Practical syntheses of triacylglycerol regioisomers containing long-chain polyunsaturated fatty acids

Article abstract of DOI:10.1007/s11746-006-1001-9

Docosahexaenoic acid (DHA, 22:6n-3) is known to protect against a range of degenerative disease conditions and aid in the development of eye and brain function in infants. In dietary lipids DHA is found primarily in the triacylglycerol (TAG) form. However, the effects of the positional distribution of DHA in TAG on lipid functional properties such as bioactivity and oxidative stability are not clearly understood. Studies on this subject for the most part are limited by a lack of regioisomerically pure TAG model compounds containing DHA or similar long-chain (LC)-polyunsaturated fatty acids (PUFA). This paper reports on the development of a practical procedure, based on chemical and enzymatic reactions, for the syntheses of regioisomerically enriched, symmetrical and unsymmetrical TAG isomers containing two palmitic acid and one of linoleic acid, linolenic acid, or DHA. 1,3-Selective acylation of glycerol with vinyl esters of fatty acids catalyzed by Candida antarctica lipase and direct coupling with fatty acids in the presence of the coupling agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine furnished 1,3-dihexadecanoyl-2-docosahexaenoyl glycerol and its unsymmetrical isomer 1,2-dihexadecanoyl-3-docosahexaenoyl glycerol in 99 and 60% yield, respectively. Critical to the success of the unsymmetrical TAG synthesis is the demonstration that PUFA-containing glycerol acetonides can readily survive appropriately tailored acid-catalyzed conditions. In this way, sufficient quantities of highly regioisomerically enriched PUFA-containing unsymmetrical monoacylglycerols (MAG) and TAG have now become routinely accessible. The methods are amenable to scale-up and could be adopted for regioenriched synthesis of a wide range of TAG. AOCS 2007.

Full text of DOI:10.1007/s11746-006-1001-9

J Amer Oil Chem Soc (2007) 84:11–21
DOI 10.1007/s11746-006-1001-9
ORIGINAL PAPER
Practical Syntheses of Triacylglycerol Regioisomers Containing
Long-chain Polyunsaturated Fatty Acids
Benjamin H. Fraser Æ Patrick Perlmutter Æ
Chakra Wijesundera
Received: 27 March 2006 / Accepted: 26 September 2006 / Published online: 19 December 2006
ꢀ AOCS 2006
Abstract Docosahexaenoic acid (DHA, 22:6n-3) is
known to protect against a range of degenerative dis-
ease conditions and aid in the development of eye and
brain function in infants. In dietary lipids DHA is
found primarily in the triacylglycerol (TAG) form.
However, the effects of the positional distribution of
DHA in TAG on lipid functional properties such as
bioactivity and oxidative stability are not clearly
understood. Studies on this subject for the most part
are limited by a lack of regioisomerically pure TAG
model compounds containing DHA or similar long-
chain (LC)-polyunsaturated fatty acids (PUFA). This
paper reports on the development of a practical pro-
cedure, based on chemical and enzymatic reactions, for
the syntheses of regioisomerically enriched, symmet-
rical and unsymmetrical TAG isomers containing two
palmitic acid and one of linoleic acid, linolenic acid, or
DHA. 1,3-Selective acylation of glycerol with vinyl
esters of fatty acids catalyzed by Candida antarctica
lipase and direct coupling with fatty acids in the
presence of the coupling agents 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride and
4-dimethylaminopyridine furnished 1,3-dihexadeca-
noyl-2-docosahexaenoyl glycerol and its unsymmetrical
isomer 1,2-dihexadecanoyl-3-docosahexaenoyl glycerol
in 99 and 60% yield, respectively. Critical to the suc-
cess of the unsymmetrical TAG synthesis is the dem-
onstration that PUFA-containing glycerol acetonides
can readily survive appropriately tailored acid-cata-
lyzed conditions. In this way, sufficient quantities of
highly regioisomerically enriched PUFA-containing
unsymmetrical monoacylglycerols (MAG) and TAG
have now become routinely accessible. The methods
are amenable to scale-up and could be adopted for
regioenriched synthesis of a wide range of TAG.
Keywords Docosahexaenoic acid Á FA positional
distribution Á MAG Á Regioisomers Á Regiospecific
syntheses Á n-3 long-chain PUFA Á TAG
Introduction
There is currently a significant interest in developing
renewable food sources containing n-3 long-chain-
polyunsaturated fatty acids (LC-PUFA; C20 and
greater) such as docosahexaenoic acid (DHA) to re-
place the declining traditional fish sources. Oilseeds are
an obvious choice as they are easily grown and pro-
cessed into oils using conventional methods. Significant
advances have already been made towards achieving
the goal of producing n-3 LC-PUFA, including DHA,
in plants [1, 2]. The continued interest in food sources
of DHA stems from clinical trials showing that DHA
assists in brain and vision development in infants [3]
and protects against coronary heart disease [4]. There
is also emerging evidence that DHA protects against
other diseases, including diseases of the retina [5] and
metabolic syndrome disorders such as type-2 diabetes
and obesity [6, 7].
B. H. Fraser Á P. Perlmutter
School of Chemistry, Monash University,
Wellington Road, Clayton, VIC 3800, Australia
C. Wijesundera (&)
CSIRO Food Futures National Research Flagship
and Food Science Australia, 671 Sneydes Road,
Werribee, VIC 3030, Australia
e-mail: Chakra.Wijesundera@csiro.au
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J Amer Oil Chem Soc (2007) 84:11–21
The beneficial health effects of DHA may be related
and regioisomerically pure compounds are rarely ob-
tained. Furthermore, as it is the symmetrical 1,3-TAG
isomers that find applications as energy supplements,
relatively little attention has been given to the prepa-
ration of the corresponding unsymmetrical 1,2-TAG.
We report here on practical procedures for the
synthesis of regioisomerically enriched, symmetrical
and unsymmetrical TAG containing two palmitoyl and
one of linoleoyl, linolenoyl, or docosahexaenoyl resi-
dues.
not only to the amounts of these acids present in the
diet but also to their positional distribution within the
triacylglycerol (TAG) molecules [8–10]. By and large,
digestion of dietary TAG takes place via hydrolysis
with pancreatic lipase, which releases fatty acids (FA)
regiospecifically from the sn-1 and sn-3 positions,
resulting in enhanced absorption of FA attached to the
sn-2 position of the TAG. This is widely believed to be
the reason why palmitic acid in human milk fat, which
is largely in the sn-2 position, is better absorbed by
infants than it is from vegetable oil sources of similar
FA composition where the saturated FA is mainly
esterified to the sn-1,3 positions [11]. Apart from a few
studies on stearic acid in cocoa butter, FA other than
palmitic acid have not been studied systematically with
respect to possible positional effects [10], and despite
the growing interest in the health effects of DHA, little
effort has been made to find out if the positional dis-
tribution of DHA in TAG affects bioactivity.
Experimental Procedures
Materials
Most reactants were purchased from the Aldrich
Chemical Company (Sydney, Australia) and were used
as supplied. Vinyl palmitate was purchased from TCI
(Tokyo, Japan). Candida antarctica (Novozym 435)
and Rhizomucor miehei (Lipozyme RM IM) lipases
were gifts of Novozymes Australia Pvt. Ltd (Sydney,
Ausltralia). DHA (>99% purity) was purchased from
Nu-Chek Prep (Elysian, Minn.). Drying agents and
inorganic salts were purchased from AJAX or BDH
chemicals. Solvents were purified as follows. Anhy-
drous diethyl ether was distilled from sodium/benzo-
phenone ketyl prior to use. Dichloromethane (DCM)
was distilled from calcium hydride. Hexane refers to
the fraction boiling between 40ꢁ and 60ꢁC.
