Sterically Hindered Triacylglycerol Analogues as Potent Inhibitors of Human Digestive Lipases

Article abstract of DOI:10.1002/chem.200305573

A novel class of inhibitors of human digestive lipases have been developed. Various sterically hindered triacylglycerols based on 2-methyl- and 2-butylglycerol, and/or 2-methyl fatty acids were synthesized. The triacylglycerol analogues were tested for their ability to form stable monomolecular films at the air/water interface by recording their surface-pressure/molecular-area compression isotherms. The inhibition of human pancreatic and gastric lipases by the sterically hindered triacylglycerol analogues was studied by using the monolayer technique with mixed films of 1,2-dicaprin, which contained variable proportions of each inhibitor. Triolein analogues that contain a butyl group at the 2-position of the glycerol backbone or methyl groups both at the 2-position of glycerol, and the α-position of each oleic acid residue were potent inhibitors; this caused a 50% decrease in HPL activity at 0.003 molar fraction.

Full text of DOI:10.1002/chem.200305573

FULL PAPER
Sterically Hindered Triacylglycerol Analogues as Potent Inhibitors of Human
Digestive Lipases
Violetta Constantinou-Kokotou,*[a] Victoria Magrioti,[a] and Robert Verger[b]
Abstract: A novel class of inhibitors of
human digestive lipases have been de-
veloped. Various sterically hindered tri-
acylglycerols based on 2-methyl- and 2-
butylglycerol, and/or 2-methyl fatty
acids were synthesized. The triacylgly-
cerol analogues were tested for their
ability to form stable monomolecular
films at the air/water interface by re-
cording their surface-pressure/molecu-
lar-area compression isotherms. The in-
hibition of human pancreatic and gas-
tric lipases by the sterically hindered
triacylglycerol analogues was studied
by using the monolayer technique with
mixed films of 1,2-dicaprin, which con-
tained variable proportions of each in-
hibitor. Triolein analogues that contain
a butyl group at the 2-position of the
glycerol backbone or methyl groups
both at the 2-position of glycerol, and
the a-position of each oleic acid resi-
due were potent inhibitors; this caused
Keywords: enzyme inhibitors
¥
enzymes ¥ lipids ¥ monolayers ¥
triacylglycerol analogues
a
50% decrease in HPL activity
at 0.003 molar fraction.
Introduction
aldehydes,[5] and trifluoromethylketones,[6] as well as 2-oxo
amide and bis(2-oxo)amide triacylglycerol analogues[7] have
been studied by the monolayer technique, and have been
shown to inhibit HPL and HGL. In this work, we propose a
novel approach for the development of lipolytic enzyme in-
hibitors. We present the synthesis of a new class of sterically
hindered triacylglycerol analogues, the study of their surface
properties, and their inhibitory effect on HPL and HGL ac-
tivities; these were measured by the monolayer technique.
Lipids constitute a large part of the biomass of living organ-
isms, and lipolytic enzymes play an essential role in the turn-
over of this material. In particular, triglycerides (triacylgly-
cerols, TAGs) constitute the major part (95%) of dietary
lipids, which are present in the human diet at a quantity of
100 150 g per day in industrially developed countries.
Human pancreatic (HPL) and gastric (HGL) lipases are es-
sential enzymes for efficient fat digestion.[1] The hydrolysis
of dietary TAGs by these enzymes to form monoacylglycer-
ols and free fatty acids is a necessary step for fat absorption
by the enterocytes. Therefore, potent and specific inhibitors
of digestive lipases are of interest, because they may find
applications as anti-obesity agents. The b-lactone that con-
tains the inhibitor tetrahydrolipstatin is already in clinical
use for the treatment of obesity.[2] Phosphonate-type inhibi-
tors have been found to irreversibly inactivate HPL and
HGL as well as microbial lipases.[3] Recently a strategy for
the rational design of lipase inhibitors was developed, which
is based on the incorporation of an activated carbonyl group
in a triacylglycerol structure. Thus, lipophilic 2-oxo amides,[4]
Results and Discussion
Design: To develop novel potent inhibitors of digestive li-
pases, we decided to maintain the natural substrate back-
bone, which contains three ester bonds, and to replace a hy-
drogen atom by incorporating an alkyl group at the carbon
atom of the sn-2 position or/and at the a-carbon atom of the
acyl residue (Scheme 1). The rationale behind the present
design was to create structures that closely resembled natu-
ral lipase substrates and give steric hindrance around the
scissile ester bonds. The replacement of the hydrogen atom
of glycerol at the sn-2 position by a methyl or a butyl group,
and the presence of a methyl group at the carbon atom next
to the carbonyl group may reduce the hydrolysis rate of
such a triacylglycerol.
[a] Assoc. Prof. V. Constantinou-Kokotou, V. Magrioti
Chemical Laboratories, Agricultural University
of Athens, Iera Odos 75, Athens 11855 (Greece)
Fax : (+30)210-5294-265
E-mail: vikon@aua.gr
Synthesis: 2-Methyl-2-propen-1-ol (1) was used as a starting
material for the synthesis of triacylglycerol analogues 3a e.
Dihydroxylation of 1 was performed by treatment with
H2O2 in H2O in the presence of a catalytic amount of
[b] Dr. R. Verger
Laboratoire de Lipolyse Enzymatique
CNRS-IFR1, UPR 9025, 31 Chemin Joseph-Aiguier
13402 Marseille, Cedex 20 (France)
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FULL PAPER
Scheme 1. General structure of the sterically hindered triacylglycerol ana-
logues that were synthesized.
