Regioselective opening of an oxirane system with trifluoroacetic anhydride. A general method for the synthesis of 2-monoacyl- and 1,3-symmetrical triacylglycerols

Article abstract of DOI:10.1016/j.tet.2005.02.034

A trifluoroacetic anhydride-catalyzed opening of the oxirane system of glycidyl esters with a simultaneous migration of the acyl group provides a new, efficient entry to either 2-monoacylglycerols (2-MAG) or 1,3-symmetrical triglycerides (1,3-STG) as potential prodrug frameworks.

Full text of DOI:10.1016/j.tet.2005.02.034

Tetrahedron 61 (2005) 3659–3669
Regioselective opening of an oxirane system with trifluoroacetic
anhydride. A general method for the synthesis of 2-monoacyl-
and 1,3-symmetrical triacylglycerols
b,c,
Stephan D. Stamatova, and Jacek Stawinski
*
*
aDepartment of Chemical Technology, University of Plovdiv, 24 Tsar Assen St., Plovdiv 4000, Bulgaria
bDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
cInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
Received 10 November 2004; revised 10 February 2005; accepted 11 February 2005
Abstract—A trifluoroacetic anhydride-catalyzed opening of the oxirane system of glycidyl esters with a simultaneous migration of the acyl
group provides a new, efficient entry to either 2-monoacylglycerols (2-MAG) or 1,3-symmetrical triglycerides (1,3-STG) as potential
prodrug frameworks.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
been identified as specific targets for a major psychoactive
ingredient of marijuana, d9-tetrahydrocannabinol. Other
2-MAG, bearing linoleoyl- or palmitoyl fragments, have
been suggested ‘entourage’ co-factors for enhancing the
endogenous cannabinoid potential of 2-AG.5
2-Monoacylglycerols (Fig. 1) have recently attracted
research interest as unique carriers of fatty acids through
intestinal mucosa,1,2 as metabolic precursors of structured
triglycerides having particular fatty acid residues at
2-position in the glycerol backbone2 as well as biomolecules
of importance to human nutrition.3 Moreover, it was found4
that a homologue from the same class of lipid mediators,
namely 2-arachidonoylglycerol (2-AG), might be an
intrinsic, natural ligand for central and peripheral can-
nabinoid receptors (CB1 and CB2), which had previously
Complementary to their dual function as carrier molecules
and biological effectors per se, 2-monoacylglycerols could
be an attractive alternative of the currently employed 1,3-
diacylglycerols as key-intermediates in the rational design
of prodrugs representing symmetrically substituted 1,3-
diacyl isosters of 2-MAG with requisite pharmaceutical
moieties at the incipient glycerol 2-position.2,6 In view of
their resemblance to endogenous triglycerides, such a type
of micromolecular vectors have already been used in order
to confer various drugs to the metabolic pathways of natural
lipids thus achieving therapeutic indices better than those of
the starting substances (e.g., higher oral bioavailability,
reduced ulcerogenity, first-pass metabolism resistance,
etc.).7 Also, 1,3-symmetrical triglycerides have founding
interesting applications (e.g., in enzymatic synthesis of
structurally modified lipids,2 molecular modeling,8 analyti-
cal studies,9 etc.), but the potential of these conjugates has
not been exploited to any significant extent due to
unsatisfactory efficacy of the known methods of their
preparation (e.g., multistep reaction sequences, lengthy
isolation and purification steps, etc.).9,10
Figure 1. Structures of 2-MAG and 1,3-STG.
Keywords: 2-Monoacylglycerols; 2-Arachidonoylglycerol; Glycidyl
arachidonate; Acyl migration; 1,3-Symmetrical triacylglycerols; Prodrugs.
In spite of high demand for isomerically pure 2-MAG for
biological studies or as starting material in the synthesis of
*
Corresponding authors. Tel.: C46 8 16 24 85; fax: C46 8 15 49 08 (J.S.);
e-mail: js@organ.su.se
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.02.034

3660
S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
other lipid derivatives, these compounds are often isolated
from natural sources,11 as chemical12 and enzymatic
methods13 for their preparations are rather inefficient.
inherent. Two chemical methods described in literature for
the synthesis of this compound are based on the same
chemistry: acylation of suitable 1,3-protected glycerol
precursors with an arachidonic acid derivative, followed
by deprotection and separation of the isomeric
arachidonoylglycerols. In the original method developed
by Martin16 and its two most recent modifications,12,17 1,3-
benzylideneglycerol is used as a substrate and, after
introduction of the arachidonoyl moiety, the acetal group
is removed using boric acid derivatives. In the other
approach,18 triisopropylsilyl (TIPS) groups are employed
for the protection of 1-and 3-hydroxyl functions of glycerol
and their removal from 1,3-bissilyl-2-arachidonoyl inter-
mediate is effected by a prolonged treatment with tetra-n-
butylammonium fluoride (TBAF) and acetic acid at low
temperature. The use of 1,3-dibenzyloxy-2-propanol as
starting material and b-chlorocatecholborane as a reagent
for cleavage of the benzyl group are the latest improvements
which do not affect the core of the same strategy.19
Searching for an alternative methodology that would
circumvent shortcomings in the present methods of
chemical synthesis of 2-monoacylglycerols, in this paper
we describe an efficient and highly regioselective trans-
formation of glycidyl esters 1–7 into 2-acyl-1,3-
bis(trifluoroacetyl)glycerol derivatives 8–14 (Chart 1),
from which 2-monoacylglycerols 15–21 can be obtained
directly without recourse to any additional purification
techniques whatsoever.14 One can also envisage tri-
glycerides 8–14 as convenient storage forms of 2-MAG
(protection of 1- and 3-hydroxyl groups as trifluoroacetyl
esters should prevent scrambling of the acyl moiety) or
starting material for the preparation of other lipid mediators,
for example, 1,3-STG 22–26.
2. Results and discussion
One should note that these methods provide only fragmen-
tary solution to synthetic drawbacks, for example, extended
reaction time, acidic or basic conditions required for the
removal of protecting groups, necessity for an aqueous
workup after each synthetic step or separation of the
intermediates from the accompanying by-products etc, that
have frequently been reported to contribute to isomerisation
and oxidative or hydrolytic side-reactions during the
synthesis of 2-MAGs. To lessen the problem of acyl
migrations, in these synthetic procedures, the deprotection
steps were either not taken to completion,18 or the produced
isomeric compounds were separated by various chromato-
graphic techniques.12,18,19 Although useful in general sense,
the above approaches have not been evaluated on substances
with other structural variations, and thus scope and
generality of these methods are unclear.
There are two problems that make synthesis of 2-MAG most
difficult. First, due to the presence of two adjacent primary
hydroxyl functions, 2-monoacylglycerols show high pro-
pensity towards isomerisation (acid, base and heat promoted
migration of an acyl group)15 and this poses severe
limitations in the choice of synthetic methods as well as
means of their isolation, storage, etc. Secondly, a problem
specific to 2-MAG with polyunsaturated systems is the
pronounced susceptibility to autoxidation affecting integrity
of the corresponding, native olefinic system that further
reducing the number of available procedures for their
preparation.
In this context, 2-AG is probably the most typical synthetic
target to which similar acute complications seem to be
Chart 1.

S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
3661
In these studies, therefore, we adopted another strategy and
investigated the regioselectivity of a trifluoroacetic
anhydride-catalyzed opening of the oxirane system14 of
glycidyl esters 1–7 to produce the corresponding 1,3-
bis(trifluoroacetyl)-2-acylglycerols as a novel approach to
the synthesis of 2-monoacylglycerols (2-MAG) and also
1,3-symmetrical triglycerides (1,3-STG).
Step C) To this end, 2-monoacylglycerols 15–18 in
dichloromethane were treated with acyl chlorides a–d
(3 equiv) in the presence of pyridine (20 equiv).
Under these conditions, the acylation proceeded without
detectable acyl migration22 and was complete within 2–4 h
to give the target products, triglycerides 22–26, in
consistently high overall yields (82–86% after silica gel
chromatography, see Section 3).
The starting materials were chosen to include model
substrates bearing bioactive acyl fragments4,11,18,20,21 with
variable chain length and different degree of unsaturation
in the acyl moiety [e.g., 2-(acetyloxymethyl)oxirane 1,
2-(oleoyloxymethyl)oxirane 2, and 2-(arachidonoyloxy-
methyl)oxirane 3] or aromatic acyl residues with electron-
withdrawing and electron-donating groups, and with
different steric requirements [e.g., 2-(benzoyloxymethyl)-
oxirane 4, 2-(4-nitrobenzoyloxymethyl)oxirane 5, and 2-(4-
methoxybenzoyloxymethyl)oxirane 6, and 2-(2,4,6-tri-
methylbenzoyloxymethyl)-oxirane 7] (see Chart 1). The
choice of palmitoyl a, oleoyl b, acetyl c, and benzoyl
d chlorides as acylating agents was justified by
accessibility of these compounds and their common use in
the synthesis of acyl bioconjugates based on glycerol
chemistry.7,21
Regarding a possible mechanism for the regioselective
epoxide opening, some additional observations are
pertinent. Thus, in preliminary model experiments it was
established that in methylene chloride at rt other carboxylic
anhydrides (e.g., acetic-, benzoic anhydride, etc) were
completely unreactive and only trichloroacetic anhydride
could act in a similar way as TFAA to give regioselectively
the isosteric 1,3-bis(trichloroacetates), although in a signifi-
cantly slower reaction (ca. 24 h for the completion). The use
of trifluoroacetic acid alone afforded variable proportions of
1-acyl- and 2-acyl glycerols under the same conditions.
