Synthesis of deuterated [D32]oleic acid and its phospholipid derivative [D64]dioleoylsn- glycero-3-phosphocholine

Article abstract of DOI:10.1002/jlcr.3088

Oleic acid and its phospholipid derivatives are fundamental to the structure and function of cellular membranes. As a result, there has been increasing interest in the availability of their deuterated forms for many nuclear magnetic resonance, infrared, mass spectroscopy and neutron scattering studies. Here, we present for the first time a straightforward, large-scale (gram quantities) synthesis of highly deuterated [D₃₂]oleic acid by using multiple, yet simple and high yielding reactions. The precursors for the synthesis of [D₃₂]oleic acid are [D₁₄]azelaic acid and [D₁₇]nonanoic acid, which were obtained by complete deuteration (>98% D) of their ¹H forms by using metal catalysed hydrothermal H/D exchange reactions. The oleic acid was producedwith ca. 94% D isotopic purity and with no contamination by the trans-isomer (elaidic acid). The subsequent synthesis of [D₆₄]dioleoyl-sn-glycero-3-phosphocholine from [D₃₂]oleic acid is also described. Copyright

Full text of DOI:10.1002/jlcr.3088

Special Issue Article
Received 22 May 2013,
Revised 12 June 2013,
Accepted 12 June 2013
Published online in 16 July 2013 Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3088
Synthesis of deuterated [D32]oleic acid and its
phospholipid derivative [D64]dioleoyl-
sn-glycero-3-phosphocholine†‡
a
a
a
a
*
Tamim A. Darwish, Emily Luks, Greta Moraes, Nageshwar R. Yepuri,
Peter J. Holden,a and Michael Jamesa,b,c
Oleic acid and its phospholipid derivatives are fundamental to the structure and function of cellular membranes. As a result,
there has been increasing interest in the availability of their deuterated forms for many nuclear magnetic resonance,
infrared, mass spectroscopy and neutron scattering studies. Here, we present for the first time a straightforward,
large-scale (gram quantities) synthesis of highly deuterated [D32]oleic acid by using multiple, yet simple and high yielding
reactions. The precursors for the synthesis of [D32]oleic acid are [D14]azelaic acid and [D17]nonanoic acid, which were
obtained by complete deuteration (>98% D) of their 1H forms by using metal catalysed hydrothermal H/D exchange
reactions. The oleic acid was produced with ca. 94% D isotopic purity and with no contamination by the trans-isomer (elaidic acid).
The subsequent synthesis of [D64]dioleoyl-sn-glycero-3-phosphocholine from [D32]oleic acid is also described.
Keywords: deuterated oleic acid; dioleoyl-sn-glycero-3-phosphocholine; deuterated phospholipid; [D17]nonanoic acid; [D14]azelaic
acid; olefinic proton
nuclear magnetic resonance (NMR),9–11 mass spectroscopy
Introduction
1
2
(MS),12 and neutron scattering13–23 studies. Although H and H
behave similarly in chemical reactions, the composition of their
nuclei results in vastly different neutron scattering properties.
The use of mixtures of deuterated and hydrogenated solvents to
manipulate neutron scattering length densities to enable contrast
variation is widespread in techniques, such as neutron
reflectometry, neutron diffraction and small angle neutron
scattering. However, this approach is limited for multicomponent
organic and biological systems containing molecules of similar
scattering length density, and the application of molecular
deuteration to solve this issue has been limited by the range of
deuterated molecules that are commonly available.
The surface of a biological cell is of great interest because it
mediates interactions and exchanges between the cell and its
surroundings. Phospholipids bearing different saturated and
unsaturated fatty acid chains are major components of biological
membranes [Figure 1(a)] and have been the focus of a vast
number of scientific studies in fields such as molecular biology,
biochemistry, chemistry, biophysics and pharmacology. Oleic
acid [Figure 1(c)] is a monounsaturated fatty acid, with a cis-
configuration about its double bond and is one of the most
widely distributed fatty acids in nature.
At the molecular level, oleic acid constitutes the unsaturated
tail component of many phospholipid molecules such as 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and
dioleoyl-sn-glycero-3-phosphocholine [DOPC—Figure 1(b)] that
are fundamental to the structure, function, order and fluidity of
cellular membranes. Oleic acid not only promotes membrane
fluidity at low temperature but also allows the cell membrane
to maintain a liquid crystalline state as the temperature
increases.1 Biophysical investigations into cellular membranes
are of vital importance in understanding fundamental
mechanisms of biomolecular transport, transcription, signalling
and receptor function. In addition, numerous neurological
disorders, neurodegenerative diseases (such as schizophrenia,
Parkinson's, Alzheimer's and prion disease)2–4 and the action of
bacterial toxins5–8 are closely associated with biochemical
interactions at cellular membranes. Therefore, there exists
substantial demand for isotopically labelled lipids and fatty acids
for many studies of membrane biology, biochemistry and
biophysics. Deuteration of phospholipids or other long-chain fatty
2
In NMR spectroscopy studies, H NMR has proven to be very
effective for the elucidation of lipid structure and motion in
aNational Deuteration Facility, Australian Nuclear Science and Technology
Organisation (ANSTO), Locked Bag 2001 Kirrawee DC NSW 2232, Australia
bSchool of Chemistry, The University of New South Wales, Kensington, Locked
Bag 2001 NSW 2052, Australia
cAustralian Synchrotron, 800 Blackburn Road, Clayton VIC 3168, Australia
*Correspondence to: Tamim A. Darwish, Australian Nuclear Science and
Technology Organisation (ANSTO), Kirrawee, DC NSW 2232, Australia.
E-mail: tamim.darwish@ansto.gov.au
† This article is published in Journal of Labelled Compounds and
Radiopharmaceuticals as a special issue on IIS 2012 Heidelberg Conference,
edited by Jens Atzrodt and Volker Derdau, Isotope Chemistry and Metabolite
Synthesis, DSAR-DD, Sanofi-Aventis Deutschland GmbH, Industriepark Höchst
G876, 65926 Frankfurt am Main, Germany.
1
2
‡ Supporting information can be found in the online version of this article.
acids is an essential prerequisite in many H and H (deuterium)
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T. A. Darwish et al.
