Synthesis of reversed structured triacylglycerols possessing EPA and DHA at their terminal positions

Article abstract of DOI:10.1016/j.tet.2015.09.034

This report describes synthesis of reversed structured triacylglycerols (TAGs) of the LML type, possessing pure EPA or DHA located at the terminal 1,3-positions of the glycerol backbone along with pure even number saturated fatty acids (C6:0–C16:0) occupying the 2-position. These compounds were synthesized by a two-step chemoenzymatic route involving a highly regioselective immobilized Candida antarctica lipase to incorporate EPA or DHA activated as acetoxime esters exclusively into the 1,3-positions of glycerol. The saturated fatty acyl groups were subsequently introduced to the remaining 2-position by EDCI coupling agent to accomplish the title compounds highly efficiently. This is the first report on reversed structured TAGs possessing the long-chain n-3 polyunsaturated fatty acids. It is anticipated that these novel compounds and their synthetic methodology will find various important uses such as analytical standards, in screening for bioactivity and in the pharmaceutical area as prodrugs.

Full text of DOI:10.1016/j.tet.2015.09.034

Tetrahedron 71 (2015) 8544e8550
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of reversed structured triacylglycerols possessing EPA and
DHA at their terminal positions
Anna V. Gudmundsdottir, Kaj-Anders Hansen, Carlos D. Magnusson,
*
Gudmundur G. Haraldsson
Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik, Iceland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2015
Received in revised form 5 September 2015
Accepted 14 September 2015
Available online 15 September 2015
This report describes synthesis of reversed structured triacylglycerols (TAGs) of the LML type, possessing
pure EPA or DHA located at the terminal 1,3-positions of the glycerol backbone along with pure even
number saturated fatty acids (C6:0eC16:0) occupying the 2-position. These compounds were synthe-
sized by a two-step chemoenzymatic route involving a highly regioselective immobilized Candida ant-
arctica lipase to incorporate EPA or DHA activated as acetoxime esters exclusively into the 1,3-positions of
glycerol. The saturated fatty acyl groups were subsequently introduced to the remaining 2-position by
EDCI coupling agent to accomplish the title compounds highly efficiently. This is the first report on re-
versed structured TAGs possessing the long-chain n-3 polyunsaturated fatty acids. It is anticipated that
these novel compounds and their synthetic methodology will find various important uses such as an-
alytical standards, in screening for bioactivity and in the pharmaceutical area as prodrugs.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Reversed structured triacylglycerols
Candida antarctica lipase
Chemoenzymatic synthesis
PUFA acetoxime esters
n-3 PUFA 1,3-Diacylglycerols
1. Introduction
TAGs are not only a source of energy but also provide essential fatty
acids for various biological roles including the n-6 and n-3 PUFAs.
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)
are by far the most predominant of the long-chain n-3 poly-
unsaturated fatty acids (n-3 PUFAs) that are characteristic of fish oil
and marine fat.1,2 Numerous beneficial effects on human health and
prevention of various diseases are attributed to EPA and DHA. They
include cardiovascular diseases, inflammation, autoimmune dis-
eases, rheumatoid arthritis, Alzheimer’s disease and related neu-
rogenerative disorders, type-2 diabetes and cancer.3e9 EPA and
DHA are precursors to various bioactive eicosanoid and docosanoid
mediators that include prostaglandins, leukotrienes, prostacyclins
and thromboxans5,6 as well as the more recently discovered highly
potent anti-inflammatory and pro-resolving protectins, resolvins
and maresins.7e9 Therefore, EPA and DHA may be regarded as
prodrugs.10 Their ethyl esters are in fact available as prescription
drugs registered as an adjuvant therapy to treat hyper-
triglyceridemia, both as a mixture of EPA and DHA11,12 as well as
virtually pure EPA.13,14
They constitute a glycerol skeleton to which three fatty acyl groups
are linked as carboxylate esters. The variety and number of differ-
ent TAG molecular species in fats and oils is countless as a result of
the great number of various fatty acids that differ in length, satu-
ration and location of carbonecarbon double bonds in unsaturated
and polyunsaturated fatty acids. This is particularly eminent in fish
oils that commonly comprise more than 50 different fatty acids.1,2
The location of fatty acids within the glycerol backbone adds fur-
ther to the complexity in terms of positional isomers that are also
named regioisomers. And, when the two fatty acids occupying the
terminal positions differ the TAGs become chiral offering the pos-
sibility of TAG enantiomers present.
The physical, sensory, nutritional and biological properties of
the TAGs must be largely influenced by the above described vari-
eties. The situation is somewhat simplified, however, by the fact
that the fatty acids are not randomly distributed in the TAGs that
are known to differ significantly in animals and plants from species
to species. Classical examples of such positionally structured TAGs
include cocoa butter15 used in chocolate manufacturing and human
milk TAGs.16 Generally in fish oil TAGs the mid-position is higher
enriched with EPA and particularly DHA, whereas in marine
mammals including whale oil and seal oil this is the other way
around.17 There are also multiple reports on enantiospecific posi-
tioning of fatty acids in animal and plant TAGs.18e20
Triacylglycerols (TAGs) are important constituents of the human
and animal diet. They are by far the largest class of nonpolar lipids
that occur ubiquitously in fats and oils of plant and animal origin.
* Corresponding author. Tel.: þ354 525 4818; fax: þ354 552 8911; e-mail
address: gghar@hi.is (G.G. Haraldsson).
http://dx.doi.org/10.1016/j.tet.2015.09.034
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

A.V. Gudmundsdottir et al. / Tetrahedron 71 (2015) 8544e8550
8545
Structured lipids usually refer to acylglycerols constituting se-
2. Results and discussion
lected fatty acids that are located at predetermined positions of the
glycerol backbone.21 Structured TAGs possessing long-chain poly-
unsaturated bioactive fatty acids such as EPA and DHA at the 2-
position and saturated medium chain (C6:0, C8:0 and C10:0) fatty
acids (MCFAs) at the terminal 1,3-positions have gained a continu-
ing interest of scientists as a result of their nutritional value and
properties.22,23 Preparation of such MLM (medium-long-medium)
type structured TAGs constituting varying enrichment levels of
MCFAs and n-3 PUFAs has been described in numerous reports.24
There are also number of reports on syntheses of MLM type
structured TAGs containing a pure MCFA at the terminal positions
2.1. Previous synthesis of normal structured MLM type TAGs
We reported a two-step chemoenzymatic process designed for
synthesis of MLM type structured TAGs possessing pure EPA or DHA
at the mid-position and pure MCFA at the end-positions.30 This
chemoenzymatic approach is demonstrated in Scheme 1 for cap-
rylic acid and DHA. In the first step glycerol was acylated exclu-
sively at the terminal positions by employing
a
highly
regioselective immobilized Candida antarctica lipase (CAL-B from
Novozymes) using the saturated fatty acids activated as vinyl esters.