PUFA such as DHA are very susceptible to oxida-
tive deterioration. For this reason, food products con-
taining DHA may develop off-flavors, thereby limiting
their useful shelf-life. Means of making oils containing
DHA more resistant to oxidation would be extremely
valuable. Consequently, a knowledge of the effects of
positional distribution of DHA on its oxidative stabil-
ity would help in designing oils for maximum stability.
The few studies conducted to elucidate the effects of
positional distribution on the oxidative stability of
DHA have produced inconclusive and contradictory
results.
Chromatography
A major impediment to elucidating the role of the
positional distribution of DHA on oil functionality is
the lack of regioisomerically pure TAG model com-
pounds in amounts required for experiments. The
synthesis of regioisomerically pure TAG containing n-
3 LC-PUFA has recently been reviewed [12]. Although
considerable efforts have been made to prepare
structured lipids containing n-3 LC-PUFA such as
DHA, with a few exceptions, these efforts have fo-
cused on symmetrical medium-chain (C8–C12) TAG
with the DHA in the sn-2 position and the medium-
chain FA in the sn-1 and sn-3 positions [13, 14]. Focus
has been placed on the medium-chain TAG due to
their usefulness as energy supplements for patients
requiring remedial nutrition. In these applications, the
medium-chain FA serve as a ready energy source and
the DHA provide the essential FA [15]. In most cases,
these structured lipids have been made by modifying
natural oils, for example by lipase-mediated intereste-
rification of fish oil with medium-chain FA using a 1,3-
selective lipase. Such processes produce mixed TAG,
The silica gel used for chromatography was a 40- to
63-lm (230–400 mesh) silica gel 60 (no. 9385; Merck,
Darmstadt, Germany). Analytical thin-layer chroma-
tography (TLC) was performed on Polygram Sil G/
UV254 plastic sheets coated with silica gel containing a
UV254 fluorescent indicator (Merck) and were visual-
ized under UV light and/or dipped in an ammonium
molybdate/cerium sulphate solution.
Proton NMR (1H NMR) Spectroscopy
1H nuclear magnetic resonance (NMR) spectra were
recorded (Monash University, Melbourne, Australia)
at 300 MHz on a Bruker AM 300 spectrometer, at
400 MHz on a Bruker Avance DRX 400 spectrometer,
and at 800 MHz on a Bruker Avance 800 spectrometer
(Bruker, Germany). Chemical shifts are given on the d
scale in parts per million (ppm). Unless otherwise
stated, spectra were measured in deuterochloroform
(CDCl3) using the residual chloroform (CHCl3,
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J Amer Oil Chem Soc (2007) 84:11–21
13
7.26 ppm) signal as an internal reference. Each reso-
nance is shown according to the following convention:
chemical shift (d ppm) [multiplicity, coupling con-
stant(s) (Hz), number of hydrogens, assignment (where
possible)]. Multiplicities are designated as ‘s’ the sin-
glet, ‘d’ the doublet, ‘t’ the triplet, ‘q’ the quartet, ‘p’
the pentuplet, and ‘m’ the multiplet.
by weight of the ethanolic solution of TAG) at 30ꢁC for
4 h. The products were separated on a silica solid phase
extraction (SPE) cartridge to isolate 2-monoacylglyc-
erol (MAG), which were trans-methylated to fatty acid
methyl esters (FAME) and analyzed by gas chroma-
tography (GC) for FA composition.
Synthesis of 1,3-Dihexadecanoylglycerol 2
Carbon NMR (13C NMR) Spectroscopy
The procedure of Halldorsson et al. [17] was followed
and enabled the synthesis of the title compound on a
scale of approximately 30 g.
13C NMR spectra were recorded at 75 MHz on a
Bruker AM 300 spectrometer or at 200 MHz on a
Bruker Avance 800 spectrometer using CDCl3, unless
otherwise stated. The spectra were referenced using
the solvent carbon signal (CDCl3 = 77.16 ppm). Two-
dimensional NMR techniques such as homonuclear
correlation spectroscopy (COSY), heteronuclear mul-
tiple quantum coherence (HMQC), heteronuclear
multiple bond coherence (HMBC), and nuclear
Overhauser effect spectroscopy (NOESY) were used
to aid the assignment of some NMR spectra.
Synthesis of 1,3-Dihexadecanoyl-2-(9,12-
octadecadienoyl)glycerol (PLP) 3
1,3-dihexadecanoylglycerol
2
(5.3 g, 9.3 mmol) in
DCM (100 ml) was mixed with 9,12-octadecadienoic
acid (linoleic acid, 3.02 g, 11.2 mmol), 4-dimethylami-
nopyridine (DMAP, 0.45 g, 3.7 mmol), and 1-(3-dim-
ethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCI, 2.14 g, 11.2 mmol). The solution was stirred at
ambient temperature overnight before most of the
DCM was removed under reduced pressure and the
residue passed through a short plug of silica (100 g
silica, 100 ml of 20:80 ether/DCM by vol. eluent). The
eluent was removed under reduced pressure, yielding
the title compound 3 (97:3, PLP:PPL by 13C NMR) as
Mass Spectrometry
Electrospray ionisation (ESI) was performed on a
Micromass Platform Quadrupole Mass Spectrometet
(Monash University, Melbourne, Australia). High-
resolution mass spectra (HRMS) were recorded on a
Bruker BioApex 47e FTMS using NaI for accurate
mass calibration. M+ refers to the molecular ion.
1
white crystals (4.81 g, 62%). MP 34–36ꢁC. H NMR
(800 MHz, CDCl3) d 0.88 (t, J 7.3 Hz, 6H, 2· palmitic
CH3), 0.89 (t, J 7.8 Hz, 3H, linoleic CH3), 1.22–1.33 (m,
64H, 32· CH2), 1.58–1.63 (m, 6H, 3 · CO2CH2CH2
CH2), 2.00–2.03–2.06 (apparent q, J 7.2 Hz 4H,
CH2CH=CHCH2CH=CHCH2), 2.31 (t, J 7.5 Hz, 2H,
2· palmitic CO2CH2CH2CH2), 2.32 (t, J 7.4 Hz, 2H,
linoleic CO2CH2CH2CH2), 2.77 (apparent t, J 7.0 Hz,
2H, CH=CHCH2CH=CH), 4.14 (dd, J 5.9, 11.9 Hz, 2H,
one of each OCH2CHCH2O), 4.30 (dd, J 4.3, 11.9 Hz,
one of each OCH2CHCH2O), 5.24–5.28 (m, 1H,
Infrared (IR) Spectroscopy
IR spectra were recorded on a Perkin Elmer 1600
Series Fourier Transform spectrometer (Perkin Elmer,
Foster City, Calif.) as neat samples, CHCl3 solutions,
or as paraffin (Nujol) mulls of solids between NaCl
plates. IR frequencies are reported in wave numbers
(cm–1), and intensities are reported qualitatively as
strong (s), medium (m) or weak (w) and/or broad (b).