Figure 1. Chemical and schematic structure of (a) a lipid bilayer membrane, (b) dioleoyl-sn-glycero-3-Phosphatidylcholine and (c) oleic acid.
model and biological membranes.9–11 However, relatively few fatty acids are well suited to such conditions. They undergo H/D
research groups have employed this technique because of the exchange when treated with alkaline D2O solution in the
difficulty in the synthesis of suitably deuterium labelled lipids. presence of a metal catalyst (Pt or Pd) at high temperatures
Although the use of commercially available deuterated (180–240°C).27,28
phospholipids with saturated acyl chains has been prevalent,
Prior to our development of a viable synthetic route to
investigations of highly biologically relevant biomimetic cellular deuterate oleic acid, it was evident that hydrothermal catalytic
membranes24 have been lacking because of the lack of H/D exchange of protonated oleic acid in Parr reactors reduced
deuterated lipids and fatty acids with unsaturated (e.g. oleoyl) the double bond to a great extent and induced migration of
chains. Previous reports in the literature have focused on site- the central double bond, scrambling the cis-configuration to
specific deuteration of oleic acid and its subsequent lipids and the more stable trans-isomer. Thus, for the synthesis of fully
derivatives.11b,25,26 With this background in mind, it becomes deuterated oleic acid, it was necessary to start from saturated
evident that there is a significant need to develop a convenient alkyl chain precursors, namely azelaic acid (diacid) and nonanoic
synthesis of completely deuterated oleic acid, from which acid, which were deuterated using hydrothermal H/D exchange
corresponding deuterated phospholipids can be produced. Here, reactions. Several routes for the stereospecific synthesis of
we report a convenient method for the production of gram unsaturated fatty acids and their derivatives have been
quantities of [D32]oleic acid (ca. 94% D isotopic purity) and its reported,29 with perhaps the simplest of these methods being
phospholipid derivative ([D64]DOPC) using a number of simple the one reported by Bergelson et al.30 in which the Wittig
and high yielding chemical reactions. This involves the reaction is employed to introduce unsaturation to the molecule.
hydrothermal metal catalysed H/D exchange reactions of azelaic The complete reaction sequence to synthesize [D32]oleic acid is
and nonanoic acids that are produced in high yields using given in Scheme 2. Specific aspects of the chemistry and issues
upscalable quantities (12–20 g ) and then conjugating the two arising from H/D scrambling during various reaction steps are
saturated alkyl chains in a cis-configuration. The overall deuteration examined and discussed in the succeeding text.
extent was measured by MS, and the deuteration level at the
different methylene units was probed using NMR spectroscopy to
validate the reaction process and monitor any D/H back exchange
Methylation of [D14]azelaic acid (1) to [D14]dimethyl azelate (2)
which could reduce the isotopic purity of the final products.
[D14]Azelaic acid was methylated to protect both ends of the
diacid by simply refluxing it in protonated MeOH with a catalytic
amount of H2SO4 to give pure [D14]dimethyl azelate (2) with 91%
yield (Scheme 2a), which did not require chromatographic
purification. The two protonated methyl groups on each end of
the molecule were used as an internal standard to calculate
the percentage deuteration at the different methylene units
along the alkyl chain as well as the overall deuterium content,
which was calculated to be 98.5% D for 2. This reaction was
achieved without any noticeable D/H back exchange on the
methylene units including in particular the alpha carbon (Cα)
positions next to the two carboxylic acid groups (~98.7% D)
(Supplementary data, Figures S1 and S6).
Results and discussions
Metal catalysed hydrothermal reactions in D2O are known to
affect H/D exchange on carbon sites in general and even on
saturated aliphatic carbons (non-acidic protons) to produce
completely or partially deuterated compounds (Scheme 1).27,28
As a result of the harsh conditions of hydrothermal reactions
(typically >200°C and >20 bar), undesirable side reactions, such
as dehalogenation, deuterium addition to multiple bonds,
hydrolysis, epimerization or the cleavage of protecting groups
can take place. However, robust molecules such as saturated
Selective hydrolysis of [D14]dimethyl azelate (2) to [D14
methyl hydrogen azelate (3)
]
Half esters (mono-esters) have often been prepared by partial
Scheme 1. Deuteration of saturated fatty acid by using metal catalysed hydrothermal
reaction.
esterification31 and direct fractional distillation of the three
J. Label Compd. Radiopharm 2013, 56 520–529
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T. A. Darwish et al.
Scheme 2. [D32]Oleic acid synthetic steps.
products of the reaction; however, this method is laborious. to be due to the reactivity of the acidic proton toward borane to
Furthermore, disproportionation of the half ester occurs with give trialkoxyboroxin intermediates which eventually liberate the
higher-boiling half esters (e.g. methyl hydrogen azelate) because alcohol.33,34 The rates of reduction being carboxylic acids >
prolonged fractional distillation at high temperatures is needed. aldehydes > ketones > olefins, imines > nitriles, amides and
In this study, the deuterated diester 2 was selectively hydrolysed epoxides > esters.33 The reduction of the acid group to alcohol
to yield the half ester 3 on the basis of the method of Rama Rao in [D14]methyl hydrogen azelate (3) was achieved using
et al.32 by using Ba(OH)2.8H2O.
dimethylsulfide borane (protonated version) in THF to give the
The method of selective hydrolysis using Ba(OH)2.8H2O relies corresponding alcohol ([D14]methyl 9-hydroxynonanoate (4))
on the insolubility of the mono-barium salt of the diester in with 96% yield. This reduction step did not lead to any D/H back
MeOH. Once the mono-barium salt ester is formed by the exchange on the deuterated methylene units and in particular
hydrolysis of the diester 2 with the barium base, it precipitates on the Cα (97.5% D), see Supplementary data, Figure S12.
out of the MeOH solution preventing further hydrolysis of the Although the deuterated borane (BD3) could be used to
second ester group, and it also prevents any significant D/H back generate 4 with two deuterium atoms on the Cα, the protonated
exchange in the basic solution. Any unreacted (unhydrolysed) version of the borane was used in this study to generate a
diester 2 remains in solution and hence the precipitated mono- protonated methylene unit next to the hydroxyl group. This
barium salt of the half ester was isolated by filtration, and the was deliberate to assist in characterizing the cis-configuration
1
white product was washed with aliquots of cold MeOH to wash of the double bond in the final product using H and 13C NMR
off the unreacted diester 2. The diester can be recovered by spectroscopy, as well as to allow probing of the extent of
dilution of the filtrates/washings with water followed by deuteration and the D/H back exchange that may occur during
extraction. The white precipitate of the mono-barium salt of the subsequent reaction steps. Unlike carbon bearing deuterium,
the half ester was acidified with HCl to pH 1 and extracted with the carbon signal of carbon-bearing hydrogen will show clearly
EtOAc. The crude product thus obtained contained 90% of the as a singlet in the 13C NMR spectrum.
