Design and synthesis of bombykol analogues for probing pheromone-binding protein-ligand interactions

Article abstract of DOI:10.1016/j.tet.2008.10.106

Mono-, difluorinated, and thioanalogues of Bombyx mori female sex pheromones (bombykol, 1) were designed according to the ab initio calculations. These rationally designated analogues were synthesized using hydroboration and Sonogashira coupling strategy

Full text of DOI:10.1016/j.tet.2008.10.106

Tetrahedron 65 (2009) 1069–1076
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design and synthesis of bombykol analogues for probing
pheromone-binding protein–ligand interactions
a
b
a,
Madina Mansurova , Vojtech Klusak , Petra Nesnerova y, Alexander Muck a,
ˇ
´
ˇ ˇ
´
a
,b,
*
´
ˇ
ˇ a
Jan Doubsky , Ales Svatos
a Max Planck Institute for Chemical Ecology, Hans-Kno¨ll-Str. 8, Jena D-07745, Germany
b
´
Institute for Organic Chemistry and Biochemistry, The Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6, CZ-16610, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 August 2008
Received in revised form 19 October 2008
Accepted 27 October 2008
Available online 8 November 2008
Mono-, difluorinated, and thioanalogues of Bombyx mori female sex pheromones (bombykol, 1) were
designed according to the ab initio calculations. These rationally designated analogues were synthesized
using hydroboration and Sonogashira coupling strategy via (5E,7Z)-undecadien-1-ol (10) as a common
intermediate. A new simplified binding assay based on nanoLC-linear ion trap ESI-MS for quantifying
complexation of the B. mori pheromone-binding protein (BmPBP) with native (1) and prepared ana-
logues was developed. The binding properties of native 1 and thioanalogue 4 with PBP were studied in
detail. The dissociation constant (KD) of 1 and 4 was determined to be 2.1ꢀ10ꢁ6 M and 2.4ꢀ10ꢁ6 M,
respectively. The similar values for both ligands correlated with ab initio calculations. The new binding
assay could be used to determine the KD of other PBPs.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Prestwich9 requires specific conditions for measurement and does
not provide binding constants of the expected magnitudes. Mass
Since Butenandt1 identified bombykol as a sex pheromone of
the silkworm moth, Bombyx mori (L.), considerable progress has
been made in understanding its pheromone chemistry and
biology.2,3 The mechanism suggests that various proteins act col-
laboratively, including pheromone receptors, pheromone-binding
proteins (PBPs), and pheromone-degrading enzymes, during the
olfactory process.4 The molecular structure of the proteins is
already firmly established; even the crystal structure of the pher-
omone-binding protein (PBP) with the native ligand is known.5
However, the limited information available on the kinetic param-
eters of individual peri-receptor events in insect antennae has
slowed construction of a permanent model, despite Kaissling’s
proposals.6,7 The role of individual proteins in sex pheromone
perception and degradation needs to be investigated further to
understand how insects recognize individual pheromone
molecules.8
spectrometric methods were employed to measure binding of
bombykol (1) to B. mori PBP (BmPBP).10 Leal’s group developed an
assay based on filtering off the non-bound pheromone from the
incubation solution containing PBP at appropriate pH and ionic
strength; the ligand was then determined by gas chromatography
(GC) and the PBP by electrospray mass spectrometry (ESI-MS).11
Other researchers use competitive binding studies with fluores-
cence readouts12 to study the pheromone binding indirectly.
Fluorescence probes, which are expected to bind to the PBP binding
site, are used (1-aminoanthracene), and the release of the fluo-
rescence probe is measured by fluorescence spectroscopy. Because
the probe is not structurally related to sex pheromone structures,
the binding can be nonspecific.
To improve assay accuracy, we may need to use an internal
standard that is structure like native bombykol but with a different
mass. This will allow the binding constants of diverse ligands to be
determined in a standardized way. We have recently been able to
identify amino acid residues that contribute significantly to the
enthalpy of binding of bombykol ligands in the BmPBP–Bom
complex.13 Here, using a similar computation method, we charac-
terize the energetic binding changes that occur when an atom is
isosterically replaced in the original ligand. Those calculations were
the basis of the set of pheromone analogues we designed. In the
following sections we describe the synthesis of the newly designed
pheromone analogues and evaluate their binding constants by si-
multaneously determining amounts of both ligand and PBP using
Determining binding constants of lipophilic pheromone ligands
in aqueous solutions of proteins is not a trivial task, as the water-
immiscible ligand is difficult to be kept in solution at the higher
concentrations needed for NMR or calorimetric assays. The com-
petitive assay for radioactive substrate analogues developed by
* Corresponding author. Tel.: þ49 (0) 3641 57 1700; fax: þ49 (0) 3641 57 1701.
ˇ
E-mail address: svatos@ice.mpg.de (A. Svatos).
y
Present address: Roche Diagnostics, Penzberg D-82377, Germany.
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.10.106

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M. Mansurova et al. / Tetrahedron 65 (2009) 1069–1076
Table 2
LC–ESI-MS. The approach speeds evaluation of biological assays
and minimizes sample handling.
Unrestrained optimization of the structures corresponding to the calculated opti-
mum benzene ligand distances
Starting
orientation
Propane
2,2-Difluoro-
propane
2-Fluoro-
propane
Dimethyl-
sulfane
2. Results and discussion
Above center
Above,
2 Å away
ꢁ3.1
ꢁ2.9
ꢁ3.4a
ꢁ3.5
ꢁ3.4
ꢁ2.6
ꢁ3.8
2.1. Quantum chemical calculation
ꢁ3.3a
2.1.1. Interaction energy change of the ligand versus Phe12
and Phe118 in the BmPBP
Interaction values of optimized structures (in kcal/mol).
a
The optimization from both orientations ended in a similar conformation.
The most important interacting partners for the middle, satu-
rated part of the ligand are aromatic side chains, namely Phe12,
Phe118 residues.13 We estimated the effect of pheromone structure
isosteric change on the interaction energy for the Phe12 and Phe118
residues. Using the X-ray structure of the BmPBP–bombykol com-
plex, the particular CH2 groups were replaced by CF2 or S and the
structures of pheromone and the two phenylalanine benzene rings
were optimized for the positions of hydrogen and fluorine atoms.
We selected sites distant from the terminal alcohol group and from
conjugated double bonds, evaluating the C4, C5, C6, C7, and C16
positions in the carbon chain. We conclude (Table 1) that none of
the modifications resulted in a large value of repulsion or attrac-
tion; however, several positions seem to be preferential.
benzene ligand distance were performed. The calculated in-
teraction energies are shown in Table 2.
Propane and 2-fluoropropane behave similarly, interacting more
strongly above the center of benzene and less strongly as the
molecule moves from the ring center. The dimethylsulfane mole-
cule interacts more strongly if above the ring plane, shifted 2 Å
sideways (Fig. 1).
The effect of solvation has been assessed using a continuum
solvation model. The results confirmed our assumption that the
CH2 group replacement by more polar groups systematically en-
hances solubility of the ligand. The DGsolv change ranged between
ꢁ2.2 and ꢁ3 kcal/mol (Table 3).
In order to gain similar or stronger affinity in comparison to the
native ligand bombykol, the modified ligands need to compensate
for the unfavorable effect of solvation (Table 3). The compensation
may arise from the enthalpic contribution to the energy of ligand–
protein interaction. The calculations on model molecules show that
the considered functional groups may enhance the enthalpic con-
tribution (Table 2). It was shown that the three model molecules
interact more strongly than propane. Interaction energies calcu-
lated by unrestrained optimization, are surprisingly similar (Table
2), though the resulting conformations are different. In addition,
the difference in behavior of 2-fluoropropane and 2,2-difluoro-
propane is also remarkable (Fig. 1), indicating that the interaction
enthalpy increases in response to contact with the aromatic ring
and the C–H bond polarized by the adjacent C–F group. This led us
to consider testing the racemic mixture of monofluorinated
bombykol. We have chosen to test the following functional groups
in the following positions in the bombykol molecule (Fig. 2): Y (C6)
for –CF2– and for the racemic mixture of –CFH–, Z (C16) for –CH2F–,
and X (C5) for –S–.