OCH2CHCH2O), 5.30–5.40 (m, 4H, 4· CH alkene). 13
C
NMR (200 MHz, CDCl3) d 14.22 (CH3), 14.26 (CH3),
22.84 (CH2), 25.02 (CH2), 25.03 (CH2), 25.79 (CH2)
27.35 (CH2), 29.21 (CH2), 29.28 (CH2), 29.29 (CH2),
29.50 (CH2), 29.51 (CH2), 29.64 (CH2), 29.78 (CH2),
29.82 (CH2), 29.84 (CH2), 29.85 (CH2), 32.08 (CH2),
34.21 (CH2), 62.24 (OCH2), 69.04 (OCH2), 128.04 (CH
alkene), 128.24 (CH alkene), 130.14 (CH alkene), 130.38
(CH alkene), 172.98 (C=O, 1C, 2-linoleic), 173.44 (C=O,
2C, 1,3-palmitic). IR (CHCl3) tmax 3010s, 2922s, 2854s,
1744s, 1465s, 1418m, 1378m, 1215s, 1170s, 1113s, 868w,
759s, 721m, 668w cm–1. MS calculated for
C53H98O6Na+ = 853.7; found: 853.7. HMS calculated for
C53H98O6Na+ = 853.7261; found: 853.7257.
Melting Points (MP)
The MP were recorded on a Kofler hot stage apparatus
and are uncorrected.
Lipase-based Positional Analysis
1
In addition to H and 13C NMR, a recently published
lipase-mediated method [16] was used to determine
regiopurity of the synthesized TAG. In brief, a mixture
of the TAG in ethanol (1:10, w/w) was shaken with the
1,3-specific immobilized Candida antarctica lipase (4%,
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Synthesis of 1,3-Dihexadecanoyl-2-(9,12,15-
octadecatrienoyl)glycerol (PLnP) 4
6H, 2· palmitic CH3), 0.97 (t, J 7.5 Hz, 3H, DHA
CH3), 1.21–1.33 (m, 50H, 25· CH2), 1.59–1.62 (m, 4H,
2· palmitic O2CCH2CH2CH2), 2.03–2.13 (m, 2H,
DHA CH=CHCH2CH2), 2.31 (t, J 7.6 Hz, 4H, 2·
palmitic O2CCH2CH2), 2.36–2.39 (m, 2H, O2CCH2
CH=CH2) 2.80–2.87 (m, 10H, DHA 5· C=CCH2C=C),
4.14 (dd, J 5.9, 11.9 Hz, 2H, one each of OCH2CH-
CH2O), 4.29 (dd, J 4.4, 11.9 Hz, 2H, one each of
OCH2CHCH2O), 5.22–5.46 (m, 13H, 12· CH alkene
and CH2CHOCH2). 13C NMR (200 MHz, CDCl3) d
14.26 (CH3), 14.42 (CH3), 20.71 (CH2), 22.8 (CH2),
22.79 (CH2), 22.84 (CH2), 25.01 (CH2), 25.70 (CH2),
25.76 (CH2), 25.79 (CH2), 29.28 (CH2), 29.42 (CH2),
29.51 (CH2), 29.63 (CH2), 29.77 (CH2), 29.81 (CH2),
29.84 (CH2), 32.08 (CH2), 34.20 (CH2), 62.18 (OCH2),
69.26 (OCH), 127.17 (CH alkene), 127.79 (CH alkene),
128.03 (CH alkene), 128.13 (CH alkene), 128.23 (CH
alkene), 128.43 (CH alkene), 128.44 (CH alkene),
128.49 (CH alkene), 128.73 (CH alkene), 129.61 (CH
alkene), 132.19 (CH alkene), 172.29 (C=O, 1C, 2-
DHA), 173.43 (C=O, 2C, 1,3-palmitic). IR (neat) tmax
3014s, 2913s, 2849s, 1732s, 1655w, 1471s, 1418s, 1376m,
1280s, 1230s, 1156s, 1107s, 1064m, 1025m, 921w, 867w,
718s cm–1. MS calculated for C57H98O6Na+ = 901.7;
found: 901.8. HMS calculated for C57H98O6H+ =
879.7442; found: 879.7445.
A similar procedure as for 3 was followed. 1,3-di-
hexadecanoylglycerol 2 (4.00 g, 7.03 mmol) in DCM
(100 ml) was reacted with 9,12,15-octadecatrienoic
acid (linolenic acid) (2.28 g, 8.24 mmol), DMAP (0.2 g,
1.64 mmol), and EDCI (1.92 g, 9.88 mmol). The
product was passed through a short plug of silica (100 g
silica, 100 ml of 20:80 ether/DCM by vol eluent) to give
the title compound 4 (98:2 PLnP:PPLn by 13C NMR)
as white crystals (5.60 g, 94%). MP 28–30ꢁC. 1H NMR
(800 MHz, CDCl3) d 0.88 (t, J 7.2 Hz, 6H, 2· palmitic
CH3), 0.98 (t, J 7.5 Hz, 3H, linolenic CH3), 1.22–1.38
(m, 56H, 28· CH2), 1.59–1.62 (m, 6H, 3· O2CCH2CH2
CH2), 2.03–2.10 (m, 4H, CH2CH=CHCH2CH
=CHCH2CH=CHCH2), 2.31 (t, J 7.4 Hz, 4H, 2· pal-
mitic O2CCH2CH2), 2.32 (t, J 6.3 Hz, 2H, linolenic
O2CCH2CH2), 2.81 (apparent t, J 5.6 Hz, 4H, CH2CH
=CHCH2CH=CHCH2CH=CHCH2), 4.14 (dd, J 5.9,
11.9 Hz, 2H, one each of OCH2CHCH2O), 4.29 (dd, J
4.3, 11.9 Hz, one each of OCH2CHCH2O), 5.25–5.28
(m, 1H, CH2CHOCH2), 5.30–5.42 (m, 6H, 6· CH al-
kene). 13C NMR (200 MHz, CDCl3) d 14.26 (CH3),
14.42 (CH3), 20.70 (CH2), 22.84 (CH2), 25.02 (CH2),
25.68 (CH2), 25.77 (CH2), 27.36 (CH2), 29.20 (CH2),
29.28 (CH2), 29.35 (CH2), 29.42 (CH2), 29.51 (CH2),
29.63 (CH2), 29.77 (CH2), 29.81 (CH2), 29.85 (CH2),
32.08 (CH2), 34.21 (CH2), 62.24 (OCH2), 69.04 (OCH),
127.26 (CH alkene), 127.92 (CH alkene), 128.38 (CH
alkene), 128.46 (CH alkene), 130.36 (CH alkene),
132.11 (CH alkene), 172.98 (C=O, 1C, 2-linolenic),
173.44 (C=O, 2C, 1,3-palmitic). I.R (neat) tmax 2927s,
2855s, 1737s, 1466m, 1379w, 1216w, 1165w, 1099w,
908s, 734s, 651s cm–1. MS calculated for
C53H96O6Na+ = 851.7; found: 851.8. HMS calculated
for C53H96O6H+ = 829.7285; found: 829.7268.
Synthesis of 1,2-Acetonide-3-(9,12-
octadecadienoyl)glycerol 7
1,2-Glycerol acetonide (solketal, 6) was synthesized
according to the procedure of Kubiak and Bruzik [18].