half ester 3, and the remainder was the diacid 1, which was
easily separated on a silica column to give 63% yield of [D14]
methyl hydrogen azelate (3). This was achieved with negligible Synthesis of the Wittig salt [D14]9-carbomethoxy-
D/H back exchange on the Cα next to the acid and the ester nonyl-triphenyl phosphonium bromide (6)
groups. On the basis of NMR spectroscopy, the deuteration level
The bromination of the alcohol 4 using Br2 and triphenyl
at Cα position decreased from 98.7% D in 2 to 97.7% D in 3
phosphine in dichloromethane was a straightforward process35
(Supplementary data, Figure S9).
that readily produced [D14]methyl 9-bromononanoate (5) with
71% yield, and no D/H back exchange was observed
(Supplementary data, Figure S15). The synthesis of [D14]9-
carbomethoxy-nonyl-triphenylphosphonium bromide (6) was
Reduction of [D14]methyl hydrogen azelate (3) to [D14
methyl 9-hydroxynonanoate (4)
]
Borane complexes are known to reduce carboxylic acid groups then achieved in 83% yield by refluxing 5 with triphenyl
faster than most other groups and are therefore the reagents phosphine in anhydrous toluene.36 This was achieved with a
of choice for the reduction of carboxylic acids. Commonly, ester small amount of D/H back exchange at Cα position, which
groups remain intact during borane reduction of a carboxylic showed reduction in the degree of deuteration from 97.7% D
acid with almost quantitative yields. This has been demonstrated to 95.3% D (Supplementary data, Figure S18).
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T. A. Darwish et al.
Synthesis of [D18]nonanal (9)
The protonated methyl capping group (C(5)H3) in this
compound was used to accurately calculate the percentage
deuteration on the different methine and methylene units along
the alkyl chain. The percentage deuteration of the two
deuterium atoms on the Cα (C(4)D2) were observed to decease
by ca. 44% and the olefinic proton C(6)H was observed to
exchange to deuterium by ca. 59%. This indicates that H/D
scrambling has occurred between these two positions (i.e. C(6)
H and C(4)D2) and most likely this was mediated by the strong
base, hexamethyldisilazide, during the ylide formation when
mixing the phosphonium bromide salt 6 and the base for 1 h
Reduction of [D17]nonanoic acid to the [D19]alcohol nonanol was
achieved in 89% yield using LiAlD4, (Supplementary data, Figures
S21 and S22). The partial oxidation of the alcohol to the
corresponding perdeuterated aldehyde 9 was then performed
using pyridinium chlorochromate in dichloromethane which
gave 43% yield of 9 with no back exchange or H/D scrambling
(Supplementary data, Figures S23–S24).
Synthesis of methyl oleate via the Wittig reaction
before the addition of the deuterated aldehyde. The degree of
2
Coupling [D18]nonanal (9) to the phosphonium bromide 6 was exchange between the two positions was confirmed by the H
achieved by first generating the ylide at À78°C under aprotic NMR spectrum of this compound, which showed an increase in
conditions and then adding the aldehyde at the coolest possible the deuteration content of the olefinic position C(6)H and a
temperature (À70°C to À80°C). To ensure the formation of Z decrease in the deuteration extent in the C(4)D2 (Supplementary
double bond of methyl oleate, this reaction was performed using data , Figure S25). There was no indication of any H/D scrambling
the Z-specific variant of the Wittig reaction reported by occurring on other sites.
Bestmann et al.37, a method that has been used widely by a
number of groups to obtain pure Z-alkene forms of related
compounds, or in some cases, specifically deuterated oleic
Saponification of the [D32]methyloleate to oleic acid (10)
acid.26,38–41 This involved the use of a mixture of dry THF and The saponification of the oleic acid methyl ester by lithium
1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H)-pyrimidinone (DMPU) as hydroxide in aqueous MeOH (a mild reagent for hydrolysis that
solvents and potassium hexamethyldisilazide (KN(SiMe3)2) as does not affect unsaturated bonds)39,43 gave [D32]oleic acid
the base. This resulted in a 60% yield of the (Z)-methyl oleate (10) as a colourless oil; 80% yield. In the 1H NMR spectrum
(cis-configuration at the olefinic centre) with no contamination (Figure 3), the relative peak area of residual protons at C(4)D2
by the (E)-isomer (elaidic acid methyl ester; trans-configuration and the olefinic signal C(6)H remained unaltered in comparison
1
at the olefinic centre). H NMR of the product showed only two with the previous reaction step (Figure 2). This shows that no
signals in the region of δH 5.0–5.5 ppm. The small signal at δH significant D/H back exchange has occurred during the
5.25 ppm was assigned to the olefinic proton residue of C(6')D, saponification process. By considering the deuteration
whereas the signal at δH 5.32 ppm was assigned to the olefinic percentage values that were calculated from the [D32]oleic acid
proton of C(6)H (Figure 2). Bernstein et al.42 demonstrated that ester in Figure 2, the overall deuteration percentage of oleic acid
the trans-isomer (elaidic acid ester) has a signal at 0.03 ppm was calculated to be 94% D.
downfield (higher chemical shift) to that of the cis-isomer
As expected, the 13C NMR spectrum in Figure 4 (decoupling
(methyl oleate). In Figure 2, no other proton signal of C(6)H only protons, 13C{1H}) showed a single resonance for the olefinic
was observed at a higher chemical shift to δH 5.32 ppm, carbon C6 indicating that only a single isomer (i.e. cis) is present.
demonstrating that there was no contamination by the trans- Signals from carbons bearing only deuterium atoms are
isomer. Further analysis of the configuration of the olefinic depleted in normal 13C{1H} NMR spectra because of the
centre will be addressed later in the discussion.
extensive coupling with 2H atoms (spin quantum number 1).
Moreover, slower relaxation rates of carbons bearing deuterium
atoms in comparison with carbons bearing only protons or a
mixture of protons and deuterium also lead to spectra with low
Figure 2. 1H NMR (CDCl3, 400 MHz) of deuterated methyl oleate showing the
different resonances with their percentage deuteration, calculated using the
proton signal of the methoxy-group C(5)H3. Signal at δH 1.58 is due to H2O in the
NMR solvent.
Figure 3. 1H NMR (400 MHz, CDCl3) of [D32]oleic acid, showing the different
resonances with their relative percentage deuteration. Residual dichloromethane
solvent signal appears at δH 5.30 ppm.
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T. A. Darwish et al.