2.1.2. Model interactions with an aromatic ring
In order to elucidate the orientation preference of these func-
tional groups toward the aromatic residues, we considered the
interactions of four model molecules (2-fluoropropane, 2,2-
difluoropropane, and dimethylsulfane were compared with pro-
pane) with benzene; three different orientations were studied:
above the ring center, above the carbon atom of the benzene ring,
and above the ring plane shifted 2 Å out of the ring (see Fig. 1).
Dissociation curves were calculated using RI-MP2/aug-cc-pVDZ ab
initio method for each complex. In addition, unrestrained optimi-
zations of the structures corresponding to the calculated optimum
Table 1
The effect of the substitution of CF2 or S group for the CH2 group in bombykol (1)
Position
C4
C5
0.9
ꢁ0.3
C6
C7
0.2
ꢁ0.6
C16
CF2
S
ꢁ0.4
ꢁ0.6
ꢁ0.2
ꢁ0.3
0.0
0.0
The interaction energies (kcal/mol) of pheromone versus Phe12 and Phe118 in na-
tive crystallographic conformation. Values are relative to the native pheromone
interactions.
2.2. Synthesis of the bombykol ligands
A simple and efficient synthetic approach was designed based
on Pd-catalyzed Sonogashira coupling, hydroboration, and hydro-
lysis steps. The synthesis of bombykol analogues 2, 3, and 4 shares
a common dienol intermediate.
Table 3
The DDGsolv for replacement of C(n) atoms in bombykol (1)
Ligand
Bombykol C(5)H2_S C(6)H2_C(6)F2 C(6)H_C(6)HF CH3_CH2F
E (kcal/mol)
0
ꢁ2.7
ꢁ3.0
ꢁ2.3
ꢁ2.2
Y
OH
X
6
5
16
Z
Compound No. X
Y
Z
1
2
3
4
5
CH2
CH2
CHF
CF2
CH2
CH2
CH3
CH3
CH3
CH3
CH2F
CH2
CH2
S
Figure 1. Four model molecules (2-fluoropropane, 2,2-difluoropropane, and dime-
thylsulfane were compared with propane) were considered to interact with benzene in
three different orientations (above the ring center, above a carbon atom of the benzene
ring, and above ring shifted 2 Å out of ring).
CH2
Figure 2. Chemical structures of rationally designed bombykol (1) analogues.

M. Mansurova et al. / Tetrahedron 65 (2009) 1069–1076
1071
a
b
I
OH
O
O
8
7
6
O
d,e
h,i
c
f,g
OH
10
9
OH
F
OTHP
OH
11
2
f
O
F
F
OTHP
h,i
X
12
3 (X= OH)
3b (X= F)
Scheme 1. (a) 2-Methylpropene; (b) DIBAL-H, I2; (c) 1-pentyne, Pd(PPh3)4, CuI; (d) Cy2BH/AcOH; (e) Ac2O, FeCl3, then NaOH; (f) PCC; (g) BrMgC5H10OTHP; (h) DAST; (i) PTSA.
The synthesis of both mono- and difluoro ligands 2 and 3 started
from protected (E)-iodohexan-6-ol (8, Scheme 1),14 which was
prepared from the corresponding alkynol 6.
2.3. Binding assay of ligands to BmorPBP
The molecular interactions of bombykol with its binding partner
PBP have recently11 been studied in an assay inwhich thepheromone
is incubated with the PBP under physiological conditions in plastic
vials equipped with molecular filter membranes; subsequently the
complex is separated from the unreacted ligand by centrifugation.
However, the GC–MS analysis of the pheromone requires hexane to
be extracted from the aqueous buffer, and the additional LC–MS
analysis of the protein increases the complexity of the analysis,
possibly leading to more experimental errors or extended analysis
times. These could be eliminated by detecting both ligands and PBPs
Only tert-butyl ether of the alcohol functionality was found to
protect (E)-iodohex-1-en-1-ol 8 at more than 90% yield. Moreover,
the tert-butoxy protecting group is highly recommended in the
chemistry of organolithium or Grignard reagents, because of the
bulk of the tert-butyl group, which efficiently impedes any chela-
tion of the metal (Li or Mg) by the oxygen of the tert-butyl ether
functionality.15 Pd-catalyzed Sonogashira coupling reaction with
pent-1-yne led to enyne 9 in a good yield. The selective reduction of
triple bond was performed with dicyclohexylborane, which is
known to be a mild reagent for cis-isomer formation.16 The iso-
meric purity of the prepared (5E,7Z)-undecadienol 10 was higher
than 96% (using NMR and GC on DB5 phase). After deprotection, the
terminal hydroxyl group in 10 was oxidized, and the obtained
aldehyde reacted with Grignard reagent, prepared from THP-pro-
tected 1-bromopentan-5-ol. For 6-fluoro ligand 2, the formed
OH-group in 11 was directly fluorinated using DAST in 80% yield.
THP protection was removed in the presence of p-toluenesulfonic
acid, resulting in an overall yield of ligand 2 of 16%. For 6,60-
difluorinated compound, alcohol 11 was converted to the corre-
sponding ketone (12) and then fluorinated as described above. It
turned out that the usual fluorination procedure was not efficient
enough for the keto-group; however, the DAST needed for the
complete fluorination of 12 led to the cleavage of THP-protecting
group and subsequently a similar level of fluorination was observed
in the terminal OH-function. Unfortunately, 1,6,6-trifluorodiene
side product (17%) was an isolated product; the expected 6,60-
difluoroalcohol 3 was obtained only in a trace amount insufficient
to fully characterize the compound to the question of how specific
are PBP–pheromone interactions.
a
b
10
OTs
OH
13
S
4
Scheme 2. (a) TsCl; (b) HSC4H8OH.
a
AcO
AcO
+
I
O
14
b
O
15
For sulfur-containing ligands, alcohol 10 was tosylated and
treated with 4-mercaptobutan-1-ol (Scheme 2). The absence of an
O-protecting group in mercaptobutanol does not affect the reaction
and does not impair the yields. Thus, thioanalogue 4 was obtained
from 10 in 87% isolated yield.
16-Fluoro compound 5 was prepared using the same strategy
described above for fluoro ligands starting from pentynol acetate
and iodoalkene 14 in 24% overall yield (Scheme 3).
AcO
O
c,d
16
F
OH
5
Scheme 3. (a) Pd(PPh3)4, CuI; (b) Cy2BH; (c) MeONa; (d) DAST; Ac2O, FeCl3, MeONa.

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M. Mansurova et al. / Tetrahedron 65 (2009) 1069–1076
in aqueous solutions at conditions similar to those of the binding
assay. In our approach, descriptions of how the binding energy
changes when atoms or functional groups are replaced in the pher-
omone may help to clarify the mechanisms of the peri-receptor
events in insect olfaction and might provide answers to the question
of how specific are PBP–pheromone interactions.
(2.5 pmol PBP and 5 pmol 1 on column). The ESI spectra of 1 display
an intense protonated molecular peak at 239.1 Da and an additional
dimer [2MþH]þ peak (inset Bol). The purity of the recombinant
protein is demonstrated in inset BmPBP by a full protein envelope
of multiplet peaks carrying charges in the range of 8–14 protons.
