Synthesis of site-specifically13C labeled linoleic acids

Article abstract of DOI:10.1016/j.tetlet.2016.08.071

Soybean lipoxygenase-1 (SLO-1) catalyzes the C–H abstraction from the reactive carbon (C-11) in linoleic acid as the first and rate-determining step in the formation of alkylhydroperoxides. While previous labeling strategies have focused on deuterium labeling to ascertain the primary and secondary kinetic isotope effects for this reaction, there is an emerging interest and need for selectively enriched¹³C isotopologues. In this Letter, we present synthetic strategies for site-specific¹³C labeled linoleic acid substrates. We take advantage of a Corey–Fuchs formyl to terminal¹³C-labeled alkyne conversion, using¹³CBr₄as the labeling source, to reduce the number of steps from a previous fatty acid¹³C synthetic labeling approach. The labeled linoleic acid substrates are useful as nuclear tunneling markers and for extracting active site geometries of the enzyme–substrate complex in lipoxygenase.

Full text of DOI:10.1016/j.tetlet.2016.08.071

Tetrahedron Letters 57 (2016) 4537–4540
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of site-specifically 13C labeled linoleic acids
Adam R. Offenbacher a,b, Hui Zhu a,b,y, Judith P. Klinman a,b,c,
⇑
a Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, United States
b Quantitative Institutes of Biosciences, University of California, Berkeley, Berkeley, CA 94720, United States
c Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA 94720, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Soybean lipoxygenase-1 (SLO-1) catalyzes the C–H abstraction from the reactive carbon (C-11) in linoleic
acid as the first and rate-determining step in the formation of alkylhydroperoxides. While previous label-
ing strategies have focused on deuterium labeling to ascertain the primary and secondary kinetic isotope
effects for this reaction, there is an emerging interest and need for selectively enriched 13C isotopologues.
In this Letter, we present synthetic strategies for site-specific 13C labeled linoleic acid substrates. We take
advantage of a Corey–Fuchs formyl to terminal 13C-labeled alkyne conversion, using 13CBr4 as the label-
ing source, to reduce the number of steps from a previous fatty acid 13C synthetic labeling approach. The
labeled linoleic acid substrates are useful as nuclear tunneling markers and for extracting active site
geometries of the enzyme–substrate complex in lipoxygenase.
Received 28 July 2016
Revised 19 August 2016
Accepted 20 August 2016
Available online 22 August 2016
Keywords:
Secondary isotope effects
Hydrogen tunneling
Lipoxygenase
Ó 2016 Elsevier Ltd. All rights reserved.
Linoleic acid
Lipoxygenases, widely represented in animals, plants, fungi, and
prokaryotes, have gained much attention in the field due to obser-
vations of enormous, non-classical primary deuterium kinetic iso-
tope effects (KIEs) in the range of 20–120 for the native enzymes.1
These enzymes catalyze the C–H abstraction from long chain
polyunsaturated fatty acids in the process of preparing diverse
(per)oxidized metabolites, important for growth, development,
and pathogenic defense in plants and various cell signaling and
inflammatory response pathways in animals.2 Importantly, soy-
bean lipoxygenase-1 (SLO-1) has emerged as one of the paradig-
matic enzymes in hydrogen tunneling, because the rate-
determining hydrogen atom abstraction in the native form is asso-
ciated with a very large primary KIE at room temperature (ca. 80),
center and are integral for efficient hydrogen wave function
overlap in SLO-1.5
Inflated, non-classical primary deuterium isotope effects
(kH/kD > 7), together with deviations in Swain–Schaad relationships
(especially for secondary D/T KIEs) and temperature independent
KIEs, are considered as kinetic signatures for full tunneling in enzy-
matic reactions occurring at room temperature.6 In the case of
lipoxygenases, many synthetic strategies have been reported for
site-specific deuterium labeled substrates, but there has also been
an emerging interest in 13C labels for these compounds. While the
tunneling properties have been ascribed to the transferred hydro-
gen, recent evidence in SLO-1 reaction points toward the tunneling
of heavy atoms in the backbone of the substrate.7 In that study, the
13C KIEs were measured from natural abundance 13C using an NMR
approach designed for small molecules by Singleton.8 To overcome
the relatively large errors associated with these measurements for
enzymatic reactions, a high precision 1H-detected 13C heteronu-
clear single quantum coherence (HSQC) 2 dimensional (2D) NMR
approach has been previously reported for the determination of
such 13C KIEs.9 This technique requires the site-specific labeling
of the carbon atom of interest and a carbon atom, adjacent to the
target carbon atom so that they are spin-coupled, as a reporter.10
While 1 dimensional (1D) 13C NMR techniques are typically easier
and faster and can be utilized to test these competitive isotope
effects,10 the HSQC approach9 may offer enhanced signal-to-noise,
which is particularly advantageous in the lipoxygenase reaction
that is limited to low (micromolar) substrate concentrations. In
addition, site-specific 13C labeling of arachidonic acid has
in a nearly temperature independent manner (D
Ea = 0.9 kcal/mol).3
As shown in Figure 1, for the SLO-1 reaction, a mononuclear, non-
heme ferric hydroxide accepts the transferred electron and proton,
respectively, generating a ferrous water cofactor and a pentadienyl
radical. Molecular oxygen is then inserted into the backbone, selec-
tively at position 13 for wild-type SLO-1.1a,4 A second round of
proton-coupled electron transfer (PCET) regenerates the active
ferric-hydroxide cofactor and yields the product, 13S-
hydroperoxyoctadecadienoic acid. Also shown in Figure 1 are two
amino acid side chains, Leu546 and Leu754, which position the
reactive carbon in proximity to the ferric-hydroxide reactive
⇑
Corresponding author.
E-mail address: klinman@berkeley.edu (J.P. Klinman).
Current address: School of Chemistry and Biochemistry, Georgia Institute of
y
Technology, Atlanta, GA 30332, United States.
http://dx.doi.org/10.1016/j.tetlet.2016.08.071
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