The use of vinyl esters secures fast and irreversible reactions under
sufficiently mild conditions to eliminate detrimental acyl-
migration side reactions.
EPA and DHA were subsequently introduced to the remaining 2-
position of the resulting 1,3-diacylglycerol (1,3-DAG) intermediates
by aid of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCI) as a chemical coupling agent in presence of dime-
thylaminopyridine (DMAP) serving both as a base and catalyst in
dichloromethane at rt. All products and intermediates were ob-
tained in excellent yields with excellent chemical and regiopurity.
and
a pure EPA or DHA at the 2-position of the glycerol
backbone.25e27
All these reports take advantage of lipases that are ideally
suited as biocatalysts for structured lipid syntheses.24 This is
based on their high regioselectivity and strong preference to act
on the primary alcohol positions of the glycerol moiety in various
esterification and transesterification reactions. The mild condi-
tions in terms of temperature under which they act are also of
crucial importance to maintain full regiocontrol by hampering
disruptive intramolecular acyl-migration side reactions that are
associated with partially acylated glycerol and polyol
syntheses.28,29
We have reported an efficient two-step chemoenzymatic
synthesis of MLM type structured TAGs constituting pure EPA and
DHA at the 2-position with a pure MCFA at the terminal positions
of the glycerol backbone starting from glycerol.30 That work was
extended to a focused library of similarly structured TAGs cover-
ing all saturated even carbon number fatty acids from
C2eC16:0.30,31 This has been further extended to similarly struc-
tured enantiopure diacylglyceryl ethers (DAGEs) of the 1-O-alkyl-
sn-glycerol type possessing pure saturated even carbon number
fatty acids located at the terminal sn-3 position of the glycerol
backbone with pure EPA or DHA at the sn-2 position.32 A com-
prehensive well-defined library of such single pure structured
lipid compounds will enable their systematic screening for vari-
ous important chemical and biological properties. And, very re-
cently, the focused library was further expanded to include
reversed structured DAGEs, this time possessing the pure EPA and
DHA acyl groups at the terminal sn-3 position with the saturated
fatty acyl groups located at the sn-2 position of the glycerol
framework.33,34
By the work disclosed herein the focused library is further di-
versified to include oppositely structured TAGs, this time possess-
ing a pure EPA or DHA acyl group at the terminal positions with the
saturated fatty acyl group located at the 2-position of the glycerol
framework. Fig. 1 illustrates the chemical structures of a normal
MLM type structured TAG (constituting EPA and capric acid, C10:0)
and a reversed structured TAG of the LML (long-medium-long) type
(possessing DHA and caprylic acid, C8:0).
2.2. Synthesis of reversed structured LML type TAGs
The intended reversed structured TAG synthesis was more of
a challenge. For that task an activated form of EPA and DHA was
needed for the enzymatic step to proceed at adequate rate under
the mild conditions needed to retard the acyl-migration side re-
action. In structured TAG synthesis it is essential to avoid such acyl
migration reactions that result in loss of regiocontrol when the
fatty acids migrate from the intended position to an unwanted
position within the glycerol framework.30,31 The use of vinyl esters
was no longer an option since the polyunsaturated fatty acids do
not tolerate the chemical conditions required for preparing such
derivatives as was discussed in details in a recent report.33 More-
over, most of the commercially available microbial lipases do not
accept EPA or DHA and their derivatives as substrates.
Both obstacles were successfully overcome by the use of EPA
and DHA activated as oxime esters on which the immobilized C.
antarctica lipase (CAL-B) acted at sufficient rate under mild enough
condition to eliminate any acyl-migration side reaction. The mild
conditions are also greatly beneficial for transformations involving
the highly labile n-3 PUFAs.35 There is no doubt that CAL-B is su-
perior to all currently known lipases in dealing with EPA and DHA
as may be witnessed from numerous reports.27,36 The 1,3-DAG in-
termediate adducts 1 (EPA) and 2 (DHA) were obtained regiopure
and in very high yields (85% for both EPA and DHA). The details
have been reported in a recent publication.33 Fig. 2 shows the
chemical structure of the acetoxime ester of DHA.
Fig. 1. Chemical structures of a normal structured MLM type TAG possessing EPA at the 2-position and capric acid (C10:0) at the 1,3-positions (top), and a reversed structured LML
type TAG possessing DHA at the 1,3-positions and caprylic acid (C8:0) at the 2-position (bottom).

8546
A.V. Gudmundsdottir et al. / Tetrahedron 71 (2015) 8544e8550
Scheme 1. The two-step chemoenzymatic synthesis of normal structured MLM type TAG shown for caprylic acid (C8:0) and DHA. Reagents and conditions: (a) C. antarctica lipase,
vinyl octanoate, CH2Cl2, rt, 3 h; (b) DHA, EDCI, DMAP, CH2Cl2, rt, 12 h.
Fig. 2. The chemical structure of a DHA acetoxime ester.
To obtain the reversed structured TAGs the pure even carbon
number saturated fatty acids ranging from C6:0 to C16:0 were in-
troduced by chemical coupling to the 2-position of the resulting
1,3-DAGs 1 and 2 derived from glycerol possessing pure EPA and
DHA at their terminal 1,3-positions. In our previous reports this
turned out to be easy, in the symmetric 1,3-DAGs possessing the
straight-chain saturated acyl groups being readily acylated with
EPA and DHA using EDCI as a coupling agent. The question
remained as to whether reversing this, with two of the long-chain
n-3 PUFAs this time occupying both terminal positions of the
glycerol skeleton, would create problems related to steric hin-
drance in the coupling reaction. The PUFAs possess number of
permanent kinks on their hydrocarbon chain as a result from each
of the double bonds affecting their shape, rigidity and bulkiness.