Additional studies (Table 1) revealed that the oxirane
system became extremely resistant towards TFAA when the
acyl group was replaced by an alkyl one (entry 1). Synthons
from the traditional pool of acylated glycerol acetals (e.g.,
entries 2 and 3) remained unaffected under the same
conditions, as well.
At first, the ring-opening of glycidyl esters 1–7 with TFAA
to produce triacylglycerols 8–14 was investigated under
various experimental conditions (type of solvents, ratio of
reactants, temperature; Chart 1, Step A). The best results
were obtained when glycidol derivatives 1–7 were allowed
to react with TFAA (4 equiv) in CH2Cl2 at rt for 1–4 h. The
rate of the epoxide opening in 1–7 was not appreciably
affected by electronic and structural features of the acyl
group present and the reactions usually were complete
within 1 h. The only exception was 2-(4-nitrobenzoyl-
oxymethyl)oxirane 5 whose conversion to 12 required ca.
4 h. 1H and 13C NMR analysis revealed that under the
investigated reaction conditions the conversion of 1–7 to
1,3-bis(trifluoroacetyl)-2-acylglycerols 8–14 did not
involve any detectable intermediates, and it was quantitative
and completely regioselective (O99%). The produced
bis(trifluoroacetyl) derivatives 8–14 could thus be
either directly used for a subsequent reactions, or isolated
(w86–96% yields, see Section 3) and stored for several
months (K20 8C, under argon) without detectable
alterations of their spectral characteristics (1H and 13C
NMR spectroscopy).
In all instances where mixtures of TFAA with up to three
equivalent excess of non-halogenated carboxylic acids or
carboxylic anhydrides were used, 1,3-bis(trifluoroacetyl)-2-
acylglycerols were still formed as the only products of the
reaction (e.g., entries 4 and 5).
Organic bases were shown to completely inhibit the epoxide
opening, irrespective of their potential to act as nucleophiles
or base catalysts, despite the presence of excess TFAA in
the reaction mixtures (entries 6, 8–10). All these reactions
could be rescued by the addition of trifluoroacetic acid
providing the expected products (entry 7). A similar effect
was observed for the reaction using tetrabutylammonium
trifluoroacetate with TFAA (entry 11).
These data are consistent with a mechanism depicted in
Scheme 1, which involves initial coordination of the
epoxide oxygen by a strongly electrophilic TFAA, followed
by the opening of the activated oxirane ring via an
intramolecular attack of the adjacent carbonyl group to
form cyclic acyliumglycerol cation A. This is apparently the
rate-determining step of the reaction as opening of the
oxirane ring does not occur under these conditions without
assistance of the neighbouring carbonyl group. The
produced acylium ion A then collapses in a fast reaction
to the corresponding 1,3-bis(trifluoroacetyl)-2-acylglycerol
by a regioselective attack of a trifluoroacetate ion on the
primary carbon atom of the dioxolane ring.
Since trifluoroacetate esters are known to undergo smooth
transesterification with alcohols,18 as the next step of this
synthetic protocol trifluoroacetyl-conjugates 8–14 in
pentane–CH2Cl2 were treated with methanol (15 equiv) in
the presence of pyridine (10 equiv) (Chart 1, Step B). The
reaction was quantitative (completion within 2–3 h) and
after removal of volatile products via evaporation, iso-
merically homogenous 2-monoacylglycerols 15–21 (purity
1
O99%, H and 13C NMR spectroscopy) were obtained in
85–99% overall yields (calculated on 1–7) without any
additional purification.
A mechanism by which amines inhibit this reaction is not
clear. It is possible that acid catalysis by traces of
trifluoroacetic acid most likely present in the reaction
mixture is essential for the formation of cyclic intermediate
A, for example, to facilitate the departure of trifluoro-
acetoxy group during opening of the oxirane ring. An
Due to high purity of the monoacylglycerols 15–21
produced, these could be directly used for the acylation to
afford 1,3-symmetrical triacylglycerols 22–26 (Chart 1,

3662
S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
Table 1. Mechanistic studies
No.
Reaction conditions: methylene chloride, rt
Reaction time (remarks)
1.
rt/24 h
rt/24 h
2.
3.
4.
rt/24 h
rt/w4 h (quantitative yield)
5.
6.
rt/w4 h (quantitative yield)
80 8C/w4 h
7.
8.
rt/w4 h (quantitative yield)
rt/24 h
9.
rt/24 h
rt/24 h
10.
11.
rt/w4 h (quantitative yield)
RZC16H33; RCOZoleoyl; 1RCOZAcetyl, benzoyl, etc. TFAKZF3CCOOK; TFAAZ(F3CCO)2O; PyZpyridine.
alternative scenario could be that due to increased acylating
properties of TFAA in the presence of bases, the carbonyl
function of the ester group is acylated and thus converted
into tetrahedral species that cannot provide an intramole-
cular nucleophilic assistance necessary for the opening of
the oxirane ring. Elucidation of these mechanistic aspects
needs further studies.
In conclusion, we have developed an efficient synthetic
strategy based on a novel, regioselective transformation of
glycidyl esters 1–7 into 2-acyl-1,3-bis(trifluoroacetyl)-
glycerols 8–14, from which 2-monoacylglycerols 15–21
can be retrieved under mild conditions. The main features
of this new synthetic protocol are: (i) highly effective
and practically quantitative, one-pot synthesis of
Scheme 1.

S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
3663
2-monoacylglycerols 15–21 under mild reaction conditions;
(ii) the produced compounds 8–14 and 15–21 are of high
purity, which alleviates problems of their additional
purification, and thus the extent of acyl migration (and of
other side-reactions) is minimized; (iii) 2-acyl-1,3-bis(tri-
fluoroacetyl)glycerols 8–14 can be envisaged either as
convenient storage forms of 2-MAG or prospective prodrug
frameworks for this class of lipid mediators; (iv) the general
strategy also introduces 2-monoacylglycerols as common
intermediates in the direct synthesis of prodrug isosters that
typically represent triglycerides 1,3-STG (e.g., 22–26); (v)
the method makes use of commercially available reactants
and it is easy to scale-up.
of the crude product by flash chromatography (CH2Cl2)
gave the title compound 1 (4.3 g, 74%) as a colorless oil.
[Found: C, 51.66; H, 7.00. C5H8O3 (116.11) requires C,
1
51.72; H, 6.94%]; Rf (system A)Z0.39; H NMR dH (in
ppm, CDCl3, 400 MHz) 4.37 (1H, dd, JZ2.9, 2.9 Hz,
OCH2CHCHaHbOCO); 3.87 (1H, dd, JZ6.6, 6.2 Hz,
OCH2CHCHaHbOCO); 3.17 (1H, m, C(O)CH2CHCH2O);
2.81 (1H, t, JZ4.4 Hz, C(O)CH2CHCHaHbO); 2.61 (1H,
dd, JZ2.6, 2.6 Hz, C(O)CH2CHCHaHbO); 2.06 (3H, s,
2-CH3); 13C NMR dC (in ppm, CDCl3, 100 MHz) 170.88
(C1); 20.90 (C2): acetyl fragment; 65.20 (C3); 49.48 (C2);
44.82 (C1): oxirane-2-methyl fragment.
3.1.2. 2-(Oleoyloxymethyl)oxirane (2). To a solution of
(G)-glycidol (1.1 g; 15 mmol) and 4-DMAP (2.2 g;
18 mmol) in CH2Cl2 (15 mL) at rt was added oleoyl
chloride (5.4 g; 18 mmol). After 4 h, the solution was
passed through a silica gel pad (w5 g) prepared in CH2Cl2.
The support was washed with CH2Cl2 (150 mL) and the
solvent evaporated in vacuo. Purification of the crude
product by flash chromatography (toluene) gave the title
compound 2 (4.4 g, 86%) as a colorless oil. [Found: C,
74.58; H, 11.28. C21H38O3 (338.52) requires C, 74.51; H,
11.31%]; Rf (system C)Z0.52; 1H NMR dH (in ppm,
CDCl3, 400 MHz) 5.34 (2H, m, CH]CH); 4.40 (1H, dd,
JZ2.9, 2.9 Hz, OCH2CHCHaHbOCO); 3.91 (1H, dd,
JZ5.9, 6.2 Hz, OCH2CHCHaHbOCO); 3.20 (1H, m,
C(O)CH2CHCH2O); 2.84 (1H, dd, JZ4.0, 4.4 Hz,
C(O)CH2CHCHaHbO); 2.63 (1H, dd, JZ2.6, 2.6 Hz,
C(O)CH2CHCHaHbO); 2.34 (2H, t, JZ7.3 Hz, 2-CH2);
2.01 (4H, m, 8-CH2, 11-CH2); 1.63 (2H, m, 3-CH2); 1.30
(20H, m, 4-7-CH2, 12-17-CH2); 0.87 (t, JZ7.0 Hz, 18-CH3,
3H); 13C NMR dC (in ppm, CDCl3, 100 MHz) 173.73 (C1);
129.95 and 130.23 (C9 andC10); 34.28 (C2); 32.12 (C16);
29.30–29.98 (C4-C7, C12-C15); 27.38 and 27.43 (C11 and
C8); 25.08 (C3); 22.90 (C17); 14.32 (C18): oleoyl fragment;
64.97 (C3); 49.60 (C2); 44.88 (C1): oxirane-2-methyl
fragment.
