Preparation of deuterium-labeled monounsaturated and saturated fatty acids for use as stable isotope metabolic tracers

Article abstract of DOI:10.1002/jlcr.1142

Synthetic routes adaptable to the preparation of a variety of d ₇-labeled mono-unsaturated and saturated fatty acids are reported. Using these reaction sequences, d₇-oleic [18:1 (9c)], d ₇-elaidic [18:1 (9t)], and d₇-stearic acid (18:0) were prepared for use in metabolism studies as stable isotope-labeled tracers. Copyright

Full text of DOI:10.1002/jlcr.1142

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1273–1285.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1142
Research Article
Preparation of deuterium-labeled monounsaturated
and saturated fatty acids for use as stable isotope
metabolic tracers
Sheldon N. Crane*, Kevin Bateman, Sebastien Gagne and Jean-Francois
Levesque
Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Highway,
Kirkland, Quebec, Canada H9H 3L1
Summary
Synthetic routes adaptable to the preparation of a variety of d7-labeled mono-
unsaturated and saturated fatty acids are reported. Using these reaction sequences,
d7-oleic [18:1 (9c)], d7-elaidic [18:1 (9t)], and d7-stearic acid (18:0) were prepared for
use in metabolism studies as stable isotope-labeled tracers. Copyright # 2006 John
Wiley & Sons, Ltd.
Received 11 September 2006; Revised 19 September 2006; Accepted 21 September
2006
Key Words: fatty acid; metabolic syndrome; stable isotope; deuterium label
Introduction
The hallmarks of metabolic syndrome}blood dyslipidaemia, elevated blood
pressure and insulin resistance/glucose intolerance, are an interrelated set of
risk factors that contribute to the development of cardiovascular diseases and
non-insulin dependent diabetes.1a,b,c It has been projected that obesity-induced
metabolic syndrome and its associated pathologies will reach epidemic
proportions in industrialized nations within the next several decades.1a,b,c
The development of therapeutic agents for the treatment of the dyslipidaemia
associated with metabolic syndrome will require delineation of the poorly
understood molecular mechanisms that underlie its origin.1d,e Unfortunately,
acute monitoring of the effects of obesity on lipid metabolism is complicated
*Correspondence to: Sheldon N. Crane, Merck Frosst Centre for Therapeutic Research, 16711
TransCanada Highway, Kirkland, Quebec, Canada H9H 3L1. E-mail: sheldon crane@merck.com
Copyright # 2006 John Wiley & Sons, Ltd.

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S. N. CRANE ET AL.
by interference from the endogenous lipid pool. This issue can be obviated by
the use of radiolabeled tracers,2a however, stable isotopes are more attractive
from handling and safety standpoints.2b We have recently reported a high-
throughput LC-MS method for the quantitative analysis of deuterium labeled
fatty acids in biological samples.3 The requirement for a minimum
incorporation of seven deuterium (d7) atoms in these tracers arose out of
the necessity to optimally discriminate the tracer from naturally occurring
fatty acids present in biological samples. In order to support our studies in the
area of metabolic syndrome-associated dyslipidaemia, we required access to a
variety of d7-labeled saturated and monounsaturated fatty acids for use as
standards in an array of in vivo and ex vivo experiments. Herein, we report the
synthetic routes that were developed to provide us with our requisite molecular
tool kit.
Synthesis of d7-labeled monounsaturated and saturated fatty acids
Our plan was to develop a flexible strategy whereby we could vary the chain
length of a late stage alkyne intermediate that could subsequently be
transformed into the corresponding series of saturated and monounsaturated
(cis- and trans-) fatty acids. We also wished to position the deuterium labels as
far as possible from the carboxylic acid functionality in order to monitor
b-oxidation along the entire length of the hydrocarbon chain. In order to meet
these requirements, we chose to assemble the fatty acid precursor from three
components as illustrated in Figure 1. The adaptable sequence of chemical
transformations that was developed to prepare both saturated and unsatu-
rated d7-labeled fatty acids is shown in Scheme 1. Scheme 2 details a similar
reaction sequence that was designed to rapidly access saturated d7-fatty acids.
C18 fatty acids were prepared as illustrative examples in both instances.
Scheme 1 starts with a Wittig reaction of phosphonium salt 1 with
d7-butyraldehyde (2), prepared via PCC oxidation of commercially available
d7-butanol, to give olefin 3 (Z/E 11:1). Reduction of the double bond in 3 was
accomplished by exposure to hydrogen (40 PSI) in the presence of Wilkinson’s
catalyst.4a,y Removal of the silyl ether protecting group in 4 with TBAF was
followed by conversion of the alcohol 5 to the corresponding bromide 6 with
HBr, and finally to iodide 7 under typical Finkelstein conditions. With the
deuterium-labeled terminal C11–C18 fragment of our fatty acid targets in hand,
we then turned our attention to the preparation of the C1–C10 fragment.