Figure 4. 13C{1H} NMR (400 MHz, CDCl3) of [D32]oleic acid showing a single resonance for the olefinic carbon of C6 and two resonances for C4, one attributed to C(4)DH
(triplet) and the other to C(4)H2 (singlet).
signal to noise ratio. H/D scrambling and back exchange at C4 residual allylic C(1)H. Similarly, 2H NMR showed only one
was confirmed by the 13C resonance of C4, which showed two deuterium signal for the allylic C(1)D (Supplementary data,
isotopically shifted signals; one was attributed to C(4)DH (triplet) Figure S26). In addition, signals of the olefinic protons of elaidic
and the other to C(4)H2 (singlet).
acid are usually slightly downfield by ~0.03 ppm (higher
The cis-configuration and trans-configuration of the olefinic chemical shift) compared with oleic acid,44 which was not
carbons in oleic acid can be distinguished by the H-1H scalar observed in Figure 3 for the olefinic proton C(6)H.
1
1
coupling, as well as by H NMR chemical shifts of the allylic C
Mass spectral analysis of the deuterated oleic acid (10)
1
(1)H. The H-1H scalar coupling of the olefinic proton signals in (Figure 5) revealed the relative abundance and mass isotopic
this case could not be determined because of deuteration and distribution of the different isotopologues, from which the
the extensive C-D coupling. The signals of the allylic protons in overall deuteration of 93.7% D can be calculated. This value
elaidic acid (trans-isomer) are usually shifted slightly upfield by matches the value calculated from 1H NMR (94% D) when
~0.05 ppm (lower chemical shift) compared with oleic acid (cis- considering the deuteration percentage at each carbon as
isomer). In Figure 3, only one signal was observed for the previously shown.
Figure 5. Electrospray ionization mass spectra in negative mode of [D32]oleic acid showing the mass distribution of the different isotopologues, which range from d28
–
d33. The distribution of the isotopologues is as follows: 5.9%, d33; 25.3%, d32; 34.8%, d31; 23.4%, d30; 8.3%, d29; 2%, d28
.
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T. A. Darwish et al.
Scheme 3. Reaction for synthesizing deuterated [D64]dioleoyl-sn-glycero-3-phosphocholine from [D32]oleic acid.
Figure 6. 1H NMR (CDCl3, 400 MHz) overlay spectra of protonated dioleoyl-sn-glycero-3-phosphocholine (DOPC) (commercial source, Avanti) and [D64]DOPC produced in
this study. Methylene units at C(7) (Cα) showed significant proton resonance in the [D64]DOPC suggesting considerable D/H back exchange. C(11')D is the proton residue of
the deuterated olefinic carbon.
Figure 7. Electrospray ionization mass spectra in positive mode of [D64]dioleoyl-sn-glycero-3-phosphocholine showing the mass distribution of the different
isotopologues, which ranges from d56–d66. The distribution of the isotopologues is as follows (M+ + Na+ + 1): 0.7%, d66; 0.5%, d65; 1.8%, d64; 7.2%, d63; 16.7%, d62; 24.1%,
d61; 23.7%, d60; 14.8%, d59, 6.7%, d58; 2.6%, d57; 1.2%, d56
.
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T. A. Darwish et al.
Synthesis of [D64]dioleoyl-sn-glycero-3-phosphocholine
(DOPS), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium
salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt)
acid (DOPA) may also be readily derived for the first time given
the current availability of deuterated oleic acid that is produced
using the method reported in this study.
[D32]Oleic acid produced using the aforementioned method was
subsequently used to synthesize deuterated [D64]DOPC. This
involved a single step, Steglich esterification, using a commercially
available compound (sn-glycero-3-phosphocholine-CdCl2, GPC),
following the method of Sing45 and is summarized in Scheme 3.
This resulted in the corresponding deuterated DOPC with 60%
yield, and ca. 91.5% deuteration of the oleoyl tails.
Experimental section
General
1
The H NMR spectrum of [D64]DOPC (Figure 6) was found to
Chemicals and reagents of the highest grade were purchased from
Sigma-Aldrich (Sydney, Australia) and were used without further
purification. Solvents were purchased from Sigma-Aldrich and Merck and
were purified by established methods.46 NMR solvents were purchased
from Cambridge Isotope Laboratories Inc. (MA, USA) and Sigma-Aldrich
and were used without further purification. D2O (99.8%) was supplied by
AECL, Canada. Thin-layer chromatography (TLC) was performed on Fluka
analytical silica gel aluminium sheets (25 F254) (product of Sigma-Aldrich).
Davisil® silica gel (LC60 Å 40–63μm ) (product of Sigma-Aldrich) was used
for bench-top column chromatography. Hydrothermal reactions were
performed in D2O at high temperatures by mixing the appropriate fatty
acid with NaOD and Pt/C (10% w/w) in a Mini Benchtop 4560 Parr reactor
(600 mL vessel capacity, 3000 psi maximum pressure, 350°C maximum
temperature) (Moline, USA). This was followed by filtering the catalyst,
acidifying the solution and then extracting the aqueous phase with EtOAc.
Thin layer chromatography was used (referenced with the protonated
compound) to estimate the purity and to develop separation protocols.
contain resonances with the same structure and chemical shift
as observed for a commercially available protonated sample
(Avanti Polar Lipids Inc. Alabama, USA). The only significant
difference between these two spectra arises from the presence
of D atoms at specific sites along the oleoyl tails. By integrating
the unlabelled trimethylamine subunit and comparing the
values with the two methylene units at the Cα positions (C(7)
D2), it was evident that the percentage deuteration at this
position had decreased to 21% D (Supplementary data, Figure
S27). This was also confirmed by 2H NMR, which showed a
diminished deuterium signal for the same methylene units
(Supplementary data, Figure S29). This is attributed to the basic
conditions of the coupling reaction which can lead to back
exchange at these relatively acidic sites. No exchange was
observed to occur at any other carbon in this molecule. By
considering the new values of the percentage deuteration of the
oleoyl chains, the overall percentage deuteration of deuterated
alkyl chains of DOPC can be calculated to be 91.9% D by NMR. This
value agrees with the percentage deuteration obtained from the
MS of [D64]DOPC by calculating the relative abundance and mass
isotopic distribution of the different isotopologues, which showed
an overall value of 91.5% D (Figure 7). The 31P NMR spectrum of
[D64]DOPC was found to be identical to that of the commercially
available protonated analogue (Supplementary data, Figure S28).