The two-step overall chemoenzymatic process is illustrated in
Scheme 2 for the synthesis of 1aef (the EPA series) and 2aef (the
DHA series) with a, b, c, d, e and f referring to C6:0, C8:0, C10:0,
C12:0, C14:0 and C16:0, respectively.
sterically hindered 2-position of a 1,3-DAG intermediate as in the
current case. This relates to the lipase incorporating two equiva-
lents of the PUFAs into the less crowded terminal positions to form
a 1,3-DAG intermediate that subsequently underwent an acyl-
migration promoted by the acidic conditions offered by the pres-
ence of free fatty acids at an elevated temperature (65 ꢀC) to form
a racemic 1(3),2-DAG with the final acylation step taking place at
the less hindered primary alcohol outer position.36
The coupling reaction was successfully conducted in
dichloromethane at room temperature under similar conditions
as previously described for the MLM type structured TAG syn-
thesis.30,31 The saturated fatty acids were used in about 10% molar
excess with approximately 1.5 molar equivalents of the EDCI
coupling agent and 1 molar equivalent of DMAP as based on the
starting 1,3-DAG adduct. The reaction was allowed to proceed
overnight, although the reaction was possibly already completed
at an earlier stage (3e4 h), like in the preparation of the MLM type
structured TAGs described before.31 The reversed LML type
Scheme 2. The two-step chemoenzymatic synthesis of LML type reversed structured TAGs. Reagents and conditions: (a) C. antarctica lipase, PUFA as an acetoxime ester, CH2Cl2, rt,
3.5 h; (b) SFA, EDCI, DMAP, CH2Cl2, rt, 12 h. (For the sake of clarity the use of SFA (saturated fatty acid) and PUFA (polyunsaturated fatty acid) in this scheme refers to the hydrocarbon
chains of these molecules being saturated or polyunsaturated.).
Earlier, we had successfully prepared homogeneous TAGs pos-
sessing pure EPA or DHA as the sole fatty acid accommodating all
three positions of the glycerol moiety by use of lipase, where
glycerol was acylated with stoichiometric amount (three molar
equivalents) of EPA or DHA as free acids.36 In that case there were
strong evidences that the introduction of the third and final
equivalent of the PUFAs was taking place at a terminal 1- or 3-
position of a 1(3),2-DAG intermediate rather than the more
structured TAG products were obtained chemically and regioiso-
merically pure as was confirmed by 1H and 13C NMR spectroscopy,
in very high to excellent yields (88e93% for the EPA series and
89e94% for the DHA series) after purification by flash chroma-
tography on short silica gel column. Table 1 shows the yields
obtained for these reversed structured TAG products, the total of
12 such reversed structured TAG derivatives, 1aef for EPA and
2aef for DHA.

A.V. Gudmundsdottir et al. / Tetrahedron 71 (2015) 8544e8550
8547
Table 1
compounds of high value for pharmaceutical and medical pur-
poses since they may be esterified with a drug at the 2-position,
making use of the methodology described herein. The resulting
prodrugs acquire the biological properties of n-3 PUFAs and the
pharmaceutical properties of the drug in one and the same mol-
ecule and may indeed improve the therapeutic value of the
drug.40,41
The reversed structured TAG products constituting a pure saturated fatty acid (SFA)
at their 2-position and EPA (1aef) or DHA (2aef) at the 1,3-positions, along with
their obtained yields
Compound
SFA
PUFA
Yield
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
C6:0
C8:0
EPA
EPA
EPA
EPA
EPA
EPA
DHA
DHA
DHA
DHA
DHA
DHA
91%
90%
88%
89%
93%
90%
89%
94%
90%
91%
90%
90%
C10:0
C12:0
C14:0
C16:0
C6:0
2.3. Regiocontrol by 1H and 13C NMR spectroscopy studies
1H NMR spectroscopy was of great importance to monitor the
progress of the reactions and confirm the chemical identity and
purity of the intermediates and products. It was also of high utility
to evaluate the extent of possible acyl-migration side reaction and
therefore the regiopurity and regiocontrol of these processes. This
has been extensively discussed and described in our previous re-
ports on the chemoenzymatic synthesis of the MLM type struc-
tured TAGs.30,31 The acyl-migration side reaction is a well-
recognized problem in dealing with syntheses that involve par-
tially acylated carbohydrates, polyols and glycerol derivatives.28e31
This is a spontaneous intramolecular process controlled by ther-
modynamics and independent of the biocatalyst. It is promoted by
various parameters that include pH, presence of acid or base, type
of solvent, immobilized enzyme carrier material and temperature,
the most crucial parameter in the current studies. As before, there
were no signs of acyl-migration taking place neither in the enzy-
matic part33 of the current synthesis nor the coupling reaction
described herein as was firmly established by the 1H NMR studies.
Despite significantly lower accuracy 13C NMR spectroscopy was
also useful as an additional support to the regiocontrol of the
syntheses described. This relates to the carbonyl carbons of each
of the three categories of fatty acids, the saturated fatty acids, EPA
and DHA, each of them displaying a distinct resonance peak that
C8:0
C10:0
C12:0
C14:0
C16:0
It is hard to see how synthesis of structured TAGs of the current
type may be brought about without a lipase. Traditional organic
synthesis is much dependent on protection-deprotection based
approaches. The use of many of the most practised protective
groups such as acetals requires mild acidic conditions for their
deprotection under which acyl-migration is unescapable. And, in
the current case involving polyunsaturated fatty acids alternative
use of benzyl based protective groups can also be ruled out since
catalytic hydrogenolysis is no longer an option when that type of
acyl groups are present. This underlines the importance of the li-
pase and use of activated PUFAs in tasks of this type to prepare
reversed structured TAGs and other related acylglycerol based
lipids.
These compounds are the first reversed structured TAGs of the
LML type possessing the long-chain n-3 PUFAs to be reported.