[8]
H2WO4 (Scheme 2). 2-Methylglycerol (2) was used as the
key intermediate for the synthesis of triesters 3a e. Glycerol
4 was used as a starting material for the synthesis of 2-butyl-
Scheme 3. Synthesis of triacylglycerol analogues based on 2-butylglycerol.
2-Methyldecanoic acid and 2-methyloleic acid were pre-
pared from decanoic and oleic acid, respectively. The fatty
acid was first treated by lithium diisopropylamide (LDA),
then
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone
(DMPU), and finally methyl iodide were added to produce
the corresponding branched fatty acid.[11]
Scheme 2. Synthesis of triacylglycerol analogues based on 2-methylglyc-
erol.
Triesters 3a e, 9a b, and 10a b were prepared by treat-
ment of the appropriate alcohol, 2, 8, and 4, respectively,
with five equivalents of the appropriate acid, and dicyclo-
hexylcarbodiimide (DCC) in the presence of 4-dimethylami-
nopyridine (DMAP).[12] In the case of triacylglycerols 10a b
(Scheme 4), quantitative esterification was achieved after an
glycerol (8) (Scheme 3). The two primary hydroxy groups of
4 were protected by the trityl group, and consequently the
secondary hydroxy group was oxidized to the corresponding
ketone 6 by NaOCl in the presence of 4-acetamido-2,2,6,6-
tetramethyl-1-piperidinyloxy
free
radical
(AcNH-
TEMPO).[9] Reaction of ketone 6 with nBuLi at 08C under
a nitrogen atmosphere produced the protected 2-butylgly-
cerol (7), which was deprotected by treatment with
TsOH¥H2O.[10] 2-Butylglycerol (8) was used as a starting ma-
terial for the synthesis of triesters 9a b.
Abstract in Greek:
Scheme 4. Synthesis of triacylglycerol analogues based on glycerol.
overnight treatment. However, in the case of triesters 3a e
and 9a b, stirring for several days was required for esterifi-
cation of the tertiary hydroxy group.
All intermediates and final products gave satisfactory ana-
lytical and spectroscopic data.
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Triacylglycerol Analogues
1133 1140
Force/area curves of triacylglycerol analogues: The use of
the monolayer technique, which is based upon a decrease in
surface-pressure due to lipid film hydrolysis, is advanta-
geous for the study of lipases inhibitors, since with conven-
tional emulsified systems it is not possible to control the ™in-
terfacial quality∫.[13] The kinetic studies of the lipase hydrol-
ysis reactions requires the lipids used as substrates to form a
stable monomolecular film at the air/water interface. Alter-
natively, lipolytic products should desorb rapidly from the
interface.[13]
To determine the film stability and the interfacial proper-
ties at the air/water interface of the synthesized triacylgly-
cerol analogues, we recorded their force/area curves. The ex-
periments were performed in the reservoir compartment of
a ™zero-order∫ trough. A force/area curve was obtained
after a small volume of lipid solution, in a volatile solvent,
was spread at the air/water or argon/water interface. Moving
a mobile barrier at a constant rate progressively reduced the
surface of the trough, and the surface pressure was continu-
ously recorded during compression.
accordance with the literature data (Table 1).[14,15] The col-
lapse pressure of the films of the triolein analogues de-
creased as the size or number of branched groups increased.
The collapse pressure for compound 9a (7.5 mNmÀ1) was
Table 1. Collapse pressures and molecular areas of triacylglycerol ana-
logues.
Compound
Collapse Pressure
Molecular Area at the Collapse
[mNmÀ1
]
Pressure [ä2 moleculeÀ1
]
Triolein
3a
3b
12.2
10.8
9.9
98
100
112
122
106
78
71
96
106
88
9a
7.5
10a
Tricaprin
3c
3d
9b
11.2
18.9
17.9
15.3
13.2
17.4
10b
Figure 1 shows the molecular area dependency for com-
pounds 3a, 3b, 9a, 10a, and the natural substrate triolein as
much lower than the collapse pressure of compound 3a
(10.8 mNmÀ1) and triolein (13 mNmÀ1), which was found to
be the highest of the series. This behavior was to be expect-
ed, since the alkyl substitutions in the triolein analogues in-
creased the hydrophobic character of triolein.
Similar conclusions can also be drawn when comparing
the molecular area dependency for compounds 3c, 3d, 9b,
10b, and tricaprin as a function of the surface pressure
(Figure 2). By comparing this series of tricaprin analogues,
an increase in the molecular area was also observed with
the size and number of branched groups. For example, at
13.3 mNmÀ1, the molecular area of 9b is 106 ä2, whereas
the molecular area of 3d is 102 ä2.
Accordingly, the collapse pressure decreased as the size
or number of branched groups increased. For example, the
collapse pressure of compound 9b (13.2 mNmÀ1), was much
lower compared with the collapse pressure of compound 3c
(17.9 mNmÀ1). As expected, when the series of trolein ana-
Figure 1. Force/area curves of the triolein analogues. The aqueous sub-
phase was composed of Tris/HCl (10mm, pH8.0), NaCl (100mm), CaCl2
(21mm), and EDTA (1mm). The continuous compression experiments
were performed in the rectangular reservoir of the zero-order trough.
a function of the surface pressure of a film spread over a
buffered subphase at pH8.0. The surface behavior of these
compounds was measured at an argon/water interface to
avoid the oxidation of the double bond. By comparing this
series of triolein analogues at a given surface pressure value,
we observed an increase in the molecular area occupied by
these compounds as the size or number of branched groups
increased. For example, at its collapse pressure of
7.4 mNmÀ1, the molecular area of 9a with a butyl group at
the sn-2 position is 122 ä2, whereas the molecular area occu-
pied by 3a with a methyl group at the sn-2 position is
109 ä2. Also, the molecular area of 3b is 112 ä2 in compari-
son with the molecular area of 10a, which is 106 ä2 at the
collapse pressure of each compound. As a reference value,
under the same experimental conditions, the molecular area
of triolein at its collapse pressure was found to be 98 ä2, in
Figure 2. Force/area curves of the tricaprin analogues. The aqueous sub-
phase was composed of Tris/HCl (10mm, pH8.0), NaCl (100mm), CaCl2
(21mm), and EDTA (1mm). The continuous compression experiments
were performed in the rectangular reservoir of the zero-order trough.