The association binding constants (KD) for sulfo ligand 4 and
bombykol 1 determined from these assays were 2.4ꢀ10ꢁ6 M and
2.1ꢀ10ꢁ6 M, respectively. From the previously published KD, the
value of ourassociation constant is closer to 1.1ꢀ10ꢁ6 M, determined
using the fluorescence assay.12 The previously published value using
a ‘cold binding assay’11 was 0.105ꢀ10ꢁ6 M. The observed differences
could be explained by different experimental setups. If we consider
the calculated binding enthalpy (ca. 20 kcal/mol)13 and the difficulty
of observing the binding of 1 with BmPBP in native electrospray
experiments,10 the nanomolar value for KD determined by Leal11
seems to be too low. Based on our native electrospray experiments,
the dissociation of 1 from PBP should be comparable with the dis-
sociation of heme from hemoglobin18 with KD¼6.2ꢀ10ꢁ6 M. The
similar value of KD correlates with the predicted effects of isosteric
replacement determined by ab initio calculations.
To determine the binding constants of BmPBP with bombykol
and its modified isomers, we used an assay based on a centrifugal
filter device that separated bound and free ligands.11 Only one an-
alytical method (LC–ESI-MS) was used to determine the amount of
PBP and ligand before and after incubation. The method is more
sensitive than earlier methods and does notrequirelargeamounts of
protein/ligand as was necessary in GC quantification11 of free ligand
or native gel assays.17 Three filtration columns were tested, and
Microcon filters were found to recover most of the PBPs (91%). To
compare, using the ZEBA column to isolate BmBPB–pheromone
complex led to 29% recovery of the PBP, using the VIVASPIN column
led to 10%. The binding of thio ligand 4 to BmPBP was studied
and results were compared with the binding constants for PBP–
bombykol complex. A detailed description of the method will be
published elsewhere. The assays were repeated three times. Anal-
yses were performed within several hours after the binding assay as
ligands are notoriously unstable when stored for long periods (24 h).
A baseline separation observed in Figure 3 for the interacting
species on a reversed-phase n-butyl-silica column with a well-de-
fined PBP peak at 17.5 min and the hydrophobic bombykol eluting
shortly thereafter at 21 min demonstrate the usefulness of the
approach. The selected ion chromatogram plotted at 239.2 and
1588.5 Da using a 1 Da window, for the molecular peaks of 1 and for
the [Mþ10H]10þ PBP multiplet, respectively (selected to maintain
comparable protein/ligand signal ratios), enables sensitive quanti-
tation in the picomolar concentration range to be performed
The uses of the developed assay could be further extended to
evaluate binding constants for similar assays of other PBPs. Con-
stants of ligand binding for the lipocaline family4 functionally
related to PBPs might be measured by this approach after minor
modifications.
3. Experimental
3.1. General
All reactions were carried out under an inert atmosphere (Ar).
All reported yields are of isolated pure products. The NMR spectra
17.5
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
BmPBP
1765.2
477.5
100
90
80
70
60
50
40
30
20
10
0
100
BmPBP
Bol
[2M+H]+
90
80
70
1588.7
[M+H]+
239.1
60
50
40
30
20
10
0
1444.5
280.3
1985.7
1324.2
1000 1200 1400 1600 1800 2000
250
350
Mass (m/z)
450
550
Mass (m/z)
Bol
21.4
0
0
2
4
6
8
10
12
14
16
18 20
Time (min)
22
24
26
28
30
32
34
36
38
Figure 3. A section of total ion current trace from LC–MS separation of a mixture after binding assay of bombykol (1) with BmPBP protein. Insets show average mass spectra under
peaks of BmPBP and (1), respectively.

M. Mansurova et al. / Tetrahedron 65 (2009) 1069–1076
1073
were recorded on Bruker AVANCE DR 400 spectrometer. All spectra
were measured in CDCl3. Chemical shifts are given in parts per
million (ppm) downfield relative to tetramethylsilane as an internal
standard. Mass spectra were obtained on MassSpec 2 or Quattro II
(both Micromass, Manchester, UK).
using three analytical determinations. The binding constants were
calculated from ligand peak areas integrated in selected ion chro-
matograms (SIMs) displayed each time as molecular ion intensities
with 0.5 Da mass window and average area values of three con-
secutive retentate analyses. The SIM peak areas of the PBP (at
a mass of 1588.5 Da, corresponding to a 10-fold protonated protein)
were used for normalizing the yield of the centrifugation and ex-
traction of the sample from the membrane columns.
d
3.1.1. Computational details
3.1.1.1. Methods. All structures were first optimized using the
density functional theory (DFT) method. The B3LYP functional19
was used in conjunction with the 6-31G** basis set.20 The un-
restrained optimization of the model complexes was performed at
the second order Møller Plesset (MP2) perturbation method level
using the aug-cc-pVDZ basis set.21 Interaction energies were
computed at the MP2/aug-cc-pVDZ level of theory. MP2 calcula-
tions were sped up by expanding the Coulomb interactions in an
auxiliary basis set, the resolution-of-identity (RI) approximation.22
The solvation effect was described using the COSMO model of an
aqueous solvent implemented in the Turbomole 5.7 program
suite23 with the default parameters.
The assay samples were analyzed using a nanoHPLC–MS system
consisting of an Agilent 1100 series autosampler and a micro-LC
pump (Agilent, Palo Alto, CA, USA) and of a linear ion trap mass
spectrometer (LTQ, Thermo-Finnigan Inc., San Jose, CA, USA). The
injected samples were first loaded onto a Nanoease trap column
(240 mmꢀ180
Nanoease nano-LC analytical column (100 mmꢀ75
packed with Symmetry 300 C4 3.5 m particles, both Waters,
Milford, MA, USA). The sample was injected from 10% MeCN–0.1%
FA at a flow rate of 12 L/min for 5 min and eluted by a linear 10–
90% acetonitrile gradient in 10 min at a flow rate of approximately
300 L/min after a passive split. The possible hydrophobic con-
mm I.D.) and then eluted onto a reversed-phase
mm I.D., both
m
m
m
taminants were then removed by an additional 90% MeCN–0.1% FA
step and the column was equilibrated at 10% MeCN–0.1% FA for
additional 12 min. The column outlet was coupled to the nano-
electrospray ionization source of the LTQ linear ion trap mass
spectrometer (Thermo-Finnigan Inc.) using a liquid junction and an
3.1.1.2. Model. The model for native interaction of pheromone and
the residues Phe12, Phe118 was built by truncating the X-ray
structure [Ref. 5, 1DQE]. Whole pheromone and benzene rings of
the phenylalanines were considered. The coordinates of the heavy
atoms were fixed at the original crystallographic positions; hy-
drogen atom positions were optimized.
uncoated fused silica nanoelectrospray needle (120 mmꢀ30
mm
I.D., Picotip, New Objective, Woburn, MA, USA).
The mass spectrometer operated with 1.7 kV set on the emitter,
42 V on the ion transfer capillary and 140 V tube lens voltages, and
140 ꢂC capillary temperature using an alternating three full mass
scanevents(100–2000 Dafollowed byan SIMscanof theBmPBPand
of the ligand). The binding constants were calculated based on Eq.1:
3.1.2. BmPBP preparation and purification
The recombinant protein was prepared as follows.24,25 Briefly,
the pheromone-binding protein from B. mori (BmPBP) was
expressed in Escherichia coli periplasm. The recombinant
pET22b(þ)/BmPBP plasmid (obtained from J. Krieger, Hohenheim
University, Germany) was transformed on E. coli BL21(DE3) ex-
pression hosts (Novagen) and the expression was performed by
growing the no induced cells at 30 ꢂC in an LB medium (2-L flasks
with 700 mL suspension culture) supplemented with 50 mg/L
ampicillin. The culture was grown until A600 values were greater
than 2. The cells were harvested by centrifugation and the peri-
plasmic fraction was obtained by the osmotic shock procedure
described in the pET System manual (Novagen). The periplasmic
fraction was loaded onto a DEAE-Toyopearl 650S (Tosoh, Tokyo,
Japan) column (diameter 2.3 cm, length 15 cm) equilibrated with
10 mM Tris–HCl, pH 8.0. The proteins were eluted with a very slow
linear gradient of 0–200 mM NaCl in 10 mM Tris–HCl, pH 8.0. The
fractions were analyzed by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and the fractions containing pure BmPBP were pooled.