3. Experimental
3.1. General
All reagents were commercial grade (Fluka, Lancaster,
Merck, Sigma) with purity O98% and were used as
provided without further purification. Solvents were dried
and distilled prior to use according to standard protocols.23
Reaction conditions were kept strictly anhydrous.
Column chromatography (CC) was carried out on silica gel
60 (70–230 mesh ASTM, Merck) using the following
mobile phases: system A, pentane–toluene–ethyl acetate
(40:50:10, v/v/v); system B, pentane–toluene–ethyl acetate
(30:20:50, v/v/v); system C, pentane–ethyl acetate (90:10,
v/v); system D, dichloromethane–methanol (90:10, v/v);
system E, pentane–toluene–ethyl acetate (60:35:5, v/v/v);
system F, pentane–ethyl acetate (70:30, v/v). Progress of the
reactions was monitored by analytical thin-layer chroma-
tography (TLC) on pre-coated glass plates of silica gel 60
F254 (Merck) using the same solvent systems as for CC. The
spots were visualized using the commercially available
3.5% molybdatophosphoric acid spray reagent (Merck) or
50% sulphuric acid followed by heating at 140 8C.
3.1.3. 2-(Arachidonoyloxymethyl)oxirane (3). To a
solution of (G)-glycidol (0.22 g; 3.0 mmol), 4-DMAP
(0.49 g; 4.0 mmol) and arachidonic acid (1.22 g;
4.0 mmol) in CH2Cl2 (15 mL) at rt was added N,N0-
dicyclohexylcarbodiimide (DCC, 0.82 g; 4.0 mmol) and
the reaction system was stirred under these conditions for
12 h. After filtration, the solution was passed through a
silica gel pad (w5 g) prepared in CH2Cl2. The support was
washed with CH2Cl2 (150 mL) and the solvent evaporated
under reduced pressure. Purification of the crude product by
flash chromatography (system C) afforded the target
compound 3 (1.03 g, 95%) as a yellowish oil. [Found: C,
76.70; H, 10.04. C23H36O3 (360.54) requires C, 76.62; H,
10.06%]; Rf (system C)Z0.33; 1H NMR dH (in ppm,
CDCl3, 400 MHz) 5.36 (8H, m, CH]CH); 4.40 (1H, dd,
JZ3.3, 2.9 Hz, OCH2CHCHaHbOCO); 3.91 (1H, dd,
JZ6.2, 6.2 Hz, OCH2CHCHaHbOCO); 3.19 (1H, m,
C(O)CH2CHCH2O); 2.82 (7H, m, 7, 10, 13-CH2, C(O)CH2-
CHCHaHbO); 2.64 (1H, dd, JZ2.6, 2.6 Hz, C(O)CH2-
CHCHaHbO); 2.36 (2H, t, JZ7.5 Hz, 2-CH2); 2.11, 2.05
(4H, m, 16-CH2, 4-CH2); 1.71 (2H, p, JZ7.5 Hz, 3-CH2);
1.31 (6H, m, 17-19-CH2); 0.88 (3H, t, JZ6.8 Hz, 20-CH3);
13C NMR dC (in ppm, CDCl3, 100 MHz) 173.47 (C1);
130.71, 129.19, 129.04, 128.80, 128.45, 128.35, 128.07 and
1H and 13C NMR spectra were recorded on a Varian
400 MHz machine and chemical shifts are reported in ppm
relative to TMS. The assignment of proton and carbon
resonances of 1–26 was done on the basis of known or
expected chemical shifts in conjunction with 1H–1H,
1H–13C, and DEPT correlated NMR spectroscopy. The
melting points were determined on a Kofler melting point
apparatus and are uncorrected.
Glycidyl esters 1–7 (see below), were obtained in one step
from (G)-glycidol (Fluka) and appropriate acyl-donors
(e.g., free fatty acids, fatty acid anhydrides, or acyl
chlorides), in 74–95% yields as described elsewhere14 or
analogously to conventional approaches.18,24 No attempts
were made to optimize these particular procedures.
3.1.1. 2-(Acetyloxymethyl)oxirane (1). To a solution of
(G)-glycidol (3.7 g; 50 mmol) and 4-dimethylamino-
pyridine (4-DMAP, 6.1 g; 50 mmol) in CH2Cl2 (15 mL) at
rt was added acetic anhydride (6.1 g; 60 mmol). After 12 h,
the solution was passed through a silica gel pad (w5 g)
prepared in CH2Cl2. The support was washed with CH2Cl2
(150 mL) and the solvent evaporated in vacuo. Purification

3664
S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
127.76 (C5-6, C8-9, C11-12, C14-15); 33.62 (C2); 31.73
(C17); 29.54 (C18); 27.43 (C4); 26.73 (C16); 25.83 (C7,
C10, C13); 24.91 (C3); 22.78 (C19); 14.28 (C20):
arachidonoyl fragment; 65.05 (C3); 49.56 (C2); 44.87
(C1): oxirane-2-methyl fragment.
(3.7 g; 30 mmol), 2,4,6-trimethylbenzoic acid (4.9 g;
30 mmol) and DCC (6.2 g; 30 mmol) at rt (reaction time:
24 h) and purified (system C) as described for 3. Yield: 3.6 g
(82%, colorless oil). [Found: C, 70.95; H, 7.40. C13H16O3
(220.26) requires C, 70.89; H, 7.32%]; Rf (system C)Z0.31;
1H NMR dH (in ppm, CDCl3, 400 MHz) 6.86 (2H, s, Aryl);
4.63 (1H, dd, JZ3.3, 3.3 Hz, OCH2CHCHaHbOCO); 4.15
(1H, dd, JZ6.2, 6.6 Hz, OCH2CHCHaHbOCO); 3.32 (1H,
m, C(O)CH2CHCH2O); 2.88 (1H, dd, JZ4.4, 4.7 Hz,
C(O)CH2CHCHaHbO); 2.71 (1H, dd, JZ2.6, 2.6 Hz,
C(O)CH2CHCHaHbO); 2.32 (6H, s, 2-CH3, 6-CH3); 2.28
(3H, s, 4-CH3); 13C NMR dC (in ppm, CDCl3, 100 MHz)
169.94 (–C(O)O); 139.83 (C4); 135.61 (C2 and C6); 130.49
(C1); 128.73 (C3 and C5); 21.37 (4-CH3); 20.11 (2-CH3 and
6-CH3): 2,4,6-trimethylbenzoyl fragment; 65.39 (C3); 49.83
(C2); 44.94 (C1): oxirane-2-methyl fragment.
3.1.4. 2-(Benzoyloxymethyl)oxirane (4). Obtained from
(G)-glycidol (1.1 g; 15 mmol), 4-DMAP (2.2 g; 18 mmol)
and benzoyl chloride (2.5 g; 18 mmol) at rt (reaction time:
4 h) and then purified (system C) in the same way as
described for 2. Yield: 2.3 g (85%, colorless oil). [Found: C,
67.50; H, 5.70. C10H10O3 (178.19) requires C, 67.41; H,
5.66%]; Rf (system C)Z0.34; 1H NMR dH (in ppm, CDCl3,
400 MHz) 8.06 (2H, m, Aryl); 7.57 (1H, m, Aryl); 7.44 (2H,
m, Aryl); 4.65 (1H, dd, JZ2.9, 2.9 Hz, OCH2CHCHaHb-
OCO); 4.17 (1H, dd, JZ6.2, 6.2 Hz, OCH2CHCHaHb-
OCO); 3.34 (1H, m, C(O)CH2CHCH2O); 2.89 (1H, dd, JZ
4.0, 4.4 Hz, C(O)CH2CHCHaHbO); 2.73 (1H, dd, JZ2.6,
2.6 Hz, C(O)CH2CHCHaHbO); 13C NMR dC (in ppm,
CDCl3, 100 MHz) 166.48 (–C(O)O); 133.44 (C4); 129.97
(C2, C6); 129.89 (C1); 128.64 (C3 and C5): benzoyl
fragment; 65.66 (C3); 49.70 (C2); 44.92 (C1): oxirane-2-
methyl fragment.
3.2. General procedure for the preparation of 2-acyl-1,3-
bis(trifluoroacetyl)glycerols 8–14 (step A)
To a solution of a glycidyl ester 1–7 (1.00 mmol), in
dichloromethane (3.0 mL), trifluoroacetic anhydride
(TFAA, 4.00 mmol), in CH2Cl2 (3.0 mL), was added at
K20 8C, and the reaction mixture was kept at rt for 1–4 h.
The solvent and unreacted TFAA were removed under
reduced pressure (bath temperature 40–60 8C). Traces of
TFAA were removed by co-evaporation with toluene (3!
25 mL) under the same conditions and the residue was kept
under vacuum at rt for 2 h to give the target compound 8–14,
in practically quantitative yield.