Solketal (3.76 g, 28.5 mmol) in DCM (40 ml) was
mixed with 9,12-octadecadienoic acid (linoleic acid)
(8.00 g, 28.5 mmol), DMAP (1.36 g, 11.2 mmol), and
EDCI (6.56 g, 20.9 mmol). The solution was stirred at
ambient temperature overnight before most of the
solvent was removed under reduced pressure, and the
residue was passed through a short plug of silica (100 g
silica, 200 ml of 20:80 ether/DCM by vol. eluent). The
solvent was then removed under reduced pressure,
yielding the title compound 7 as a colorless oil (11.2 g,
100%). 1H NMR (300 MHz, CDCl3) d 0.88 (t, J 7.0 Hz,
3H, linoleic CH3), 1.27–1.41 (m, 16H, 8· CH2), 1.36 (s,
3H, acetonide CH3), 1.42 (s, 3H, acetonide CH3), 1.58–
1.69 (m, 2H, O2CCH2CH2CH2), 2.00–2.09 (m, 4H,
CH2CH=CHCH2CH=CHCH2), 2.33 (t, J 7.5 Hz, 2H,
linoleic CO2CH2CH2CH2), 2.75 (apparent t, 5.7 Hz,
2H, CH=CHCH2CH=CH), 3.72 (dd, J 6.2, 8.4 Hz, 1H,
one of OCH2CHCH2O), 4.03–4.18 (m, 3H, thee of
OCH2CHCH2O), 4.29 (m, 1H, CH2CHOCH2), 5.26–
5.42 (m, 4H, alkene CH). 13C NMR (75 MHz, CDCl3)
d 14.2 (CH3), 22.7 (CH2), 25.0 (CH2), 25.5 (CH3), 25.8
Synthesis of 1,3-Dihexadecanoyl-2-(3,6,9,12,15,
18-docosahexaenoyl)glycerol (PDP) 5
A similar procedure as for 3 was followed. 1,3-di-
hexadecanoylglycerol 2 (4.00 g, 7.04 mmol) in DCM
(100 ml) was mixed with 3,6,9,12,15,18-DHA (2.89 g,
8.8 mmol), DMAP (0.36 g, 3.08 mmol), and EDCI
(1.48 g, 7.72 mmol). The solution was stirred at ambi-
ent temperature overnight before the DCM was re-
moved under reduced pressure. The residue was
purified by flash chromatography (140 g silica, 2 l of
10:90 ethyl acetate/hexane by vol. eluent) to afford the
title compound 5 (97:3, PDP:PPD, by 13C NMR) as a
white semi-crystalline solid (6.19 g, 99%). MP 24–
1
26ꢁC. H NMR (800 MHz, CDCl3) d 0.88 (t, J 7.2 Hz,
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15
(CH2), 26.8 (CH3), 27.3 (CH2), 29.2 (CH2), 29.3 (CH2),
29.5 (CH2), 29.7 (CH2), 31.6 (CH2), 34.2 (CH2), 64.7
(OCH2), 66.5 (OCH2), 73.8 (OCH), 109.9 (O2C(CH3)2
acetonide), 128.0 (CH alkene), 128.2 (CH alkene),
130.1 (CH alkene), 130.3 (CH alkene), 173.6 (C=O li-
noleic). MS calculated for C24H42O4Na+ = 417.3;
found: 417.3. HMS calculated for C24H42O4Na+ =
417.2981; found: 417.2986.
ether/DCM by vol. eluent) to obtain the title com-
pound 9 as a colorless oil (7.51 g, 75%). 1H NMR
(300 MHz, CDCl3) d 0.97 (t, J 7.5 Hz, 3H, DHA CH3),
1.37 (s, 3H, CH3 acetonide), 1.43 (s, 3H, CH3 aceto-
nide), 2.00–2.12 (m, 4H, CH=CHCH2CH2CH3), 2.41
(d, J 4.9 Hz, 2H, DHA O2CCH2C=CH2), 2.80–2.90
(m, 10H, DHA 5· CH=CHCH2CH=C), 3.73 (dd, J
6.1, 8.4 Hz, 1H, one of OCH2CHCH2O), 4.04–4.20
(m, 3H, three of OCH2CHCH2O), 4.27 (m, 1H,
CH2CHOCH2), 5.29–5.44 (m, 12H, alkene CH). 13C
NMR (75 MHz, CDCl3) d 14.4 (CH3), 20.7 (CH2), 22.9
(CH2), 25.5 (CH3), 25.7 (CH2), 25.7 CH2), 25.8 (CH2),
25.8 (CH2), 26.8 (CH3), 34.1 (CH2), 64.8 (OCH2), 66.5
(OCH2), 73.8 (OCH), 110.0 (O2C(CH3)2 acetonide),
127.2 (CH alkene), 127.2 (CH alkene), 127.9 (CH al-
kene), 128.0 (CH alkene), 128.2 (CH alkene), 128.3
(CH alkene), 128.4 (CH alkene), 128.5 (CH alkene),
128.7 (CH alkene), 129.6 (CH alkene), 132.2 (CH
alkene), 173.0 (C=O DHA). IR (neat) tmax 3013s,
2964s, 2933s, 1715s, 1654w, 1455m, 1380s, 1371s, 1258s,
1215s, 1157s, 1084s, 1058s, 990m, 928m, 843m, 792w,
711s cm–1. MS calculated for C28H42O4Na+ = 465.3;
found: 465.3. HMS calculated for C28H42O4Na+ =
465.2981; found: 465.2977.
Synthesis of 1,2-Acetonide-3-(9,12,
15-octadecatrienoyl)glycerol 8
A similar procedure as for 7 was followed. 1,2-Aceto-
nideglycerol 6 (3.76 g, 28.5 mmol) in DCM (40 ml) was
reacted with 9,12,15-octadecatrienoic acid (linolenic
acid) (8.36 g, 30.0 mmol), DMAP (1.44 g, 11.8 mmol),
and EDCI (6.88 g, 22.3 mmol), and the product was
purified by passage through a short plug of silica (100 g
silica, 200 ml of 20:80 ether/DCM by vol. eluent) to
obtain the title compound 8 as a colorless oil (10.7 g,
1
96%). H NMR (300 MHz, CDCl3) d 0.94 (t, J 7.5 Hz,
3H, linolenic CH3), 1.22–1.31 (m, 8H, 4· CH2), 1.33 (s,
3H, CH3 acetonide), 1.39 (s, 3H, CH3 acetonide), 1.54–
1.61 (m, 2H, CO2CH2CH2CH2), 2.00–2.09 (m, 4H,
CH2CH=CHCH2CH=CHCH2CH=CHCH2), 2.30 (t, J
7.7 Hz, 2H, linolenic O2CCH2CH2CH2), 2.77 (appar-
ent t, 5.6 Hz, 4H, CH2CH=CHCH2CH=CHCH2
CH=CHCH2), 3.69 (dd, J 6.2, 8.4 Hz, 1H, one of
OCH2CHCH2O), 4.00–4.11 (m, 3H, thee of
OCH2CHCH2O), 4.25 (m, 1H, CH2CHOCH2), 5.26–
5.33 (m, 6H, alkene CH). 13C NMR (75 MHz, CDCl3)
d 13.2 (CH3), 19.5 (CH2), 23.8 (CH2), 24.4 (CH3), 24.5
(CH2), 24.6 CH2), 25.6 (CH3), 26.2 (CH2), 28.1 (CH2),
28.6 (CH2), 33.1 (CH2), 63.5 (OCH2), 65.4 (OCH2),
72.7 (OCH), 108.8 (O2C(CH3)2 acetonide), 126.1 (CH
alkene), 126.7 (CH alkene), 127.2 (CH alkene), 127.3
(CH alkene), 129.2 (CH alkene), 130.9 (CH alkene),
172.5 (C=O linolenic). I.R (neat) tmax 3010s, 2932s,
2856s, 1742s, 1654w, 1456m, 1380s, 1371s, 1214s, 1161s,
1086s, 1058s, 918w, 843m, 792w, 721m cm–1. MS cal-
culated for C24H40O4Na+ = 415.3; found: 415.4. HMS
calculated for C24H40O4Na+ = 415.2824; found:
415.2829.