There is little doubt that such compounds will become an impor-
tant complementary to the already existing focused library of well
defined chemically and regiopure lipid compounds.30e34 The re-
versed structured TAGs may certainly offer unique opportunities in
terms of screening for biological activity of EPA and DHA in relation
to their location at the end-positions versus the mid-position in
TAGs bearing in mind the difference between fish oil and marine
mammal TAGs.17 Ackman pointed out a possible relevance to that in
relation to a lower incidence of cardiovascular diseases among
Greenland Inuits consuming seal fat rather than much fish in their
diet.37
depends upon their location at the terminal 1,3-positions (
the 2-position ( ) of the TAG glycerol backbone. The carbonyl
carbons of all the saturated fatty acids C6:0eC16:0 investigated in
the current work were observed to resonate at 172.8 ppm when
a) or
b
d
located at the 2-position of the TAGs. The corresponding reso-
nance value for EPA located at the 1,3-positions remained at
d
173.0 ppm and that for DHA at these positions at d 172.5 ppm. As
can be noticed in the experimental section these values were quite
consistently obtained for these fatty acids. This compares to
d
173.3 ppm for the saturated fatty acids when located at the 1,3-
positions and 172.6 ppm for EPA and 172.1 ppm for DHA when
d
d
The reversed structured TAGs may also find value as important
chemical standards and fine chemicals for various purposes. These
structured TAGs as well as their preparation methodology may also
find important use in the pharmaceutical area as prodrugs offering
the possibility of attaching an active drug molecule to the 2-
position of the glycerol backbone in a combination with bioactive
EPA or DHA already within the same molecule. Interestingly
enough, EPA and DHA have been described as prodrugs in relation
to their serving as precursors to drug-like bioactive eicosanoids,
resolvins and protectins.10 Finally, the straightforward and simple
methodology may also become of use in introducing radiolabelled
molecules into the acylglycerol structure.
located at the 2-position as obtained for normal structured MLM
type TAGs from previous studies.30,31 Table 2 lists the character-
istic 13C NMR chemical shift values for these carbonyl groups.
Some deviations from previously reported data for MLM struc-
tured TAGs30 may be noticed that are most likely related to the
current data being derived from a 400 MHz instrument as com-
pared to 250 MHz instrument (as based on protons) in the pre-
vious case. These differences affecting EPA when located at the b-
position and DHA at the
decimal digits.
a-position were evident in the second
For rational design of acylglycerol prodrugs labile drugs such as
L-Dopa and some non-steroidal anti-inflammatory drugs
(NSAIDs) have been incorporated into the 2-position of 1,3-DAGs
to increase drugs absorption and delivery by the intestinal lym-
phatic system following oral administration.38,39 Up to now their
syntheses, however, have been primarily confined to saturated
acyl chains due to lack of efficient methods to insert other types of
more labile fatty acids into the end-positions of the glycerol
backbone. Symmetric 1,3-DAGs comprised of EPA and DHA are
Table 2
The 13C NMR chemical shift values characteristic for the carbonyl carbons of satu-
rated fatty acids (C6:0eC16:0), EPA and DHA depending on their location at the 1,3-
positions (
a
) or 2-position (
b) of the glycerol backbone of structured TAGs
Fatty acid type
d
(ppm), C]O (ppm), b C]O
a
d
C6:0eC16:0
EPA
DHA
173.3
173.0
172.5
172.8
172.6
172.1

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A.V. Gudmundsdottir et al. / Tetrahedron 71 (2015) 8544e8550
3. Conclusion
2.14e2.04 (m, 8H, ]CCH2CH3 and ]CCH2CH2), 1.70 (q (br), 4H, J
7.5 Hz, CH2CH2COO in EPA), 1.65e1.58 (m, 2H, CH2CH2COO in FA),
1.35e1.27 (m, 4H, CH2), 0.97 (t, 6H, J 7.5 Hz, CH3 in EPA), 0.90 (t, 3H, J
A novel approach was recently disclosed to incorporate EPA and
DHA exclusively into the terminal positions of glycerol and enan-
tiopure 1-O-alkylglycerols by use of EPA and DHA activated as
acetoxime esters in a highly regioselective transesterification pro-
cess catalyzed by immobilized C. antarctica lipase B.33 This resulted
in reversed structured 1,3-DAGs, possessing EPA and DHA at their
end-positions, and enantiopure MAGEs, possessing EPA and DHA at
their sn-1 or sn-3 end-position, in very high to excellent yields
(85e93%). A subsequent introduction of saturated fatty acids into
their 2-position resulted in novel reversed structured TAGs and
DAGEs by use of chemical coupling. This was very recently reported
for the reversed structured DAGEs, which were obtained in very
high to excellent yields (85e98%),34 and is disclosed in the current
report for the similarly structured TAGs that were obtained in
88e94% yields.
7.0 Hz, CH3) ppm; 13C NMR (100 MHz, CDCl3)
d 173.0 (2), 172.9,
132.0 (2), 128.9 (2), 128.8 (2), 128.6 (2), 128.3 (2), 128.2 (4), 128.1 (2),
127.9 (2), 127.0 (2), 68.8, 62.2 (2), 34.1, 33.4 (2), 31.2, 26.5 (2), 25.6
(6), 25.5 (2), 24.7 (2), 24.6, 22.3, 20.6 (2), 14.3 (2), 13.9 ppm; FTIR:
nmax 3012 (s, CH), 2932 (vs, CH), 2873 (s, CH), 1743 (vs, C]O) cmꢁ1
.
HRMS m/z calcd for
C
49H74O6 (MþNHþ4 ) 776.5824, found
776.5806 amu.
4.1.2. Synthesis of 1,3-dieicosapentaenoyl-2-octanoylglycerol
(1b). The same procedure was followed as for 1a except using
1,3-dieicosapentaenoylglycerol 1 (40 mg, 0.061 mmol), DMAP
(8 mg, 0.065 mmol), EDCI (20 mg, 0.104 mmol) and octanoic acid
(9 mg, 0.062 mmol). The product 1b (43 mg, 0.055 mmol) was
afforded as clear oil, yield 90%. 1H NMR (400 MHz, CDCl3):
d
5.43e5.29 (m, 20H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.30
4. Material and methods
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.15 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.77 (m, 16H, ]CCH2C]), 2.33 (t, 4H, J 7.6 Hz,
CH2COO in EPA), 2.31 (t, 2H, J 7.5 Hz, CH2COO in FA), 2.14e2.04 (m,
8H, ]CCH2CH3 and ]CCH2CH2), 1.70 (q (br), 4H, J 7.5 Hz,
CH2CH2COO in EPA),1.65e1.58 (m, 2H, CH2CH2COO in FA),1.35e1.27
(m, 8H, CH2), 0.97 (t, 6H, J 7.5 Hz, CH3 in EPA), 0.88 (t, 3H, J 7.0 Hz,
4.1. General
1H and 13C NMR spectra were recorded on a Bruker Avance 400
spectrometer using CDCl3 as a solvent. Chemical shifts (d) are re-
ported in parts per million (ppm) and the coupling constants (J) in
Hertz (Hz). The following abbreviations are used to describe the
multiplicity: s, singlet; d, doublet; t, triplet; dd, doublet of doublets;
m, multiplet. In assignments for the 1H NMR spectra FA refers to the
saturated fatty acyl part of the molecule. The number of carbon
nuclei behind each 13C signal is indicated in parentheses after each
chemical shift value, when there is more than one carbon re-
sponsible for the peak. Infrared (IR) spectra for all the products as
neat liquids were conducted on a Nicolet Avatar 360 FTIR (E.S.P.)