3.1.5. 2-(4-Nitrobenzoyloxymethyl)oxirane (5). Obtained
from (G)-glycidol (1.1 g; 15 mmol), 4-DMAP (2.2 g;
18 mmol) and 4-nitrobenzoyl chloride (3.3 g; 18 mmol) at
rt (reaction time: 5 h) and then purified (system F) as
described for 2 and 4. Yield: 2.8 g (85%, yellowish crystals,
mp 60.3–61.9 8C, from system F; a commercial sample from
Fluka: mp 59.9–62 8C). [Found: C, 53.75; H, 3.97; N, 6.19.
C10H9NO5 (223.18) requires C, 53.82; H, 4.06; N, 6.28%];
Rf (system F)Z0.42; 1H NMR dH (in ppm, CDCl3,
400 MHz) 8.28 (4H, m, Aryl); 4.74 (1H, dd, JZ2.6,
2.9 Hz, OCH2CHCHaHbOCO); 4.18 (1H, dd, JZ6.6,
6.6 Hz, OCH2CHCHaHbOCO); 3.37 (1H, m, C(O)CH2-
CHCH2O); 2.93 (1H, t, JZ4.4 Hz, C(O)CH2CHCHaHbO);
2.74 (1H, dd, JZ2.9, 2.6 Hz, C(O)CH2CHCHaHbO); 13C
NMR dC (in ppm, CDCl3, 100 MHz) 164.63 (–C(O)O);
150.92 (C4); 135.22 (C1); 131.15 (C2, C6); 123.85 (C3 and
C5): 4-nitrobenzoyl fragment; 66.70 (C3); 49.43 (C2);
44.91 (C1): oxirane-2-methyl fragment.
If necessary, the thus obtained intermediate could addition-
ally be purified by flash, solid-phase filtration through a
silica gel pad (w 5 g) utilizing an appropriate eluant (e.g.,
toluene or dichloromethane).
3.2.1. 2-Acetyl-1,3-bis(trifluoroacetyl)glycerol (8).
Obtained from 2-(acetyloxymethyl)oxirane (1; 0.116 g;
1.00 mmol) and trifluoroacetic anhydride (0.840 g;
4.00 mmol) according to the above general procedure.
After 1 h, solid-phase filtration using dichloromethane as
eluant gave 8 as a colorless oil (0.280 g, 86%). [Found: C,
33.20; H, 2.50. C9H8O6F6 (326.15) requires C, 33.14; H,
2.47%]; Rf (system C)Z0.45; 1H NMR dH (in ppm, CDCl3,
400 MHz) 5.40 (1H, m, CH2CHCH2); 4.63 (2H, dd, JZ4.4,
4.0 Hz, C(O)OCHbHaCHCHaHbOCO); 4.46 (2H, dd, JZ
5.5, 5.5 Hz, C(O)OCHbHaCHCHaHbOCO); 2.10 (3H, s,
2-CH3); 13C NMR dC (in ppm, CDCl3, 100 MHz) 170.07
(C1); 20.62 (C2): acetyl fragment; 157.19 (C1, q, JZ
43.5 Hz); 114.49 (C2, q, JZ285.3 Hz): trifluoroacetyl
fragment; 67.45 (C2); 64.86 (C1, C3): glycerol fragment.
3.1.6. 2-(4-Methoxybenzoyloxymethyl)oxirane (6).
Obtained from (G)-glycidol (1.5 g; 20 mmol), 4-DMAP
(2.9 g; 24 mmol) and 4-methoxybenzoyl chloride (4.1 g;
24 mmol) at rt (reaction time: 6 h) and purified (system F)
identically as described for 5. Yield: 3.5 g (83%, colorless
oil). [Found: C, 63.55; H, 5.75. C11H12O4 (208.21) requires
1
C, 63.45; H, 5.81%]; Rf (system F)Z0.51; H NMR dH (in
ppm, CDCl3, 400 MHz) 8.01 (2H, m, Aryl); 6.90 (2H, m,
Aryl); 4.62 (1H, dd, JZ2.9, 2.9 Hz, OCH2CHCHaHbOCO);
4.12 (1H, dd, JZ6.2, 6.2 Hz, OCH2CHCHaHbOCO); 3.85
(3H, s, CH3O); 3.32 (1H, m, C(O)CH2CHCH2O); 2.88 (1H,
dd, JZ4.0, 4.4 Hz, C(O)CH2CHCHaHbO); 2.71 (1H, dd,
JZ2.6, 2.6 Hz, C(O)CH2CHCHaHbO, 1); 13C NMR dC (in
ppm, CDCl3, 100 MHz) 166.23 (–C(O)O); 163.78 (C4);
132.03 (C2 and C6); 122.24 (C1); 113.89 (C3 and C5);
55.67 (4-OCH3): 4-methoxybenzoyl fragment; 65.39 (C3);
49.83 (C2); 44.94 (C1): oxirane-2-methyl fragment.
3.2.2. 2-Oleoyl-1,3-bis(trifluoroacetyl)glycerol (9).
Obtained by reacting 2-(oleyloxymethyl)oxirane (2;
0.169 g; 0.50 mmol) and trifluoroacetic anhydride
(0.420 g; 2.00 mmol) for 1 h. Solvents were evaporated in
vacuo to give 9 as a yellowish/colorless oil (0.274 g, 100%).
[Found: C, 54.62; H, 7.00. C25H38O6F6 (548.57) requires C,
1
54.74; H, 6.98%]; Rf (system A)Z0.59; H NMR dH (in
ppm, CDCl3, 400 MHz) 5.41 (1H, m, OCH2CHOCO);
5.34 (2H, m, CH]CH); 4.63 (2H, dd, JZ4.0, 4.4 Hz,
C(O)OCHbHaCHCHaHbOCO); 4.46 (2H, dd, JZ5.5,
5.5 Hz, C(O)OCHbHaCHCHaHbOCO); 2.34 (2H, t,
3.1.7. 2-(2,4,6-Trimethylbenzoyloxymethyl)oxirane (7).
Obtained from (G)-glycidol (1.5 g; 20 mmol), 4-DMAP

S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
3665
JZ7.3 Hz, 2-CH2); 2.00 (4H, m, 8-CH2, 11-CH2); 1.62 (2H,
m, 3-CH2); 1.30 (20H, m, 4-7-CH2, 12-17-CH2); 0.88 (3H, t,
JZ6.6 Hz, 18-CH3); 13C NMR dC (in ppm, CDCl3,
100 MHz) 172.72 (C1); 130.25 and 129.90 (C9 and C10);
34.06 (C2); 32.12 (C16); 29.14–29.98 (C4-C7, C12-C15);
27.42 and 27.36 (C11 and C8); 24.87 (C3); 22.89 (C17);
14.30 (C18): oleoyl fragment; 157.18 (C1, q, JZ43.5 Hz);
114.51 (C2, q, JZ285.3 Hz): trifluoroacetyl fragment;
67.15 (C2); 64.94 (C1, C3): glycerol fragment.
ppm, CDCl3, 100 MHz) 163.73 (–C(O)O); 151.28 (C4);
133.92 (C1); 131.22 (C2 and C6); 124.08 (C3 and C5):
4-nitrobenzoyl fragment; 157.21 (C1, q, JZ44.2 Hz);
114.46 (C2, q, JZ285.3 Hz): trifluoroacetyl fragment;
69.02 (C2); 64.77 (C1, C3): glycerol fragment.
3.2.6. 2-(4-Methoxybenzoyl)-1,3-bis(trifluoroacetyl)gly-
cerol (13). Obtained from 2-(4-methoxybenzyloxymethyl)-
oxirane (6; 0.208 g; 1.00 mmol) and trifluoroacetic
anhydride (0.840 g; 4.00 mmol) for 2 h. Solid-phase
filtration employing toluene as the mobile phase provided
13 as a colorless oil (0.401 g, 96%). [Found: C, 43.13; H,
2.92. C15H12O7F6 (418.25) requires C, 43.08; H, 2.89%]; Rf
(system A)Z0.50; 1H NMR dH (in ppm, CDCl3, 400 MHz)
7.92 (2H, m, Aryl); 6.86 (2H, m, Aryl); 5.62 (1H, tt, JZ4.8,
5.1 Hz, CH2CHCH2); 4.73 (2H, dd, JZ4.4, 4.4 Hz,
C(O)OCHbHaCHCHaHbOCO); 4.62 (2H, dd, JZ5.3,
5.1 Hz, C(O)OCHbHaCHCHaHbOCO); 3.87 (3H, s,
4-CH3OC6H4); 13C NMR dC (in ppm, CDCl3, 100 MHz)
165.08 (–C(O)O); 164.38 (C4); 132.21 (C2 and C6); 114.17
(C3 and C5); 55.73 (4-CH3): 4-methoxybenzoyl fragment;
157.22 (C1, q, JZ43.5 Hz); 114.53 (C2, q, JZ284.6 Hz):
trifluoroacetyl fragment; 67.57 (C2); 65.03 (C1, C3):
glycerol fragment.