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V. Constantinou-Kokotou et al.
FULL PAPER
logues and tricaprin analogues were compared, a decrease
in molecular area, and an increase in the collapse pressure
was observed when the length of the acyl chain was de-
creased.
Pancreatic and gastric lipase activity on monomolecular
films that contain triacylglycerol analogues: The inhibition
of HPL and HGL was studied by means of the monomolec-
ular film technique with mixed films of 1,2-dicaprin that
contain variable proportions of each synthetic triacylglycerol
analogue.[13] The inhibition studies for HPL were performed
at a constant surface pressure of 14 mNmÀ1 for the triolein
analogues, and 17 mNmÀ1 for the tricaprin analogues. The
inhibition studies for HGL were performed at a constant
surface pressure of 17 mNmÀ1 for triolein analogues and
20 mNmÀ1 for the tricaprin analogues. At these surface
pressure values, HPL and HGL were fully active and linear
kinetics were recorded. These surface pressures were chosen
to be slightly higher than the collapse pressure of the films
for the pure triacylglycerol analogues. It is known that in a
mixed film of 1,2-dicaprin that contains a small fraction of
inhibitor, the film is stabilized even at a surface pressure
higher than the collapse pressure value of the pure inhibitor.
Figure 4. Effect of increasing the concentration of tricaprin analogues on
the remaining activity of HPL on the 1,2-dicaprin monolayer maintained
at a constant surface pressure (17 mNmÀ1). The aqueous subphase was
composed of Tris/HCl (10mm, pH8.0), NaCl (100mm), CaCl2 (21mm),
and EDTA (1mm). The kinetics of hydrolysis were recorded for 20 min.
high surface pressures, the inhibitory capacity of the sterical-
ly hindered triglyceride analogues could be improved. The
molar fraction values (a50) for HPL obtained with all the tri-
acylglycerol analogues are summarized in Table 2.
The inhibition of lipolytic enzymes are best quantified in
7,13]
terms of surface molar fraction of inhibitor.[4
Thus, re-
maining lipase activity was plotted as a function of the sur-
face molar-fraction (a) of inhibitor. The data that was ob-
tained for HPL, by using the triolein analogues and the tri-
caprin analogues, are presented in Figure 3 and 4, respec-
tively. A 50% decrease of HPL activity was observed
when 0.003 (9a), 0.003 (3b), and 0.008 (3a) molar fractions
(a50) of the inhibitors were mixed with a monolayer of 1,2-
dicaprin. The a50 is defined as the molar fraction of inhibitor
which reduces by 50% of the initial rate of lipolysis. It is im-
portant to note that when we studied the inhibitory effect of
3a on HPL at 17 mNmÀ1, we observed an even higher inhib-
itory effect (a50 =0.006). This observation indicates that at
All the compounds were also tested as potential inhibitors
Table 2. Inhibition values of triacylglycerol analogues on HPL with the
monolayer technique.
Compound [mNmÀ1
]
Surface Pressure
a50 [%]
3a
3a
3b
9a
10a
3c
3d
9b
10b
14
17
14
14
14
17
17
17
17
0.008
0.006
0.003
0.003
0.012
0.118
0.049
0.143
0.078
of HGL, and the data obtained with triolein and tricaprin
analogues, are presented in Figure 5 and 6, respectively. The
molar fraction values (a50) for HGL obtained with all the
triacylglycerol analogues are summarized in Table 3. A 50%
decrease of HGL activity was observed when 0.009 (9a) and
0.017 (3b) molar fractions of the inhibitors were mixed with
a monolayer of 1,2-dicaprin.
As shown from the data, the length of the acyl chain influ-
ences the potency of the inhibition. For both HPL and HGL
the presence of oleoyl chains (C18:1) gives rise to a better
inhibition capacity relative to the decanoyl chain (C10:0).
Comparison of the data obtained for compounds 9a and 9b
shows that the change from an oleoyl chain to a decanoyl
chain may result in up to a 50-fold decrease in the a50 value
for HPL. Both digestive lipases seem to be better inhibited
with the most hindered molecules, such as compounds 9a
(one butyl group at the glycerol C-2), and 3b (one methyl
Figure 3. Effect of increasing the concentration of triolein analogues on
the remaining activity of HPL on the 1,2-dicaprin monolayer maintained
at a constant surface pressure (14 mNmÀ1). The aqueous subphase was
composed of Tris/HCl (10mm, pH8.0), NaCl (100mm), CaCl2 (21mm),
and EDTA (1mm). The kinetics of hydrolysis were recorded for 20 min.
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Triacylglycerol Analogues
1133 1140
group at C-2 together with one methyl group at the a-posi-
tion of each acyl chain).
Among all the compounds tested in this study, the triolein
analogue 9a, bearing a butyl group at the 2-position of the
glycerol backbone, was shown to be the most potent inhibi-
tor, which caused a 50% decrease in HPL and HGL activity
at 0.003 and 0.009 molar fractions, respectively. The triolein
analogue 3b, bearing methyl groups both at the 2-position
of the glycerol, and at the a-position of each oleoyl chain,
presented an equal inhibitory activity against HPL, though
lower (a50 0.017) against HGL. The triolein analogue 3a
based on 2-methylglycerol, also proved to be a potent inhib-
itor of HPL and HGL, which caused a 50% decrease in
their activity at 0.008 and 0.029 molar fraction, respectively.