Spectrophotometer on a ZnSe plate. The high-resolution mass
spectra (HRMS) were acquired on a Bruker micrOTOF-Q mass
spectrometer equipped with an atmospheric pressure chemical
ionization chamber (APCI) or an E-spray ionization chamber (ESI).
All data analysis was done on a Bruker software.
CH3) ppm; 13C NMR (100 MHz, CDCl3)
d 173.0 (2), 172.8, 132.0 (2),
128.9 (2), 128.8 (2), 128.6 (2), 128.3 (2), 128.2 (2), 128.1 (4), 127.8 (2),
127.0 (2), 68.8, 62.2 (2), 34.2, 33.4 (2), 31.6, 29.0, 28.9, 26.5 (2), 25.6
(6), 25.5 (2), 24.9, 24.7 (2), 22.6, 20.6 (2), 14.3 (2), 14.1 ppm; FTIR:
nmax 3012 (s, CH), 2930 (vs, CH), 2857 (s, CH), 1740 (vs, C]O) cmꢁ1
.
HRMS m/z calcd for
C
51H78O6 (MþNHþ4 ) 804.6137, found
804.6134 amu.
4.1.3. Synthesis of 1,3-dieicosapentaenoyl-2-decanoylglycerol
(1c). The same procedure was followed as for 1a except using
1,3-dieicosapentaenoylglycerol 1 (70 mg, 0.106 mmol), DMAP
(13 mg, 0.107 mmol), EDCI (30 mg, 0.156 mmol) and decanoic acid
(20 mg, 0.116 mmol). The product 1c (76 mg, 0.093 mmol) was
afforded as clear oil, yield 88%. 1H NMR (400 MHz, CDCl3):
All chemicals and solvents were used without further purifica-
tion unless otherwise stated. EDCI (1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride) was obtained from Sigma-
eAldrich (Steinheim, Germany). Hexanoic acid (98%, zur synthese)
and decanoic acid (98%, zur synthese) were obtained from Merck
(Germany) and hexadecanoic acid (99%) from Fluka (Switzerland).
Octanoic acid (>99.5%), dodecanoic acid (>99.5%), tetradecanoic
acid (>99.5%) and 4-dimethylaminopyridine (DMAP, 99%) were
obtained from Acros Organics (Geel, Belgium). CH2Cl2 was obtained
HPLC grade from SigmaeAldrich (Steinheim, Germany) and dried
over CaH2 under nitrogen atmosphere when needed. Column
chromatography was performed on Silica gel 60 (Silicycle, Ontario).
Reactions were monitored by TLC on Silica gel 60 F254 (Silicycle,
Ontario), with detection by quenching of fluorescence, rhodamine
6G in CH3OH and/or with phosphomolybdic acid in ethanol.
d 5.43e5.29 (m, 20H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.30
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.15 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.77 (m, 16H, ]CCH2C]), 2.35e2.29 (m, 6H,
CH2COO in EPA and FA), 2.14e2.04 (m, 8H, ]CCH2CH3 and ]
CCH2CH2), 1.70 (q (br), 4H, J 7.5 Hz, CH2CH2COO in EPA), 1.65e1.58
(m, 2H, CH2CH2COO in FA), 1.35e1.27 (m, 12H, CH2), 0.97 (t, 6H, J
7.5 Hz, CH3 in EPA), 0.88 (t, 3H, J 7.0 Hz, CH3) ppm; 13C NMR
(100 MHz, CDCl3)
d 173.0 (2), 172.8, 132.0 (2), 128.9 (2), 128.8 (2),
128.6 (2), 128.3 (2), 128.2 (2), 128.1 (4), 127.8 (2), 127.0 (2), 68.8, 62.2
(2), 34.2, 33.4 (2), 31.9, 29.4, 29.3 (2), 29.1, 26.5 (2), 25.6 (6), 25.5 (2),
24.9, 24.7 (2), 22.7, 20.5 (2), 14.3 (2), 14.1 ppm; FTIR: nmax 3012 (s,
CH), 2926 (vs, CH), 2855 (s, CH), 1743 (vs, C]O) cmꢁ1. HRMS m/z
calcd for C53H82O6 (MþNHþ4 ) 832.6450, found 832.6451 amu.
4.1.4. Synthesis of 1,3-dieicosapentaenoyl-2-dodecanoylglycerol
(1d). The same procedure was followed as for 1a except using
1,3-dieicosapentaenoylglycerol 1 (43 mg, 0.065 mmol), DMAP
(8 mg, 0.065 mmol), EDCI (21 mg, 0.110 mmol) and dodecanoic acid
(15 mg, 0.075 mmol). The product 1d (49 mg, 0.058 mmol) was
afforded as clear oil, yield 89%. 1H NMR (400 MHz, CDCl3):
4.1.1. Synthesis of 1,3-dieicosapentaenoyl-2-hexanoylglycerol
(1a). To a solution of 1,3-dieicosapentaenoylglycerol 1 (41 mg,
0.062 mmol), DMAP (10 mg, 0.082 mmol) and EDCI (21 mg,
0.109 mmol), in dry CH2Cl2 (1 mL) was added hexanoic acid (9 mg,
0.077 mmol) and the resulting solution stirred at rt overnight under
nitrogen atmosphere. The reaction mixture was then passed
through a short silica column chromatography (CH2Cl2) to afford
product 1a (43 mg, 0.057 mmol) as clear oil, yield 91%. 1H NMR
d
5.43e5.29 (m, 20H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.30
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.15 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.77 (m, 16H, ]CCH2C]), 2.35e2.29 (m, 6H,
CH2COO in EPA and FA), 2.14e2.04 (m, 8H, ]CCH2CH3 and ]
CCH2CH2), 1.70 (q (br), 4H, J 7.5 Hz, CH2CH2COO in EPA), 1.65e1.58
(m, 2H, CH2CH2COO in FA), 1.35e1.27 (m, 16H, CH2), 0.97 (t, 6H, J
7.5 Hz, CH3 in EPA), 0.88 (t, 3H, J 6.8 Hz, CH3) ppm; 13C NMR
(400 MHz, CDCl3):
d 5.43e5.29 (m, 20H, ]CH), 5.29e5.24 (m, 1H,
CH2CHCH2), 4.31 (dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.15 (dd, 2H, J
11.9, J 6.0 Hz, CH2OCO), 2.86e2.79 (m,16H, ]CCH2C]), 2.33 (t, 4H, J
7.6 Hz, CH2COO in EPA), 2.31 (t, 2H, J 7.5 Hz, CH2COO in FA),
(100 MHz, CDCl3)
d 173.0 (2), 172.8, 132.0 (2), 128.9 (2), 128.8 (2),

A.V. Gudmundsdottir et al. / Tetrahedron 71 (2015) 8544e8550
8549
128.6 (2), 128.3 (2), 128.2 (2), 128.1 (4), 127.9 (2), 127.0 (2), 68.8, 62.2
(2), 34.2, 33.4 (2), 31.9, 29.6 (2), 29.5, 29.3 (2), 29.1, 26.5 (2), 25.6 (6),
HRMS m/z calcd for
828.6142 amu.