3.2.3. 2-Arachidonoyl-1,3-bis(trifluoroacetyl)glycerol
(10). Obtained from 2-(arachidonyloxymethyl)oxirane (3;
0.180 g; 0.50 mmol) and trifluoroacetic anhydride (0.420 g;
2.00 mmol) for 1 h. Evaporation of solvents followed by
solid-phase filtration (toluene) afforded 10 as a yellowish oil
(0.267 g, 94%). [Found: C, 56.93; H, 6.30. C27H36O6F6
(570.57) requires C, 56.84; H, 6.36%]; Rf (system A)Z0.57;
1H NMR dH (in ppm, CDCl3, 400 MHz) 5.36 (9H, m,
CH]CH, CH2CHCH2); 4.63 (2H, dd, JZ4.2, 4.4 Hz,
C(O)OCHbHaCHCHaHbOCO); 4.46 (2H, dd, JZ5.5,
5.5 Hz, C(O)OCHbHaCHCHaHbOCO); 2.81 (6H, m, 7, 10,
13-CH2); 2.36 (2H, t, JZ7.5 Hz, 2-CH2); 2.12, 2.05 (4H, m,
16-CH2, 4-CH2, 4H); 1.70 (2H, p, JZ7.3 Hz, 3-CH2); 1.31
(6H, m, 17-19-CH2); 0.89 (3H, t, JZ6.9 Hz, 20-CH3); 13C
NMR dC (in ppm, CDCl3, 100 MHz) 172.51 (C1); 130.72,
129.44, 128.83, 128.68, 128.54, 128.22, 128.03 and 127.73
(C5-6, C8-9, C11-12, C14-15); 33.41 (C2); 31.73 (C17);
29.53 (C18); 27.43 (C4); 26.57 (C16); 25.81 (C7, C10,
C13); 24.70 (C3); 22.78 (C19); 14.26 (C20): arachidonoyl
fragment; 157.17 (C1, q, JZ43.5 Hz); 114.50 (C2, q, JZ
285.3 Hz): trifluoroacetyl fragment; 67.22 (C2); 64.92 (C1,
C3): glycerol fragment.
3.2.7. 2-(2,4,6-Trimethylbenzoyl)-1,3-bis(trifluoro-
acetyl)glycerol (14). Obtained from 2-(2,4,6-Trimethyl-
benzyloxymethyl)oxirane (7; 0.110 g; 0.50 mmol) and
trifluoroacetic anhydride (0.420 g; 2.00 mmol) for 2 h.
Solid-phase filtration (toluene) afforded 14 as a colorless
oil (0.201 g, 93%). [Found: C, 47.52; H, 3.70. C17H16O6F6
(430.30) requires C, 47.45; H, 3.75%]; Rf (system A)Z0.53;
1H NMR dH (in ppm, CDCl3, 400 MHz) 6.88 (2H, m, Aryl);
5.69 (1H, m, CH2CHCH2); 4.74 (2H, dd, JZ3.8, 3.8 Hz,
C(O)OCHbHaCHCHaHbOCO); 4.56 (2H, dd, JZ5.9,
6.0 Hz, C(O)OCHbHaCHCHaHbOCO); 2.28, 2.29 (9H, s,
s, CH3-C6H2); 13C NMR dC (in ppm, CDCl3, 100 MHz)
168.97 (–C(O)O); 140.56 (C4); 135.74 (C2 and C6); 129.25
(C1); 128.91 (C3 and C5); 21.36 (4-CH3); 19.86 (2-,
6-CH3): 2,4,6-trimethylbenzoyl fragment; 157.16 (C1, q,
JZ43.5 Hz); 114.48 (C2, q, JZ285.3 Hz): trifluoroacetyl
fragment; 67.96 (C2); 65.31 (C1, C3): glycerol fragment.
3.2.4. 2-Benzoyl-1,3-bis(trifluoroacetyl)glycerol (11).
Obtained by reacting 2-(benzyloxymethyl)oxirane (4;
0.089 g; 0.50 mmol) and trifluoroacetic anhydride
(0.420 g; 2.00 mmol) for 1 h. Solvents were removed
under reduced pressure to give 11 as a colorless oil
(0.194 g, 100%). [Found: C, 43.43; H, 2.60. C14H10O6F6
(388.22) requires C, 43.31; H, 2.60%]; Rf (system A)Z0.55;
1H NMR dH (in ppm, CDCl3, 400 MHz) 8.00 (2H, m, Aryl);
7.60 (1H, m, Aryl); 7.47 (2H, m, Aryl); 5.66 (1H, m,
CH2CHCH2); 4.75 (2H, dd, JZ4.4, 4.4 Hz, C(O)OCHbHa-
CHCHaHbOCO); 4.63 (2H, dd, JZ5.1, 5.3 Hz, C(O)OCHb-
HaCHCHaHbOCO); 13C NMR dC (in ppm, CDCl3,
100 MHz) 165.44 (–C(O)O); 134.16 (C4); 130.05 (C2,
C6); 128.88 (C3 and C5); 128.66 (C1): benzoyl fragment;
157.22 (C1, q, JZ43.5 Hz); 114.52 (C2, q, JZ285.3 Hz):
trifluoroacetyl fragment; 67.92 (C2); 64.97 (C1, C3):
glycerol fragment.
3.3. General procedure for the preparation of 2-mono-
acyglycerols 15–21 (step B)
To a solution of 8–14 (1.00 mmol) in pentane–CH2Cl2 (3:1,
v/v. 5.0 mL), a mixture of pyridine (10.0 mmol) and
methanol (15.0 mmol) in the same solvent (5.0 mL) was
added at K20 8C, and the reaction mixture was left at rt for
2–3 h. Solvents were evaporated under reduced pressure
(bath 40–60 8C) and the residue was kept under vacuum at rt
for 2–3 h to give the target 2-monoacylglycerol 15–21.
3.2.5. 2-(4-Nitrobenzoyl)-1,3-bis(trifluoroacetyl)glycerol
(12). Obtained from 2-(4-nitrobenzyloxymethyl)oxirane (5;
0.112 g; 0.50 mmol) and trifluoroacetic anhydride (0.420 g;
2.00 mmol) for 4 h. Solid-phase filtration using dichloro-
methane as eluant gave 12 (0.187 g, 86%) as yellowish
crystals, mp 104.9 - 105.8 8C (from CH2Cl2). [Found: C,
39.00; H, 2.05; N, 3.23. C14H9NO8F6 (433.22) requires C,
The product could also be retrieved from the corresponding
bis(trifluoroacetyl)-intermediate 8–14 directly in a one-pot
procedure.
1
38.82; H, 2.09; N, 3.23%]; Rf (system A)Z0.44; H NMR
3.3.1. 2-Acetylglycerol (15). Synthesized in a one-pot
procedure comprising:
dH (in ppm, CDCl3, 400 MHz) 8.30 (2H, m, Aryl); 8.20 (2H,
m, Aryl); 5.69 (1H, m, CH2CHCH2); 4.79 (2H, dd, JZ4.2,
4.2 Hz, C(O)OCHbHaCHCHaHbOCO); 4.65 (2H, dd, JZ
5.5, 5.3 Hz, C(O)OCHbHaCHCHaHbOCO); 13C NMR dC (in
Step A. Reaction of 2-(acetyloxymethyl)oxirane (1; 0.116 g;
1.00 mmol) and trifluoroacetic anhydride (0.840 g;

3666
S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
4.00 mmol) in CH2Cl2 at rt for 1 h followed by evaporation
of volatile products in vacuo to give 2-acetyl-1,3-bis(tri-
fluoroacetyl)glycerol 8 as described above.
ppm, CDCl3, 400 MHz) 8.04 (2H, m, Aryl); 7.56 (1H, m,
Aryl); 7.43 (2H, m, Aryl); 5.16 (1H, tt, JZ4.8, 4.8 Hz,
OCH2CHOCO); 3.95 (4H, d, JZ4.9 Hz, OCH2CHCH2O);
13C NMR dC (in ppm, CDCl3, 100 MHz) 166.89 (–C(O)O);
133.57 (C4); 129.99 (C2 and C6); 128.66 (C1, C3, C5):
benzoyl fragment; 75.97 (C2); 62.60 (C1, C3): glycerol
fragment.
Step B. Direct treatment of the thus obtained intermediate 8
with pyridine (0.79 g; 10 mmol) and methanol (0.48 g;
15 mmol) in pentane–CH2Cl2 (3:1, v/v) at rt for 2 h and then
removing solvents under reduced pressure to give the title
compound 15 as a colorless oil (0.134 g, 100%). [Found: C,
44.71; H, 7.57. C5H10O4 (134.13) requires C, 44.77; H,
7.51%]; Rf (system D)Z0.33; 1H NMR dH (in ppm, CDCl3,
400 MHz) 4.89 (1H, tt, JZ4.8, 4.8 Hz, OCH2CHOCO);
3.80 (4H, d, JZ4.6 Hz, OCH2CHCH2O); 2.11 (3H, s,
2-CH3); 13C NMR dC (in ppm, CDCl3, 100 MHz) 171.58
(C1); 21.32 (C2): acetyl fragment; 75.31 (C2); 62.30 (C1,
C3): glycerol fragment.
3.3.5. 2-(4-Nitrobenzoyl)glycerol (19). Obtained from
2-(4-nitrobenzyloxymethyl)oxirane
(5;
0.112 g;
0.50 mmol) identically with 17 and 18. Yield: 0.109 g
(90%, yellowish crystals, mp 115.9–117.8 8C, from
pentane–CH2Cl2Z3:1, v/v). [Found: C, 49.75; H, 4.67; N,
5.78. C10H11NO6 (241.20) requires C, 49.80; H, 4.60; N,
5.81%]; Rf (system B)Z0.15; 1H NMR dH (in ppm,
CD3OD, 400 MHz) 8.31 (4H, m, Aryl); 5.18 (1H, tt, JZ
4.9, 4.9 Hz, OCH2CHOCO); 3.83 (4H, m, OCH2CHCH2O);
13C NMR dC (in ppm, CD3OD, 100 MHz) 164.76 (–C(O)O);
150.88 (C4); 135.95 (C1); 130.80 (C2 and C6); 123.34 (C3
and C5): 4-nitrobenzoyl fragment; 77.20 (C2); 60.47 (C1,
C3): glycerol fragment.