Synthesis of 3-(9,12-Octadecadienoyl)glycerol 10
An adaptation of the procedure of Kubiac and Bruzik
[18] was used. A solution of 1,2-acetonide-3-(9,12-oc-
tadecadienoyl)glycerol 7 (14.0 g, 33.5 mmol) in meth-
anol/DCM (2:1 by vol., 150 ml) was mixed with
Amberlyst-H+ resin (5 g). The mixture was stirred at
ambient temperature for 2 days before the resin was
filtered off and the solvent removed under reduced
pressure. The residual oil was purified by flash chro-
matography (150 g silica, 2 l of 60:40 ethyl acetate/
hexanes by vol.) to yield the title compound 10 as a
1
clear oil (9.75 g, 78%). H NMR (200 MHz, CDCl3) d
0.89 (t, J 6.8 Hz, 3H, linoleic CH3), 1.19–1.46 (m, 14H,
7· CH2), 1.55–1.76 (m, 2H, CO2CH2CH2CH2), 2.04
(m, 4H, CH2CH=CHCH2CH=CHCH2), 2.35 (t, J
7.2 Hz, 2H, CO2CH2CH2CH2), 2.50 (bs, 2H, 2· OH),
2.77 (apparent t, J 5.1 Hz, 2H, CH=CHCH2CH=CH),
3.58 (dd, J 5.8, 11.1 Hz, 2H, one each of OCH2CH-
CH2O), 3.70 (dd, 3.9, 11.5 Hz, 2H, one of each
OCH2CHCH2O), 3.88–4.21 (m, 1H, CH2CHOCH2),
5.31–5.47 (m, 4H, alkene CH). 13C NMR (75 MHz,
CDCl3) d 13.6 (CH3), 22.7 (CH2), 25.0 (CH2), 25.4
(CH2), 25.8 (CH2), 27.3 (CH2), 29.2 (CH2), 29.3 (CH2),
29.5 (CH2), 29.7 (CH2), 31.7 (CH2), 33.5 (CH2), 34.3
(CH2), 63.5 (OCH2), 65.3 (OCH2), 71.6 (OCH), 128.0
(CH alkene), 128.2 (CH alkene), 130.1 (CH alkene),
130.4 (CH alkene), 174.4 (C=O linoleic). IR (neat) tmax
Synthesis of 1,2-Acetonide-3-(3,6,9,12,15,
18-docosahexaenoyl)glycerol 9
A similar procedure as for 7 was followed. 1,2-Aceto-
nide glycerol (solketal, 6) (3.00 g, 22.7 mmol) in DCM
(100 ml) was reacted with DHA (8.22 g, 25.0 mmol),
DMAP (1.26 g, 10.0 mmol), and EDCI (5.76 g,
18 mmol). The product was purified by passage
through a plug of silica (100 g silica, 100 ml of 20:80
123

16
J Amer Oil Chem Soc (2007) 84:11–21
3421s, 3010s, 2929s, 2856s, 1731s, 1648w, 1466m, 1378w,
1247w, 1181w, 1120w, 1050w, 909s, 734s, 649m cm–1.
MS calculated for C21H38O4Na+ = 377.3; found: 377.3.
HMS calculated for C21H38O4Na+ = 377.2668; found:
377.2670.
7.5 Hz, 3H, DHA CH3), 2.02–2.12 (m, 2H, CH=
CHCH2CH2CH3), 2.26 (bs, 1H, OH), 2.39–2.45 (m,
4H, DHA O2CCH2C=CH2 and CH=CHCH2CH2CH3),
2.68 (bs, 1H, OH), 2.79–2.89 (m, 10H, DHA 5·
CH=CHCH2CH=C), 3.58 (dd, J 5.8, 11.5 Hz, 1H, one
of OCH2CHCH2O), 3.68 (dd, J 3.9, 11.5 Hz, 1H, one of
OCH2CHCH2O), 3.88–3.95 (m, 1H, one of OCH2CH
CH2O), 4.08–4.23 (m, 2H, one of OCH2CHCH2O and
OCH2CHCH2O), 5.28–5.45 (m, 12H, alkene CH). 13C
NMR (75 MHz, CDCl3) d 13.2 (CH3), 19.5 (CH2), 21.7
(CH2), 24.5 (CH3), 24.6 (CH2), 24.6 CH2), 33.0 (CH2),
62.3 (OCH2), 64.3 (OCH2), 69.2 (OCH), 126.0 (CH
alkene), 126.7 (CH alkene), 126.9 (CH alkene), 126.9
(CH alkene), 127.1 (CH alkene), 127.3 (CH alkene),
127.3 (CH alkene), 127.4 (CH alkene), 127.6 (CH
alkene), 128.6 (CH alkene), 131.0 (CH alkene),
172.5 (C=O DHA). IR (neat) tmax 3045s, 3013s, 2963s,
2932s, 1740s, 1654w, 1442m, 1391m, 1266m, 1161m,
1120m, 1053m, 988m, 928m, 792w, 708s cm–1. MS
calculated for C25H38O4Na+ = 425.3; found: 425.5.
HMS calculated for C25H38O4Na+ = 425.2668; found:
465.2667.
Synthesis of 3-(9,12,15-Octadecatrienoyl)glycerol
11
A similar procedure as for 10 was used. A solution of 1,2-
acetonide-3-(9,12,15-octadecatrienoyl)glycerol 8 (8.1 g,
20.6 mmol) in methanol/DCM (2:1 by vol., 120 ml) was
mixed with Amberlyst-H+ resin (3.5 g) and stirred at
ambient temperature for 2 days. The product was
recovered and purified by flash chromatography (140 g
silica, 2 l of 60:40 ethyl acetate/hexanes by vol.) to yield
1
the title compound 11 as a clear oil (5.07 g, 69%). H
NMR (300 MHz, CDCl3) d 0.94 (t, J 7.5 Hz, 3H, lino-
lenic CH3), 1.21–1.41 (m, 8H, 4· CH2), 1.54–1.68 (m, 2H,
CO2CH2CH2CH2), 2.01–2.08 (m, 4H, CH2CH=CHCH2
CH=CHCH2CH=CHCH2), 2.33 (t, J 7.4 Hz, 2H, lino-
lenic O2CCH2CH2CH2), 2.62 (bs, 1H, OH), 2.79
(apparent t, 5.6 Hz, 4H, CH2CH=CHCH2CH=CH
CH2CH=CHCH2), 2.98 (bs, 1H, OH), 3.57 (dd, J 5.9,
11.5 Hz, 1H, one of OCH2CHCH2O), 3.67 (dd, J 3.8,
11.5 Hz, 1H, one of OCH2CHCH2O), 3.90 (m, 1H,
CH2CHOCH2), 4.10–4.20 (m, 2H, two of OCH2CH-
CH2O), 5.28–5.40 (m, 6H, alkene CH). 13C NMR
(75 MHz, CDCl3) d 14.4 (CH3), 20.7 (CH2), 25.0 (CH2),
25.6 (CH2), 25.7 (CH2), 27.3 CH2), 29.2 (CH2), 29.3
(CH2), 29.7 (CH2), 34.2 (CH2), 34.4 (CH2), 63.5 (OCH2),
65.2 (OCH2), 70.4 (OCH), 127.2 (CH alkene), 127.9 (CH
alkene), 128.4 (CH alkene), 128.4 (CH alkene), 130.3
(CH alkene), 132.1 (CH alkene), 174.4 (C=O linoleic).
IR (neat) tmax 3396s, 3011s, 2930s, 2855s, 1739s, 1654w,
1462s, 1392m, 1244m, 1178s, 1120s, 1056s, 932w, 866w,
714m cm–1. MS calculated for C21H36O4Na+ = 375.3;
found: 375.4. HMS calculated for C21H36O4Na+ =
375.2511; found: 375.2511.