C
53H78O6 (MþNHþ4 ) 828.6137, found
25.5 (2), 24.9, 24.7 (2), 22.7, 20.5 (2), 14.3 (2), 14.1 ppm; FTIR: nmax
3012 (s, CH), 2925 (vs, CH), 2854 (s, CH), 1743 (vs, C]O) cmꢁ1
.
4.1.8. Synthesis of 1,3-didocosahexaenoyl-2-octanoylglycerol
(2b). The same procedure was followed as for 1a except using
1,3-didocosahexaenoylglycerol 2 (41 mg, 0.058 mmol), DMAP
(10 mg, 0.082 mmol), EDCI (22 mg, 0.115 mmol) and octanoic acid
(9 mg, 0.062 mmol). The product 2b (45 mg, 0.054 mmol) was
afforded as light yellow oil, yield 94%. 1H NMR (400 MHz, CDCl3):
HRMS m/z calcd for
C
55H86O6 (MþNHþ4 ) 860.6763, found
860.6802 amu.
4.1.5. Synthesis of 1,3-dieicosapentaenoyl-2-tetradecanoylglycerol
(1e). The same procedure was followed as for 1a except using
1,3-dieicosapentaenoylglycerol 1 (73 mg, 0.110 mmol), DMAP
(10 mg, 0.082 mmol), EDCI (32 mg, 0.167 mmol) and tetradecanoic
acid (25 mg, 0.109 mmol). The product 1e (89 mg, 0.102 mmol) was
afforded as clear oil, yield 93%. 1H NMR (400 MHz, CDCl3):
d
5.44e5.29 (m, 24H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.31
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.16 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.78 (m, 20H, ]CCH2C]), 2.39e2.37 (m, 8H,
CH2CH2COO in DHA), 2.32 (t, 2H, J 7.5 Hz, CH2COO in FA), 2.11e2.03
(m, 4H, ]CCH2CH3), 1.65e1.57 (m, 2H, CH2CH2COO in FA),
1.34e1.23 (m, 8H, CH2), 0.97 (t, 6H, J 7.5 Hz, CH3 in DHA), 0.88 (t, 3H,
d
5.43e5.29 (m, 20H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.30
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.15 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.77 (m, 16H, ]CCH2C]), 2.35e2.29 (m, 6H,
CH2COO in EPA and FA), 2.14e2.04 (m, 8H, ]CCH2CH3 and ]
CCH2CH2), 1.70 (q (br), 4H, J 7.5 Hz, CH2CH2COO in EPA), 1.65e1.57
(m, 2H, CH2CH2COO in FA), 1.35e1.27 (m, 20H, CH2), 0.97 (t, 6H, J
7.5 Hz, CH3 in EPA), 0.88 (t, 3H, J 6.8 Hz, CH3) ppm; 13C NMR
J 6.9 Hz, CH3) ppm; 13C NMR (100 MHz, CDCl3)
d 172.8, 172.5 (2),
132.0 (2), 129.5 (2), 128.5 (2), 128.3 (4), 128.2 (2), 128.1 (4), 128.0 (2),
127.9 (2), 127.6 (2), 127.0 (2), 68.8, 62.2 (2), 34.2, 33.9 (2), 31.6, 29.0,
28.9, 25.6 (8), 25.5 (2), 24.9, 22.6 (3), 20.5 (2), 14.3 (2), 14.0 ppm;
FTIR: nmax 3013 (s, CH), 2929 (vs, CH), 2857 (s, CH), 1743 (vs, C]O)
cmꢁ1. HRMS m/z calcd for C55H82O6 (MþNHþ4 ) 856.6450, found
856.6461 amu.
(100 MHz, CDCl3)
d 173.0 (2), 172.8, 132.0 (2), 128.9 (2), 128.8 (2),
128.6 (2), 128.3 (2), 128.2 (2), 128.1 (4), 127.8 (2), 127.0 (2), 68.8, 62.2
(2), 34.2, 33.4 (2), 31.9, 29.7, 29.6 (3), 29.5, 29.3 (2), 29.1, 26.5 (2),
25.6 (6), 25.5 (2), 24.9, 24.7 (2), 22.7, 20.5 (2), 14.3 (2), 14.1 ppm;
FTIR: nmax 3012 (s, CH), 2924 (vs, CH), 2854 (s, CH), 1743 (vs, C]O)
cmꢁ1. HRMS m/z calcd for C57H90O6 (MþNHþ4 ) 888.7076, found
888.7104 amu.