3.3.2. 2-Oleoylglycerol (16). Synthesized in a one-pot
procedure from 2-(oleyloxymethyl)oxirane (2; 0.338 g;
1.00 mmol), (Step B: rt/3 h), as described for 15. Yield:
0.356 g (100%, yellowish/colorless oil). [Found: C, 70.71;
H, 11.35. C21H40O4 (356.55) requires C, 70.74; H, 11.31%];
Rf (system B)Z0.32; 1H NMR dH (in ppm, CDCl3,
400 MHz) 5.34 (2H, m, CH]CH); 4.92 (1H, tt, JZ4.8,
4.8 Hz, OCH2CHOCO); 3.83 (4H, d, JZ4.8 Hz, OCH2-
CHCH2O); 2.36 (2H, t, JZ7.7 Hz, 2-CH2); 1.99 (4H, m,
8-CH2, 11-CH2); 1.63 (2H, m, 3-CH2); 1.30 (20H, m, 4-7-
CH2, 12-17-CH2); 0.87 (3H, t, JZ7.0 Hz, 18-CH3); 13C
NMR dC (in ppm, CDCl3, 100 MHz) 174.29 (C1); 130.26
and 129.92 (C9 and C10); 34.55 (C2); 32.12 (C16); 29.98–
29.29 (C4-C7, C12-C15); 27.43 and 27.37 (C8 and C11);
25.16 (C3); 22.90 (C17); 14.32 (C18): oleoyl fragment;
75.22 (C2); 62.70 (C1, C3): glycerol fragment.
3.3.6. 2-(4-Methoxybenzoyl)glycerol (20). Obtained from
2-(4-Methoxybenzyloxy-methyl)oxirane (6; 0.104 g;
0.50 mmol) in a synonymous way as described for 17–19.
Yield: 0.107 g (95%, white crystals, mp 78.9–80.1 8C, from
pentane–CH2Cl2Z3:1, v/v). [Found: C, 58.48; H, 6.30.
C11H14O5 (226.23) requires C, 58.40; H, 6.24%]; Rf (system
1
B)Z0.17; H NMR dH (in ppm, CDCl3, 400 MHz) 7.95
(2H, m, Aryl); 6.85 (2H, m, Aryl); 5.12 (1H, tt, JZ4.8,
4.8 Hz, OCH2CHOCO); 3.93 (4H, d, JZ4.8 Hz, OCH2-
CHCH2O); 3.85 (3H, s, 4-CH3OC6H4); 13C NMR dC (in
ppm, CDCl3, 100 MHz) 166.70 (–C(O)O); 163.94 (C4);
132.09 (C2, C6); 122.25 (C1); 113.93 (C3 and C5); 55.69
(4-CH3): 4-methoxybenzoyl fragment; 75.79 (C2); 62.72
(C1, C3): glycerol fragment.
3.3.3. 2-Arachidonoylglycerol (17). Synthesized in two
steps: (a) treatment of 2-(arachidonyloxymethyl)oxirane (3;
0.180 g; 0.50 mmol) with trifluoroacetic anhydride
(0.420 g; 2.00 mmol) in CH2Cl2 (reaction time: rt/1 h); b)
isolation of intermediate 10 by solid-phase filtration
(toluene) followed by its hydrolysis (reaction time: rt/3 h)
in pentane–CH2Cl2 (3:1, v/v) using pyridine (0.39 g;
5.0 mmol) and methanol (0.24 g; 7.5 mmol). Yield:
0.175 g (92%, yellowish oil). [Found: C, 73.00; H, 10.18.
C23H38O4 (378.56) requires C, 72.98; H, 10.12%]; Rf
(system B)Z0.33; 1H NMR dH (in ppm, CDCl3, 400 MHz)
5.37 (8H, m, CH]CH); 4.92 (1H, tt, JZ4.6, 4.6 Hz,
OCH2CHOCO); 3.82 (4H, d, JZ4.8 Hz, OCH2CHCH2O);
2.81 (6H, m, 7, 10, 13-CH2); 2.39 (2H, t, JZ7.6 Hz, 2-CH2);
2.13, 2.05 (4H, m, 4-CH2, 16-CH2); 1.73 (2H, p, JZ7.3 Hz,
3-CH2); 1.32 (6H, m, 17-19-CH2); 0.88 (3H, t, JZ6.8 Hz,
20-CH3); 13C NMR dC (in ppm, CDCl3, 100 MHz) 174.03
(C1); 130.76, 129.26, 128.99, 128.85, 128.54, 128.32,
128.06 and 127.75 (C5-6, C8-9, C11-12, C14-15); 33.90
(C2); 31.74 (C17); 29.54 (C18); 27.43 (C4); 26.71 (C16);
25.83 (C7, C10, C13); 24.97 (C3); 22.78 (C19); 14.29
(C20): arachidonoyl fragment; 75.28 (C2); 62.67 (C1, C3):
glycerol fragment.
3.3.7. 2-(2,4,6-Trimethylbenzoyl)glycerol (21). Obtained
from 2-(2,4,6-trimethylbenzyloxymethyl)oxirane (7;
0.110 g; 0.50 mmol) identically with 17–20. Yield:
0.113 g (95%, white crystals, mp 85.9–90.5 8C, from
pentane–CH2Cl2Z3:1, v/v). [Found: C, 65.50; H, 7.69.
C13H18O4 (238.29) requires C, 65.53; H, 7.61%]; Rf (system
1
B)Z0.35; H NMR dH (in ppm, CDCl3, 400 MHz) 6.85
(2H, m, Aryl); 5.18 (1H, tt, JZ4.8, 4.8 Hz, OCH2CHOCO);
3.93 (4H, m, OCH2CHCH2O); 2.28, 2.30 (9H, s, s, CH3-
C6H2); 13C NMR dC (in ppm, CDCl3, 100 MHz) 170.35
(–C(O)O); 139.83 (C4); 135.28 (C2 and C6); 130.77 (C1);
128.69 (C3 and C5); 21.34 (4-CH3); 19.97 (2-, 6-CH3):
2,4,6-trimethylbenzoyl fragment; 75.88 (C2); 62.59 (C1,
C3): glycerol fragment.
3.4. General procedure for the preparation of 1,3-
symmetrical triacylglycerols 22–26 (step C)
A solution of 2-monoacylglycerol 15–18 (1.00 mmol) and
pyridine (20.0 mmol), in dichloromethane (10.0 mL), was
treated with a solution of acyl chloride a–d (3.00 mmol) in
dichloromethane (10.0 mL) at K20 8C, and the reaction
mixture was kept at rt for 2–4 h. Solvents were removed
under reduced pressure. The residue was taken in dichloro-
methane (10.0 mL), solution was passed through a dichloro-
methane-filled aluminium oxide pad (w20 g) and the
3.3.4. 2-Benzoylglycerol (18). Obtained from 2-(benzyloxy-
methyl)oxirane (4; 0.089 g; 0.50 mmol) in the same way as
described for 17. Yield: 0.094 g (96%, colorless oil).
[Found: C, 61.19; H, 6.20. C10H12O4 (196.20) requires C,
1
61.22; H, 6.16%]; Rf (system B)Z0.21; H NMR dH (in

S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
3667
support was washed with the same solvent (w150 mL).
Dichloromethane was removed under reduced pressure and
triglyceride formed 22–26 was isolated in pure state by flash
column chromatography (CC).
acetyl chloride (c; 0.118 g; 1.50 mmol) in CH2Cl2 (rt/2 h),
as described for 22–23. Purification of the crude product by
flash CC (system A) afforded the title compound 24
(0.187 g, 85%) as a colorless oil. [Found: C, 68.20; H,
10.10. C25H44O6 (440.63) requires C, 68.15; H, 10.07%]; Rf
(system A)Z0.37; 1H NMR dH (in ppm, CDCl3, 400 MHz)
5.33 (2H, m, CH]CH); 5.26 (1H, m, OCH2CHOCO); 4.27
(2H, dd, JZ4.4, 4.4 Hz, C(O)OCHbHaCHCHaHbOCO);
4.14 (2H, dd, JZ6.0, 6.0 Hz, C(O)OCHbHaCHCHaHb-
OCO); 2.32 (2H, t, JZ7.3 Hz, 2-CH2); 2.06 (6H, s,
CH3CO); 1.99 (4H, m, 8-CH2, 11-CH2); 1.62 (2H, m,
3-CH2); 1.30 (20H, m, 4-7-CH2, 12-17-CH2); 0.87 (3H, t,
JZ7.0 Hz, 18-CH3); 13C NMR dC (in ppm, CDCl3,
100 MHz) 173.12 (C1); 130.25 and 129.90 (C10 and C9);
34.39 (C2); 32.11 (C16); 29.97–29.22 (C4-C7, C12-C15);
27.43 and 27.37 (C8 and C11); 25.08 (C3); 22.88 (C17);
14.32 (C18): oleoyl fragment; 170.70 (C1); 20.89 (C2):
acetyl fragment; 68.95 (C2); 62.53 (C1, C3): glycerol
fragment.