Exposure of commercially available 2-decyn-1-ol to NaH in 1,3-diaminopro-
y Freshly prepared catalyst was used in this reaction in order to minimize the presence of rhodium oxides
that are generated from Wilkinson’s catalyst that has been exposed to atmospheric oxygen. These rhodium
oxide species are capable of scrambling the deuterium labels in the final product as a consequence of
competitive double bond isomerization during the hydrogenation reaction. For an in-depth discussion of
this phenomenon. See references 4b and 4c.
Copyright # 2006 John Wiley & Sons, Ltd.
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PREPARATION OF DEUTERIUM-LABELED FATTY ACIDS
1275
D D
CHO
X
D3C
I–
D D
D D
D3C
H
D D
D3C
+
CO2H
n
D D
OR2
Ph3P
m
m
m
D D
n
OR2
M
n
Figure 1. Strategy for the preparation of a variety of d7-labeled saturated and
monounsaturated fatty acids
I–
PPh3
a
I
CD3
TBSO
TBSO
HO
D
D D
CD3
1
D
c
d
D
D
TBSO
X
D D
CD3
O
H
D D
TBSO
4
D D
b
3
CD3
e
D D
D D
D D
CD
2
3
g
h
D D
HO
RO
i
5 X = OH
8 R = H
9 R = TBS
f
6
7
X = Br
X = I
j
D D
D D
o,p,m
CD3
CD3
TBSO
D D
HO2C
D D
D D
10
13
k
n,l
l,m
m
D D
CD3
HO2C
HO2C
CD3
TBSO
HO
D D
D D
D D
12
15
11
14
D D
CD3
D D
CD3
D D
Scheme 1. Conditions : (a) PPh3, CH3CN, reflux, >99%; (b) PCC, CH2Cl2; (c)
NaHMDS, THF, ꢀ788C, 65%; (d) 40 PSI H2, (PPh3)3RhCl, PhH, >99%; (e)
TBAF, THF, 94%; (f) 48% HBr, conc H2SO4, 1008C, 89%; (g) NaI, acetone,
reflux, 89%; (h) 1,3-diaminopropane, NaH, 708C, 88%; (i) TBSCI, DMAP,
Et3N, CH2Cl2, 75%; (j) n-BuLi, THF, hexanes, HMPA, –788–08C, 86% for
X ¼ I; (k) 1 atm H2, Lindlar catalyst, quinoline, hexanes, >99%; (l) TBAF,
THF, 97%; (m) Jones oxidation (71% for 12, 65% for 13; 93% for 15); (n) 40
PSI H2, 10% Pd/C, EtOAc, >99%; (o) TBAF, THF, 78%; and (p) LiAIH4,
diglyme, 1608C, 90%
pane promoted an alkyne zipper reaction that resulted in isomerization of the
internal alkyne to the terminal position to give 8.5 Protection of the alcohol in
8 as a TBS-ether in 9 was followed by removal of the acetylenic hydrogen with
n-BuLi to generate the corresponding lithium acetylide, that was coupled to
Copyright # 2006 John Wiley & Sons, Ltd.
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1276
HO
S. N. CRANE ET AL.
THPO
THPO
a,b,c
d
Ph3P
I–
Br
D3C
D D
D
D
16
17
e
D D
THPO
D3C
D D
f,g
CD3
D
D
HO2C
D D
15
18
Scheme 2. Conditions: (a) dihydropyran, TsOH, CH2Cl2, 89%; (b) NaI,
acetone, reflux, >99%; (c) PPh3, CH3CN, reflux, >99%; (d) NaHMDS, THF,
2, –788–08C, 78%; (e) 40 psi H2, (PPh3)3 RhCl, PhH, 90%; (f) MeOH, TsOH,
508C, 90%; and (g) Jones oxidation, 97%
iodide 7 to give 10 in 86% yield.6 The coupling reaction could also be
conducted with the corresponding bromide 6, albeit in a more modest yield
(59%). In the latter case, the product (10) was not easily separated from
unreacted 9 using conventional silica gel chromatography. Key intermediate
10 could then be converted into the corresponding cis-, trans-, or saturated
fatty acid depending on the reaction conditions chosen for reduction of the
triple bond. Lindlar reduction of the alkyne in 10, followed by removal of the
TBS protecting group, and Jones oxidation afforded d7-oleic acid (12).
Liberation of the alcohol in 10 with TBAF, followed by conversion of the
alkyne to the trans-alkene with LiAlH4 in refluxing glyme,7 and subsequent
Jones oxidation, gave d7-elaidic acid (13). Reduction of 10 with 40 PSI of
hydrogen in the presence of Pd/C, followed by the same deprotection/
oxidation sequence yielded d7-stearic acid (15).
A more efficient approach leading to d7-labeled saturated fatty acids, such as
d7-stearic acid (15), is detailed in Scheme 2. Essentially, the sequence of events
in this instance is identical to the first phase of Scheme 1 leading to
intermediate 4. Use of a THP instead of a TBS protecting group allowed us to
minimize the number of chromatographic purifications, and more readily
access multi-gram quantities of d7-stearic acid (14).