On comparing the series of triolein analogues, it seems
that the analogue that contains a methyl group at the 2-posi-
tion of the glycerol, presents a comparable inhibition level
against both HPL and HGL as compared to the analogue
that contains a methyl group at the a-position of each
oleoyl residue. It is clear that the introduction of a methyl
substituent, both at the 2-position and a-position, resulted
in molecules with enhanced inhibitory potency. However,
the presence of a butyl group at the 2-position causes an
equal (for HPL) or even better (for HGL) inhibitory effect
relative to the methyl substituent.
Figure 5. Effect of increasing the concentration of triolein analogues on
the remaining activity of HGL on the 1,2-dicaprin monolayer maintained
at a constant surface pressure (17 mNmÀ1). The aqueous subphase was
composed of CH3COONa (10mm, pH5.0), NaCl (100mm), CaCl2
(21mm), and EDTA (1mm). The kinetics of hydrolysis were recorded for
20 min.
The tricaprin analogues are relatively weak inhibitors for
HPL and showed a better inhibitory capacity against HGL.
This observation is in line with the known short and
medium chain specificity of HGL. Among the series of tri-
caprin analogues, compound 9b that contains 2-butylglycer-
ol backbone exhibited the most potent inhibition capacity
(a50 0.036) against HGL.
Up to now, the best synthetic inhibitor of HPL reported
in the literature is O-hexadecyl-O-(p-nitrophenyl) n-undecyl
phosphonate, with an a50 value of 0.003.[16] When we tested
this compound by using HPL under our experimental condi-
tions (at 15 mNmÀ1), it exhibited an a50 value of 0.006 molar
fraction. In the case of HGL, the highest inhibition was ob-
tained with O-undecyl-O-(p-nitrophenyl) n-decyl phospho-
nate, which exhibited an a50 value of 0.008.[16] From the pres-
ent study, the triolein analogue 9a, bearing the butyl group
at the 2-position, shows a similar inhibitory effect relative to
the most potent synthetic inhibitors reported for HPL and
HGL.[16]
Figure 6. Effect of increasing the concentration of tricaprin analogues on
the remaining activity of HGL on the 1,2-dicaprin monolayer maintained
at a constant surface pressure (20 mNmÀ1). The aqueous subphase was
composed of CH3COONa (10mm, pH5.0), NaCl (100mm), CaCl2
(21mm), and EDTA (1mm). The kinetics of hydrolysis were recorded for
20 min.
During the early seventies it was demonstrated that syn-
thetic triacylglycerols, substituted by a methyl group either
at the a-position of the acyl residue or at the C-2 position of
the glycerol backbone, presented reduced ability to be hy-
drolyzed by porcine pancreatic lipase by using a simple
monolayer assay.[17] Using the pH-stat titration assay, it was
also reported that 2-methylglycerol trioleate was hydrolyzed
at a relative rate of 1% relative to triolein.[18] Taking into
account the fact that tetrahydrolipstatin bears a lactone ring,
it comes as no surprise that poor lipase substrates (that con-
tain a carboxylic ester function) behave as potent competi-
tive lipase inhibitors.[19] For the first time, we demonstrated
by using the monolayer technique, that triolein analogues
containing a methyl or a butyl group at the sn-2 position of
the glycerol backbone, or/and at the a-position of the acyl
Table 3. Inhibition values of triacylglycerol analogues on HGL with the
monolayer technique.
Compound
Surface Pressure [mNmÀ1
]
a50 [%]
3a
3b
9a
10a
3c
3d
9b
10b
17
17
17
17
20
20
20
20
0.029
0.017
0.009
0.028
0.071
0.055
0.036
0.078
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V. Constantinou-Kokotou et al.
FULL PAPER
OH); 13C NMR: d=143.8 (aromatic C), 126.8 128.9 (aromatic CH), 86.6
(C(C6H5)3), 70.2 (CHOH), 64.5 ppm (CH2O); elemental analysis calcd
(%) for C41H36O3 (576.723): C 85.39, H 6.29; found C 85.23, H 6.38.
residue, act as potent inhibitors of human pancreatic and
gastric lipases. It is apparent that the development of steric
hindrance around the ester bonds of triacylglycerols results
in competitive inhibitors of digestive lipases.
1,3-Bis(triphenylmethoxy)-2-propanone (6): A solution of NaBr (590 mg,
5.7 mmol) in water (2.6 mL), and subsequently AcNH-TEMPO (10.6 mg,
0.05 mmol) was added at 08C to a solution of compound 5 (3.0 g,
2.6 mmol) in CH2Cl2 (15.6 mL). A solution of NaOCl (0.21 g, 2.86 mmol),
and NaHCO3 (1.3 g, 15.6 mmol) in H2O (5 mL) was added dropwise to
the resulting biphasic system at 08C over a period of 1 h under vigorous
stirring. After stirring for 30 min at room temperature, EtOAc (50 mL)
and water (20 mL) were added. The organic layer was washed with 10%
aqueous citric acid (25 mL), which contained KI (0.15 g), 10% aqueous
Na2S2O3 (25 mL), brine, and dried over Na2SO4. The solvent was evapo-
rated under reduced pressure, and the crude product was purified by
column chromatography by using petroleum ether/EtOAc 8/2 to yield 6
(2.77 g, 93%) as a yellowish solid. M.p 183 1848C; 1H NMR: d=7.20
7.40 (m, 30H; aromatic CH), 3.97 ppm (s, 4H; CH2O); 13C NMR: d=
204.6 (CO), 143.1 (aromatic C), 127.5 128.9 (aromatic CH), 87.3
[C(C6H5)3], 68.9 ppm (CH2O); elemental analysis calcd (%) for C41H34O3
(574.7): C 85.69, H 5.96; found C 85.78, H 5.86.