4538
A. R. Offenbacher et al. / Tetrahedron Letters 57 (2016) 4537–4540
Figure 1. Mechanism of linoleic acid peroxidation by soybean lipoxygenase-1. The
first and final steps of the reaction are associated with proton-coupled electron
transfer (PCET) processes. The important, conserved aliphatic residues (Leu546 and
Leu754) that position the reactive carbon (C-11 of linoleic acid) against the active
site ferric hydroxide cofactor in SLO-1 are modeled for reference.
previously served to assign the magnetic resonance spectral contri-
butions of the delocalized radicals, generated upon reaction with
prostaglandin H synthase.11 While arachidonic acid is the preferred
substrate of mammalian lipoxygenases2a and exhibits rate
constants and deuterium KIE for the reaction with SLO-1 that is
comparable to linoleic acid, the latter is considered the
physiological substrate for the plant enzyme.12 This manuscript
describes synthetic strategies for generating site-specific 13C
enriched linoleic acids. These labeled substrates can be applied to
evaluate the extent of tunneling in the substrate backbone from
13C KIEs by 1D 13C or 2D HSQC NMR techniques, to assign magnetic
resonance signatures of the substrate radicals in the lipoxygenase
reaction, and/or to resolve the elusive donor–acceptor distances
in various lipoxygenase enzymes using magnetic resonance tech-
niques sensitive to electron-nuclear couplings.13
Scheme 1. Synthesis of 11-[13C]-1. The asterisks denote the position of the 13C
label.
hydrogenation to linoleic acid, 11-[13C]-1. The hydrogenation reac-
tion was performed with standard Lindlar catalyst and H2. Further,
our hydrogenation reactions were carried out in the presence of
quinolones which enhance selectivity for 9Z, 12Z alkene produc-
tion.15 HPLC analyses of the purified final products indicated only
linoleic acid (C18:2
D
9,12) with no detectable amounts of oleic
Our first target is the isotopologue with 13C enriched at the
reactive carbon, C-11, of linoleic acid (11-[13C]-1), which, as shown
in Scheme 1, was synthesized in two parts with an unlabeled frag-
ment, 2-(dec-9-yn-1-yloxy)tetrahydro-2H-pyran (3) and a 13C
labeled fragment, 1-bromo-2-octyne (1-[13C]-4). As shown for
example in Scheme 1, the two fragments 3 and 4 were joined via
a Cu(I)-assisted Grignard coupling reaction. A similar strategy
was previously used in our laboratory with 1,1-[2H2]-4 and 9-decy-
noic acid that produced low yields of the desired, stable intermedi-
ate in the synthesis of monodeuterated or dideuterated linoleic
acids.14 To overcome these previous pitfalls, we started with
commercially available 9-decyn-1-ol (2), which we protected
through the overnight reaction of 3,4-dihydro-2H-pyran (DHP) to
generate 3. In this manner, only 1 equiv of the ethylmagnesium
bromide (EtMgBr) is required to deprotonate the terminal alkyne,
3. This strategy produced reasonable yields (ca. 70%) of the desired
11-[13C]-2-(octadeca-9,12-diyn-1-yloxy)tetrahydro-2H-pyran,
11-[13C]-5. In the course of arachidonic acid synthesis for prosta-
glandin H synthase substrates, Peng et al. followed a similar
approach that led to high yields (86%).11 One caveat is that a
fraction of the starting material 3 is virtually inseparable from
product 11-[13C]-5 with silica gel purifications. We maintained
the contaminating starting material from the coupling step to the
end, because it did not interfere with the final steps. In our final
purification step, linoleic acid could be separated cleanly and easily
from this impurity using a reverse phase C18 column.
(C18:1
D
9) or stearic (C18:0) acids, consistent with no over-reduc-
tion. 1H NMR spectra were consistent with cis,cis-9,12 isomer. As
an alternative approach, described by our group previously,16 cat-
echolborane can also be used to achieve reduction selectivity.15
The 13C labeled 1-bromo-2-octyne fragment, 1-[13C]-4, was syn-
thesized as described in Scheme 2. First, 2-octyn-1-ol (10) was
generated from the deprotonation of the commercially available
1-heptyne 8 with EtMgBr that was subsequently reacted with
13C-paraformaldehyde ([13C]-9). This route has been described
previously for the synthesis of 1-[2H]-10 and 1,1-[2H2]-10.14,17
Bromination of 1-[13C]-10 compounds to the corresponding
desired 1-[13C]-4 was carried out cleanly with Ph3P/Br2.
As depicted in Scheme 3, the linoleic acids, 10-[13C]-11,11-
[2H2]-1 and 10,11-[13C2]-11,11-[2H2]-1 were also synthesized in
two parts, with a common 13C labeled C-1 to C-10 fragment (2).
The label (12C or 13C) at reactive carbon in the final product, 1,
was controlled by the nature of the carbon isotope in the 1-
bromo-2-octyne (3) fragment. In addition, deuterium was also
included at the reactive carbon (C-11) for both linoleic acids in
Scheme 3. Our lab has previously shown that for the WT SLO-1
enzyme chemistry is partially rate limiting with the protium sub-
strate under certain conditions, but becomes fully rate limiting
with deuterated substrate.1a Therefore, dideuterated substrates
would be required to isolate the intrinsic KIE of the reactive
carbon from NMR competitive measurements.
After the coupling that generates 11-[13C]-3, the linoleic acid
substrate could be completed (Scheme 1) by deprotection of
11-[13C]-5, liberating the corresponding alcohol, 11-[13C]-6, followed
by oxidation to the acid, 11-[13C]-7, with Jones reagent and
The protection of the 1,9-nonanediol (11) resulted in a mixture
of starting material 11, desired single protected 9-((tetrahydro-2H-
pyran-2-yl)oxy)nonan-1-ol (12) and fully protected species.
Because the starting material 11 was inexpensive and the desired