Conclusion
A synthetic route for the preparation of a diverse array of physiologically
relevant monounsaturated and fully saturated d7-labeled fatty acids from a
common alkyne intermediate has been developed. A conceptually related
chemical sequence was also devised to rapidly access d7-labeled, saturated fatty
acids. We plan to use these stable isotope-labeled compounds as tools to study
Copyright # 2006 John Wiley & Sons, Ltd.
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PREPARATION OF DEUTERIUM-LABELED FATTY ACIDS
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lipid metabolism in animal models of obesity, diabetes and atherosclerosis.
The results of these investigations will be reported in due course.
Experimental section
General
Unless specified otherwise, all reagents were obtained commercially and used
without further purification. Jones reagent [2.7 M] was prepared by carefully
adding 18 M H2SO4 (23 ml) to a solution of chromic acid (26.7 g, 267 mmol) in
water (40 ml), and adjusting the final volume to 100 ml in a volumetric flask
with water. n-Butyl-2,2,3,3,4,4,4-d7-alcohol was purchased from CDN
Isotopes (Pointe Claire, Quebec, Canada). Proton (1H NMR) and carbon
(13C NMR) magnetic resonance spectra were recorded on a Bruker instrument
at the indicated frequency. Spectra were recorded in CDCl3, unless specified
otherwise, using residual solvent as internal standard. Signal multiplicity was
designated according to the following abbreviations: s ¼ singlet, d ¼ doublet,
dd ¼ doublet of doublets, ddd ¼ doublet of doublet of doublets, t ¼ triplet,
q ¼ quartet, dt ¼ doublet of triplets, td ¼ triplet of doublets, dq ¼ doublet of
quartets, m ¼ multiplet; br ¼ broad. High-resolution mass spectra were
recorded on a Micromass QTof Premiere from Waters (Manchester, England)
coupled to a Waters Acquity UPLC. Reactions were carried out with
continuous stirring under a positive pressure of nitrogen except where noted.
Flash chromatography was performed with silica gel 60, 230–400 mesh.
(4-{tert-Butyl(dimethyl)silyl}oxy)butyl)(triphenyl)phosphonium iodide (1)
A solution of tert-butyl(4-iodobutoxy)dimethylsilane (25.5 g, 81.2 mmol) and
triphenylphosphine (21.3 g, 81.3 mmol) were heated together in dry acetonitrile
(100 ml) at 858C for 24 h. The mixture was evaporated, and the residue was
triturated with anhydrous ether to afford the title compound as a colorless
1
powder (44.2 g, 95%). H NMR (CD2Cl2, 400 MHz) d 7.93–7.73 (15 H, m),
3.71 (2 H, t, J ¼ 5:2 Hz), 3.57–3.45 (2 H, m), 1.87–1.80 (4 H, m), 0.84 (9 H, s),
0.03 (6 H, s).
2,2,3,3,4,4,4-d7-Butyraldehyde, 20 wt% in CH2Cl2 (2)
A solution of n-butyl-2,2,3,3,4,4,4-d7-alcohol (21.80 g, 270 mmol) in CH2Cl2
(20 ml) was added dropwise at RT over 10 min to a slurry of PCC (73 g,
340 mmol, 1.25 eq.) and Celite (20 g) in CH2Cl2 (500 ml). The reaction mixture
was then stirred at RT (mild spontaneous reflux controlled with a condenser)
for 3 h prior to removal of the supernatant solution by decantation, and
passage through a silica gel plug (6 ꢁ 9 cm) that had been prepacked using
CH2Cl2. The plug was then washed with additional CH2Cl2 (125 ml), and the
eluate was concentrated by fractional distillation under nitrogen through a
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12 ꢁ 0.75 inch Vigreux column using an oil bath (max temperature 608C). The
residue in the distillation flask (40.44 g, 3.7:1 CH2Cl2 : aldehyde by 1H NMR,
1
38% yield) was used without further purification in subsequent reactions. H
NMR (acetone-d6, 500 MHz) d 9.74 (1 H, s), 5.63 (s, residual CH2Cl2).
6,6,7,7,8,8,8-d7-tert-Butyl(dimethyl)[(4Z)-oct-4-en-1-yloxy]silane (3)
A solution of NaHMDS (1 M in THF, 22 ml) was added to a 08C solution of 1
(12.67 g, 22.0 mmol) in THF (30 ml). The resulting orange solution was stirred
at this temperature for 25 min prior to cooling to ꢀ788C, and rapid addition of
the 20 wt % solution of 2 (8.23 g, 21 mmol). After 20 min at ꢀ788C, the
reaction was quenched by the addition of 1 M pH 7 phosphate buffer (0.20 ml,
1:1 K3PO4/KH2PO4) followed by warming to RT. Solvents were removed by
rotary evaporation, and the residue was stirred with 1:1 hexanes/H2O. The
solid triphenylphosphine oxide by product was removed by suction filtration,
and the organic phase of the filtrate was separated, washed with water, and
dried over Na2SO4. Concentration in vacuo and flash chromatography on
silica gel eluting with 5/95 ether/hexanes afforded the title compound
1
(Rf ¼ 0:50; 1/99 ether/hexanes) as a colorless oil (3.35 g, 65%, Z:E 11:1). H
NMR (400 MHz) d 5.40 (2 H, m), 3.63 (2 H, t, J=6.5 Hz), 2.12–2.05 (2 H, m),
1.63–1.45 (2 H, m), 0.92 (9 H, s), 0.07 (6 H, s); discernable signals for the
E-isomer: d 5.42 (2 H, m).