Conclusion
By using the monolayer technique we have demonstrated
that sterically hindered triacylglycerol analogues are potent
human digestive lipase inhibitors. These triglyceride ana-
logues are easily prepared and are chemically similar to the
natural substrates of lipases. Our results indicate that an
alkyl group at the sn-2 position of the glycerol backbone, or/
and at the a-position of the acyl residue are important sub-
stituents for the design and synthesis of potent inhibitors of
digestive lipases. This might be useful for the inhibition of
the hydrolysis and absorption of dietary fats in humans,
which could be used as agents for the treatment of obesity.
1-(Triphenylmethoxy)-2-[(triphenylmethoxy)methyl]-2-hexanol (7):
A
solution of nBuLi in hexane (2.0m, 3.0 mL, 6.0 mmol) was added to a solu-
tion of compound 6 (2.6 g, 45 mmol) in dry THF (18 mL) at 08C under a
nitrogen atmosphere. The solution was stirred for an additional 10 min,
and then poured into a saturated solution of NH4Cl. The aqueous layer
was washed three times with Et2O, and then the organic layers were
washed with brine, dried (Na2SO4), and concentrated in vacuo. The resi-
due was purified by column chromatography by using petroleum ether/di-
Experimental Section
1
Materials and methods: Compounds 2-methyl-2-propen-1-ol, decanoic
acid, oleic acid, hexadecanoic acid, AcNH-TEMPO, and silica gel (230
400 mesh) were purchased from Aldrich. 1,2-Dicaprin was purchased
from Sigma. Analytical TLC plates (silica gel 60 F254), and silica gel 60
(70 230 mesh) were purchased from Merck. HPL, co-lipase, and HGL
were purified in the laboratory by using previously described procedures
by Verger et al.[13] THF, toluene, and Et2O were dried by standard proce-
dures, and stored over molecular sieves or Na. Petroleum ether was used
and had a b.p. of 40 608C. Et3N was distilled from ninydrin. All other
solvents were of reagent grade and used without further purification.
Melting points were determined on a B¸chi 530 apparatus and are uncor-
rected. 1H NMR, 13C NMR spectroscopy, and COSY (correlation spec-
troscopy) were obtained in CDCl3, except when otherwise noted, by
using a Varian Mercury (200 MHz) spectrometer. FAB mass spectra were
obtained on a VG 70-SE mass spectrometer (Manchester, UK). Elemen-
tal analyses were performed on a Perkin Elmer 2400 instrument.
chloromethane/EtOAc 85:10:5 to yield 7 (1.8 g, 63%) as a yellow oil; H
NMR: d=7.20 7.50 (m, 30H; aromatic CH), 3.31 (m, 4H; CH2O), 2.33
(s, 1H; OH); 1.40 1.60 (m, 2H; CCH2), 1.10 1.35 (m, 4H; CH2),
0.89 ppm (m, 3H; CH3); 13C NMR: d=143.8 (aromatic C), 126.9 128.9
(aromatic CH), 86.5 (C(C6H5)3), 74.0 (COH), 65.6 (CH2O), 34.3 (CH2C),
24.1 (CH2), 23.2 (CH2), 14.0 ppm (CH3); elemental analysis calcd (%) for
C45H44O3 (632.8): C 85.41, H 7.01; found C 85.63, H 6.85.
2-Butyl-1,2,3-propanetriol (8): pTsOH¥H2O (12 mg, 0.06 mmol) was
added to a solution of compound 7 (300 mg, 0.47 mmol) in methanol
(4.0 mL), and dichloromethane (10 mL), the solution was then stirred
overnight. The solvents were evaporated and water was added. The aque-
ous layer was washed three times with EtOAc and was concentrated in
vacuo to give a yellowish oil. The residue was passed through a short
plug af silica gel with chloroform, and then chloroform/methanol 9:1.
The solvents were concentrated in vacuo to yield 8 (29 mg, 42%) as a
yellow oil; 1H NMR: d=3.52 3.85 (m, 6H; CH2O, 2îOH), 2.65 (b, 1H;
OH), 1.20 1.52 (m, 6H; 3îCH2), 0.92 ppm (d, J=6.8 Hz, 3H; CH3); 13C
NMR: d=74.2 (COH), 66.8 (CH2O), 34.4 (CH2C), 25.1 (CH2), 23.3
(CH2), 14.0 ppm (CH3); elemental analysis calcd (%) for C7H16O3
(148.2): C 56.73, H 10.88; found C 56.55, H 10.93.
2-Methyl-1,2,3-propenetriol (2): Alcohol 1 (10 g, 139 mmol) and H2WO4
(263 mg) were dissolved in twice-distilled H2O (10 mL) by heating to
708C. The temperature was maintained at 70 778C by the addition of
30% H2O2 (exothermic). After a consumption of 18.3 g, the reaction mix-
ture was kept for 1 h at 758C, and in order to destroy residual H2O2, for
another 2.5 h at 978C. The reaction mixture was cooled to room tempera-
ture, filtered, and passed through a Dowex 1-X4 column (27 mL; OHÀ
form; equilibrated in twice-distilled H2O) in order to remove H2WO4.