The BmPBP was precipitated using trichloroacetic acid and stored
at ꢁ80 ꢂC. Prior to use, the fatty acids were removed by delipida-
tion.26 The obtained solution was aliquoted, vacuum-dried, and
stored at ꢁ20 ꢂC prior to use.
h
i
h
i h
i
KD
¼
PBPtotal ꢀ PBPfree = PBPcomplex
(1)
3.1.4. Synthesis of ligands
3.1.4.1. 1-tert-Butoxyhex-5-yne (7). Amberlyst (1.25 g) was added
to the reaction mixture of 5-hexyn-1-ol (6, 5 g, 0.05 mol) in hex-
anes (25 mL) at 0 ꢂC. 2-Methylpropene was bubbled into the mix-
ture for 14 h at room temperature. The resin was filtered off, K2CO3
(1 g) was added, and the solvent was evaporated. Flash chroma-
tography (FC) on silica gel, eluting with CH2Cl2, provided the
product (7.8 g, 100%). 1H NMR (400 MHz, CDCl3):
d
¼3.28 (t, 2H, 1-
CH2), 2.14 (m, 2H, 4-CH2), 1.86 (t, 1H, 6-CH), 1.54 (m, 4H, 2-CH2, 3-
CH2), 1.11 (br s, 9H, 3ꢀCH3). 13C (100 MHz, CDCl3):
¼84.9 (C-5),
d
72.8 (Cquad), 68.6 (C-6), 61.3 (C-1), 30.1 (C-2), 27.9 (3ꢀCH3), 25.8 (C-
3), 18.7 (C-4). EIMS, m/z 154, [M]ꢃþ
.
3.1.4.2. (E)-6-tert-Butoxy-1-iodohex-1-ene (8). A solution of alkyne
7 (4 g, 26 mmol) in hexanes (30 mL) was treated with DIBAL-H
(40 mL, 1 M in hexanes) at 55 ꢂC for 5 h and cooled till ꢁ45 ꢂC, and
a solution of iodine (8 g, 1.1 equiv) in THF (30 mL) was added over
60 min. The resulting mixture was allowed to warm to room tem-
perature and stirred at those conditions for 14 h, then poured into
6 M NaOH, and stirred until the brown color disappeared. The or-
ganic layer was separated, and the aqueous layer was extracted with
hexanes (3ꢀ10 mL). The combined organic extracts were washed
with water (3ꢀ10 mL) and dried over Na2SO4. FC on silica gel, eluted
with CH2Cl2–hexanes (1:3), gave 8 (6.74 g, 93%). 1H NMR (400 MHz,
3.1.3. Binding assay and LC–MS analysis
A 1
L of 5
monium acetate, pH 7) in a glass vial insert silanized by dime-
thylsilyl dichloride. The sample was incubated in shaker
(300 rpm) at 27 ꢂC for 1 h. An aliquot (5
L) of the reaction mixture
mL aliquot of 10
mM ethanolic ligand solution was added to
99
m
m
M delipidated protein solution in 10 mM buffer (am-
a
m
was taken for assay A and the rest was transferred to a washed
Microcon YM-10 10 kDa cut-off membrane column (Millipore,
Billerica, MA, USA) and centrifuged (17,950 g/4 ꢂC/10 min). The fil-
CDCl3):
d
¼6.43 (m, 1H, 2-CH), 5.91 (dt, 1H, 1-CH, J12¼14.3 Hz,
J13¼1.4 Hz), 3.26 (m, 2H, 6-CH2), 2.01 (m, 2H, 3-CH2), 1.45 (m, 4H, 4-
ter was washed with another 95
was weighed and diluted to total volume 95
solution (5 L) was diluted with 45 L of an eluent solution (ace-
mL of buffer, pH 7. The retentate
CH2, 5-CH2),1.11 (br s, 9H, 3ꢀCH3).13C (100 MHz, CDCl3):
¼146.9 (C-
d
m
L. Part of the retentate
2), 74.9 (C-1), 72.8 (Cquad), 61.5 (C-6), 36.3 (C-3), 30.3 (C-5), 28.0
(3ꢀCH3), 25.8 (C-4). EIMS, m/z (rel int.): 280 (5, [M]ꢃþ), 207 (75,
[MꢁOPg]þ).
m
m
tonitrile–0.1% formic acid 1:10 v/v) and analyzed by LC–MS. The
experiments were performed with three assay replicates each

1074
M. Mansurova et al. / Tetrahedron 65 (2009) 1069–1076
3.1.4.3. (E)-11-tert-Butoxy-undec-6-en-4-yne(9). Vinyliodide 8 (2.2 g,
8 mmol) was injected at room temperature into a solution of
Pd(MeCN)2Cl2 (450 mg, 5 mol %) in anhydrous piperidine (30 mL)
under argon atmosphere. The resulting mixture was stirred for an
additional hour. Then a solution of pentyne (2 mL, 2 equiv) in
piperedine (5 mL) and CuI (350 mg, 10 mol %) as solid were added.
After 18 h stirring under argon, the mixture was poured into water
(20 mL) and extracted into ether (2ꢀ20 mL). Extracts were washed
with aq HCl (1 M, 10 mL), satd aq NaHCO3 (10 mL), brine (2ꢀ20 mL),
and water (2ꢀ10 mL), and dried over Na2SO4. FC on silica gel, eluting
with CH2Cl2–hexanes (1:5), gave 9 (1.6 g, 68%). 1H NMR (400 MHz,
d
¼98.9 (C-20), 67.4 (C-1), 62.4 (C-60), 33.7 (C-5), 32.6 (C-4), 30.8 (C-
2), 28.9 (C-30), 25.5 (C-3), 25.0 (C-50), 19.7 (C-40). GC–MS, m/z (rel
int.): 252, 250 [M]ꢃþ
.
3.1.4.6. (10E,12Z)-1-(Tetrahydro-2H-pyran-2-yloxy)hexadeca-10,12-
dien-6-ol (11). Alcohol 10 (850 mg, 5.1 mmol) in CH2Cl2 (5 mL) was
added to the suspension of PCC (1.3 g, 1.2 equiv) and anhydrous
NaOAc (98 mg) in CH2Cl2 (6 mL). The resulting mixture was stirred
at room temperature for 90 min, then poured into ether (200 mL),
and filtered through the column with sand/Celite/coal/sand to give
(5E,7Z)-undecadien-1-al (630 mg, 76%), which was used in the next
CDCl3):
d
¼5.95 (m,1H, 7-CH), 5.40 (d,1H, 6-CH, J67¼14.0 Hz), 3.25 (m,
step without additional purification. 13C (100 MHz, CDCl3):
(CHO), 131.7 (C-5), 129.7 (C-6), 127.5 (C-7), 125.3 (C-8), 42.2 (C-2),
31.1 (C-9), 24.7 (C-10), 21.9 (C-3), 20.8 (C-4), 14.1 (C-11).
d¼201.4
2H, 11-CH2), 2.18 (m, 2H, 3-CH2), 2.01 (m, 2H, 4-CH2), 1.46 (m, 6H,
3ꢀCH2), 1.10 (br s, 9H, 3ꢀCH3), 0.91 (m, 3H, 1-CH3). 13C (100 MHz,
CDCl3):
d
¼143.4 (C-7), 110.4 (C-6), 88.9 (C-4), 79.7 (C-5), 72.8 (Cquad),
One small crystal of iodine was added to a dry Mg (273 mg) and
the mixture was stirred under heat till the glass walls turned violet.