The latter compound could also be obtained via a one-pot
procedure by conveniently combining steps A, B and C.
3.4.1. 1,3-Dipalmitoyl-2-acetylglycerol (22). Synthesized
in a one-pot procedure by consecutively performing:
Step A. Transformation of 2-(acetyloxymethyl)oxirane (1;
0.058 g; 0.50 mmol) by means of trifluoroacetic anhydride
(0.420 g; 2.00 mmol) to 2-acetyl-1,3-bis(trifluoroacetyl)-
glycerol 8 (reaction time: rt/1 h).
Step B. Direct hydrolysis of the thus obtained intermediate 8
with pyridine (0.39 g; 5.0 mmol) and methanol (0.24 g;
7.5 mmol) to 2-acetylglycerol 15 (reaction time: rt/2 h).
Step C. Treatment of 15 in the presence pyridine (0.79 g;
10.0 mmol) with palmitoyl chloride (a; 0.412 g; 1.50 mmol)
(reaction time: rt/4 h). Finally, purification of the crude
triglyceride by flash column chromatography (system E)
gave the title compound 22 (0.254 g, 83%) as a white solid,
mp 58.2–60.9 8C (from system E). [Found: C, 72.74; H,
11.60. C37H70O6 (610.97) requires C, 72.74; H, 11.55%]; Rf
(system E)Z0.36; 1H NMR dH (in ppm, CDCl3, 400 MHz)
5.24 (1H, m, OCH2CHOCO); 4.29 (2H, dd, JZ4.2, 4.2 Hz,
C(O)OCHbHaCHCHaHbOCO); 4.15 (2H, dd, JZ5.9,
5.9 Hz, C(O)OCHbHaCHCHaHbOCO); 2.31 (4H, t, JZ
7.3 Hz, 2-CH2); 2.07 (3H, s, CH3CO); 1.60 (4H, m, 3-CH2);
1.28 (48H, m, 4-15-CH2); 0.87 (6H, t, JZ6.8 Hz, 16-CH3);
13C NMR dC (in ppm, CDCl3, 100 MHz) 173.54 (C1); 34.26
(C2); 32.14 (C14); 29.91–29.32 (C4-13); 25.07 (C3); 14.33
(C16): palmitoyl fragment; 170.28 (C1); 21.09 (C2): acetyl
fragment; 69.40 (C2); 62.23 (C1, C3): glycerol fragment.
3.4.4. 1,3-Dibenzoyl-2-arachidonoylglycerol (25).
Obtained according to the general procedure from 2-ara-
chidonoylglycerol (17; 0.189 g; 0.50 mmol), pyridine
(0.79 g; 10.0 mmol), and benzoyl chloride (d; 0.211 g;
1.50 mmol) in CH2Cl2 (reaction time: rt/4 h). The crude
triglyceride was purified by flash CC (system A) to give the
target compound 25 (0.252 g, 86%) as a colorless oil.
[Found: C, 75.63; H, 7.95. C37H46O6 (586.78) requires C,
1
75.74; H, 7.90%]; Rf (system A)Z0.60; H NMR dH (in
ppm, CDCl3, 400 MHz) 8.03 (4H, m, Aryl); 7.57 (2H, m,
Aryl); 7.44 (4H, m, Aryl); 5.60 (1H, m, OCH2CHOCO);
5.36 (8H, m, CH]CH); 4.63 (2H, dd, JZ4.4, 4.4 Hz,
C(O)OCHbHaCHCHaHbOCO); 4.52 (2H, dd, JZ5.9,
5.9 Hz, C(O)OCHbHaCHCHaHbOCO); 2.90–2.70 (6H, m,
7, 10, 13-CH2); 2.37 (2H, t, JZ7.5 Hz, 2-CH2); 2.07 (4H, m,
16-CH2, 4-CH2); 1.70 (2H, p, JZ7.3 Hz, 3-CH2); 1.30 (6H,
m, 17-19-CH2); 0.88 (3H, t, JZ6.8 Hz, 20-CH3); 13C NMR
dC (in ppm, CDCl3, 100 MHz) 172.96 (C1); 130.71, 129.72,
129.72, 128.96, 128.80, 128.47, 128.32, 128.07, 127.76
(C5-6, C8-9, C11-12, C14-15); 33.85 (C2); 31.73 (C17);
29.54 (C18); 27.43 (C4); 26.70 (C16); 25.86, 25.83 and
25.80 (C13, C7 and C10); 24.98 (C3); 22.78 (C19); 14.29
(C20): arachidonoyl fragment; 166.26 (–C(O)O); 133.53
(C4); 129.95 (C6, C2); 129.10 (C1); 128.71 (C5, C3):
benzoyl fragment; 69.23 (C2); 63.18 (C1, C3): glycerol
fragment.
3.4.2. 1,3-Dioleoyl-2-acetylglycerol (23). Synthesized in a
one-pot, three-step procedure from 2-(acetyloxymethyl)-
oxirane (1; 0.058 g; 0.50 mmol), pyridine (0.79 g;
10.0 mmol), and oleoyl chloride (b; 0.451 g; 1.50 mmol)
as described for 22. The crude product was purified by flash
CC (system E) to give the title compound 23 (0.271 g, 82%)
as a colorless oil. [Found: C, 74.32; H, 11.22. C41H74O6
(663.04) requires C, 74.27; H, 11.25%]; Rf (system E)Z
0.35; 1H NMR dH (in ppm, CDCl3, 400 MHz) 5.35 (4H, m,
CH]CH); 5.24 (1H, m, OCH2CHOCO); 4.30 (2H, dd, JZ
4.4, 4.4 Hz, C(O)OCHbHaCHCHaHbOCO); 4.14 (2H, dd,
JZ5.9, 5.9 Hz, C(O)OCHbHaCHCHaHbOCO); 2.31 (4H, t,
JZ7.5 Hz, 2-CH2); 2.07 (3H, s, CH3CO); 2.00 (8H, m,
8-CH2, 11-CH2); 1.61 (4H, m, 3-CH2); 1.30 (40H, m, 4-7-
CH2, 12-17-CH2); 0.87 (6H, t, JZ6.9 Hz, 18-CH3); 13C
NMR dC (in ppm, CDCl3, 100 MHz) 173.49 (C1); 130.23,
129.93 (C9, C10); 34.24 (C2); 32.12 (C16); 29.98–29.29
(C4-C7, C12-C15); 27.43 and 27.38 (C8 and C11); 25.06
(C3); 22.90 (C17); 14.32 (C18): oleoyl fragment; 170.26
(C1); 21.09 (C2): acetyl fragment; 69.40 (C2); 62.24 (C1,
C3): glycerol fragment.
3.4.5. 1,3-Dioleoyl-2-benzoylglycerol (26). Obtained in a
one-pot procedure from 2-(benzyloxymethyl)oxirane (4;
0.089 g; 0.50 mmol), pyridine (0.79 g; 10.0 mmol), and
oleoyl chloride (b; 0.451 g; 1.50 mmol) followed by
purification of the crude product (CC system E) as described
for 22–24. Overall yield of 26: 0.308 g (85%, colorless oil).
[Found: C, 76.32; H, 10.60. C46H76O6 (725.11) requires C,
1
76.20; H, 10.56%]; Rf (system E)Z0.43; H NMR dH (in
ppm, CDCl3, 400 MHz) 8.04 (2H, m, Aryl); 7.58 (1H, m,
Aryl); 7.44 (2H, m, Aryl); 5.52 (1H, m, OCH2CHOCO);
5.35 (4H, m, CH]CH); 4.39 (2H, dd, JZ4.4, 4.4 Hz,
C(O)OCHbHaCHCHaHbOCO); 4.33 (2H, dd, JZ6.0,
6.0 Hz, C(O)OCHbHaCHCHaHbOCO); 2.31 (4H, t, JZ
7.5 Hz, 2-CH2); 1.99 (8H, m, 8-CH2, 11-CH2); 1.60 (4H, m,
3-CH2); 1.30 (40H, m, 4-7-CH2, 12-17-CH2); 0.88 (6H, t,
JZ7.0 Hz, 18-CH3); 13C NMR dC (in ppm, CDCl3,
3.4.3. 1,3-Diacetyl-2-oleoylglycerol (24). Synthesized in a
one-pot procedure from 2-(oleyloxymethyl)oxirane (2;
0.169 g; 0.50 mmol), pyridine (0.79 g; 10.0 mmol), and

3668
S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
Ghyselburton, J.; Maloteaux, J. M.; Dumont, P. J. Pharm.
Pharmacol. 1993, 45, 186–191. Garzon-Aburbeh, A.;
Poupaert, J. H.; Claesen, M.; Dumont, P.; Atassi, G. J. Med.
Chem. 1983, 26, 1200–1203. Paris, G. Y.; Garmaise, D. L.;
Cimon, D. G.; Swett, L.; Carter, G. W.; Young, P. J. Med.
Chem. 1979, 22, 683–687.
100 MHz) 173.53 (C1); 130.21, 129.96 (C10, C9); 34.28
(C2); 32.12 (C16); 29.98–29.29 (C12-C15, C4-C7); 27.44,
27.38 (C8 and C11); 25.07 (C3); 22.90 (C17); 14.33 (C18):
oleoyl fragment; 165.84 (–C(O)O); 133.53 (C4); 130.01
(C2, C6); 129.82 (C1); 128.65 (C3, C5): benzoyl fragment;
70.00 (C2); 62.36 (C1, C3): glycerol fragment.
