, 1399 (2000).
4. B. Borhan, M.L. Souto, H. Imai, Y. Shichida, and K.
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5. R.R. Rando. Chem. Rev. 101, 1881 (2001).
6. N. Nesnas, R.R. Rando, and K. Nakanishi. Tetrahedron, 58,
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same temperature, the mixture was poured into a satd aq. so-
lution of NaHCO3 (20 mL) and the resultant mixture was
extracted with CHCl3 (20 mL). The organic layer was
washed with a satd aq. NH4Cl solution, water and brine, fol-
lowed by drying over MgSO4. After concentration in vacuo,
purification of the residue by silica gel column chromatogra-
phy (methanol–CHCl3, 7:93) afforded the biotinylated
bromoacetate 1 (1.6 mg, 1.77 µmol, 66%) as a colorless oil.
1H NMR (CDCl3, ppm) δ: 6.53, 6.13 (each 1H, d, J =
11.4 Hz), 6.50, 6.12 (each 1H, d, J = 15.7 Hz), 5.86 (1H, t, J
= 5.0 Hz), 5.58 (1H, t, J = 7.3 Hz), 5.51 (1H, brs), 5.16 (1H,
d, J = 8.6 Hz), 5.10 (1H, m), 4.87 (1H, brs), 4.77 (2H, d, J =
7.3 Hz), 4.51 (1H, dd, J = 4.7, 7.8 Hz), 4.32 (1H, dd, J =
4.7, 7.6 Hz), 4.23 (1H, m), 3.83 (2H, s), 3.23 (2H, qn, J =
6.1 Hz), 3.16 (1H, m), 2.92 (1H, dd, J = 4.7, 12.6 Hz), 2.73
(1H, d, J = 12.6 Hz), 2.51 (2H, t, J = 7.0 Hz), 2.39 (2H, t, J
= 6.5 Hz), 2.10 (1H, m), 2.14 (2H, t, J = 6.5 Hz), 2.10
(1H, m), 1.92 (3H, s), 1.88–1.30 (16H), 1.72 (3H, s), 1.45
(3H × 3, s), 1.10, 1.08 (each 3H, s).
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Acknowledgement
We acknowledge the National Institutes of Health (GM
36564) for their generous support of this research.
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© 2006 NRC Canada