4.1.9. Synthesis of 1,3-didocosahexaenoyl-2-decanoylglycerol
(2c). The same procedure was followed as for 1a except using
1,3-didocosahexaenoylglycerol 2 (42 mg, 0.059 mmol), DMAP
(11 mg, 0.090 mmol), EDCI (15 mg, 0.078 mmol) and decanoic acid
(13 mg, 0.075 mmol). The product 2c (46 mg, 0.053 mmol) was
afforded as light yellow oil, yield 90%. 1H NMR (400 MHz, CDCl3):
4.1.6. Synthesis of 1,3-dieicosapentaenoyl-2-hexadecanoylglycerol
(1f). The same procedure was followed as for 1a except using 1,3-
dieicosapentaenoylglycerol 1 (70 mg, 0.106 mmol), DMAP (9 mg,
0.074 mmol), EDCI (29 mg, 0.151 mmol) and hexadecanoic acid
(27 mg, 0.105 mmol). The product 1f (86 mg, 0.095 mmol) was
afforded as clear oil, yield 90%. 1H NMR (400 MHz, CDCl3):
d
5.44e5.29 (m, 24H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.31
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.16 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.78 (m, 20H, ]CCH2C]), 2.39e2.37 (m, 8H,
CH2CH2COO in DHA), 2.31 (t, 2H, J 7.5 Hz, CH2COO in FA), 2.11e2.04
(m, 4H, ]CCH2CH3),1.65e1.57 (m, 2H, CH2CH2COO in FA),1.34e1.21
(m, 12H, CH2), 0.97 (t, 6H, J 7.5 Hz, CH3 in DHA), 0.88 (t, 3H, J 6.9 Hz,
d
5.43e5.29 (m, 20H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.30
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.15 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.77 (m, 16H, ]CCH2C]), 2.35e2.29 (m, 6H,
CH2COO in EPA and FA), 2.14e2.04 (m, 8H, ]CCH2CH3 and ]
CCH2CH2), 1.70 (q (br), 4H, J 7.5 Hz, CH2CH2COO in EPA), 1.65e1.57
(m, 2H, CH2CH2COO in FA), 1.34e1.20 (m, 24H, CH2), 0.97 (t, 6H, J
7.5 Hz, CH3 in EPA), 0.88 (t, 3H, J 6.8 Hz, CH3) ppm; 13C NMR
CH3) ppm; 13C NMR (100 MHz, CDCl3)
d 172.8, 172.5 (2), 132.0 (2),
129.5 (2), 128.5 (2), 128.3 (4), 128.2 (2), 128.1 (4), 128.0 (2), 127.8 (2),
127.6 (2), 127.0 (2), 68.8, 62.2 (2), 34.2, 33.9 (2), 31.8, 29.4, 29.3 (2),
29.1, 25.6 (8), 25.5 (2), 24.9, 22.6 (3), 20.5 (2), 14.3 (2), 14.1 ppm;
FTIR: nmax 3013 (s, CH), 2926 (vs, CH), 2855 (s, CH), 1743 (vs, C]O)
cmꢁ1. HRMS m/z calcd for C57H86O6 (MþNHþ4 ) 884.6763, found
884.6752 amu.
(100 MHz, CDCl3)
d 173.0 (2), 172.8, 132.0 (2), 128.9 (2), 128.8 (2),
128.6 (2), 128.3 (2), 128.2 (2), 128.1 (4), 127.8 (2), 127.0 (2), 68.8, 62.2
(2), 34.2, 33.4 (2), 31.9, 29.7 (5), 29.6, 29.5, 29.4, 29.3, 29.1, 26.5 (2),
25.6 (6), 25.5 (2), 24.9, 24.7 (2), 22.7, 20.6 (2), 14.3 (2), 14.1 ppm;
FTIR: nmax 3012 (s, CH), 2924 (vs, CH), 2853 (s, CH), 1743 (vs, C]O)
cmꢁ1. HRMS m/z calcd for C59H94O6 (MþNHþ4 ) 916.7389, found
916.7363 amu.
4.1.10. Synthesis of 1,3-didocosahexaenoyl-2-dodecanoylglycerol
(2d). The same procedure was followed as for 1a except using
1,3-didocosahexaenoylglycerol 2 (70 mg, 0.098 mmol), DMAP
(10 mg, 0.082 mmol), EDCI (28 mg, 0.146 mmol) and dodecanoic
acid (20 mg, 0.100 mmol). The product 2d (80 mg, 0.089 mmol) was
afforded as light yellow oil, yield 91%. 1H NMR (400 MHz, CDCl3):
4.1.7. Synthesis of 1,3-didocosahexaenoyl-2-hexanoylglycerol
(2a). The same procedure was followed as for 1a except using
1,3-didocosahexaenoylglycerol 2 (70 mg, 0.098 mmol), DMAP
(10 mg, 0.082 mmol), EDCI (28 mg, 0.146 mmol) and hexanoic acid
(14 mg, 0.121 mmol). The product 2a (71 mg, 0.092 mmol) was
afforded as light yellow oil, yield 89%. 1H NMR (400 MHz, CDCl3):
d 5.44e5.30 (m, 24H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.31
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.16 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.78 (m, 20H, ]CCH2C]), 2.41e2.37 (m, 8H,
CH2CH2COO in DHA), 2.31 (t, 2H, J 7.5 Hz, CH2COO in FA), 2.11e2.03
(m, 4H, ]CCH2CH3), 1.65e1.57 (m, 2H, CH2CH2COO in FA),
1.34e1.24 (m, 16H, CH2), 0.97 (t, 6H, J 7.5 Hz, CH3 in DHA), 0.88 (t,
d
5.44e5.29 (m, 24H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.31
3H, J 6.8 Hz, CH3) ppm; 13C NMR (100 MHz, CDCl3)
d 172.8, 172.5 (2),
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.16 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.80 (m, 20H, ]CCH2C]), 2.41e2.37 (m, 8H,
CH2CH2COO in DHA), 2.32 (t, 2H, J 7.5 Hz, CH2COO in FA), 2.11e2.04
(m, 4H, ]CCH2CH3), 1.66e1.58 (m, 2H, CH2CH2COO in FA),
1.38e1.26 (m, 4H, CH2), 0.97 (t, 6H, J 7.5 Hz, CH3 in DHA), 0.89 (t, 3H,
132.0 (2), 129.5 (2), 128.6 (2), 128.3 (4), 128.2 (2), 128.1 (4), 128.0 (2),
127.9 (2), 127.6 (2), 127.0 (2), 68.8, 62.3 (2), 34.2, 33.9 (2), 31.9, 29.6
(2), 29.5, 29.3 (2), 29.1, 25.6 (8), 25.5 (2), 24.9, 22.7, 22.6 (2), 20.6 (2),
14.3 (2), 14.1 ppm; FTIR: nmax 3013 (s, CH), 2925 (vs, CH), 2854 (s,
CH), 1743 (vs, C]O) cmꢁ1. HRMS m/z calcd for C59H90O6 (MþNHþ4 )
912.7076, found 912.7062 amu.