A. R. Offenbacher et al. / Tetrahedron Letters 57 (2016) 4537–4540
4539
Scheme 2. Synthesis of fragment 1-[13C]-4 using 13C-paraformaldehyde, [13C]-9.
The asterisks denote the position of the 13C label.
compound 12 was easily separable with silica gel purification, we
could prepare this compound on a relatively large scale. As illus-
trated in Scheme 4, oxidation of the alcohol 12 to the correspond-
ing 9-((tetrahydro-2H-pyran-2-yl)oxy)nonan-1-ol (13) was carried
out with pyridinium chlorochromate (PCC) and sodium acetate to
maintain the THP protecting group. The highlight within this syn-
thesis is the preparation of 2-(10-[13C]-dec-9-yn-1-yloxy)tetrahy-
dro-2H-pyran (10-[13C]-3). This step was accomplished by a
formyl-to-ethynyl conversion, first described by Corey and Fuchs,18
using the aldehyde 13 and triphenylphosphine-carbon tetrabro-
mide-13C. The inclusion of 1 equiv of anhydrous triethylamine19
during the addition of the aldehyde 13 to the stirring triph-
enylphosphine-carbon tetrabromide solution in CH2Cl2 was essen-
tial to produce the desired 2-((10-[13C]-10,10-dibromodec-9-en-1-
yl)oxy)tetrahydro-2H-pyran (10-[13C]-14). Due to the cost of the
13C isotopologue, we reduced the starting equivalents of 13C–car-
bontetrabromide from the recommended 2 equiv18,19 to 1 equiv
in this Letter. Even with only 1 equiv of 13CBr4 (and 2 equiv
Ph3P), we were able to cleanly produce the desired 10-[13C]-14 in
very satisfactory yields (ca. 90%).
Scheme 4. Synthesis of common fragment 10-[13C]-3.
generated in situ, with natural abundance 1-bromo-2-octyne (4)
were not successful and upon work up gave only the alkyne 10-
[
13C]-3. We therefore reacted the dibromoalkene, 10-[13C]-14, with
excess LDA at ꢀ70 °C to ensure conversion to the lithium alkyne
salt, which was warmed to room temperature and quenched to iso-
late the desired 2-(10-[13C]-dec-9-yn-1-yloxy)tetrahydro-2H-
pyran (10-[13C]-3) in good yield (90%; Scheme 4).
For the synthesis of 1,1-[2H2]-10 and 1-[13C]-1,1-[2H2]-10, the
2H and/or 13C isotopologues were introduced onto 1-heptyne 8
using paraformaldehyde-d2 and paraformaldehyde-13C-d2, respec-
tively, forming the corresponding labeled 2-octyn-1-ol (1,1-[2H2]-
10 and 1-[13C]-1,1-[2H2]-10). The route is comparable to that
described for the synthesis of 1-[13C]-10 in Scheme 2.
Paraformaldehyde allows us to add not only 13C at the reactive car-
bon, but also deuterium. The compound 1,1-[2H2]-10 has also been
prepared previously via reduction of the commercially available
methyl 2-octynoate with LiAlD4.16 Reduction of 2-octynoate with
LiAlD4 produces slightly better yields of the 1,1-[2H2]-2-octyn-1-
ol, but requires either additional purification or reaction steps to
remove unwanted side products. For the synthesis of the deu-
terium and 13C labeled 2-octyn-1-ol, the most direct route is
through the use of paraformaldehyde-d2-13C, as outlined here. As
illustrated in Scheme 2, bromination of these 2-octyn-1-ol (10)
labels generated the respective labeled 1-bromo-2-octyne (4).
The coupling and final steps for these labeled compounds are iden-
tical to those described above in Scheme 1 for 11-[13C]-1 and gave
similar yields and purities for final products.
It has been suggested that alkyne lithium salts couple with
appropriate electrophiles in a two-step, one pot synthesis.19 It
could be envisioned that addition of 2 equiv of a strong base such
as lithium diisopropylamide (LDA) would sequentially transform
10-[13C]-14 to a lithium alkyne salt. Subsequent addition of 4
would, in principle, act as a proper electrophile and undergo car-
bon–carbon bond formation, thus generating the desired 9,12-
diyne. However, attempts to directly couple the lithium alkyne salt,
Unimolecular rate (kcat) and Michaelis (KM) constants were
measured from the reaction of the linoleic acids synthesized here
with WT SLO-1 at 30 °C (see Table 1). Michaelis–Menton saturation
curves were plotted from measuring the rate of product formation,
monitored spectrophotometrically from the absorbance at
234 nm,20 as a function of substrate concentration (2–80
l
M).
The kinetic parameters of 10-[13C]-11,11-[2H2]-1 and 10,11-
13C2]-11,11-[2H2]-1 are compared to the commercially available
[
perdeuterated substrate, extracted from algal cells.21 It is
important to note that the kcat for the perdeuterated substrate
(kcat = 5.7 sꢀ1) is taken from a recent report22 and the apparent
slight elevation of this value in reference to the kcat magnitudes
for the deuterated, carbon-13 labeled substrates, reported here
(Table 1), is well within the typical variation from different enzyme
Scheme 3. General strategy outline for 2H, 13C isotope labeling of linoleic acid
substrates. The asterisks denote the position of the 13C label. For details about the
synthetic steps, refer to Scheme 1.

