A R T I C L E S
Meyer and Klinman
column. Activity was usually detected in fractions 7-13 when
collecting 10 mL fractions. The collected active fractions were then
dialyzed overnight against 4 L of 20 mM Bis-Tris (pH 6.0). The
dialyzed solution was centrifuged at 27000g in an SS-34 rotor for
at each vinylic position, R
p
. These partial conversion reactions
usually required about 1 and 10 mg of SLO-1 to achieve the
appropriate fractional conversion of combinatorially labeled linoleic
acid and 11,11-d2-LA, respectively. The large amount of SLO-1
was necessary, given that SLO-1 typically becomes progressively
inactive over time, and these reactions typically took place over
24 h.
2
0 min to remove a white particulate that forms during dialysis.
The supernatant was injected onto an FPLC system with a 10 mL
Mono-S column using the same loading buffer as for the SP-
sepharose column. A salt gradient for protein elution went from
The sample reactions were quenched when the reaction appeared
to have stopped and the fractional conversion was near the desired
0
.00 to 0.25 M NaCl in 20 mM Bis-Tris (pH 6.0) buffer. FPLC
purification utilized a flow rate of 2.5 mL/min. Most activity was
obtained in fractions 18-25 when collecting 5 mL fractions. SLO-1
activity was assayed by observing the formation of 13-hydroperoxy-
3
2
5% completion mark, as determined by absorbance for HPOD at
34 nm. The quenching process involved the addition of about 600
mL of diethyl ether followed by the addition of 3.9 g of NaBH
36.9 g of KH PO , and 584.4 g of NaCl. The NaBH was used to
reduce 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid to 13-
hydroxy-9,11-(Z,E)-octadecadienoic acid (HOD). The KH PO
4
,
9
,11-(Z,E)-octadecadienoic acid (HPOD) at 234 nm upon the
1
2
4
4
addition of 2 µL of a column fraction to 498 µL of 100 µM linoleic
acid in 0.1 M borate buffer (pH 9.0).
2
4
Synthesis of Labeled Linoleic Acids. The full synthesis of
linoleic acid (11,11-h2-LA) and 11,11-d2-LA with approximately
acidified the solution to aid in extraction of linoleic acid and the
reduced product, as did the added salt (NaCl). We note that the
KH PO should be added slowly, as NaBH evolves hydrogen
2 4 4
4
% deuterium label in the 9, 10, 12, and 13 positions is described
in ref 23 and the Supporting Information. The primary concern was
to generate a low but measurable level of deuterium labeling in
the vinylic positions of the linoleic acid isotopomers so that an
insignificant number of the individual molecules would have two
deuterium labels in the vinylic positions. The deuterium labeling
at these positions was necessary for the ultimate quantitative
D-NMR measurements which yielded the raw data used to compute
KIEs. The vinylic labeling was achieved by reducing 9,12-
octadecadienoic acid with 1.5 equiv of catecholborane, followed
by acetolysis in a mixture of 12% (mol/mol) d-acetic acid and 88%
rapidly in the presence of acid and foaming can occur due to the
soapy nature of the substrate and product. The quenched reactions
were separated into three approximately equivalent volumes and
extracted in a 4 L separatory funnel with six 200 mL volumes of
diethyl ether. After extraction, the combined ether layers were
washed with about 200 mL of brine. The ether was then removed
using rotary evaporation. The residual oil containing linoleic acid
and/or HOD was methylated using diazomethane prepared via the
addition of N-methyl-N-nitrosourea to a biphasic mixture of 4.5
mL of 15% KOH and 10 mL of diethyl ether. The methyl esters of
the linoleic acid and HOD were separated via flash chromatography
using 200 mesh neutral silica as the stationary phase in a 2.5 in. ×
(
mol/mol) acetic acid. This reduction method is ideally suited to
the experiments described herein because it does not lead to
scrambling of the hydrogen or deuterium atoms into the 8, 11, and
1
1
4 in. column. The mobile phase consisted of (in order) 1000 mL
24
4 positions, as do some heterogeneous metallic catalyst methods.
of 20% diethyl ether/80% hexanes, then 500 mL of 30% diethyl
ether/70% hexanes, then 500 mL of 50% diethyl ether/50% hexanes.
About 160 fractions of about 10 mL were collected. The methyl
linoleate typically eluted from fraction 15 to fraction 30, and the
methyl ester of HOD (MHOD) eluted between fractions 100 and
Additionally, over-reduction is prevented by both steric and
2
5,26
electronic properties of the hydroboration product.