Synthesis of 1,2-Dihexadecanoyl-3-(9,12-
octadecadienoyl)glycerol (PPL) 13
A similar procedure as for 3 was followed. 3-(9,12-
ctadecadienoyl)-Glycerol 10 (4.00 g, 10.8 mmol) in
DCM (80 ml) was reacted with hexadecanoic acid
(palmitic acid) (5.52 g, 21.6 mmol), DMAP (1.04 g,
8.6 mmol), and EDCI (4.96 g, 25.9 mmol). The prod-
uct was purified by passage through a short plug of
silica (100 g silica, 100 ml of 20:80 ether/DCM by vol.
eluent) to obtain the title compound 13 (92:8 PPL:PLP
by 13C NMR) as a white solid (7.88 g, 88%). MP 25–
1
28ꢁC. H NMR (800 MHz, CDCl3) d 0.88 (t, J 7.3 Hz,
6H, 2· CH3 palmitic), 0.89 (t, J 7.8 Hz, 3H, CH3 lino-
leic) 1.23–1.38 (m, 64H, 32· CH2), 1.57–1.63 (m, 6H, 3·
CO2CH2CH2CH2), 2.03–2.06 (m, 4H, CH2CH=CH
CH2CH=CHCH2), 2.31 (t, J 7.2 Hz, 6H, 2· CO2CH2
CH2CH2), 2.32 (t, J 7.4 Hz, 3H, CO2CH2CH2CH2),
2.77 (apparent t, J 7.0 Hz, 2H, CH=CHCH2CH=CH),
4.15 (dd, J 6.0, 11.9 Hz, one of each of OCH2CH
CH2O), 4.29 (dd, J 4.1, 11.9 Hz, one of each of
OCH2CHCH2O), 5.25–5.28 (m, 1H, OCH2CHCH2O),
5.30–5.41 (m, 4H, alkene CH). 13C NMR (200 MHz,
CDCl3) d 14.21 (CH3), 14.26 (CH3), 22.72 (CH2), 22.84
(CH2), 25.79 (CH2), 27.35 (CH2), 29.24 (CH2), 29.28
(CH2), 29.50 (CH2), 29.64 (CH2), 29.66 (CH2), 29.77
(CH2), 29.78 (CH2), 29.82 (CH2), 29.86 (CH2), 31.68
(CH2), 32.08 (CH2), 34.38 (CH2), 62.27 (OCH2), 69.03
(OCH), 128.05 (CH alkene), 128.22 (CH alkene),
130.16 (CH alkene), 130.37 (CH alkene), 173.03 (C=O,
Synthesis of 3-(3,6,9,12,15,
18-docosahexaenoyl)glycerol 12
A similar procedure as for 10 was used. A solution of
1,2-acetonide-3-(3,6,9,12,15,18-docosahexaenoyl)glyc-
erol 9 (4.50 g, 10.2 mmol) in 2:1 MeOH/DCM (90 ml)
was mixed with Amberlyst-H+ resin (2 g). The mixture
was stirred at ambient temperature overnight before
the resin was filtered off, and solvent was removed
under reduced pressure. The residual oil was purified
by flash chromatography (ethyl acetate/hexane, 60:40
by vol.) to yield the title compound 12 as a clear oil
(3.60 g, 88%). 1H NMR (300 MHz, CDCl3) d 0.97 (t, J
123

J Amer Oil Chem Soc (2007) 84:11–21
17
1C, 2-palmitic), 173.40 (C=O, 1C, 3-linoleic), 173.44
(C=O, 1C, 1-palmitic). IR (neat) tmax 3009m, 2925s,
2854s, 1746s, 1466s, 1378m, 1236s, 1163s, 1116m, 722w
cm–1. MS calculated for C53H98O6Na+ = 853.7; found:
853.7. HRMS calculated for C53H98O6Na+ = 853.7261;
found: 853.7265.
hexadecanoic acid (palmitic acid) (5.10 g, 19.7 mmol),
DMAP (0.99 g, 7.98 mmol) and EDCI (4.62 g,
24.0 mmol). The product was purified by passage
through a short plug of silica (100 ml of 20:80 ether/
DCM by vol.) to obtain the title compound 15 (90:10
PPL:PLP by 13C NMR) as a white/yellow solid (7.01 g,
1
˚
89%). MP 21–23C. H NMR (800 MHz, CDCl3) d 0.88
Synthesis of 1,2-Dihexadecanoyl-3-(9,12,15-
octadecatrienoyl)glycerol (PPLn) 14
(apparent t, J 7.2 Hz, 6H, 2· palmitic CH3), 0.97 (t, J
7.3 Hz, 3H, DHA CH3), 1.22–1.34 (m, 50H, 25· CH2),
1.58–1.63 (m, 4H, 2· palmitic O2CCH2CH2CH2), 2.06–
2.10 (m, 2H, DHA CH=CHCH2CH2), 2.31 (t, J 7.4 Hz,
2H, palmitic O2CCH2CH2), 2.32 (t, J 7.3 Hz, 2H,
A similar procedure as for 3 was followed. A solution
of 3-(9,12,15-octadecatrienoyl)-Glycerol 11 (5.07 g,
14.4 mmol) in DCM (90 ml) was reacted with hexa-
decanoic acid (palmitic acid) (8.25 g, 31.8 mmol),
DMAP (1.59 g, 12.9 mmol), and EDCI (7.44 g,
38.7 mmol). The product was purified by passage
through a short plug of silica (100 ml of 20:80 ether/
DCM by vol.) to yield the title compound 14 (88:12
PPLn:PLnP by 13C NMR) as a white solid (10.3 g,
86%). MP 23–25ꢁC. 1H NMR (800 MHz, CDCl3) d
0.88 (apparent t, J 7.0 Hz, 6H, 2· palmitic CH3), 0.98
(t, J 7.5 Hz, 3H, linolenic CH3), 1.16–1.31 (m, 56H, 28·
CH2), 1.56–1.58 (m, 6H, 3· O2CCH2CH2CH2), 2.03–
2.10 (m, 4H, CH2CH=CHCH2CH=CHCH2CH=
CHCH2), 2.28–2.33 (m, 4H, 2· palmitic O2CCH2CH2),
2.81 (apparent t, J 5.6 Hz, 4H, CH2CH=CHCH2CH=
CHCH2CH=CHCH2), 4.14 (dd, J 6.0, 12.0 Hz, 2H, one
each of OCH2CHCH2O), 4.29 (dd, J 7.8, 12.0 Hz, one
each of OCH2CHCH2O), 5.25–5.41 (m, 7H, 6· CH
alkene and CH2CHOCH2). 13C NMR (200 MHz,
CDCl3) d 14.26 (CH3), 14.42 (CH3), 20.70 (CH2), 22.84
(CH2), 24.99 (CH2), 25.02 (CH2), 25.06 (CH2), 25.68
(CH2), 25.77 (CH2), 27.36 (CH2), 29.24 (CH2), 29.27
(CH2), 29.33 (CH2), 29.43 (CH2), 29.45 (CH2), 29.52
(CH2), 29.63 (CH2), 29.66 (CH2), 29.74 (CH2), 29.78
(CH2), 29.82 (CH2), 29.86 (CH2), 32.08 (CH2), 34.18
(CH2), 34.21 (CH2), 34.38 (CH2), 62.27 (OCH2), 69.03
(OCH), 127.27 (CH alkene), 127.90 (CH alkene),
128.40 (CH alkene), 128.45 (CH alkene), 130.39 (CH
alkene), 132.11 (CH alkene), 173.03 (C=O, 1C, 2-pal-
mitic), 173.40 (C=O, 1C, 3-linolenic), 173.44 (C=O, 1C,
1-palmitic). IR (neat) tmax 3011s, 2926s, 2854s, 1747s,
1465w, 1377w, 1236m, 1162s, 1115m, 721w cm–1. MS
calculated for C53H96O6Na+ = 851.7105; found: 851.8.
