Synthesis of linoleic acids combinatorially labeled at the vinylic positions as substrates for lipoxygenases

Article abstract of DOI:10.1016/j.tetlet.2008.04.023

Mammalian lipoxygenases have been implicated in a number of inflammation-related human diseases. Soybean lipoxygenase-1 is the archetypical example of known lipoxygenases. Here we report the synthesis of linoleic acid and (11,11)-d2-linoleic acid which are combinatorially labeled at the vinylic positions (9, 10, 12, and 13). Combinatorial labeling schemes allow for the simultaneous determination of KIEs in enzymatic reactions using NMR. Substrates are, thus, available as probes of detailed mechanism in kinetic isotope effect (KIE) studies of lipoxygenases.

Full text of DOI:10.1016/j.tetlet.2008.04.023

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3600–3603
Synthesis of linoleic acids combinatorially labeled at the
vinylic positions as substrates for lipoxygenases
*
*
Matthew P. Meyer , Judith P. Klinman
Department of Chemistry, University of California, Berkeley, CA 94720, United States
Received 6 August 2007; revised 1 April 2008; accepted 2 April 2008
Available online 6 April 2008
Abstract
Mammalian lipoxygenases have been implicated in a number of inflammation-related human diseases. Soybean lipoxygenase-1 is the
archetypical example of known lipoxygenases. Here we report the synthesis of linoleic acid and (11,11)-d2-linoleic acid which are com-
binatorially labeled at the vinylic positions (9, 10, 12, and 13). Combinatorial labeling schemes allow for the simultaneous determination
of KIEs in enzymatic reactions using NMR. Substrates are, thus, available as probes of detailed mechanism in kinetic isotope effect
(
KIE) studies of lipoxygenases.
Ó 2008 Elsevier Ltd. All rights reserved.
Human lipoxygenases which catalyze the oxidation of
arachidonic acid have been implicated in a number of
leads to the formation of a radical which may be in one
of three configurations. Energetics favor the formation of
1
a
1b
diseases, including asthma, atherosclerosis, colorectal
1
c
1d
1a
cancer, prostate cancer, and arthritis. Because these
enzymes catalyze the same basic reaction as plant lipoxy-
genases, mechanistic studies of the easily obtainable soy-
bean lipoxygenase-1 (SLO-1) are of importance to the
1
3 12 10
HS HR
9
C H
C H14COOH
7
1
5
11
2
HO Fe(III)
design of mechanistic inhibitors. Soybean Lipoxygenase-
1
1
(SLO-1) catalyzes the oxidation of linoleic acid (1) to
3-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (13-
O
1
0
9
O
1
2
1
3 12 10
9
12
10
1
3
9
R'
R
R
R'
1
3
(
S)-HPOD) (Fig. 1). This conversion proceeds by the initial
2
a
2b
R
2c
R'
rate-determining abstraction of the pro-S hydrogen atom
from the methylene at C11. Following this, in an ordered
fashion, is the combination of molecular oxygen with the
H2O Fe(II)
3
intermediate radical. Transfer of an electron and proton
1
2
10
9
OO
from the active site to the resulting peroxyl radical (3)
regenerates the active Fe(III) form of the enzyme and
results in product (4). The initial rate-determining step
C H
C H14COOH
3
4
5
11 13
7
HR
H
O
Fe(II)
H
HOO
*
C H
C H14COOH
7
Corresponding authors. Present address: School of Natural Sciences,
5
11
University of California, Merced, CA 95344, United States. Tel.: +1 209
HR
2
2
28 2982; fax: +1 209 228 4053 (M.P.M.); tel.: +1 510 642 2668; fax: +1
09 643 6232 (J.P.K.).
HO
Fe(III)
E-mail addresses: mmeyer@ucmerced.edu (M. P. Meyer), klinman@
berkeley.edu (J. P. Klinman).
Fig. 1. Consensus mechanism for oxidation of linoleic acid catalyzed by
SLO-1.
0
040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.023