After cooling, THF (2 mL) was added followed by 1/5 of the 5-O-
tetrahydropyranoxy-1-bromopentane solution (1.4 g, 5.6 mmol,
dissolved in 4 ml THF). The resulting mixture was stirred at 65 ꢂC
for 30 min. Then the rest of 5-O-tetrahydropyranoxy-1-bromo-
pentane was added carefully, and the mixture was stirred under
refluxing for an additional 2.5 h. The reaction was allowed to cool;
the reaction solution was added dropwise by syringes into a mix-
ture of aldehyde (630 mg, 3.8 mmol) in THF (6 mL) cooled till 0 ꢂC.
After 15 min the ice-bath was removed, and the resulting mixture
was stirred at room temperature for 19 h. Ice-cold NH4Cl (10 mL) was
added, the organic phase was separated, and the aqueous one was
extracted into pentane and ether (1:1, 100 mL). The combined or-
ganic extracts were washed with NH4Cl (3ꢀ50 mL) and water
(20 mL), and dried over Na2SO4. FC on silica gel, eluted with
CH2Cl2–pentane (1:3/100%), furnished 11 (810 mg, 64%). 1H
61.7 (C-11), 34.0 (C-8), 30.3 (C-10), 27.9 (3ꢀCH3), 26.3 (C-9), 23.0 (C-
2), 22.7 (C-3), 13.9 (C-1). EIMS, m/z (rel int.): 222 (5, [M]ꢃþ), 149 (75,
[MꢁOPg]þ).
3.1.4.4. (5E,7Z)-Undecadien-1-ol (10). A dicyclohexylborane sus-
pension was prepared in THF (15 mL) from borane–DMS complex
(2 M, 3 mL) and cyclohexene (488 mg, 2.2 equiv). The obtained
white suspension was treated at 0 ꢂC with a solution of enyne 9
(600 mg, 2.7 mmol) in THF (5 mL). The mixture was warmed to
room temperature and stirred for 4 h. The formed vinylborane was
hydrolyzed with glacial AcOH (2.5 mL) at room temperature for
14 h; the reaction mixture was then neutralized with NaOH (20%,
5 mL), and carefully treated with aq H2O2 (30%, 2.5 mL). The crude
product was extracted with hexanes (3ꢀ50 mL) and dried over
Na2SO4. FC on silica gel, eluted with CH2Cl2–hexanes (1:3), gave
O-tert-butoxy-undec-5-(E)-7-(Z)-dien-1-ol (560 mg, 93%). 1H NMR
(400 MHz, CDCl3):
d
¼6.23 (dd, 1H, 7-CH, J76¼15.0 Hz, J78¼11.0 Hz),
NMR (400 MHz, CDCl3):
d
¼6.19 (dd, 1H, 12-CH, J1211¼15.0 Hz,
5.88 (t, 1H, 6-CH, J65¼15.0 Hz), 5.58 (m, 1H, 8-CH), 5.21 (m, 1H, 5-
J1213¼11.0 Hz), 5.83 (t, 1H, 11-CH, J1110¼15.0 Hz), 5.52 (m, 1H,
13-CH), 5.20 (m, 1H, 10-CH), 4.45 (m, 1H, CH-THP), 3.74 (m,
1H, CHH-THP), 3.60 (m, 1H, CHH-THP), 3.50 (m, 1H, 6-CH), 3.36 (m,
1H, 1-CHH), 3.28 (m, 1H, 1-CHH), 2.00 (m, 4H, 14-CH2, 9-CH2), 1.55
(m, 2H, CH2), 1.29 (m, 18H, 9ꢀCH2), 0.79 (m, 3H, 16-CH3). 13C
CH), 3.26 (m, 2H, 1-CH2), 2.05 (m, 2H, 9-CH2), 1.77 (m, 2H, 4-CH2),
1.40 (m, 6H, 3ꢀCH2), 1.10 (br s, 9H, 3ꢀCH3), 0.81 (m, 3H,11-CH3). 13
C
(100 MHz, CDCl3):
d
¼134.6 (C-5), 130.1 (C-6), 129.1 (C-7), 126.2 (C-
8), 72.7 (Cquad), 61.7 (C-1), 31.9 (C-4), 30.3 (C-2), 27.9 (3ꢀCH3), 26.0
(C-9), 25.9 (C-10), 23.0 (C-3), 14.4 (C-11). EIMS, m/z (rel int.): 224 (4,
[M]ꢃþ), 167 (63, [MꢁPg]þ), 151 (70, [MꢁOPg]þ).
(100 MHz, CDCl3):
d
¼134.0 (C-10), 130.1 (C-11), 128.7 (C-12), 126.0
(C-13), 98.9 (C-20), 71.7 (C-6), 67.5 (C-1), 62.4 (C-60), 37.4 (C-7), 37.0
(C-5), 32.8 (C-14), 30.8 (C-9), 29.7 (2C, C-15, 30) 26.3, 25.4 (3C), 22.9
(C-2, 8, 4, 3, 50), 19.7 (C-40), 13.7 (C-16). EIMS m/z (rel int.): 338 (47,
[M]ꢃþ), 320 (40, [MꢁH2O]þ).
To a solution of protected dienol (2 g, 9 mmol) in ether (45 mL),
acetic anhydride (4.5 mL) and FeCl3 (167 mg) were added under
argon. The dark brown solution was stirred at room temperature for
23 h and allowed to settle. Na2HPO4 (16 mL) was added, and the
mixture was stirred for an additional 2 h. The organic phase was
separated; the aqueous one was washed with ether (3ꢀ20 mL). The
combined extracts were washed with water (20 mL) and brine
(20 mL), dried over Na2SO4, and concentrated. The dark brown
residue was dissolved in methanol (30 mL), and NaOH (1.6 g) in
water (9 mL) was added. The resulting mixture was stirred at room
temperature overnight, then poured into the ether (3ꢀ20 mL). The
combined organic extracts were washed with water (20 mL) and
brine (15 mL), and dried over Na2SO4. FC on silica gel, eluting with
3.1.4.7. (10E,12Z)-6-Fluoro-undeca-10,12-dien-1-ol (2). Alcohol 11
(160 mg, 0.47 mmol) was dissolved in CH2Cl2 (2.5 mL) and cooled to
ꢁ78 ꢂC. Then DAST (76
mL,1.2 equiv) was added and the mixture was
stirred for 1 h, then poured into water (20 mL), and extracted with
CH2Cl2 (20 mL). After the mixture was dried, a small amount of silica
was added, and the solvents were evaporated. The residue was ap-
plied on top of a column prepacked with silica gel. Elution with
CH2Cl2–pentane (4:1) provided the fluorinated THP protected 2
(128 mg, 80%). 1H NMR (400 MHz, CDCl3):
d
¼6.30 (dd, 1H, 12-CH,
CH2Cl2, gave 10 (1.14 g, 75%). 1H NMR (400 MHz, CDCl3):
d
¼6.24 (dd,
J1211¼15.0 Hz, J1213¼11.0 Hz), 5.99 (t, 1H, 11-CH, J1110¼15.0 Hz), 5.68
(dt, 1H, 13-CH, J1312¼11.0 Hz), 5.48 (m, 1H, 10-CH), 4.60 (m, 1H, CH-
THP), 4.49 (m, 1H, 6-CHF, JHF¼44 Hz), 3.89 (m, 1H, CHH-THP), 3.75
(m,1H, CHH-THP), 3.53 (m,1H,1-CHH), 3.42 (m,1H,1-CHH), 2.15 (m,
4H, 9-CH2, 14-CH2), 1.73 (m, 2H, CH2), 1.35–1.67 (m, 18H, 9ꢀCH2),
1H, 7-CH, J76¼15.0 Hz, J78¼11.0 Hz), 5.88 (t, 1H, 6-CH, J65¼15.0 Hz),
5.57 (m, 1H, 8-CH), 5.24 (m, 1H, 5-CH), 3.57 (m, 2H, 1-CH2), 2.06 (m,
3H, 9-CH2, OH),1.50 (m, 2H, 4-CH2),1.35 (m, 6H, 3ꢀCH2), 0.85 (m, 3H,
11-CH3). 13C (100 MHz, CDCl3):
d¼134.3 (C-5), 130.5 (C-6), 129.0 (C-
7), 126.4 (C-8), 63.1 (C-1), 32.9 (C-4), 32.6 (C-2), 30.1 (C-9), 25.9 (C-
10), 23.2 (C-3), 14.1 (C-11). EIMS, m/z (rel int.): 168 [M]ꢃ
0.94 (m, 3H,16-CH3).13C (100 MHz, CDCl3):
d¼133.7 (C-10),130.2 (C-
þ
.