HMS calculated for C53H96O6H+ = 829.7285; found:
829.7270.
palmitic O2CCH2CH2), 2.38 (d,
J 2.8 Hz, 2H,
O2CCH2CH=CH2) 2.80–2.87 (m, 10H, DHA 5·
C=CCH2C=C), 4.14 (m, 2H, one each of OCH2CH-
CH2O), 4.29 (dd, J 4.2, 11.9 Hz, 2H, one each of
OCH2CHCH2O), 5.26–5.43 (m, 13H, 12· CH alkene
and CH2CHOCH2). 13C NMR (200 MHz, CDCl3) d
14.26 (CH3), 14.42 (CH3), 20.71 (CH2), 22.77 (CH2),
22.85 (CH2), 25.02 (CH2), 25.05 (CH2), 25.70 (CH2),
25.75 (CH2), 25.79 (CH2), 29.24 (CH2), 29.28 (CH2),
29.43 (CH2), 29.44 (CH2), 29.52 (CH2), 29.64 (CH2),
29.66 (CH2), 29.78 (CH2), 29.80 (CH2), 29.82 (CH2),
29.85 (CH2), 32.0 (CH2), 34.05 (CH2), 34.20 (CH2), 34.37
(CH2), 62.24 (OCH2), 62.44 (OCH2), 68.99 (OCH),
127.17 (CH alkene), 128.14 (CH alkene), 128.03 (CH
alkene), 128.15 (CH alkene), 128.24 (CH alkene),
128.26 (CH alkene), 128.41 (CH alkene), 128.43 (CH
alkene), 128.54 (CH alkene), 128.73 (CH alkene),
129.63 (CH alkene), 132.19 (CH alkene), 172.70 (C=O,
1C, 3-DHA), 173.02 (C=O, 1C, 2-palmitic), 173.43
(C=O, 1C, 1-palmitic). I.R (neat) tmax 3014s, 2925s,
2854s, 1747s, 1466s, 1377w, 1235m, 1154s, 1159m, 721m
cm–1. MS calculated for C57H98O6Na+ = 901.7; found:
901.8. HMS calculated for C57H98O6H+ = 879.7442;
found: 879.7448.
Results and Discussion
Limited studies have been conducted on regiospecific
synthesis of TAG containing n-3 LC-PUFA in combi-
nation with FA of a chain length C16 or greater [19–21].
Using a modification of the method of Awl et al. [20],
Endo et al. [19] prepared approximately 1-g quantities
of several TAG isomers containing DHA and eicosa-
pentaenoic acid (EPA) by acylation of the appropriate
MAG and diacylglycerol (DAG) in the presence of
1,1-dicyclohexylcarbodiimide (DCC) and 4-DMAP.
Although excellent regioisomeric purity was obtained
by this method, it is not suitable for the preparation of
larger amounts required for functionality studies as the
starting MAG and DAG are expensive and not readily
Synthesis of 1,2-Dihexadecanoyl-3-(3,6,9,12,15,
18-docosahexaenoyl)glycerol (PPD) 15
A similar procedure as for 3 was followed. A solution of
1-(3,6,9,12,15,18-docosahexaenoyl)glycerol 12 (3.60 g,
8.94 mmol) in DCM (100 ml) was reacted with
123

18
J Amer Oil Chem Soc (2007) 84:11–21
available in large quantities. Fauconnot et al. [21] also
recently published syntheses of structured TAG pairs
containing DHA and arachidonic acid using the (ben-
zotriazol-1-yloxy)-tripyrrolidinophosphonium hexaflu-
orophosphate (PyBOP) coupling reagent. Again, the
regioisomeric purity appeared to be excellent, but the
synthesis requires MAG and DAG starting materials
that are not readily available. The coupling reactions
were carried out on small scales (<100 mg) and gave
variable yields (20–60%). The results disclosed in the
present paper address this issue of availability of MAG
and DAG on larger scales (5–10 g) for functionality
studies.
mers 3–5 in excellent yields (94–99%, with the excep-
tion of PLP which was obtained in 62% yield) and
regiopurity (97–98% according to 13C NMR; Table 1).
The starting materials for this synthetic sequence are
readily available, and the intermediates and products
are easily isolated by crystallization or silica gel chro-
matography. In this manner, symmetrical TAG iso-
mers containing linoleic, linolenic, and DHA acids
were prepared on scales of up to 10 g.
Unsymmetrical TAG Synthesis
For the synthesis of the unsymmetrical TAG isomers
13–15 (Fig. 1), glycerol was protected, under standard
conditions, as the 1,2-acetonide 6. The unprotected
sn-3 hydroxyl was then coupled, using EDCI, with the
desired PUFA to give esters 7–9 in excellent yields. At
this stage we experimented with suitable conditions for
efficient removal of the acetonide protecting group. A
number of groups have investigated this reaction.
Working with saturated FA chains, Bornscheuer and
Yamane [22] reported that acetonide hydrolysis cata-
lyzed by boric acid produced MAG in good yield but
Symmetrical TAG Synthesis
We synthesized the symmetrical TAG isomers 3–5
(Fig. 1) using the chemoenzymatic procedure of Hall-
dorsson et al. [17]. Regioselective acylation of glycerol
1 with vinyl palmitate in the presence of Candida
antarctica lipase (CAL) gave the corresponding 1,3-
dipalmitate 2. It was then coupled, using EDCI, to the
desired PUFA to give the symmetrical 1,3-TAG iso-
Fig. 1 Regiospecific
O
OH
OH
Candida antarctica
syntheses of symmetrical and
unsymmetrical
triacylglycerols (TAG)
H+
vinyl palmitate
O
DCM
91%
acetone
95%
OH
OH
EDCI, DMAP
PUFA, DCM
glycerol
1,2-acetonide 6
glycerol 1
O
O
O
PUFA = linoleic 7
= linolenic 8
= DHA 9
(CH2)14CH3
O
OH
O
PUFA
O
(CH2)14CH3
O
O
Amberlyst
MeOH/DCM
2
EDCI, DMAP
PUFA, DCM
OH
PUFA = linoleic 10
= linolenic 11
= DHA 12
OH
O
PUFA
O
O
O
(CH2)14CH3
PUFA
PUFA = linoleic 3
= linolenic 4
= DHA 5
O
EDCI, DMAP
palmitic acd
DCM
O
O
O
(CH2)14CH3
O
Symmetrical TAG
O
(CH2)14CH3
(CH2)14CH3
PUFA = linoleic 13
= linolenic 14
= DHA 15
O
O
O
O
PUFA
Unsymmetrical TAG
123

J Amer Oil Chem Soc (2007) 84:11–21
19
Table 1 Yields, regiopurities, melting points, and characteristic 13C NMR data for synthetic triacylglycerol (TAG) regioisomers
TAGa
Yield (%)b
MP (ꢁC)
13C C=O shifts (ppm)
Regiopurity
by enzymatic
analysisc (%)
Regiopurity
by high field
13C NMR (%)
PLP
62
94
99
69
57
60
34–36
28–30
24–26
25–28
23–25
21–23
172.98, 173.44
172.98, 173.42
172.29, 173.43
173.03, 173.40, 173.44
173.03, 173.40, 173.44
172.70, 173.02, 173.43
94.8
98.0
96.6
86.7
84.1
82.2
97
98
97
92
88
90
PLnP
PDP
PPL
PPLn
PPD
a
P, Palmitic acid; L, linoleic acid; Ln, linolenic acid; D, docosahexaenoic acid
Overall yield from PUFA
b
c
According to the method of Shimada et al. [16]
that the reaction was slow. Aqueous HCl (50%) is
predominantly located in the 2-position of glycerol in
the parent oil is randomly distributed in the three
glycerol positions in the randomized oils. We also ob-
served that with increased unsaturation from linoleic,
linolenic to DHA corresponded to a decrease in the
melting point of the TAG.