J 7.0 Hz, CH3) ppm; 13C NMR (100 MHz, CDCl3)
d 172.8, 172.5 (2),
132.0 (2), 129.5 (2), 128.5 (2), 128.3 (4), 128.2 (2), 128.1 (4), 128.0 (2),
127.9 (2), 127.6 (2), 127.0 (2), 68.8, 62.2 (2), 34.1, 33.9 (2), 31.2, 25.6
(8), 25.5 (2), 24.5, 22.6 (2), 22.3, 20.5 (2), 14.3 (2), 13.9 ppm; FTIR:
4.1.11. Synthesis of 1,3-didocosahexaenoyl-2-tetradecanoylglycerol
(2e). The same procedure was followed as for 1a except using
1,3-didocosahexaenoylglycerol 2 (41 mg, 0.058 mmol), DMAP
nmax 3013 (s, CH), 2932 (vs, CH), 2873 (s, CH), 1742 (vs, C]O) cmꢁ1
.

8550
A.V. Gudmundsdottir et al. / Tetrahedron 71 (2015) 8544e8550
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503e509.
(10 mg, 0.082 mmol), EDCI (15 mg, 0.078 mmol) and tetradecanoic
acid (14 mg, 0.061 mmol). The product 2e (48 mg, 0.052 mmol) was
afforded as light yellow oil, yield 90%. 1H NMR (400 MHz, CDCl3):
4. Holub, D. J.; Holub, B. J. Mol. Cell Biochem. 2004, 263, 217e225.
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d
5.44e5.30 (m, 24H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.31
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.16 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.77 (m, 20H, ]CCH2C]), 2.39e2.37 (m, 8H,
CH2CH2COO in DHA), 2.31 (t, 2H, J 7.5 Hz, CH2COO in FA), 2.11e2.04
(m, 4H, ]CCH2CH3), 1.65e1.57 (m, 2H, CH2CH2COO in FA),
1.33e1.23 (m, 20H, CH2), 0.97 (t, 6H, J 7.5 Hz, CH3 in DHA), 0.88 (t,
7. Serhan, C. N. Nature 2014, 510, 92e101.
8. Serhan, C. N.; Petasis, N. A. Chem. Rev. 2012, 111, 5922e5943.
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3H, J 6.8 Hz, CH3) ppm; 13C NMR (100 MHz, CDCl3)
d 172.8, 172.5 (2),
132.0 (2), 129.5 (2), 128.6 (2), 128.3 (4), 128.2 (2), 128.1 (4), 128.0 (2),
127.9 (2), 127.6 (2), 127.0 (2), 68.8, 62.3 (2), 34.2, 33.9 (2), 31.9, 29.7
(2), 29.6 (2), 29.5, 29.3 (2), 29.1, 25.6 (8), 25.5 (2), 24.9, 22.7, 22.6 (2),
20.6 (2), 14.3 (2), 14.1 ppm; FTIR: nmax 3013 (s, CH), 2924 (vs, CH),
2854 (s, CH), 1743 (vs, C]O) cmꢁ1. HRMS m/z calcd for C61H94O6
(MþNHþ4 ) 940.7389, found 940.7402 amu.
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4.1.12. Synthesis of 1,3-didocosahexaenoyl-2-hexadecanoylglycerol
(2f). The same procedure was followed as for 1a except using 1,3-
didocosahexaenoylglycerol 2 (42 mg, 0.059 mmol), DMAP (10 mg,
0.082 mmol), EDCI (16 mg, 0.083 mmol) and hexadecanoic acid
(15 mg, 0.059 mmol). The product 2f (50 mg, 0.053 mmol) was
afforded as light yellow oil, yield 90%. 1H NMR (400 MHz, CDCl3):
ꢀ
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d
5.44e5.30 (m, 24H, ]CH), 5.29e5.24 (m, 1H, CH2CHCH2), 4.31
(dd, 2H, J 11.9, J 4.3 Hz, CH2OCO), 4.16 (dd, 2H, J 11.9, J 6.0 Hz,
CH2OCO), 2.88e2.80 (m, 20H, ]CCH2C]), 2.41e2.37 (m, 8H,
CH2CH2COO in DHA), 2.31 (t, 2H, J 7.5 Hz, CH2COO in FA), 2.11e2.04
(m, 4H, ]CCH2CH3), 1.65e1.57 (m, 2H, CH2CH2COO in FA),
1.33e1.22 (m, 24H, CH2), 0.97 (t, 6H, J 7.5 Hz, CH3 in DHA), 0.88 (t,
3H, J 6.8 Hz, CH3) ppm; 13C NMR (100 MHz, CDCl3)
d 172.8, 172.5 (2),
132.0 (2), 129.5 (2), 128.6 (2), 128.3 (4), 128.2 (2), 128.1 (4), 128.0 (2),
127.9 (2), 127.6 (2), 127.0 (2), 68.8, 62.3 (2), 34.2, 33.9 (2), 31.9, 29.7
(5), 29.6, 29.5, 29.3 (2), 29.1, 25.6 (8), 25.5 (2), 24.9, 22.7, 22.6 (2),
20.6 (2), 14.3 (2), 14.1 ppm; FTIR: nmax 3013 (s, CH), 2924 (vs, CH),
2853 (s, CH), 1743 (vs, C]O) cmꢁ1. HRMS m/z calcd for C63H98O6
(MþNHþ4 ) 968.7702, found 968.7727 amu.
29. Compton, D. L.; Laszlo, J. A.; Appell, M.; Vermillion, K. E.; Evans, K. O. J. Am. Oil
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The Icelandic Research Fund (Project grant number 080023021)
and Lysi hf (CDM) in Iceland are acknowledged for financial
support. Adalheidur Dora Albertsdottir at University of Iceland is
acknowledged for HRMS measurements and Olav Thorstad of
Pronova Biocare ASA in Norway for pure EPA and DHA.
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References and notes
1. Hjaltason, B.; Haraldsson, G. G. In Modifying Lipids for Use in Food; Gunstone, F.
D., Ed.; Woodhead Publishing: Cambridge UK, 2006; Chapter 4, pp 56e79.