Preparation of Wilkinson’s catalyst ((Ph3P) RhCl)8
3
ꢂ
RhCl3 3H2O (1.50 g, 5.70 mmol) and PPh3 (9.00 g, 34.0 mmol, freshly
recrystallized from ethanol) were heated together under a nitrogen atmosphere
in nitrogen saturated (dispersion tube, 30 min) 95% ethanol (220 ml) for 2 h.
The reaction mixture was then cooled to 408C, and the solid was collected by
suction filtration (glass frit) under a nitrogen atmosphere. The solid was
washed in succession with nitrogen saturated absolute ethanol (2 ꢁ ) and
nitrogen saturated anhydrous ether (3 ꢁ ), and was dried under a stream of
nitrogen, then under hi-vacuum to yield a maroon powder (4.77 g, 90%).
6,6,7,7,8,8,8-d7-tert-Butyl(dimethyl)(octyloxy)silane (4)
Wilkinson’s catalyst (380 mg, 3 mol %) was suspended in nitrogen saturated
benzene (70 ml) under a nitrogen atmosphere in a rubber septum capped Parr
flask. Compound 3 (3.35 g, 13.5 mmol) was then added as a solution in
nitrogen saturated benzene (10 ml), and the mixture was shaken under an
atmosphere of hydrogen gas (40 PSI) for 16 h. Following confirmation of the
1
completion of the reaction by H NMR, the benzene was evaporated and the
residue was purified by flash chromatography on silica gel eluting with 5/95
ether/hexanes to yield the title compound (Rf ¼ 0:78; 2/98 ether/hexanes) as a
Copyright # 2006 John Wiley & Sons, Ltd.
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PREPARATION OF DEUTERIUM-LABELED FATTY ACIDS
1
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colorless oil (3.38 g, >99%). H NMR (400 MHz) d 3.62 (2H, t, J=6.6 Hz),
1.55–1.49 (2H, m), 1.34–1.26 (6H, m), 0.92 (9H, s), 0.07 (6H, s).
6,6,7,7,8,8,8-d7-Octan-1-ol (5)
TBAF (20 ml, 1 M in THF) was added to a solution of 4 (3.38 g, 13.5 mmol) in
THF (40 ml) with stirring at RT for 2 h. The THF was then evaporated, and
the residue was taken up in ether, and washed with water and brine. The
organic phase was dried over Na2SO4/MgSO4, and filtered. Concentration of
the filtrate, and flash chromatography of the residue on silica gel eluting with
1/3 ethyl acetate/hexanes gave the title compound (Rf ¼ 0:42; 1/4 ethyl
acetate/hexanes) as a colorless oil (1.73 g, 94%). 1H NMR (400 MHz, acetone-
d6) d 3.53 (2H, q, J=6.2 Hz), 3.38 (1H, t, J=5.3 Hz), 1.59–1.46 (2H, m), 1.41–
1.27 (6H, m).
6,6,7,7,8,8,8-d7-1-Bromooctane (6)
A mixture of 5 (1.73 g, 12.6 mmol), 48% HBr (16 g) and conc H2SO4 (4 g) were
heated together with rapid stirring at 1008C for 1.5 h. The mixture was then
cooled to RT, poured onto crushed ice and extracted with hexanes (2 ꢁ ). The
organic phase was washed with water and saturated NaHCO3 solution, and
dried over Na2SO4/MgSO4. Rotary evaporation of the solvents afforded a
faint-yellow oil (2.23 g, 89%) that was used directly in the next step. Rf=0.86
1
(1/3 ethyl acetate/hexanes); H NMR (400 MHz) d 3.21 (2H, t, J=7.0 Hz),
1.83 (2H, quintet, J=6.9 Hz), 1.46–1.36 (2H, m), 1.36–1.23 (4H, m).
6,6,7,7,8,8,8-d7-1-Iodooctane (7)
Compound 6 (1.05 g, 5.25 mmol) was heated with NaI (1.23 g, 8.20 mmol) in
refluxing acetone (15 ml) for 2 h. The mixture was then poured into water and
extracted with hexanes (2 ꢁ ). The combined extracts were washed with water,
dried over Na2SO4/MgSO4, and concentrated. Flash chromatography on silica
gel eluting with hexanes provided the title compound (Rf=0.69) as a colorless
1
oil (1.15 g, 89%). H NMR (400 MHz) d 3.21 (2 H, t, J=7.0 Hz), 1.84 (2H,
quintet, J=7.0 Hz), 1.45–1.36 (2H, m), 1.36–1.23 (4H, m).