reported to be unsuitable because it results in high
concentrations of DAG and TAG in the reaction
mixture. In our experience, strong acids (e.g. HCl) are
unsuitable for this purpose, particularly for compounds
containing PUFA because of significant by-product
formation, including the cleavage of the PUFA ester
linkage and acyl migration and reesterification. Kodali
[23] synthesized 1-MAG using dimethylboron bromide
at –50ꢁC to cleave the acetonide group, but we con-
sidered this inappropriate for larger scale synthesis
because of the high cost of the reagent and the low
reaction temperature required. The method of Kubiak
et al. [18] was an attractive option for larger scale
syntheses. It involves cleavage of acetonide groups
with H+-amberlyst resin/methanol at ambient temper-
ature. Thus, the treatment of esters 7–9 with H+-
amberlyst resin/methanol gave MAG 10–12 in good
yields, with no evidence of ester cleavage nor a
reaction with the highly sensitive methylene inter-
rupted alkene functionality. Evidence of some acyl
migration in the 13C NMR was observed and is
discussed in detail later. A final coupling of two pal-
mitic acid units onto the sn-1 and sn-2 positions of
MAG was achieved with EDCI and afforded regioen-
riched unsymmetrical TAG 13–15 (Fig. 1) in good
yields (57–69%) and high regiopurity (82–92%
according to 13C NMR; Table 1).
13C NMR Spectra
The regioassignment of the symmetrical TAG (PLP,
PLnP, PDP) and unsymmetrical TAG (PPL, PPLn,
PPD) targets were primarily made from high-field
(200 MHz) 13C NMR spectroscopy. For the symmet-
rical TAG 3–5 only two carbonyl resonances were
observed, as expected, which included a signal inte-
grating for one carbon in the range 172.29–172.98 ppm
(1C, sn-2 PUFA carbonyl) and a single signal further
downfield integrating for two carbons in the range
173.42–173.44 ppm [2C, sn-1(3) palmitic carbonyls]
(Table 1). For each unsymmetrical TAG 13–15, three
distinct carbonyl resonances were observed for each, as
expected. The sn-2 carbonyl (palmitic) resonances
came in the upfield range of 172.70–173.03 ppm, while
the two distinct sn-1,3 (PUFA or palmitic) carbonyl
resonances were in the range of 173.02–173.44 ppm
(Table 1). This carbonyl data is in agreement with that
reported by Haraldsson and co-workers [17, 24] and
Bergana and Lee [25] for similarly structured TAG.
The 13C NMR alkene region was also useful when
assigning the regioposition of the unsaturated carbon
chain on the glycerol backbone. Specifically, for the
PLP/PPL pair, the C-9 (130.14) and C-12 (128.04) PLP
resonances were lower than that for the C-9 (130.16)
and C-12 (128.05) resonances of PPL, whereas the C-10
(128.24) and C-13 (130.38) PLP resonances were
higher than that for the C-10 (128.22) and C-13
(128.24) carbons of PPL. A similar pattern was ob-
served for the PLnP/PPLn pair. This trend in differ-
ence of alkene carbon shifts for an unsaturated chain in
Melting Properties of TAG Regioisomers
The melting points of the various TAG isomers fol-
lowed a predictable trend. The 1,3-symmetrical TAG
with the PUFA in the middle position had slightly
higher melting points than the corresponding unsym-
metrical 1,2-TAG isomers where the PUFA are lo-
cated in one of the outer positions. This concurs with
the fact that randomized oils have a lower solid fat
content than the parent oil because the PUFA
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J Amer Oil Chem Soc (2007) 84:11–21
the sn-2 versus sn-3 position is in agreement with the
work of Wollenberg [26] on TAG-containing linoleic
and linolenic acid.
in the context that 3-MAG intermediates 10–12 are
precursors to TAG 13–15 (Fig. 1) and that MAG are
known to isomerize faster than 1,2- or 1,3-DAG [30].
1
13C and H NMR analysis of the glycerol 1,2-aceto-
Regiopurity
nide-3-PUFA intermediates 7–9 showed that they were
free from the corresponding 1,3-isomers, but following
amberlyst-catalyzed methanolysis a small amount of
the 2-MAG isomer could be detected. Acyl migrations
catalyzed by H+-amberlyst resin have been observed
previously [31]. Equilibrium ratios of MAG are known
to be approximately 90:10 in favor of the 1-MAG iso-
mer [32], which suggests something near equilibrium
was reached during the acetonide cleavage reaction.
An equilibrium ratio of 1,3- and 1,2-DAG is generally
60:40 favoring the 1,3-DAG [32], which explains the
relatively low amount of migration during syntheses of
the symmetrical 1,3-TAG.
The regiopurity of each TAG was first determined by
13C NMR (Table 1). The regiopurities of the PLP and
PPL isomers were 97 and 92% based on the ratio of the
carbonyl signals at 173.44 ppm (PLP) and 173.4 ppm
(PPL), respectively. Similarly, the regiopurities of
PLnP and PPLn were determined to be 98 and 88%,
respectively, and those of PDP and PPD, 97 and 90%,
respectively. For the PLnP/PPLn pair the carbonyl
resonances at 172.98 (PLnP) and 173.03 (PPLn) were
used, and for the PDP/PPD pair, the resonances at
172.29 (PDP) and 172.7 (PPD) were used.
Further evidence for the high regiopurity of the
synthesized TAG was provided by enzyme-based
positional analysis. Lipase-based methods are gener-
ally considered to be unsuitable for positional analysis
of EPA and DHA because they have been shown to
resist hydrolysis by lipase [24]. However, Shimada et al.
[16] recently reported that immobilized CAL acts on
saturated and unsaturated C14–C24 FA to a similar
degree, subsequently proposing that this lipase could
be used for positional analysis of DHA-containing oils.
Analysis of the synthetic TAG according to the
method of Shimada et al. [16] gave regiopurities of 82–
98% (Table 1). The actual purities of the compounds
were probably better than those indicated by the li-
pase-based measurements, particularly for the unsym-
metrical isomers, considering the likelihood of a small
amount of acyl migration during isolation of the 2-
MAG by chromatography on silica. Furthermore, we
have recently shown (unpublished) that CAL lipase
releases DHA from TAG more slowly than other FA,
resulting in an underestimation of the regiopurity of
TAG isomers in which the DHA is located at sn-1(3).
The synthesis of unsymmetrical TAG via glycerol
1,2-acetonide and the removal of the protecting group
by hydrolysis with H+-amberlyst resin/methanol is
readily amenable to scale-up. This affords a practical
method for preparing unsymmetrical TAG containing
PUFA in quantities required for functionality studies.
The synthesis has the potential of being made enan-
tioselective, as both enantiomers of glycerol 1,2-ace-
tonide (solketal) are readily available from sugar
precursors.
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