2
1H NMR (400 MHz), 13C NMR (100.6MHz) and H NMR (61.4MHz) spectra
were recorded on a Bruker 400MHz spectrometer at 298 K (Sydney,
Australia). Chemical shifts, in parts per million, were referenced to the
residual signal of the corresponding NMR solvent. Deuterium NMR was
performed using the probe's lock channel for direct observation. In 1H
NMR measurements, the protonated group within the molecule was used
as an internal standard to calculate the percentage deuteration of the other
deuterated sites within the molecule. This was achieved by comparing the
relative areas of the 1H peaks of the protonated group and the proton
residue of the deuterated groups. Alternatively, in some cases,
methylsulfonylmethane or dichloromethane was used as internal standard
to quantify the extent of deuteration. The overall deuterium content
Conclusions
(percentage) of
a compound was calculated from the individual
determinations at the different deuterated positions. This was performed
by taking the sum of the percentage deuteration of each position
multiplied by the number of protons/deuterium atoms at each of these
positions and then dividing the answer by the total number of protons/
deuterium in the compound (excluding any exchangeable protons, e.g.
carboxylic acid and alcohol protons).
Electrospray ionization mass spectra (ESI-MS) (MA, USA) were recorded
on a 4000 QTrap AB Sciex spectrometer. The overall percentage deuteration
of the molecules was calculated by MS using the isotope distribution
analysis of the different isotopologues. This was calculated taking into
consideration the 13C natural abundance, whose contribution was
subtracted from the peak area of each M+ 1 isotopologue to allow for
accurate estimation of the percentage deuteration of each isotopologue.
We have developed a straightforward and effective method for
the production of highly deuterated [D32]oleic acid in multiple
gram quantities, starting from deuterated fatty acids that can
be readily produced by hydrothermal exchange reactions on a
large scale. The overall deuteration level of the final product is
very high (ca. 94% D), making this a very effective molecule for
use in chemical or biomolecular studies using Fourier transform
infrared spectroscopy, 2H NMR or neutron scattering techniques.
It is worth noting that the difference in scattering length density
values (SLD) of protonated oleic acid (SLD = 0.08 × 10À6 AÀ2
versus the deuterated [D32]oleic acid produced in this study
(SLD = 7.07 × 10À6 À2) is quite significant, which makes this
)
A
deuterated form very suitable for contrast variation techniques
in neutron scattering studies of oleic acid. Characterisation by
1H, 2H and 13C NMR spectroscopies confirms the presence of
only the cis-isomer and reveals that certain sites within the
molecule were susceptible to exchange during the different
synthetic steps. Specifically, one of the olefinic protons, which
was selectively protonated, was observed to exchange and gain
deuterium to 59% D content, whereas the degree of deuteration
on the Cα next to the carboxylic acid group decreased to 56% D.
Subsequently, the synthesis of [D64]DOPC from [D32]oleic acid
has been achieved, which demonstrates that similarly labelled
lipid species of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
Synthesis of the deuterated materials
Perdeuteration of azelaic acid
A mixture of azelaic acid (12 g, 63.7 mmol), Pt/activated carbon (10% Pt)
(0.38 g, 0.195 mmol) and 40% w/w NaOD (13 g, 127 mmol) in D2O
(120 mL) was loaded into the Parr pressure reactor. The contents of the
reactor were degassed by purging with N2 gas and then sealed and
heated to 220°C (23 bar), with constant stirring for 3 days. The reactor
was cooled to room temperature, and the contents were filtered through
a short plug of Celite to remove the catalyst, which was further washed
with H2O (100 mL). The aqueous filtrate was acidified to pH 2 using 1 M
HCl. The white solid (azelaic acid) that was formed upon acidification
(DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) was extracted from the aqueous solution using EtOAc (3 × 100 mL). The
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Copyright © 2013 John Wiley & Sons, Ltd.
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T. A. Darwish et al.
organic layers were combined and dried with anhydrous Na2SO4, and the waxy white solid (17.5 g, 63% yield). 1H NMR (400 MHz, CDCl3): δH 1.25
solvent was removed under vacuum to give 11.5 g of white solid of
deuterated [D14]azelaic acid (92% D). The aforementioned quantity of
acid (11.5 g) was reloaded into the reactor, and the aforementioned
(0.089 H, br s, residual 3 × CH2), 1.56 (0.065 H, br s, residual 2 × CH2),
2.26 (0.047 H, br s, residual CH2), 2.30 (0.044 H, br s, residual CH2), 3.66
(3.000 H, s, CH3). 2H NMR (61.4 MHz, CDCl3): δD 1.25 (6.3D, s, 3 × CD2),
method was repeated using fresh reagents to give 10.5 g of a mixture 1.55 (4.0D, s, 2 × CD2), 2.25–2.28 (4.0D, br s , 2 × CD2). 13C{1H} NMR
of products which was shown to contain (ca. 1 g) of deuterated octanoic (100.6 MHz, CDCl3,): δC 23.5 (m, 3 × CD2), 27.5 (m, 2 × CD2), 33.1 (m,
acid (98% D) in addition to deuterated [D14]azelaic acid (1). The mixture 2 × CD2), 51.4 (s, CH3), 174.3 (s, CO ester), 179.8 (s, CO acid)
was washed with light petroleum to remove completely the deuterated (Supplementary data, Figures S9–S11).
octanoic acid leaving behind the target compound, deuterated azelaic
acid in a pure form (9.5 g, 74% yield on the basis of the original amount of
Preparation of compound 4
the protonated azelaic acid used; 98.3% D). 1H NMR (400 MHz, MeOD-d4,
methylsulfonylmethane as internal standard): δH 1.29 (0.322 H, br s,
residual 3 × CH2,), 1.55 (0.158 H, br s, residual 2 × CH2,), 2.24 (0.161 H, br
s, residual 2 × CH2-COOH), 3.00 (6.000 H, s, 2 × CH3, internal standard). 2H
NMR (61.4 MHz, MeOH-d4): δD 1.28 (6.0D s, 3 × CD2,), 1.53 (3.9D, s,
2 × CD2,), 2.23 (4.0D s, 2 × CD2). (Supplementary data, Figures S1–S3).