M. P. Meyer, J. P. Klinman / Tetrahedron Letters 49 (2008) 3600–3603
3601
the fully delocalized pentadienyl radical (2a). The regio-
specificity of the oxygen combination with the radical has
led to the proposal of an allyl radical delocalized over posi-
tions 11–13 (2b). Finally, data collected from EPR experi-
ation of NMR signals while minimizing any double label-
ing in these positions. Double labeling is minimized in
9
order to avoid spurious contributions to the measured iso-
tope effects from breakdowns in the rule of the geometric
1
0
ments suggest a radical delocalized over positions 9–11
mean. Because the oxidation of linoleic acid catalyzed
by lipoxygenases is thought to occur largely by a tunneling
mechanism, it is instructive to measure secondary deute-
rium isotope effects as a function of the tunneling entity.
For this reason, combinatorially labeled linoleic acids were
synthesized separately with protium (1a) and deuterium
(1b) at the methylene central to the pentadiene moiety.
The tail fragments (7a and 7b) of these targets were syn-
thesized in high yield from the commercially available
4
(
2c). Work by Knapp et al. indicates that the protein con-
trols the regio- and stereospecificity of O insertion, sug-
2
gesting that any of the three proposed intermediates are
5
possible.
Secondary deuterium kinetic isotope effects (KIEs) can
be potent probes of structural changes that occur in iso-
6
tope-sensitive reaction steps. However, given the generally
small magnitude of these isotope effects, it is useful to per-
form the experiments competitively. Competitive isotope
effects can be carried out in three basic ways. The remote
label method has been widely used in determining second-
11
methyl 2-octynoate (Scheme 1). The reduction step typi-
cally resulted in crude yields in excess of 95%; whereas,
1
2
the bromination step was essentially quantitative. The
7
ary KIEs in enzymatic systems. This method requires the
reduction of propargylic alcohols with LiAlH is known
4
1
3
synthesis of molecules where tritium is specifically incorpo-
rated into the remote position at tracer levels and reports
the presence of a deuterium in the position of interest.
While this approach is very accurate, impurities containing
the tritium label can adversely affect the result of the mea-
surement. Isotope ratio mass spectrometry can be used to
accurately measure isotope effects if the reactant or product
being analyzed can be selectively converted into a volatile
molecule, where the isotopic site of interest in the original
to give a (Z)-allylic alcohol. Some of this undesired prod-
uct (typically less than 1% for LiAlD and 2–10% for
4
LiAlH ) was formed from over-reduction and was removed
4
by reacting the crude product with m-chloroperoxybenzoic
1
4
acid in chloroform. The resulting epoxide formed from
the allylic alcohol was then separated from the propargylic
alcohol using flash chromatography (see Supplementary
data). The presence of the undesired (Z)-allylic alcohol
can be minimized from the outset by using a smaller excess
8
analyte is present. This technique is usually applied to
of LiAlL and by adding the ester starting material slowly
4
investigations of heavy atom isotope effects (C, O, and
N). A third possibility involves the use of NMR-active
nuclei as isotopic probes. Although NMR is not as quan-
to the LiAlL /diethyl ether slurry.
4
Because no labeling is performed on the head fragment
(11) of the target molecules prior to the alkyne coupling
step, the synthesis of these fragments utilized inexpensive
starting materials and lower-yield reactions that were
performed on a larger scale (Scheme 2). Synthesis of
9-decyn-1-ol (10) can be performed on technical grade
9-decen-1-ol since contaminating isomers with internal
double bonds will not yield terminal alkynes at the end
of the synthesis and will not undergo coupling. The
efficiency of this scheme lies in the purification steps. The
9-decyn-1-ol (10) is distilled away from brominated con-
taminants. No purification step is undertaken immediately
following bromination. The Jones oxidation results in the
desired 9-decynoic acid with an expected ester condensa-
9
titatively accurate as scintillation counting or isotope ratio
mass spectrometry, several isotope effects can be deter-
mined simultaneously. One reason for this approach to
have only seen a wide application in small molecule organic
reactions is the large amounts of sample needed for accu-
rate analysis of isotopomers at natural abundance. This
constraint can be circumvented through combinatorial
labeling schemes, where only positions of interest in the
substrate are labeled (Fig. 2).
The synthetic approach to combinatorially labeled lino-
leic acids taken here is optimally suited to the measurement
of multiple secondary deuterium: the incorporation of
0
15
<5% deuterium at positions 8, 9 and 11, 12 allows optimiz-
tion product, 9 -decynyl 9-decynoate. The methylation
L'
C5H11
L' L'
L'
C7H14CO2H
L'
HOO
C5H11
L' L'
L'
C7H14CO2H
3
5%
LiAlL4 / Et2O
O
(
-78 º C addition)
L
L
L
L
L
OMe
(-45 º C, 4h)
OH
1
1
4a (L=H, L'=4% D / 96% H)
4b (L=D, L'=4% D / 96% H)
Compare deuterium
content in labeled
L') sites
9
8 % (L=H)
00% (L=D)
5
1
6
6
a (L=H)
b (L=D)
(
L'
C5H11
L' L'
L'
C7H14CO2H
L'
L' L'
L
L'
C7H14CO2H
HOO
1
00%
Ph P:, Br /
3 2
C5H11
CH Cl , 0 oC
L
L
L
L
2
2
Br
1
1
4a (L=H, L'=4% D / 96% H)
4b (L=D, L'=4% D / 96% H)
100%
7
a (L=H)
b (L=D)
7
Fig. 2. Conceptual depiction of secondary kinetic isotope effect experi-
ments for which 1a and 1b were synthesized.
Scheme 1. Synthesis of fragment 7.