11), 128.6 (C-12), 126.2 (C-13), 98.9 (C-10), 94.3 (d, C-6, JCF¼165 Hz)
67.5 (C-1), 62.4 (C-50), 35.1 (d, C-7, JCF¼21 Hz), 34.6 (d, C-5, JCF¼21 Hz)
32.6 (C-14), 30.8 (C-9), 29.7 (C-20), 29.6 (C-14), 26.2, 25.5, 24.9, 25.0
(d, JCF¼4 Hz) (d, JCF¼4 Hz) 22.9 (C-2,15, 8, 4, 3, 40),19.7 (C-30),13.7 (C-
16). EIMS, m/z (rel int.): 340 (25, [M]ꢃþ), 322 (35, [MꢁH2O]ꢃþ).
To a solution of THP ether (100 mg, 0.29 mmol) in methanol
(2 mL) PTSA monohydrate (76 mg, 2 equiv) was added, and resulting
mixture was stirred at room temperature for 1 h, then diluted with
3.1.4.5. 2-(5-Bromopentyloxy)-tetrahydro-2H-pyran. 5-Bromopent-
an-1-ol (1 g, 6 mmol), prepared from 1,5-pentandiol,27 was pro-
tected with DHP (0.54 mL, 1 equiv). FC on silica gel, eluted with
CH2Cl2–hexanes (2:3), provided the product (1.5 g, 99%). 1H NMR
(400 MHz, CDCl3):
d
¼4.50 (m, 1H, CH-20), 3.86 (m, 2H, CH2-60), 3.60
(m, 4H, CH2-1, CH2-5), 1.55 (m, 12H, 6ꢀCH2). 13C (100 MHz, CDCl3):

M. Mansurova et al. / Tetrahedron 65 (2009) 1069–1076
1075
ether(10 mL), washedwithwater(3ꢀ10 mL), anddriedoverNa2SO4.
FC onsilica gel, eluted withCH2Cl2, furnished2 (70 mg, 93%).1HNMR
0.28 mmol) and mercaptobutanol (35
m
L, 1.5 equiv) in THF (1 mL)
were added at 0 ꢂC. The suspension was stirred at 0 ꢂC for 1 h, and
at room temperature for 19 h, then poured into the ice-cold water
(5 mL), and extracted into the ether (3ꢀ5 mL). The combined or-
ganic phases were washed with HCl (0.5 M, 3 mL) and water
(3ꢀ3 mL), and dried over Na2SO4. FC on silica gel, eluting with
CH2Cl2–pentane (1:3), furnished 4 (85 mg, 87%). 1H NMR (400 MHz,
(400 MHz, CDCl3):
d
¼6.25 (dd, 1H, 12-CH, J1211¼15.0 Hz,
J1213¼11.0 Hz), 5.89 (t, 1H, 11-CH, J1110¼15.0 Hz), 5.58 (dt, 1H, 13-CH,
J¼11.0 Hz), 5.27 (m, 1H, 10-CH), 4.45 (m, 1H, 6-CHF, JCF¼40 Hz) 3.58
(m, 2H,1-CH2), 3.37 (s,1H, OH), 2.06 (m, 4H, 9-CH2,14-CH2),1.32 (m,
14H, 7ꢀCH2), 0.85(m, 3H,16-CH3).13C(100 MHz, CDCl3):
¼134.1(C-
d
10), 130.6 (C-11),129.0 (C-12),126.6 (C-13), 94.6 (d, C-6, JCF¼166 Hz)
CDCl3):
d
¼6.21 (m, 1H, 70-CH), 5.88 (t, 1H, 60-CH, J65¼15.0 Hz), 5.57
63.3 (C-1), 35.2 (C-7), 35.0 (C-5), 33.0 (2C) 31.5, 30.9, 30.1, 26.0, 23.3
(m, 1H, 80-CH), 5.26 (m, 1H, 50-CH), 3.60 (m, 2H, 1-CH2), 2.45 (m, 4H,
4-CH2,10-CH2), 2.06 (m, 4H, 40-CH2, 90-CH2),1.54 (2H, CH2),1.42 (2H,
CH2), 1.34 (m, 4H, 2ꢀCH2), 1.20 (m, 2H, CH2), 0.85 (m, 3H, 110-CH3).
(C-2, 3, 4, 8, 9, 14, 15), 19.7 (C-16). EIMS, m/z (rel int.): 256 [M]ꢃþ
.
3.1.4.8. (10E,12Z)-1-(Tetrahydro-2H-pyran-2-yloxy)hexadeca-10,12-
dien-6-on (12). Alcohol 11 (200 mg, 0.59 mmol) was oxidized as
described for 10. Filtration through sand/Celite/charcoal/sand fur-
13C (100 MHz, CDCl3):
d
¼133.8 (C-50), 130.1 (C-60), 128.6 (C-70),
126.1 (C-80), 62.4 (C-1), 32.6 (C-40), 32.6 (C-20), 32.6 (C-90), 31.9 (C-4,
C-10), 29.7 (CH2), 28.6 (CH2), 25.6 (C-100), 22.9 (C-30), 13.8 (C-110).
nished 12 (180 mg, 90%). 1H NMR (400 MHz, CDCl3):
d¼6.20 (dd,
EIMS, m/z (rel int.): 256 [M]ꢃþ
.
1H, 12-CH, J1211¼15.0 Hz, J1213¼11.0 Hz), 5.88 (t, 1H, 11-CH,
J1110¼15.0 Hz), 5.54 (m, 1H,13-CH), 5.27 (m, 1H, 10-CH), 4.49 (m, 1H,
CH-THP), 3.79 (m, 1H, CHH-THP), 3.66 (m, 1H, CHH-THP), 3.40 (m,
1H, 1-CHH), 3.30 (m, 1H, 1-CHH), 2.05 (m, 4H, 9-CH2, 14-CH2), 1.51
(m, 2H, CH2), 1.29 (m, 18H, 9ꢀCH2), 0.85 (m, 3H, 16-CH3). 13C
3.1.4.12. (E)-11-tert-Butoxy-1-iodoundec-1-ene (14). Iodide 14 was
prepared by hydroalumination–iodination sequence starting from
commercially available undecen-10-yn-1-ol in 94% overall yield as
described for iodide 8. 1H NMR (400 MHz, CDCl3):
d¼6.41 (m, 1H, 2-
(100 MHz, CDCl3):
d
¼211.4 (C-6), 133.6 (C-10), 130.8 (C-11), 128.9
CH), 5.90 (dt, 1H, 1-CH, J1110¼14.0 Hz), 3.24 (m, 2H, 11-CH2), 1.96 (m,
2H, 3-CH2), 1.43 (m, 4H, 2ꢀCH2), 1.19 (m, 10H, 5ꢀCH2), 1.01 (br s, 9H,
(C-12), 126.9 (C-13), 99.3 (C-20), 66.2 (C-1), 62.8 (C-60), 37.4 (C-7),
37.0 (C-5), 32.8 (C-9), 30.8 (C-30) 29.7 (2C, C-2, 14) 26.3, 25.4, 22.9
(3C) (C-3, 4, 8, 15, 50) 19.7 (C-40), 14.1 (C-16). EIMS m/z (rel int.): 336
(35, [M]þ), 318 (100, [MꢁH2O]ꢃþ).