2
Reactions for KIE Determination. Secondary H KIEs were
determined for the oxidation of 11,11-d2-LA and linoleic acid, each
2
labeled with approximately 4% H at the 9, 10, 12, and 13 positions.
1
40.
These reactions were carried out between 21 and 23 °C. A similar
protocol was used to measure 13C KIEs with natural abundance
substrates. A 12 L three-neck round-bottomed flask was charged
with 10 L of 0.1 M borate (pH 9.0). To this was added
approximately 0.63 mL of the combinatorially labeled 11,11-d2-
LA or linoleic acid dissolved in 100 mL (20.3 mM stock
concentration). This mixture was allowed to stir slowly for about
Assignment of 13C and H Peaks for MHOD. In order to
2
13
2
interpret the quantitative NMR measurements of C and H spectra,
it was necessary to unambiguously assign the resonances to the
2
1
correct position on the MHOD molecule. Since H and H spectra
have the same chemical shift dispersion, it is possible to assign the
2
H spectrum from coupling associations and other information in
1
1
the H NMR spectra. H NMR spectra of 11-d-MHOD and MHOD
allowed for the unambiguous assignment of the H resonances at
1
h before spectrophotometrically determining the initial concentra-
tion of linoleic acid at 234 nm, which was typically in the range of
65-170 µM. Three aliquots of 500 µL were taken before reaction
was initiated by the addition of SLO-1. These aliquots were diluted
:2 with 0.1 M borate (pH 9.0). A 495 µL aliquot of this diluted
1
the 9, 11, and 13 positions. The 13 position was assigned as the
resonance at 3.21 ppm (dt, J ) 6, 6 Hz, 1H). The 11 position was
assigned to the resonance at 6.37 ppm (dd, J ) 11, 15 Hz, 1H) by
virtue of its disappearance in the spectrum of 11-d-MHOD. The 9
position at 5.31 ppm (dt, J ) 7, 11 Hz, 1H) was assigned on the
basis of the fact that, of the four olefinic resonances, it was the
only one for which the splitting pattern did not simplify upon going
from 11-d-MHOD to MHOD. Finally, the 10 and 12 positions were
assigned on the basis of shared couplings between the 9 and 10
positions and the 12 and 13 positions. The resonance assigned to
the 12 position is 5.60 ppm (dd, J ) 6, 15 Hz, 1H) and is obviously
coupled with the 13 position as well as the 11 position. The
resonance assigned to the 10 position is at 5.92 ppm (dd, J ) 11,
1
1
solution was added to a cuvette, the absorbance was zeroed, and 5
µL of approximately 1 mg/mL SLO-1 in 0.1 M borate (pH 9.0)
was added to the cuvette. The absorbance was followed at 234 nm.
The final absorbance (corrected for the small absorbance of SLO-
1
) was a check on the initial concentration of substrate in the
reaction. Two types of reactions were performed for each isotopi-
cally labeled substrate. Two standard reactions of 10 L were taken
to 100% conversion so that NMR on this product could be used to
report on the initial fraction of labeling (R
1
3
0
) at each position (9,
0, 12, and 13). These standard reactions typically required about
mg of SLO-1 to complete the consumption of the combinatorially
1
1 Hz, 1H). These couplings are consonant with a resonance
coupled to both the 9 and 11 positions. The couplings and chemical
shifts of the lines associated with MHOD are available in the
Supporting Information. The assignments made via couplings and
differences in the spectra for 11-d-MHOD and MHOD were
confirmed using a short TOCSY experiment.
labeled linoleic acid and about 30 mg of SLO-1 to completely
consume the combinatorially labeled 11,11-d2-LA. Three sample
reactions of 10 L were taken to approximately 35% conversion for
the eventual NMR determination of the fraction of deuterium label
Assignments of 13C NMR lines relied upon the use of HMQC,
since the proton resonances were already accurately assigned. All
(
(
23) Meyer, M. P.; Klinman, J. P. Tetrahedron Lett. 2008, 49, 3600.
24) Thomas, A. F. Deuterium Labeling in Organic Chemistry; Meredith
Corp.: New York, 1971.
1
3
C NMR spectra were obtained at 125.77 MHz in d6-DMSO at a
constant temperature of 23.2 °C. A total of 12 resonances were
assigned out of the 19 possible: OMe, 51.1 ppm; C1, 173.2 ppm;
(
25) Brown, H. C.; Chandrasekharan, J. J. Org. Chem. 1983, 48, 5080.
(
26) Brown, H. C.; Gupta, S. K. J. Am. Chem. Soc. 1972, 94, 4370.
4
32 J. AM. CHEM. SOC. 9 VOL. 133, NO. 3, 2011