4540
A. R. Offenbacher et al. / Tetrahedron Letters 57 (2016) 4537–4540
Table 1
Drs. Shenshen Hu, Eric Koehn, John Latham, Jianyu Zhang, and
Gaëlle Deshayes for helpful discussions.
Kinetic analysis of the 2H, 13C labeled substrates with WT SLO-1a
Linoleic acid
kcat (sꢀ1
)
KM (lM)
[2H31]-1b
5.7 (0.1)
4.5 (0.2)
4.7 (0.2)
4.5 (0.2)
7.8 (0.5)
7.7 (1.3)
7.8 (1.0)
7.6 (1.0)
Supplementary data
11,11-[2H2]-1c
10-[13C]-11,11-[2H2]-1d
10,11-[13C2]-11,11-[2H2]-1d
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.08.
071.
a
Reactions were conducted with 3.5 nM WT SLO, prepared as previously
described,5 at 30 °C in 0.1 M borate, pH 9.0 buffer. Substrates were prepared at
1 mM working stocks in 0.1 M sodium borate (pH 9.0) buffer. Prior to use, the
concentration of the effective substrate was confirmed enzymatically. Each kinetic
References and notes
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preparations. Importantly, the KM values for all substrates are
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10-[13C]-11,11-[2H2]-1 and 10,11-[13C2]-11,11-[2H2]-1, as bona fide
lipoxygenase substrates.
While there are several reports that describe routes to synthe-
size lipoxygenase substrates (linoleic and arachidonic acids) that
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C
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Acknowledgements
Financial support was provided by the National Institutes of
Health GM118117-01 to J.P.K.; A.R.O was supported by NIH post-
doctoral fellowship F32 GM113432. The authors thank Prof. Caro-
lyn Bertozzi for the use of the Biotage HPLC. The authors also thank
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