Borane-methyl sulfide complex (ca. 10 M) (100 mmol, 10 mL) was added
dropwise to a stirred solution of 3 (17.5 g, 81 mmol) in anhydrous THF
(100 mL) at 0°C under an inert atmosphere. The solution was maintained
at 0°C for 2 h and then allowed to warm up gradually to room
temperature overnight (18 h). MeOH (12 mL) was added dropwise and
the mixture was stirred continuously for a further 2 h. The solution was
then sparged gently with N2 and the solvent removed in vacuo. Water
and diethyl ether were added to the residue; the diethyl ether layer
Perdeuteration of nonanoic acid
[D17]Nonanoic acid was prepared according to the method described and the aqueous layer were extracted twice more with diethyl ether.
previously for [D14]azelaic acid and on a similar scale (i.e. 12 g of The combined organic layers were washed sequentially with NaHCO3,
protonated reagent). In this case, the by-product produced as a result brine and H2O, dried over anhydrous Na2SO4 and the solvent removed
of decarboxylation was deuterated octane, which was removed under in vacuo. The resulting clear liquid required no further purification
high vacuum to give pure [D17]nonanoic acid (10 g, 75% yield on the (15.7 g, 96% yield). 1H NMR (400 MHz, CDCl3): δH 1.25 (0.115 H, br s,
basis of the original amount of the protonated nonanoic acid; 98.3% residual 4 × CH2), 1.33 (1.066 H, br s, OH), 1.51–1.56 (0.091 H, m, residual
D). 1H NMR (400 MHz, CDCl3, dichloromethane as internal standard): δH
2 × CH2), 2.26 (0.050 H, br s, residual CH2), 3.61 (2.157 H, s, CH2-OH,),
0.82 (0.058 H, br s, residual CH3), 1.23 (0.192 H, br m, residual 5 × CH2), 3.65 (3.000 H, s, CH3). 2H NMR (61.4 MHz, CDCl3): δD 1.24 (8.0D, s,
1.58 (0.038 H, br s, residual CH2), 2.30 (0.038 H, br s, CH2-COOH), 5.29 4 × CD2), 1.53 (4.0D, br m, 2 × CD2), 2.25 (1.8D, s, CD2). 13C {1H} NMR
(2.000 H, s, CH2, internal standard). 2H NMR (61.4 MHz, CDCl3): δD 0.81 (100.6 MHz, CDCl3): δC 24.1 (m, 4 × CD2), 27.7 (m, 2 × CD2), 33.1 (m, CD2),
(3.0D, s, CD3), 1.21 (10.2D, m, 5 × CD2), 1.56 (2.0D, s, CD2), 2.29 (2.0D, s, 51.4 (s, CH3), 62.8 (s, CH2-OH), 174.3 (s, CO). (Supplementary data, Figures
CD2-COOH,). (Supplementary data, Figures S4 and S5).
S12–S14).
Preparation of compound 2
Preparation of compound 5
[D14]Azelaic acid (30 g, 148 mmol), anhydrous MeOH (150 mL) and
Triphenylphosphine (30.2 g, 115 mmol) was dissolved in dry DCM
concentrated H2SO4 (3 mL) was refluxed under N2 for 48 h. The solvent (140 mL) under a N2 atmosphere. The solution was cooled to 0°C, and
was evaporated until a small amount of MeOH remained. This was then
poured on ice and water and extracted three times with diethyl ether.
bromine (5.8 mL, 113 mmol) was added dropwise, ensuring that the
solution remained cold. After stirring for 20 min at 0°C, pyridine
The organic layer was washed with 10% NaHCO3, followed by brine (10.6 mL, 131 mmol) was added and the reaction stirred for a further
and dried over anhydrous sodium sulfate. Evaporation of the solvent 5 min. Compound 4 (15.7 g, 77 mmol) was dissolved in dry DCM (40 mL)
gave [D14]dimethyl azelate (2) (31 g, 91%), which was used without and added by syringe to the cold solution. The mixture was left stirring
further purification. Integration of the two methyl groups and comparing
them with the residual proton signals of the deuterated sites gave an
at 0°C for 2 h, before allowing it to warm to room temperature and then
stirring in air for a further 30 min. The salts were removed by vacuum
overall isotopic purity of 98.5% D. 1H NMR (400 MHz, CDCl3): δH 1.24 filtration and the solvent from the filtrate removed in vacuo to yield the
(0.096 H, br s, residual 3 × CH2), 1.54 (0.055 H, br s, residual 2 × CH2), crude product. Crude material was adsorbed on silica and purified by
2.24 (0.051 H, br s, residual 2 × CH2), 3.64 (6.000 H, s, 2 × CH3). 2H NMR flash chromatography (eluent: light petroleum/dichloromethane) to yield
1
(61.4 MHz, CDCl3): δD 1.22 (5.9D, s, 3 × CD2), 1.54 (4.0D, s, 2 × CD2), 2.22 a colourless liquid (14.6 g, 71% yield). H NMR (400 MHz, CDCl3): δH 1.25
(4.0D , s, 2 × CD2). 13C{1H} NMR (100.6 MHz, CDCl3) δC 23.7 (m, 3 × CD2), (0.117 H, br s, residual 3 × CH2), 1.36 (0.037 H, br s, residual CH2), 1.56
27.5 (m, 2 × CD2), 33.2 (m, 2 × CD2), 51.3 (s, 2 × CH3), 174.3 (s, 2 × CO (masked by the H2O signal, residual CH2 integration not available), 1.81
carbonyl). (Supplementary data, Figures S6–S8).
(0.045 H, br s, residual CH2), 2.26 (0.046 H, br s, residual CH2), 3.38
(1.980 H, s, CH2-Br), δ 3.66 (3.000 H, s, CH3). 2H NMR (61.4 MHz, CDCl3):
δD 1.24 (6.0D, s, 3 × CD2), 1.35 (2.3D, s, CD2), 1.55 (2.1D, s, CD2), 1.79
(2.2D, s, CD2,), 2.25 (2.0D, s, CD2). 13C{1H} NMR (100.6 MHz, CDCl3): δC
23.0–32.0 (m, all CD2), 33.7 (s, CH2-Br), 51.4 (s, OCH3), δ 174.3 (s, C = O).
(Supplementary data, Figures S15–S17).
Preparation of compound 3
This compound was prepared following a modified literature procedure
by Rao et al.32 A solution of Ba(OH)2.8H2O (20.8 g, 66 mmol) in anhydrous
MeOH (500 mL) was added to a solution of 2 (30 g, 130 mmol) in
anhydrous MeOH (50 mL) and left stirring at room temperature for 24 h.