The eluate was set to pH 6.0 by adding some drops of 30% H2SO4, con-
centrated, and it was then dried in vacuo to yield 2 (7.3 g, 49%) as a yel-
lowish opaque oil; 1H NMR (D2O): d=3.28 (s, 4H; CH2), 0.93 ppm (s,
3H; CH3); 1H NMR (CD3OD): d=4.88 (b, 1H; OH), 3.40 (s, 4H; CH2),
1.09 ppm (s, 3H; CH3); 13C NMR (D2O): d=73.2 (COH), 65.9 (CH2OH),
19.8 ppm (CH3); MS (FAB): m/z (%): 75 (100) [C3H7O2+], 57 (82)
[C3H5O+], 43 (40) [C2H3O+]; elemental analysis calcd (%) for C4H10O3
(106.1): C 45.27, H 9.50; found: C 45.02, H 9.56.
General procedure for the synthesis of 2-methyl acids: A solution of di-
isopropylamine (4.12, 29 mmol) in anhydrous THF (25 mL) was charged
in a flask under a nitrogen atmosphere. After cooling at 08C, n-butyllithi-
um (18.1 mL, 29 mmol, 1.6m in hexane) was slowly added to keep the
temperature at 08C. After standing at this temperature for an additional
20 min, the appropriate acid (14 mmol) was added dropwise, while main-
taining the reaction temperature below 08C. A milky-white solution
formed, and after 30 min, DMPU (1.7 mL, 14 mmol) was added, the mix-
ture was then stirred at room temperature for 1 h. It was then cooled
again at 08C, and CH3I (0.9 mL, 15 mmol) was added rapidly. The reac-
tion was completed by stirring the mixture at room temperature over-
night. The product was recovered by neutralization with ice, cooled 10%
HCl, followed by extraction with Et2O (3î30 mL). 444 The combined or-
ganic layers were washed with water and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo to obtain the corresponding 2-methyl
acid.
1,3-Bis(triphenylmethoxy)-2-propanol (5): Trityl chloride (5.0 g,
18 mmol), triethylamine (3.3 mL, 24 mmol), and a catalytic amount of 4-
(dimethylamino)pyridine (122 mg, 1.0 mmol) was added to a mixture of
glycerol 4 (920 mg, 10 mmol) in dichloromethane (50 mL). The mixture
was stirred at room temperature overnight. The organic layer was
washed with brine, 10% citric acid, brine, 5% NaHCO3, brine, dried
(Na2SO4), and concentrated in vacuo to give a yellowish solid. The resi-
due was purified by column chromatography by using petroleum ether/
EtOAc 9:1 and 8:2 as an eluent to yield 5 (2.85 g, 55%) as a yellowish
solid. M.p. 160 1638C; 1H NMR: d=7.20 7.50 (m, 30H; aromatic CH),
3.98 (m, 1H; CHOH), 3.31 (m, 4H; CH2O), 2.15 ppm (d, J=5.6 Hz, 1H;
2-Methyldecanoic acid: Yellowish oil. Yield 2.10 g (80%); 1H NMR: d=
10.50 (b, 1H; COOH), 2.30 2.50 (m, 1H; CH), 1.58 1.80 (m, 2H;
CH2CH), 1.20 1.55 (m, 12H; 6îCH2), 1.15 1.20 (m, 3H; CHCH3), 0.88
1.00 ppm (m, 3H; CH2CH3); 13C NMR: d=183.6 (COOH), 39.4 (CH),
33.5 (CH2), 31.9 (CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.1 (CH2),
27.1 (CH2), 22.6 (CH2), 16.8 (CHCH3), 14.1 ppm (CH2CH3); elemental
1138
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 1133 1140

Triacylglycerol Analogues
1133 1140
analysis calcd (%) for C11H22O2 (186.3): C 70.92, H 11.90; found: C 71.03,
H 11.69.
analysis calcd (%) for C61H112O6 (941.5): C 77.81, H 11.99; found: C
77.89, H 11.93.
(Z)-2-Methyl-9-octadecenoic acid: Yellow oil. Yield 3.40 g (82%); 1H
NMR: d=11.50 (b, 1H; COOH), 5.33 (m, 2H; CH=), 2.30 2.50 (m, 1H;
CH), 1.95 2.15 (m, 4H; CH2CH=), 1.58 1.80 (m, 2H; CH2CH), 1.25 1.55
(m, 20H; 10îCH2), 1.15 1.20 (m, 3H; CHCH3), 0.88 1.00 ppm (m,
3CH; CH2CH3); 13C NMR: d=183.6 (COOH), 130.0 (CH=), 129.7 (CH=),
39.4 (CH), 34.1 (CH2), 33.5 (CH2), 31.9 (CH2), 29.8 (CH2), 29.6 (CH2),
29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.1 (CH2), 27.2 (CH2), 27.1 (CH2),
22.7 (CH2), 16.8 (CHCH3), 14.1 ppm (CH2CH3); elemental analysis calcd
(%) for C19H39O2 (296.5): C 76.97, H 12.24; found: C 76.83, H 12.32.
1,1-Bis{[(1-oxodecyl)oxy]methyl}pentyl ester of decanoic acid, (9b): Yel-
lowish oil. Yield 4.22 g (69%); 1H NMR: d=4.37 (dd, 1J=11.8 Hz, 2J=
31.2 Hz, 4H; 2îCH2O), 2.22 2.45 (m, 6H; 3îCH2CO), 1.85 2.10 (m,
2H; CCH2), 1.50 1.70 (m, 6H; 3îCH2), 1.15 1.45 (m, 40H; 20îCH2),
0.88 ppm (m, 12H; 4îCH3); 13C NMR: d=180.0 (CO), 173.8 (CH), 81.6
(C), 66.5 (CH2O), 34.5 (CH2), 34.2 (CH2), 31.8 (CH2), 29.4 (CH2), 29.2
(CH2), 29.1 (CH2), 29.0 (CH2), 24.9 (CH2), 24.6 (CH2), 23.1 (CH2), 22.6
(CH2), 14.1 ppm (CH3); elemental analysis calcd (%) for C37H70O6
(610.9): C 72.74, H 11.55; found: C 72.89, H 11.43.