3ꢀCH3). 13C (100 MHz, CDCl3):
d
¼147.0 (C-2), 74.4 (C-1), 72.6
(Cquad), 61.9 (C-11), 36.3 (C-3), 31.8, 31.0, 29.7, 29.5, 29.1 (4ꢀCH2),
27.8 (3ꢀCH3), 26.5, 22.8 (2ꢀCH2). EIMS, m/z (rel int.): 352 (10,
[M]ꢃþ), 279 (70, [MꢁOPg]þ).
3.1.4.9. (10E,12Z)-6,6-Difluoro-undeca-10,12-dien-1-ol (3). To a so-
lution of ketone 12 (180 mg, 0.54 mmol) in CH2Cl2 (4 mL) DAST (1 g,
10 equiv) was added; the mixture was stirred under reflux for 62 h,
then poured into water (20 mL), and extracted with CH2Cl2 (20 mL).
After the mixture was dried, a small amount of silica was added, and
the solvents were evaporated. The residue was applied on top of
a column packed with silica gel. Elution with CH2Cl2–pentane (4:1)
provided trifluorinated by-product 1,6,6-trifluoro-(10E,12Z)-unde-
cadiene (3b) (30 mg, 17%) and traces of deprotected 3 (0.5 mg, 2%).
3.1.4.13. (E)-16-tert-Butoxyhexadec-6-en-4-ynylacetate(15). Acetate
15 was prepared from iodide 14 and 1-acetyl-pent-4-yne via
Sonogashira coupling as described for enyne 9 in 75% yield. 1H NMR
(400 MHz, CDCl3):
d
¼5.97 (m, 1H, 6-CH), 5.38 (d, 1H, 7-CH,
J1110¼14.0 Hz), 4.09 (t, 2H, 1-CH2), 3.25 (t, 2H, 16-CH2), 2.32 (m, 2H,
3-CH2), 1.98 (m, 5H, Ac-Hs, 13-CH2), 1.78 (m, 2H, CH2) 1.48 (m, 4H,
2ꢀCH2), 1.20 (m, 10H, 5ꢀCH2), 1.11 (br s, 9H, 3ꢀCH3). 13C (100 MHz,
CDCl3):
d
¼171.5 (CO), 144.3 (C-7), 109.9 (C-6), 87.2 (C-5), 80.4 (C-4),
1H NMR (400 MHz, CDCl3) for 3b:
d¼6.20 (dd, 1H, 12-CH,
72.8 (Cquad), 63.6 (C-1), 62.0 (C-16), 33.4 (C-8), 31.1, 29.9, 29.8, 29.8,
29.5, 29.2, 28.3, 28.0 (3ꢀCH3), 26.6, 26.0, 19.6. EIMS, m/z (rel int.):
350 (15, [M].þ), 281 (65, [MꢁOPg]þ).
J1211¼15.0 Hz, J1213¼11.0 Hz), 5.89 (dt, 1H, 11-CH2, J1110¼15.0 Hz),
5.54 (m, 1H, 13-CH), 5.27 (m, 1H, 10-CH), 4.36 (dt, 2H, 1-CH2, JHF¼47
Hz, J12¼7 Hz) 2.05 (m, 4H, 9-CH2,14-CH2),1.29-1.75 (m,14H, 7ꢀCH2),
0.85 (m, 3H,16-CH3). EIMS m/z 276 [M]ꢃþ. 1H NMR (400 MHz, CDCl3)
3.1.4.14. (4Z,6E)-16-tert-Butoxyhexadeca-4,6-dienyl acetate (16).
Acetate 16 was prepared by hydroboration–hydrolysis of enyne 15
for 3:
d
¼6.20 (dd, 1H, 12-CH, J1211¼15.0 Hz, J1213¼11.0 Hz), 5.89 (dt,
1H, 11-CH2, J1110¼15.0 Hz), 5.54 (m, 1H, 13-CH), 5.27 (m, 1H, 10-CH),
as described for 10 in 65% yield. 1H NMR (400 MHz, CDCl3):
d
¼6.00
3.62 (m, 3H, 1-CH2, OH), 2.05 (m, 4H, 9-CH2, 14-CH2), 1.10-1.75 (m,
(m,1H,12-CH), 5.75 (t,1H,11-CH, J1110¼14.0 Hz), 5.46 (dt,1H,10-CH,
J1011¼14.0 Hz), 5.01 (m, 1H, 13-CH), 3.84 (t, 2H, 16-CH2), 3.08 (t, 2H,
1-CH2), 2.01 (m, 2H,14-CH2),1.85 (m, 2H, 4-CH2),1.81 (s, 3H, Ac-Hs),
1.50 (m, 4H, 2ꢀCH2), 1.20 (m, 14H, 7ꢀCH2), 1.10 (br s, 9H, 3ꢀCH3).
14H, 7ꢀCH2), 0.85 (m, 3H, 16-CH3). EIMS m/z (rel int.): 274 [M]ꢃþ
.
3.1.4.10. 1-Tosyl-(5E,7Z)-undeca-5,7-diene (13). The mixture of al-
cohol 10 (110 mg, 0.65 mmol), tosyl chloride (187 mg, 2 equiv), and
13C (100 MHz, CDCl3):
d
¼171.4 (CO), 135.8 (C-7), 130.1 (C-6), 128.2
triethylamine (34
mL) was stirred in pyridine (1.3 mL) at room
(C-5), 125.6 (C-4), 72.7 (Cquad), 64.2 (C-1), 62.0 (C-16), 33.4 (C-8),
31.1, 29.9, 29.8, 29.7, 29.6, 28.9 (CH2), 27.9 (3ꢀCH3), 26.6, 26.0, 24.4,
21.6 (CH2). EIMS, m/z (rel int.): 352 (15, [M]ꢃþ), 279 (70, [MꢁOPg]þ).
temperature for 20 h. The reaction mixture was poured into cold
water (5 mL) and extracted with ether (2ꢀ5 mL). The combined
extracts were washed with HCl (2 M, 3 mL), water (5 mL), NaHCO3
(5 mL), water (2ꢀ5 mL), and brine (3 mL), dried over Na2SO4, and
concentrated in vacuo. FC on silica gel, eluted with CH2Cl2–pentane
3.1.4.15. (10E,12Z)-16-Fluorohexadeca-10,12-dien-1-ol (5). The depro-
tection of acetate 16 was performed by standard procedure using
NaOH (4 equiv) and H2O in methanol in 99% yield. 1H NMR (400 MHz,
(1:3), gave 13 (95 mg, 60%). 1H NMR (400 MHz, CDCl3):
d
¼7.71 (d,
2H, Ph, J¼8.0 Hz), 7.26 (d, 2H, Ph, J¼8.0 Hz), 6.20 (dd, 1H, 7-CH,
J76¼15.0 Hz, J78¼11.0 Hz), 5.85 (t, 1H, 6-CH, J65¼15.0 Hz), 5.49 (m,
1H, 8-CH), 5.26 (m, 1H, 5-CH), 3.96 (m, 2H, 1-CH2), 2.35 (br s, 3H,
Ph-CH3), 2.00 (m, 4H, 9-CH2, 4-CH2), 144.6 (Ph), 1.48 (m, 6H,
CDCl3):
d
¼6.20 (m, 1H, 12-CH), 5.91 (t, 1H, 11-CH, J1110¼14.0 Hz), 5.62
(dt, 1H, 10-CH, J1110¼14.0 Hz), 5.25 (m, 1H, 13-CH), 3.59 (t, 2H, 16-CH2),
3.25 (t, 2H, 1-CH2), 2.19 (m, 2H, 14-CH2), 2.01 (m, 2H, 4-CH2), 1.59 (m,
2H, CH2), 1.44 (m, 4H, 2ꢀCH2), 1.21 (m, 10H, 5ꢀCH2), 1.11 (br s, 9H,
3ꢀCH2), 0.85 (m, 3H, 11-CH3). 13C (100 MHz, CDCl3):
d
¼144.6
3ꢀCH3). 13C (100 MHz, CDCl3):
¼135.7 (C-10), 129.8 (C-11), 129.1 (C-
d
(Ph),133.3 (C-5), 133.1 (Ph), 130.4 (C-6), 129.7 (2C, Ph), 128.5 (C-7),
127.7 (2C, Ph), 126.4 (C-8), 70.4 (C-1), 31.6 (C-4), 30.0 (C-2), 28.3 (C-
9), 25.1 (C-10), 22.6 (C-3), 21.6 (Ph-Me), 14.1 (C-11). EIMS, m/z (rel
int.): 322 (10, [M]ꢃþ), 151 (50, [MꢁOTs]þ).