8. Pleiss, J.; Scheib, H.; Schmid, R. D. Biochimie 2000, 82,
1043–1052.
3.4.6. 2-Oleoyl-1,3-bis(trichloroacetyl)glycerol. Obtained
from 2-(oleyloxymethyl)oxirane (2; 0.169 g; 0.50 mmol)
and trichloroacetic anhydride (0.617 g; 2.00 mmol) accord-
ing to the general procedure (reaction time: rt/24 h). Flash
CC using toluene–EtOAc (95:5, v/v) as eluant gave the pure
trichloroacetylated product (0.317 g, 98%) as a colorless oil.
[Found: C, 46.44; H, 6.00. C25H38O6Cl6 (647.28) requires
C, 46.39; H, 5.92%]; Rf (system A)Z0.64; 1H NMR dH (in
ppm, CDCl3, 400 MHz) 5.47 (1H, m, OCH2CHOCO); 5.34
(2H, m, CH]CH); 4.64 (2H, dd, JZ4.4, 4.4 Hz,
C(O)OCHbHaCHCHaHbOCO); 4.50 (2H, dd, JZ5.7,
5.5 Hz, C(O)OCHbHaCHCHaHbOCO); 2.34 (2H, t, JZ
7.7 Hz, 2-CH2); 2.00 (4H, m, 8-CH2, 11-CH2); 1.62 (2H, m,
3-CH2); 1.30 (20H, m, 4-7-CH2, 12-17-CH2); 0.87 (3H, t,
JZ7.1 Hz, 18-CH3); 13C NMR dC (in ppm, CDCl3,
100 MHz) 172.69 (C1); 130.27 and 129.90 (C10 and C9);
34.16 (C2); 32.12 (C16); 29.98–29.22 (C12-C15, C4-C7);
27.44 and 27.37 (C8 and C11); 24.91 (C3); 22.90 (C17);
14.33 (C18): oleoyl fragment; 161.74 (s, C1): trichloro-
acetyl fragment; 67.70 (C2); 65.99 (C1, C3): glycerol
fragment.
9. Mank, A. P. J.; Lok, C. M.; Ward, J. P. Chem. Phys. Lipids
1989, 50, 63–70.
10. Rose, W. G. J. Am. Chem. Soc. 1947, 69, 1384–1387. Bentley,
P. H.; McCrae, W. J. Org. Chem. 1970, 35, 2082. Ikeda, I.; Gu,
X. P.; Miyamoto, I.; Okahara, M. J. Am. Oil Chem. Soc. 1989,
66, 822–824. Paltauf, F.; Hermetter, A. Prog. Lipid Res. 1994,
33, 239–328. Haftendorn, R.; Schwarze, G.; Ubrich-Hofmann,
R. Chem. Phys. Lipids 2000, 104, 57–66. Gras, J. L.; Bonfanti,
J. F. Synlett 1999, 1835–1837.
11. Di Marzo, V.; Melck, D.; De Petrocellis, L.; Bisogno, T.
Prostaglandins Other Lipid Mediat. 2000, 61, 43–61.
12. Seltzman, H. H.; Fleming, D. N.; Hawkins, G. D.; Carroll, F. I.
Tetrahedron Lett. 2000, 41, 3589–3592.
13. Schmid, P. C.; Schwartz, K. D.; Smith, C. N.; Krebsbach, R. J.;
Berdyshev, E. V.; Schmid, H. H. O. Chem. Phys. Lipids 2000,
104, 185–191. Wongsakui, S.; Prasertsan, P.; Bornscheuer, U.;
H-Kittikun, A. Eur. J. Lipid Sci. Technol. 2003, 105, 68–73.
14. Stamatov, S. D.; Stawinski, J. Tetrahedron Lett. 2002, 43,
1759–1761.
15. Under acidic conditions (e.g., 0.03 N perchloric acid) the
equilibrium between isomeric acylglycerols is rapidly estab-
lished (ca. 10 min) yielding usually ca. 9:1 mixture of 1-acyl-
and 2-acylglycerols; see Martin, J. J. Am. Chem. Soc. 1953, 75,
5483–5486.
Acknowledgements
16. Martin, J. B. J. Am. Chem. Soc. 1953, 75, 5482–5483.
17. Suhara, Y.; Takayama, H.; Nakane, S.; Miyashita, T.; Waku,
K.; Sugiura, T. Chem. Pharm. Bull. 2000, 48, 903–907.
18. Han, L. N.; Razdan, R. K. Tetrahedron Lett. 1999, 40,
1631–1634.
We are indebted to Prof. P. J Garegg for his interest in this
work. Financial support from the Swedish Natural Science
Research Council and the Swedish Foundation for Strategic
Research is gratefully acknowledged.
19. Cartoni, A.; Margonelli, A.; Angelini, G.; Finazzi-Agro, A.;
Maccarrone, M. Tetrahedron Lett. 2004, 45, 2723–2726.
20. Pop, E. Curr. Opin. Chem. Biol. 1999, 3, 418–425. Sugiura, T.;
Yoshinaga, N.; Waku, K. Neurosci. Lett. 2001, 297, 175–178.
Di Marzo, V.; Melck, D.; Bisogno, T.; De Petrocellis, L.
Trends Neurosci. 1999, 22, 80. Mechoulam, R.; Fride, E.; Di
Marzo, V. Eur. J. Pharmacol. 1998, 359, 1–18. Sugiura, T.;
Kondo, S.; Kishimoto, S.; Miyashita, T.; Nakane, S.; Kodaka,
T.; Suhara, Y.; Takayama, H.; Waku, K. J. Biol. Chem. 2000,
275, 605–612. Murphy, L. L.; Munoz, R. M.; Adrian, B. A.;
Villanua, M. A. Neurobiol. Dis. 1998, 5, 432–446. Sugiura, T.;
Waku, K. Chem. Phys. Lipids 2000, 108, 89–106. Melck, D.;
De Petrocellis, L.; Orlando, P.; Bisogno, T.; Laezza, C.;
Bifulco, M.; Di Marzoo, V. Endocrinology 2000, 141,
118–126. Han, S.-Y.; Cho, S.-H.; Kim, S.-Y.; Seo, J.-T.;
Moon, S.-J.; Jhon, G.-J. Bioorg. Med. Chem. Lett. 1999, 9,
59–64.
References and notes
1. Small, D. M. Ann. Rev. Nutr. 1991, 11, 413–434.
2. Iwasaki, Y.; Yamane, T. J. Mol. Catal. B: Enzymatic 2000, 10,
129–140.
3. Innis, S. M.; Dyer, R.; Nelson, C. M. Lipids 1994, 29,
541–545.
4. Sugiura, T.; Kondo, S.; Sukagawa, A.; Nakane, S.; Shinoda,
A.; Itoh, K.; Yamashita, A.; Waku, K. Biochem. Biophys. Res.
Commun 1995, 215, 89–97. Mechoulam, R.; Benshabat, S.;
Hanus, L.; Ligumsky, M.; Kaminski, N. E.; Schatz, A. R.;
Gopher, A.; Almog, S.; Martin, B. R.; Compton, D. R.;
Pertwee, R. G.; Griffin, G.; Bayewitch, M.; Barg, J.; Vogel, Z.
Biochem. Pharmacol. 1995, 50, 83–90.
21. Mank, A. P. J.; Ward, J. P.; VanDorp, D. A. Chem. Phys.
Lipids 1976, 16, 107–114. Lok, C. M. Chem. Phys. Lipids
1978, 22, 323–337.
5. Ben-Shabat, S.; Fride, E.; Sheskin, T.; Tamiri, T.; Rhee, M. H.;
Vogel, Z.; Bisogno, T.; De Petrocellis, L.; Di Marzo, V.;
Mechoulam, R. Eur. J. Pharmacol. 1998, 353, 23–31.
6. Kurz, M.; Scriba, G. K. E. Chem. Phys. Lipids 2000, 107,
143–157.
22. Isomeric purity of triglycerides can be assessed by examining
13C NMR spectra in the region of absorption of carbonyl
carbons. For acetyl-containing derivatives, both 1H or 13C
NMR can be used for this purpose as methyl protons and the
carbonyl carbons of the acetyl groups have distinctive
chemical shifts depending on the position of the acetyl group
7. Deverre, J. R.; Loiseau, P.; Puisieux, F.; Gayral, P.;
Letourneux, Y.; Couvreur, P.; Benoit, J. P. Arzneimittel-
Forschung/Drug Research 1992, 42-2, 1153–1156. Lambert,
D. M.; Neuvens, L.; Mergen, F.; Gallez, B.; Poupaert, J. H.;

S. D. Stamatov, J. Stawinski / Tetrahedron 61 (2005) 3659–3669
3669
in the glycerol backbone. For example, for compounds 23 and
24, the relevant chemical shifts in CDCl3 are: 2.07 ppm (CH3);
170.26 ppm (1-C) and 2.06 ppm (CH3-) and 170.70 ppm
(1-C), respectively.
23. Purification of Laboratory Chemical; Perrin, D. D., Armarego,
W. L. F., Eds.; Pergamon: New York, 1988.
24. Neises, B.; Steglich, W. Angew. Chem. Int. Ed. Engl. 1978, 17,
522–524.