Dec-9-yn-1-ol ð8)
In a 250 ml round bottom flask fitted with a reflux condenser, sodium hydride
(6.44 g, 60 wt% in mineral oil, 160 mmol, washed free of the mineral oil with
hexanes (3 ꢁ 20 ml)) was suspended in 1,3-diaminopropane (150 ml, distilled
from barium oxide). The mixture was then heated carefully, to control
foaming, at 558C for 1 h. The resulting clear, brown solution was cooled to
RT, and 2-decyn-1-ol (3.14 g, 20 mmol) was added dropwise followed by
stirring at 558C. After 18 h at 558C, the reaction vessel contents were cooled to
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RT, poured into ice water, and extracted with ether (3 ꢁ ). The combined
extracts were washed with 0.5 M HCl and brine, and then were dried over
Na2SO4/MgSO4. Concentration in vacuo afforded the title compound as a
yellow oil (2.75 g, 88%) that was used directly in the next step. Rf ¼ 0:45 (3/7
ethyl acetate/hexanes); 1H NMR (500 MHz, acetone-d6) d 3.54 (2H, q,
J=6.1 Hz), 3.40 (1H, t, J=5.0 Hz, OH), 2.32 (1H, t, J=2.7 Hz), 2.19 (2H, td,
J=7.0, 2.5 Hz), 1.55–1.49 (4H, m), 1.34–1.28 (8H, m).
tert-Butyl(dimethyl)(dec-9-yn-1-yloxy)silane (9)
A solution of 8 (2.75 g, 17.9 mmol), tert-butyldimethylsilyl chloride (2.98 g,
19.9 mmol) and DMAP (0.20 g, 1.6 mmol) in CH2Cl2 (50 ml) was treated with
triethylamine (3.0 ml, 22 mmol), and stirred at RT for 16 h. The mixture was
then poured into water, and the layers were separated. The aqueous phase was
extracted with additional CH2Cl2, and the combined organics were washed
with water, dried over Na2SO4, and concentrated. Flash chromatography on
silica gel eluting with 4/96 ether/hexanes provided the title compound
1
(Rf ¼ 0:54; 2/98 ether/hexanes) as a faint-yellow oil (3.54 g, 75%). H NMR
(500 MHz, acetone-d6) d 3.65 (2H, t, J=6.4 Hz), 2.32 (1H, t, J=2.7 Hz), 2.19
(2H, td, J=6.9, 2.6 Hz), 1.55–1.49 (4H, m), 1.45–1.31 (8H, m), 0.92 (9H, d,
J=2.7 Hz), 0.07 (6H, s).
16,16,17,17,18,18,18-d7-tert-Butyl(octadec-9-yn-1-yloxy)dimethylsilane (10)
A solution of 9 (574 mg, 2.14 mmol) in THF (3.0 ml) was treated dropwise at
ꢀ788C with n-BuLi (2.5 M in hexanes, 0.94 ml, 1.1 equiv). The resulting yellow
solution was stirred at ꢀ788C for 1 h prior to warming to 08C. Compound 7
(580 mg, 2.35 mmol) was then added dropwise as a solution in HMPA (1.2 ml).
After an additional 10 min of stirring at 08C, 1 M pH 7 phosphate buffer (1:1
KH2PO4:K3PO4) was added to quench the reaction. The mixture was
extracted with hexanes (2 ꢁ ), and the combined organics were washed with
water, dried over Na2SO4/MgSO4 and concentrated. Flash chromatography of
the residue on silica gel eluting with 1/99 ether/hexanes yielded the title
compound (Rf ¼ 0:36) as a colorless oil (709 mg, 86%). 1H NMR (400 MHz) d
3.62 (2H, t, J=6.6 Hz), 2.16 (4H, t, J=6.9 Hz), 1.56–1.46 (6H, m), 1.38–1.28
(14H, m), 0.92 (9H, s), 0.07 (6H, s).
16,16,17,17,18,18,18-d7-Oleic acid (12)
A mixture of 10 (150 mg, 390 mmol), Lindlar catalyst (5% Pd on CaCO3
poisoned with lead, 40 mg) and quinoline (0.03 ml) in hexanes (8 ml) was
stirred under an atmosphere of hydrogen (balloon) until the reaction was
judged complete (4 h) by TLC analysis. The mixture was then filtered through
Celite, and concentrated. Purification of the residue by flash chromatography
Copyright # 2006 John Wiley & Sons, Ltd.