3602
M. P. Meyer, J. P. Klinman / Tetrahedron Letters 49 (2008) 3600–3603
Br2 / CH Cl
catalyst. If one is using a reduction method to label vinylic
2
2
OH
OH
positions, this method poses two obstacles. First, adsorbed
hydrogen on the platinum surface must be displaced by the
label or mixture of label and carrier if the degree of labeling
is to be controlled. Second, metal-mediated reductions tend
1
00 % crude yield
8
9
Br
40% KOH,
2
2
5
5 % purified yield
to exchange hydrogens at allylic positions. If this tech-
nique were used to make substrates for isotope effect mea-
surements at the vinylic positions, it could lead to two
situations whereby isotope effects would be incorrectly
measured. If label is mistakenly incorporated at a site that
has an intrinsically larger normal isotope effect (i.e., the
pro-S-11-position), the isotope effect on the vinylic posi-
tions would be overestimated. Likewise, mistaken incorpo-
ration of label at positions with inherently lower normal or
inverse isotope effects would lead to underestimation of the
isotope effect at the vinylic positions. This problem is par-
ticularly troublesome in isotope effects measured using
scintillation counting and mass spectrometry.
Br
1
2
.) Jones Oxidation
.) H2SO4 / MeOH
OH
7
2% purified yield
10
OMe
1
1
O
Scheme 2. Synthesis of fragment 11.
step following oxidation allows one to reclaim this byprod-
uct as the methyl ester via transesterification. The product,
-decynoate (11), is then purified by flash chromatography
Additional advantages of using catecholborane follow.
First, one can ensure full reduction of the alkyne to the
alkene by using 1.5 or more equivalents of catecholbora-
9
over silica (see Supplementary data).
1
6,17
18
The coupling
and reduction steps (Scheme 3)
1
8
ne. Second, over-reduction does not occur since the
resulting alkenylborane is electron poor and sterically hin-
dered. These aspects of the catecholborane reduction are
quite critical, since separation of compounds that differ
only in one element of unsaturation would require an
HPLC separation step, which severely limits the amount
of material that can be purified. For the combinatorial
labeling undertaken in this synthesis, the label was incorpo-
rated using a mixture of acetic acid and readily available
deuterated acetic acid for acetolysis of the alkenylborane.
In order to achieve approximately 4% labeling, the deuter-
ated acetic acid had to be present at 20% in the acetolysis
mixture, implying a primary isotope effect of about 5 for
acetolysis of internal alkenylcatecholboranes.
which conclude this synthesis illustrate its advantages over
other similar syntheses. The most likely alternative to a
simple Cu(I) coupling is a Grignard-mediated Cu(I) cou-
1
9–21
pling.
The primary advantage of the coupling reaction
used here is the tolerance of groups that are labile in the
presence of Grignard reagents. While this method has been
particularly useful for some couplings, an attempt to cou-
ple 9-decynoic acid with 7a led to very small yields of the
desired product.
The reduction step, using catecholborane, offers a num-
ber of benefits in this synthesis. Reduction of alkynes to
(
Z)-alkenes is most commonly performed using Lindlar’s
1eq. 7a (L=H) or 7b (L=D) / 0.95 eq. 11
7
5
9% 2eq. CuI, 2eq. NaI, 1.5eq. K2CO3 / DMF
L
L
O
OMe
1
2a (L=H), 12b (L=D)
1
.) catecholborane, 1.5 eq.
7% 2.) HOAc (8.8 eq.)/
DOAc (1.2 eq.)
L' L'
O
L'
L'
OMe
L
L
1
1
3a (L=H, L'=4% D / 96% H), 13b (L=D, L'=4% D / 96% H)
1
00% 30% KOH, reflux
L' L'
O
L'
L'
OH
L
L
4a (L=H, L'=4% D / 96% H), 14b (L=D, L'=4% D / 96% H)
Fig. 3. Comparison of the temperature dependence for kcat for SLO-1
catalyzed oxidation of d31-linoleic acid (filled circles) and 14b (filled
diamonds).
Scheme 3. Coupling of fragments 7 and 11; subsequent reduction of
diyne.

M. P. Meyer, J. P. Klinman / Tetrahedron Letters 49 (2008) 3600–3603
3603
To confirm the viability of the newly synthesized combi-
natorially labeled linoleic acid, 14b, the temperature depen-
dence of the unimolecular rate constant, kcat, was tested
against data obtained with perdeuteriolabeled d31-linoleic
acid for SLO-1. Figure 3 shows that the unimolecular rate
constant has a similar temperature dependence for both
substrates, an indication that synthesis-dependent impuri-
ties are not influencing the enzyme-catalyzed oxidation of
the synthesized substrate. If any differences are to be noted
for the substrates, it would be that the d31-labeled sub-
strate has a higher rate constant on average. This may be
due to incomplete deuterium incorporation, since this sub-
strate was isolated from a perdeuterated set of fatty acids
isolated from plants grown on deuterated sources.
The synthesis included in this letter provides a means by
which alkenes may be combinatorially labeled for use in
the simultaneous determination of isotope effects. Aside
from the utility of the target molecules in enzyme studies,
the synthesis of molecules containing (Z,Z)-pentadienyl
moieties has been improved upon.
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Acknowledgments
1
1
This work was supported in part by GM25765 from the
National Institutes of Health. Matt Meyer also thanks the
NIH for funding under GM64218.
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Experimental procedures and spectral characterization
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