General procedure for the synthesis of triacylglycerol analogues 3a e,
9a b, and 10a b: The appropriate acid (50 mmol) was added in a solu-
tion of the appropriate alcohol (10 mmol) in dichloromethane (65 mL),
and the solution was cooled to 08C. Then, a catalytic amount of DMAP
was added (122 mg, 1.0 mmol), followed by the addition of a solution of
DCC (10.3 g, 50 mmol) in dichloromethane (60 mL). The solution was
stirred for 30 min at 08C for several nights at room temperature. The re-
action mixture was filtered through a small pad of celite, and the solvent
was evaporated under reduced pressure. The residue was purified by
flash column chromatography by using petroleum ether/Et2O 95:5, and
9:1 as an eluent.
2-Methyl-2,3-bis{[(Z)-2-methyl-1-oxo-8-heptadecenyl]oxy}propyl ester of
9-octadecenoic acid, (Z)-(10a): Yellowish oil. Yield 6.68 g (72%); 1H
NMR: d=5.25 5.45 (m, 7H; 3îCH=, CHO), 4.35 (m, 4H; 2îCH2O),
2.30 2.55 (m, 3H; 3îCH), 1.90 2.10 (s, 12H; 6îCH2CH=), 1.58 1.75
(m, 6H; 3îCH2CH), 1.25 1.55 (m, 60H; 30îCH2), 1.15 1.20 (m, 9H;
3îCHCH3), 0.88 1.00 ppm (m, 9H; 3îCH2CH3); 13C NMR: d=176.0
(CO), 130.0 (CH=), 129.7 (CH=), 68.9 (CHO), 62.0 (CH2O), 39.5 (CH),
39.4 (CH), 33.6 (CH2), 31.9 (CH2), 29.7 (CH2), 29.5 (CH2), 29.4 (CH2),
29.3 (CH2), 29.1 (CH2), 27.2 (CH2), 27.1 (CH2), 22.7 (CH2), 16.9 (CH3),
14.1 ppm (CH3); elemental analysis calcd (%) for C60H110O6 (927.5): C
77.70, H 11.95; found: C 77.59, H 12.03.
2-Methyl-2,3-bis{[(Z)-1-oxo-8-heptadecenyl]oxy}propyl ester of 9-octade-
cenoic acid, (Z)-(3a): Yellowish oil. Yield 7.01 g (78%); 1H NMR: d=
2-Methyl-2,3-bis[(1-oxodecyl)oxy]propyl ester of decanoic acid, (10b):
Yellowish oil. Yield 4.78 g (80%); 1H NMR: d=5.38 (m, 1H; CHO),
4.35 (m, 4H; 2îCH2O), 2.35 2.55 (m, 3H; 3îCH), 1.55 1.75 (m, 6H;
3îCH2CH), 1.20 1.50 (m, 36H; 18îCH2), 1.10 1.20 (m, 9H; 3î
CHCH3), 0.88 1.00 ppm (m, 9H; 3îCH2CH3); 13C NMR: d=176.2
(CO), 68.8 (CHO), 62.1 (CH2), 39.5 (CH), 39.4 (CH), 33.6 (CH2), 31.8
(CH2), 29.5 (CH2), 29.4 (CH2), 29.2 (CH2), 27.2 (CH2), 22.6 (CH2), 16.9
(CH3), 14.1 ppm (CH3); elemental analysis calcd (%) for C36H68O6
(596.9): C 72.44, H 11.48; found: C 72.59, H 11.35.
1
2
5.33 (6H, m, 3îCH2), 4.31 (dd, J=11.4 Hz, J=25.4 Hz, 4H; 2îCH2O),
2.30 (m, 6H; 3îCH2), 1.99 (m, 12H; 6îCH2), 1.62 (m, 6H; 3îCH2),
1.49 (s, 3H; CH3), 1.27 (s, 72H; 36îCH2), 0.87 ppm (t, J=6.2 Hz, 9H;
3îCH3); 13C NMR: d=173.0 (CH), 172.7 (CO), 130.1 (CH), 130.0 (CH),
129.8 (CH), 79.6 (C), 64.7 (CH2), 35.1 (CH2), 34.1 (CH2), 31.9 (CH2), 29.7
(CH2), 29.5 (CH2), 29.3 (CH2), 29.1 (CH2), 27.1 (CH2), 24.9 (CH2), 22.6
(CH2), 18.9 (CH3), 14.1 ppm (CH3); elemental analysis calcd (%) for
C58H106O6 (899.5): C 77.45, H 11.88; found: C 77.38, H 12.05.