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PREPARATION OF DEUTERIUM-LABELED FATTY ACIDS
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on silica gel eluting with 1/99 ether/hexanes afforded 16,16,17,17,18,18,18-d7-
tert-butyl[(9Z)-octadec-9-en-1-yloxy]dimethylsilane (11) as a colorless oil (150mg,
1
99%); Rf 0.64 (2/98 ether/hexanes); H NMR (400 MHz) d 5.37 (2H, m), 3.62
(2H, t, J=6.6 Hz), 2.09–1.99 (4H, m), 1.58–1.49 (4H, m), 1.43–1.23 (16H, m),
0.92 (9H, s), 0.07 (6H, s). A solution of this compound (150 mg, 400 mmol) in
THF (2 ml) was treated with TBAF (1 M in THF, 2.5 ml) with stirring at RT
for 3 h. The mixture was then partitioned between ether and water, and the
layers were separated. The aqueous phase was extracted with additional ether,
and the combined extracts were washed with brine, before drying over
Na2SO4/MgSO4. Concentration of the filtrate, and flash chromatography on
silica gel eluting with 1/4 ethyl acetate/hexanes gave 16,16,17,17,18,18,18-d7-
(9Z)-octadec-9-en-1-ol as a colorless oil (107 mg, 97%); Rf 0.47 (1/3 ethyl
acetate/hexanes); 1H NMR (400 MHz) d 5.42–5.36 (2H, m), 3.73–3.61 (2H, m),
2.10–1.98 (4H, m), 1.61–1.55 (4H, m), 1.40–1.24 (16H, m). A solution of this
alcohol (100 mg, 360 mmol) in acetone was cooled to 08C, and treated with
Jones reagent (2.7 M, 0.29 ml, 800 mmol) with stirring at this temperature for
20 min. Water was added, and the mixture was extracted with ether (2 ꢁ ). The
combined extracts were washed with water (2 ꢁ ) and brine, and then were
dried over Na2SO4/MgSO4. The solvents were evaporated, and the residue was
purified by flash chromatography on silica gel (1/4 ethyl acetate/hexanes
containing 0.5% acetic acid) to yield the title compound as a colorless oil
(75 mg, 71%); Rf 0.41; 1H NMR (400 MHz) d 5.41–5.33 (2H, m), 2.37 (2H, t,
J = 7.5 Hz), 2.03 (4H, m), 1.68 (2H, m), 1.42–1.23 (16H, m); 13C NMR
(100 MHz) d 180.5, 130.0, 129.7, 34.1, 30.7 (quintet, J ¼ 19 Hz), 29.8, 29.7,
29.5, 29.3, 29.14, 29.07 (2C), 29.04, 27.22, 27.16, 24.7, 21.4 (quintet,
J ¼ 19 Hz), 12.9 (septet, J ¼ 19 Hz); MS (-APCI) 288.2; HRMS calculated
for C18H26D7O2 (M-H) 288.2925, found 288.2940.
16,16,17,17,18,18,18-d7-Elaidic acid (13)
A solution of 10 (174 mg, 450 mmol) in THF (5 ml) was treated with TBAF
(1 M in THF, 1.2 ml) with stirring at RT for 2 h. The mixture was then diluted
with water, and extracted with ether (2 ꢁ ). The combined organics were
washed with brine, dried over Na2SO4/MgSO4, and concentrated. The residue
was purified by flash chromatography on silica gel eluting with 1/3 ethyl
acetate/hexanes (Rf ¼ 0:41) to afford 16,16,17,17,18,18,18-d7-octadec-9-yn-1-ol
1
as a colorless oil (104 mg, 87% yield); H NMR (400 MHz) d 3.66 (2H, t,
J ¼ 6:6 Hz), 2.16 (4H, t, J ¼ 6:8 Hz), 1.64–1.54 (2H, m), 1.54–1.45 (4H, m),
1.45–1.25 (14H, m). A solution of this alkynol (36 mg, 130 mmol) was dissolved
in diglyme (3.5 ml), and treated with LiAlH4 (62 mg, 1600 mmol, 12 eq.). The
resulting slurry was then heated at 160 8C for 40 h. The excess hydride was
quenched at RT by the addition of ethyl acetate followed by 2 M HCl. The
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mixture was partitoned between ethyl acetate and water, and the layers were
separated. The organic phase was washed with brine, and dried over Na2SO4/
MgSO4. Concentration by rotary evaporation, and flash chromatography of
the residue on silica gel eluting with 1/3 ethyl acetate/hexanes (Rf ¼ 0:47) gave
16,16,17,17,18,18,18-d7-(9E)-octadec-9-en-1-ol as a colorless, waxy solid (32 mg,
90%); 1H NMR (500 MHz, acetone-d6) d 5.42 (2H, m), 3.56–3.52 (2H, m), 3.40
(1H, m, OH), 2.04–1.98 (4H, m), 1.54–1.47 (4H, m), 1.37–1.29 (16H, m). A
solution of this alcohol (32 mg, 110 mmol) in acetone (2ml) was treated with a
2.7 M solution of Jones reagent (0.10 ml, 270 mmol) with stirring at RT for
15 min. The reaction mixture was then diluted with water and extracted with
ether (2ꢁ ). The combined extracts were washed with water until no orange
color persisted in the organic phase, followed by a brine wash, and drying over
Na2SO4/MgSO4. The organics were concentrated, and the residue was purified
by flash chromatography on silica gel eluting with 1/4 ethyl acetate/hexanes
containing 0.5% acetic acid (Rf ¼ 0:37) to afford the title compound as a
1
colorless, waxy solid (22 mg, 65%). H NMR (400MHz) d 11.00 (1H, v br),
5.38 (2H, m), 2.37 (2H, t, J¼ 7:5 Hz), 2.03–1.95 (2H, m), 1.67–1.61 (2H, m),
1.43–1.24 (18H, m); 13C NMR (100MHz) 180.0, 130.5, 130.2, 34.0, 32.6, 32.5,
30.8 (quintet, J ¼ 19 Hz), 29.7, 29.45, 29.36, 29.2, 29.1, 29.02, 28.96, 28.9, 24.7,
21.4 (quintet, J¼ 19 Hz), 21.9 (septet, J¼ 19 Hz); MS (-APCI) 288.1; HRMS
calculated for C18H26D7O2 (M-H) 288.2925, found 288.2940.