The resulting white precipitate of the barium salt of the half ester was
Preparation of compound 6
collected by suction filtration. The filtrate was concentrated and refiltered Triphenylphosphine (44.1 g, 168 mmol) was added to a flame dried
to give a second crop. Both crops were washed with cold MeOH Schlenk flask and then gently put under vacuum to dry any residual
(2 × 50 mL), and the MeOH washings and the filtrate were combined
and diluted with water and extracted with dichloromethane (DCM) to
moisture. Under a N2 atmosphere, dry toluene (250 mL) was added and
the solution stirred to ensure that it was completely dissolved.
recover unreacted starting material 2. The white solid (combined) was Compound 5 (14.6 g, 55 mmol) was dissolved in dry toluene (2 × 50 mL)
suspended in H2O and 50% HCl was added to bring it to ca. pH 1, before and added to the aforementioned solution. The resulting mixture was
extracting with diethyl ether. The combined diethyl ether layers were refluxed for 68 h at 120°C under a N2 atmosphere. On cooling, an oily
washed with brine, dried over anhydrous Na2SO4 and the solvent layer was formed, which solidified upon standing. The toluene was
removed in vacuo. The crude material was purified on a silica column decanted off and the solid washed with toluene (2 mL) with gentle
(eluent: dichloromethane/EtOAc) giving the desired compound 3 as a heating and then cooling before decanting the toluene. The crude
J. Label Compd. Radiopharm 2013, 56 520–529
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T. A. Darwish et al.
material was purified on a silica column (eluent: dichloromethane/MeOH) and 5.32 (0.407 H, br s, CH = CD). 2H NMR (61.4 MHz, CDCl3): δD 0.81
to give a pale yellow oil (25.0 g, 83% yield). 1H NMR (400 MHz, CDCl3): δH (3.5D, br s, CD3), 1.21 (23.8D, br s, 10 × CD2), 1.55 (1.64D, br s, CD2), 1.94
1.17 (0.108 H, br s, residual 4 × CH2), 1.56 (0.094 H, br s, residual 2 × CH2), (3.8D, br s, 2 × CD2-C = C), 2.25 (1.2D, br s, CD2COO,) 5.36 (1.6D, br s, CH =CD).
2.20 (0.093 H, br s, residual CH2), 3.62 (3.000 H, s, CH3), 3.69 (2.013 H, d, JH- (Figures 2 and S25 in the Supplementary data).
P = 12.9 Hz, CH2-P), 7.70–7.81 (15.897 H, m, PPh3). 2H NMR (61.4 MHz,
The aforementioned methyl ester (3.6 g, 10 mmol) was dissolved in
CDCl3): δD 1.10–1.52 (12.0D, br m, 6 × CD2), 2.19 (2.1D, br m, CD2). 13C N2-purged MeOH (250 mL). Lithium hydroxide monohydrate (3.95 g,
{1H} NMR (100.6 MHz, CDCl3,): δC 22.5 (d, JC-P = 50 Hz, CH2-P), 27.0–28.0 94 mmol) dissolved in N2-purged water (100 mL) was added to the methyl
(m, all CD2), 51.4 (s, CH3), 118.4 (d, JC-P = 85.6 Hz, Ph), 130.5 (d, JC- ester solution with stirring. Initially, the reaction mixture was cloudy but
P = 12.3 Hz, Ph), 133.7 (d, JC-P = 9.98 Hz, Ph), δ 135.0 (d, JC-P = 3 Hz, Ph), δ eventually the solution became clear. The hydrolysis reaction was
174.4 (s, CO). (Supplementary data, Figures S18–S20).
monitored by TLC by using protonated oleic acid as reference. The
reaction reached completion after 24 h. HCl (1 M) was added to the
reaction mixture to acidify it to pH 1, where the solution became turbid
again indicating the liberation of the free acid. The turbid aqueous
solution was extracted three times with diethyl ether. The organic phase
was washed once with brine and then dried over anhydrous Na2SO4.
The solvent was evaporated under reduced pressure to give pale yellow
oil. The oil was purified on a silica column [eluant DCM : EtOAc (10:4)] to
give a colourless oil of deuterated oleic acid (10) (2.5 g, 80% yield). 1H
NMR (400 MHz, CDCl3): δH 0.82 (0.036 H, br s, residual CH3), 1.23 (0.279 H,
br m , residual 10 × CH2,), 1.58 (0.029, br s, residual CH2), 1.95 (0.104 H, br
s, residual 2 × CH2-C = C), 2.32 (0.890 H, br m, residual CH2COO-), 5.25
(0.045 H, br s, residual CH = CD) and 5.32 (0.407 H, br s, CH = CD). 2H NMR
(61.4 MHz, CDCl3): δD 0.81 (4.5D, br s, CD3), 1.21 (23.8D, br s, 10 × CD2),
1.57 (1.9D, br s, CD2), 1.94 (4.5D, br s, 2 × CD2-C = C), 2.30 (1.2D, br s,
CD2COO-) 5.36 (1.4D, br s, CH = CD). 13C{1H} NMR (100.6 MHz, CDCl3): δC
12.9 (m), 21.5 (m), 23.7 (m), 26.2 (m), 28.0 (br m), 30.6 (m), 33.3 (m),
Preparation of compound 8
Anhydrous THF (ca. 220 mL) was added under
a dry and inert
atmosphere into a flask containing lithium aluminium deuteride (6.5 g,
15 mmol) cooled in an ice bath. Nonanoic acid-d17 (17.1 g, 98 mmol) in
ca. 50 mL of anhydrous THF was added slowly, and the resulting mixture
was refluxed overnight with vigorous stirring. Water (ca. 50 ml) was
added very slowly to decompose the excess lithium aluminium
deuteride, followed by addition of dilute sulfuric acid (1 M) to dissolve
the resulting precipitate. The mixture was extracted with diethyl ether,
the extract washed with NaHCO3 and dried with anhydrous MgSO4.
The solvent was removed in vacuo, and the crude material was purified
on a silica column (eluent: dichloromethane/EtOAc, 9:1) to give a
colourless oil (14.3 g, 89% yield). 1H NMR (400 MHz, CDCl3): δH 0.87 (br
s, residual CH3), 1.26 (br s, residual CH2), 1.41 (s, OH), 1.52 (br s, residual
CH2), 3.60 (br s, residual CH2-OH). 2H NMR (61.4 MHz, CDCl3): δD 0.81
(3.0D, br s, CD3), 1.20 (12.3D, br s, 6 × CD2), 1.50 (2.2D, br s, CD2), 3.59
(1.8D, br s, CD2-OH). (Supplementary data, Figures S21 and S22).
129.5 (s), 179.2 (s). ESI-MS: 5.9%, d33; 25.3%, d32; 34.8%, d31; 23.4%, d30
8.3%, d29; 2%, d28. (Figures 3–5 and Figure S26 in Supplementary data).