12), 125.7 (C-13), 72.8 (Cquad), 62.8 (C-16), 62.1 (C-1), 33.2 (C-9), 33.0,
32.0, 31.1, 29.9 (2C), 29.8, 28.0 (3ꢀCH3), 26.6, 24.4, 23.0. EIMS, m/z (rel
t
int.): 236 (75, [MꢁOtBu].þ), 253 (25, [Mꢁ Bu]þ), 295 (40, [MꢁCH3]),
310 (35, [M]þ). The fluorination of terminal hydroxy group in
deacetylated 16 was performed with DAST as described for 2 in 64%
3.1.4.11. 4-((5E,7Z)-Undeca-5,7-dienylthio)butan-1-ol (4). To a sus-
pension of NaH (14 mg, 2 equiv) in THF (1 mL) tosylate 14 (90 mg,
yield. 1H NMR (400 MHz, CDCl3):
11-CH, J1110¼14.0 Hz), 5.64 (dt, 1H, 10-CH, J1110¼14.0 Hz), 5.25 (m, 1H,
d
¼6.19 (m, 1H, 12-CH), 5.93 (t, 1H,

1076
M. Mansurova et al. / Tetrahedron 65 (2009) 1069–1076
3. Tegoni, M.; Campanacci, V.; Cambillau, C. Trends Biochem. Sci. 2004, 29, 257–
264.
4. Krieger, J.; Brier, H. Transduction Mechanism of Olfactory Sensory Receptors.
In Insect Pheromone Biochemistry and Molecular Biology; Blomquist, G. J.,
Vogt, R., Eds.; Elsevier Academic: London, 2003.
5. Sandler, B. H.; Nikonova, L.; Leal, W. S.; Clardy, J. Chem. Biol. 2000, 7, 143–151.
6. Kaissling, K. E. Chem. Senses 2004, 29, 529–531 Erratum 747.
7. Kaissling, K. E.; Rospars, J. P. Chem. Senses 2001, 26, 125–150.
8. Kaissling, K. E.; Priesner, E. Naturwissenschaften 1970, 57, 23–28.
9. Du, G.; Prestwich, G. D. Biochemistry 1995, 34, 8726–8732.
13-CH), 4.39 (dt, 2H, 16-CH2, JHF¼47 Hz, J1516¼6 Hz) 3.25 (t, 2H, 1-
CH2, J12¼7 Hz), 2.23 (m, 2H,14-CH2), 2.02 (m, 2H, 4-CH2), 1.68 (m, 4H,
2ꢀCH2), 1.49 (m, 4H, 2ꢀCH2), 1.21 (m, 8H, 4ꢀCH2), 1.11 (br s, 9H,
3ꢀCH3). 13C (100 MHz, CDCl3):
¼136.0 (C-10), 130.2 (C-11), 128.1 (C-
d
12), 125.6 (C-13), 83.8 (d,C-16, JCF¼164 Hz) 72.8 (Cquad), 62.0 (C-1), 33.8
(C-9), 33.3, 31.1, 30.9, 30.7, 29.9, 29.6, 28.0 (3ꢀCH3), 26.6, 25.0, 23.7. EIMS,
ꢃ
ꢃ
t
m/z (rel int.): 312 (35, [M] þ), 297 (10, [MꢁCH3]þ), 255 (25, [Mꢁ Bu]þ).
The deprotection of (4Z,6E)-16-tert-butoxy-1-fluorohexadeca-
4,6-diene was performed in two steps as described for 10 in 51%
ˇ
10. Oldham, N. J.; Krieger, J.; Breer, H.; Fischedick, A.; Hoskovec, M.; Svatos, A.
Angew. Chem., Int. Ed. 2000, 39, 4341–4343.
overall yield. Compound 5: 1H NMR (400 MHz, CDCl3):
d
¼6.19 (m,
11. Leal, W. S.; Chen, A. M.; Ishida, Y.; Chiang, V. P.; Erickson, M. L.; Morgan, T. I.;
Tsuruda, J. M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5386–5391.
12. Campanacci, V.; Krieger, J.; Bette, S.; Sturgis, J. N.; Lartigue, A.; Cambillau, C.;
Breer, H.; Tegoni, M. J. Biol. Chem. 2001, 276, 20078–20084.
1H, 12-CH), 5.93 (t, 1H, 11-CH, J1110¼14.0 Hz), 5.64 (dt, 1H, 10-CH,
J1110¼14.0 Hz), 5.20 (m, 1H, 13-CH), 4.39 (dt, 2H, 16-CH2, JHF¼47 Hz,
J1516¼7 Hz) 3.57 (t, 2H, 1-CH2), 2.22 (m, 2H, 14-CH2), 2.02 (m, 2H, 4-
CH2), 1.65 (m, 2H, CH2), 1.49 (m, 4H, 2ꢀCH2), 1.22 (m, 10H, 5ꢀCH2).
ˇ
´ ˇ
ˇ
13. Klusa´k, V.; Havlas, Z.; Rulı´sek, L.; Vondrasek, J.; Svatos, A. Chem. Biol. 2003, 10,
331–340.
ˇ
ˇ
14. Hoskovec, M,; Saman, D.; Svatos, A. Collect. Czech. Chem. Commun. 2001, 66,
1682–1690.
13C (100 MHz, CDCl3):
d
¼135.9 (C-10), 130.2 (C-11), 128.1 (C-12),
125.6 (C-13), 83.8 (d, C-16, JCF¼164 Hz) 63.5 (C-1), 33.2 (2C), 29.9,
15. Alexakis, A.; Gardette, M.; Colin, S. Tetrahedron Lett. 1988, 29, 2951–2954.
16. Bown, H. C.; Korytnyk, W. J. Am. Chem. Soc. 1966, 82, 3866–3870.
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29.8 (3C), 29.7, 29.6, 26.1, 23.5 EIMS, m/z (rel int.): 256 [M]ꢃþ
.
Acknowledgements
Phys. Rev.
B 1988, 37, 785–789; Becke, A. D. J. Chem. Phys. 1993, 98,
5648–5652.
We thank Sybille Koch and Sybille Lorenz for sector-field EIMS
analysis and Alfredo Iban˜ez for KD calculations. We gratefully ac-
knowledge the financial support by the Max Planck Society.
20. Hehre, W. J.; Radom, L.; Schleyer, P. V.; Pople, J. A. Ab Initio Molecular Orbital
Theory; Wiley-Interscience: New York, NY, 1986.
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