16,16,17,17,18,18,18-d7-Octadeca-1-ol (14)
A solution of 10 (2.26 g, 5.84 mmol) in ethyl acetate (50 ml) was added to 10%
Pd/C (1.14 g) in a Parr flask under nitrogen. The reaction vessel contents were
then shaken under a hydrogen atmosphere (40 PSI) for 6 h. Filtration through
Celite and concentration of the filtrate afforded a colorless solid that was taken
up in THF (20 ml), and treated with TBAF (1 M in THF, 4 ml) with stirring at
RT for 2 h. The mixture was then partitioned between ether and water, and the
layers were separated. The aqueous phase was extracted with additional ether,
and the combined extracts were washed with brine before drying over Na2SO4/
MgSO4. Evaporation of the solvents, and flash chromatography on silica gel
eluting with 1/4 ethyl acetate/hexanes (Rf=0.38) afforded the title compound
1
as a colorless powder (1.57 g, 97%). H NMR (acetone-d6, 500 MHz) d 3.54
(2H, m), 3.38 (1H, t, J=5.3 Hz), 1.56–1.47 (2H, m), 1.31 (26H, br s).
16,16,17,17,18,18,18-d7-Stearic acid (15)
A solution of 14 (703 mg, 2.64 mmol) in acetone (35 ml) was treated with Jones
reagent (2.7 M, 1.5 ml, 4.0 mmol) with stirring at RT for 30 min. Water
(100 ml) was then added, and the mixture was stirred until all the chromium
salts had dissolved. The remaining solid was collected by suction filtration, and
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PREPARATION OF DEUTERIUM-LABELED FATTY ACIDS
1283
washed well with water before being dissolved in ether and dried over Na2SO4/
MgSO4. Concentration in vacuo afforded the title compound as a colorless
powder (718 mg, 93%). 1H NMR (acetone-d6, 500 MHz) d 10.41 (1H, br), 2.30
(2H, t, J=7.42 Hz), 1.68–1.56 (2H, m), 1.32 (24H, br s); 13C NMR (CDCl3,
100 MHz) d 180.4, 34.1, 30.8 (pentet, J=19 Hz), 29.70, 29.68, 29.65, 29.63,
29.60, 29.5, 29.4, 29.2, 29.13, 29.07, 24.7, 21.4 (pentet, J=19 Hz), 12.9 (pentet,
J=19 Hz); MS (-APCI) 290.5; HRMS calculated for C18H28D7O2 (M-H)
290.3082, found 290.3075.
Triphenyl[14-tetrahydro-2H-pyran-2-yloxy)tetradecyl]phosphonium iodide (16)
Toluene-4-sulfonic acid monohydrate (0.50 g, 2.6 mmol) was added to a
solution of 14-bromotetradecan-1-ol (9.90 g, 33.8 mmol) and freshly distilled
dihydropyran (4.6 ml, 50 mmol) in CH2Cl2 (35 ml) with stirring at RT for
20 min. The reaction vessel contents were then mixed with saturated NaHCO3
aqueous solution, and the layers were separated. The organic phase was
washed with water, dried over Na2SO4, and concentrated. Flash chromato-
graphy of the residue on silica gel eluting with CH2Cl2 afforded 2-[(14-
bromotetradecyl)oxy]tetrahydro-2H-pyran as a faint-yellow syrup (11.31 g,
1
89%); H NMR (acetone-d6, 500 MHz) d 4.58–4.55 (1H, narrow m), 3.80
(1H, m), 3.69 (1H, dt, J=9.6, 6.7 Hz), 3.51 (2H, t, J=6.81 Hz), 3.45 (1H, m),
3.35 (1H, dt, J=9.6, 6.4 Hz), 1.91–1.82 (2H, m), 1.80 (1H, m), 1.64 (1H, m),
1.59–1.43 (8H, m), 1.32 (18H, br s). A solution of this compound (11.31 g,
30 mmol) in acetone (80 ml) was treated with sodium iodide (6.0 g, 40 mmol)
with heating at reflux for 1.5 h. The mixture was then cooled to RT,
partitioned between water and ether, and the layers were separated. The
aqueous phase was extracted with additional ether, and the combined organics
were dried over Na2SO4/MgSO4. Concentration in vacuo afforded the iodide
as a faint-yellow syrup (12.72 g, >99%). A solution of this compound in
acetonitrile (50 mL) was treated with triphenylphosphine (7.95 g, 30.0 mmol,
1 eq.) followed by heating at reflux for 16 h. The bulk of the acetonitrile was
removed by rotary evaporation under reduced pressure, and the residue was
placed under high vacuum (P51 mmHg) with heating at 608C to remove the
remaining solvent, and to afford the title compound as a thick, yellow syrup
1
(20.58 g, >99%); H NMR (acetonitrile-d3, 500 MHz) d 7.92–7.87 (3H, m),
7.76–7.69 (12H, m), 4.54 (1H, m), 3.81 (1H, m), 3.68 (1H, dt, J=9.6, 6.8 Hz),
3.48 (1H, m), 3.36 (1H, dt, J=9.6, 6.5 Hz), 3.24–3.14 (2H, m), 1.78 (1H, m),
1.70–1.59 (3H, m), 1.57–1.44 (8H, m), 1.40–1.22 (18H, m).