;
Synthesis of [D64]dioleoyl-sn-glycero-3-phosphocholine (11)
Preparation of compound 9
from deuterated oleic acid (10)
Pyridinium chlorochromate (13.8 g, 64 mmol) and celite (14 g) were
added in one portion to a stirred solution of compound 8 (7.1 g,
43.5 mmol) in dry DCM (110 mL). The suspension was stirred vigorously
under N2 atmosphere for 18 h, and then the dark mixture was filtered
through a plug of florisil. Diethyl ether (400 mL) was run through the
plug. The ether washing was evaporated under vacuum to give a pale
orange oil (7.5 g). The oil was distilled under reduced pressure (11 mbar)
and fractions boiling between 70°C and 90°C were collected to give the
aldehyde 9 as clear oil (3 g, 43% yield). 1H NMR (400 MHz, CDCl3): δH
0.82 (br s, residual CH3), 1.23 (br m, residual 5 × CH2), 1.57 (br s, residual
CH2 masked by H2O), 2.37 (br s, residual CH2). 2H NMR (61.4 MHz, CDCl3):
δD 0.81 (3.0D, br s, CD3), 1.21 (9.8D, br s, 5 × CD2), 1.56 (2.0D, br s, CD2),
2.35 (1.7D, br s, CD2), 9.79 (0.8D, br s, CDO). (Supplementary data, Figures
S23 and S24).
[D64]Dioleoyl-sn-glycero-3-phosphocholine was synthesised according to
modified literature procedure45
where the oleoyl chains are
a
,
deuterated. In a 50-mL single-necked round-bottomed flask containing
sn-glycero-phosphocholine-CdCl2 complex (60 mg, 0.136 mmol) was
added 105 mg (0.335 mmol) of 10 dissolved in 10 ml of alcohol free
chloroform (anhydrous). The resulting suspension was vigorously stirred
and 4-dimethylamino pyridine (48 mg, 0.393 mmol) was added followed
by dicyclohexylcarbodiimide (81 mg, 0.393 mmol). The contents of the
flask were then degassed with N2, stoppered, protected from light and
stirred for 4 days at room temperature. The progress of the reaction
was monitored by TLC on silica gel CHCl3-CH3OH-H2O 65:25:4. When
the reaction was complete, chloroform was added, and the mixture
was filtered through a Celite pad (product of Sigma-Aldrich), which was then
washed with 15 mL more of chloroform. The chloroform was removed under
reduced pressure at room temperature, and the residue was dissolved in 5 mL
of CHCl3/CH3OH 1:1 and passed through a silica column, which was
preconditioned with CHCl3. The fractions containing phospholipids were
Preparation of compound 10
Phosphonium salt 6 (9.6 g, 18.3 mmol) was dissolved in dry 1,3-Dimethyl- combined, and the solvent was removed in vacuo at 25°C. This vacuum-
3,4,5,6-tetrahydro-2(1H)-pyrimidinone (60 mL) under a dry and inert dried fraction was then dissolved in a minimal volume of CHCl3/CH3OH/
atmosphere and then dry THF (160 mL) was added. The solution was H2O 65:25:4 and further purified on a column of silica gel by using the
cooled to À78°C and a 0.5 M solution of potassium hexamethyldisilazide same solvent system. Fractions were analyzed by TLC, and those fractions
(40 mL, 20 mmol) was added dropwise over a period of 30 min, when a
containing a product with an Rf identical to that of commercially
deep orange colour developed. The reaction mixture was stirred for 1 h available protonated DOPC were combined and the solvent was
at À78°C. Aldehyde 9 (3.0 g, 0.0187 mol) was then added (neat) dropwise removed in vacuo at 25°C. The phospholipid thus obtained (70 mg,
over 30 min. The reaction mixture was stirred at À78°C for 2 h, and then 60%) gave 31P NMR spectra identical to that of the commercially sourced
allowed to come to room temperature over night while stirring. The protonated sample. 1H NMR (400 MHz, CDCl3): δH 0.83 (0.073 H, br m,
reaction was then quenched with water (30 mL) and extracted with residual CH3), 1.23 (1.170 H, br m, residual 10 × CH2), 1.55 (0.046 H, br m,
diethyl ether (4 × 30 mL). The combined organic layers were washed residual CH2), 1.95 (br m, masked by the H2O/OH signal), 2.26 (3.175 H,
sequentially with 0.5 M HCl, saturated NaHCO3 and brine, and dried over br m, residual CH2COO-), 3.37 (9.000 H, s, N(CH3)3), 3.81 (1.946 H, b m,
anhydrous MgSO4. The solvent was evaporated under reduced pressure CH2-CH2-N), 3.96 (1.994 H, m, CH2-CH2-N), 4.12 (1.032H, m, -O-CH2-C(O)
to give a pale yellow oil which was purified on a silica column (eluent: H-CHH-O-P), 4.34 (1.776 H, m, -O-CH2-C(O)H-CH2-O-P), 4.39 (1.090 H, m,
dichloromethane/hexane, 1:1) to give a colourless oil (3.6 g, 60% yield).
-O-CH2-CH-CHH-O-P), 5.21 (0.978H, br m, -O-CH2-C(O)H-CH2-O-P), 5.25
1H NMR (400 MHz, CDCl3): δH 0.82 (0.069 H, br s, residual CH3), 1.23 (0.081 H, b s, residual -CH = CD-) and 5.32 (0.822 H, br s, CH = CD). 2H
(0.337 H, br m, residual 10 × CH2), 1.57 (masked by H2O signal), 1.95 NMR (61.4 MHz, CDCl3): δD 0.81 (3.0D, br s, CD3), 1.20 (17.3D, br s,
(0.139 H, br s, residual 2 × CH2-C = C,), 2.27 (0.873 H, br m, residual 10 × CD2), 1.52 (1.6D, br s, CD2), 1.94 (3.1D, br s, 2 × CD2-C = C), 2.33
CH2COO-), 3.66 (3.000 H, s, O-CH3), 5.25 (0.044 H, br s, residual CH = CD) (0.2D, br m, CD2COO) 5.37 (1.0D, br s, CH = CD). 31P NMR (161.9 MHz,
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 520–529

T. A. Darwish et al.
CDCl3) δP 0.498 (s). ESI-MS: 0.7%, d66; 0.5%, d65; 1.8%, d64; 7.2%, d63
16.7%, d62; 24.1%, d61; 23.7%, d60; 14.8%, d59, 6.7%, d58; 2.6%, d57
1.2%, d56. (Figures S27–S29 in Supplementary data)
;
;
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Acknowledgments
The authors acknowledge the National Deuteration Facility at
the Australian Nuclear Science and Technology Organization
(ANSTO); the operation of which was partially funded by the
National Collaborative Research Infrastructure Strategy (NCRIS).
The assistance of Ms Marie Gillon in preparing the deuterated
fatty acid precursors for this work is gratefully acknowledged.
[22] J. F. Hunt, P. D. McCrea, G. Zaccaı̈, D. M. Engelman, J. Mol. Biol. 1997,
Conflict of Interest
273, 1004–1019.
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The authors did not report any conflict of interest.
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