16,16,17,17,18,18,18-d7 -2-[(14Z)-Octadec-14-en-1-yloxy]tetrahydro-2H-pyran (17)
NaHMDS (1 M in THF, 31 ml) was added over a period of 5 min to a solution
of 16 (20.58 g, 30 mmol) in THF (100 ml) at 08C. The resulting cloudy, orange
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S. N. CRANE ET AL.
suspension was stirred at 08C for an additional 15 min prior to cooling to
ꢀ788C. d7-Butyraldehyde (2) (20 wt% in CH2Cl2, 3.00 g, 38 mmol) was then
added rapidly via syringe with stirring at ꢀ788C for 2 h, then at 08C for 15 min.
The reaction was quenched by the addition of 1 M pH 7 phosphate buffer (1:1
K3PO4/K2HPO4) and the THF was removed by rotary evaporation under
reduced pressure. Water and hexanes were added with rapid stirring at RT,
and the bulk of the solid triphenylphosphine oxide by-product was removed
by suction filtration. The layers were separated, and the organic phase was
washed with brine, and dried over Na2SO4/MgSO4. Concentration of the
organics, and flash chromatography on silica gel eluting with 1/99 ether/
hexanes (Rf ¼ 0:88, hexanes) gave the title compound as a colorless syrup
(8.46 g, 78%); 1H NMR (acetone-d6, 500 MHz) d 5.43–5.32 (2H, m), 4.56 (1H,
narrow m), 3.80 (1H, m), 3.69 (1H, dt, J=9.6, 6.7 Hz), 3.45 (1H, m), 3.35 (1H,
dt, J=9.6, 6.4 Hz), 2.04 (2H, overlapped m), 1.80 (1H, m), 1.65 (1H, m), 1.59–
1.48 (6H, m), 1.32 (20H, br s).
16,16,17,17,18,18,18-d7-2-(Octadecyloxy)tetrahydro-2H-pyran (18)
Wilkinson’s catalyst (960 mg, 4 mol%) was suspended in nitrogen saturated
benzene (80 ml) under a nitrogen atmosphere in a rubber septum capped Parr
flask. Compound 17 (8.46 g, 23.5 mmol) was then added as a solution in
nitrogen saturated benzene (20 ml), and the mixture was shaken under an
atmosphere of hydrogen gas (50 PSI) for 4 days. Following confirmation of the
1
completion of the reaction by H NMR, the benzene was evaporated and the
residue was purified by flash chromatography on silica gel eluting with 5/95
ethyl acetate/hexanes (Rf=0.43) to yield the title compound as a colorless
1
syrup (7.63 g, 90%). H NMR (acetone-d6, 500 MHz) d 4.56 (1H, narrow m),
3.83–3.76 (1H, m), 3.69 (1H, dt, J=9.6, 6.7 Hz), 3.45 (1H, m), 3.35 (1H, dt,
J=9.6, 6.4 Hz), 1.80 (1H, m), 1.65 (1H, m), 1.59–1.44 (4H, m), 1.43–1.24
(28H, s).
16,16,17,17,18,18,18-d7-Stearic acid (15) from scheme 2
Compound 18 (7.63 g, 21.0 mmol) was heated at 508C in methanol (150 ml)
containing toluene-4-sulfonic acid monohydrate (190 mg, 1.00 mmol). After
30 min at this temperature, the mixture was cooled to RT, and treated with a
saturated aqueous solution of NaHCO3 (10 ml), followed by concentration by
rotary evaporation under reduced pressure. The solid residue was suspended in
water, and collected by suction filtration. Drying under a stream of dry
nitrogen, and then under high vacuum overnight afforded 14 as a colorless
powder (5.28 g, 90%); Rf=0.38 (1/4 ethyl acetate/hexanes). A solution of 14
(5.28 g, 19.1 mmol) in acetone (250 ml) was added slowly over 15 min to a
solution of Jones reagent (2.7 M, 16 ml, 43 mmol) in acetone (100 ml) at RT.
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PREPARATION OF DEUTERIUM-LABELED FATTY ACIDS
1285
After stirring at RT for an additional 45 min, water (500 ml) was added, and
the mixture was stirred until all the chromium salts had dissolved. The
remaining solid was collected by suction filtration, and was washed with water
until no coloration was visible in the washings. The solid in the suction funnel
was then dissolved in ether, and this solution was washed with water, brine,
and was dried over Na2SO4/MgSO4. Concentration in vacuo afforded the title
compound as a colorless powder (5.40 g, 97%).
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J Label Compd Radiopharm 2006; 49: 1273–1285
DOI: 10.1002/jlcr