Monomolecular film experiments–Force/area curves: Surface pressure-
area curves were measured in the rectangular reservoir compartment of
the ™zero-order∫ trough (14.8 cm wide and 24.9 cm long). Before each ex-
periment the trough was at first washed with tap water, then gently
brushed in the presence of distilled ethanol, washed again with plenty of
tap water, and finally rinsed with twice-distilled water. The lipidic film as
a solution in CHCl3 (approximately 1 mgmLÀ1) was spread with a Hamil-
ton syringe over an aqueous subphase of Tris/HCl (10mm, pH 8.0), NaCl
(100mm), CaCl2 (21mm), and EDTA (1mm). The above-mentioned
buffer solution was prepared with twice-distilled water, and filtered
through a 0.22 mm millipore membrane. Before each utilization, residual
surface-active impurities were removed by sweeping and suction of the
surface. The force/area curves were automatically recorded upon a con-
2-Methyl-2,3-bis{[(Z)-2-methyl-1-oxo-8-heptadecenyl]oxy}propyl ester of
9-octadecenoic acid, (Z)-(3b): Yellowish oil. Yield 4.52 g (48%); 1H
NMR: d=5.33 (m, 6H; 3îCH=), 4.33 (m, 4H; 2îCH2O), 2.30 2.50 (m,
3H; 3îCH), 1.90 2.10 (s, 12H; 6îCH2CH=), 1.58 1.80 (m, 6H; 3î
CH2CH), 1.25 1.55 (m, 60H; 30îCH2), 1.15 1.20 (m, 9H; 3îCHCH3),
0.88 1.00 ppm (m, 9H; 3îCH2CH3); 13C NMR: d=176.0 (CO), 175.6
(CO), 130.0 (CH=), 129.6 (CH=), 79.6 (C), 64.6 (CH2O), 40.3 (CH), 39.5
(CH), 33.6 (CH2), 31.9 (CH2), 29.7 (CH2), 29.4 (CH2), 29.3 (CH2), 29.1
(CH2), 27.2 (CH2), 27.1 (CH2), 24.9 (CH2), 22.7 (CH2), 18.9 (CH3), 17.0
(CH3), 14.1 ppm (CH3); elemental analysis calcd (%) for C61H112O6
(941.5): C 77.81, H 11.99; found: C 77.69, H 12.07.
2-Methyl-2,3-bis[(1-oxodecyl)oxy]propyl ester of decanoic acid, (3c): Yel-
lowish oil. Yield 4.15 g (73%); 1H NMR: d=4.33 (dd, 1J=11.6 Hz, 2J=
25.2 Hz, 4H; 2îCH2O), 2.32 (m, 6H; 3îCH2), 1.61 (m, 6H; 3îCH2),
1.51 (s, 3H; CH3), 1.27 (b, 42H; 21îCH2), 0.88 ppm (t, 9H J=6.4 Hz;
3îCH3); 13C NMR: d=173.1 (CO), 79.6 (C), 64.9 (CH2), 64.7 (CH2),
35.1 (CH2), 34.1 (CH2), 31.8 (CH2), 29.4 (CH2), 29.5 (CH2), 29.1 (CH2),
24.1 (CH2), 22.6 (CH2), 18.9 (CH3), 14.1 ppm (CH3); elemental analysis
calcd (%) for C34H64O6 (568.9): C 71.79, H 11.34; found: C 71.63, H
11.38.
tinuous compression rate at 0.5 cmminÀ1
.
Enzyme kinetic experiments: The inhibition experiments were performed
by using the monolayer technique. The surface pressure of the lipid film
was measured by means of the platinum Wilhelmy plate technique cou-
pled with an electromicrobalance. The principle of this method has been
described previously by Verger et al.[13]
For the inhibition studies the method of ™mixed monomolecular films∫
was used. This method involved the use of a ™zero-order∫ trough, which
consisted of two compartments: a reaction compartment, in which mixed
films of substrate and inhibitor are spread, and a reservoir compartment,
in which only pure films of substrate are spread. The two compartments
are connected to each other by narrow surface channels. HPL (final con-
centration 8.3 and 4.8 ngmLÀ1 for 14 and 17 mNmÀ1, respectively), and
HGL (final concentration 113 and 56 ngmLÀ1 for 17 and 20 mNmÀ1, re-
spectively) were injected into the subphase of the reaction compartment,
in which efficient stirring was applied. In the case of HPL, the aqueous
subphase was composed of Tris/HCl (10mm, pH8.0) NaCl (100mm),
CaCl2 (21mm), and EDTA (1mm). In the case of HGL the aqueous sub-
phase was composed of CH3COONa/HCl (10mm, pH5.0), NaCl
(100mm), CaCl2 (21mm), and EDTA (1mm). Due to the lipolytic action
of the enzyme, the surface pressure decreased, and a mobile barrier
moved over the reservoir compartment to compress the film, and thus
kept the surface pressure constant. The surface pressure was measured
on the reservoir compartment. The surface and volume of the reaction
compartment was 100 cm2 and 120 mL, repsectively. The reservoir com-
partment was 14.8 cm wide and 24.9 cm long. The lipidic films were
2-Methyl-2,3-bis[(2-methyl-1-oxodecyl)oxy]propyl ester of decanoic acid,
(3d): Yellowish oil. Yield 3.48 g (57%); 1H NMR: d=4.35 (m, 4H; 2î
CH2O), 2.35 2.55 (m, 3H; 3îCH), 1.58 1.75 (m, 6H; 3îCH2CH), 1.51
(s, 3H; CH3), 1.20 1.48 (m, 36H; 18îCH2), 1.05 1.20 (m, 9H; 3î
CHCH3), 0.88 1.00 ppm (m, 9H; 3îCH2CH3); 13C NMR: d=176.0
(CO), 79.6 (C), 64.6 (CH2O), 40.3 (CH), 39.5 (CH), 33.7 (CH2), 31.8
(CH2), 29.5 (CH2), 29.4 (CH2), 29.2 (CH2), 27.2 (CH2), 22.6 (CH2), 18.9
(CH3), 17.0 (CH3), 14.1 ppm (CH3); elemental analysis calcd (%) for
C37H70O6 (610.9): C 72.74, H 11.55; found: C 72.59, H 11.63.
2-{[(Z)-1-oxo-8-heptadecenyl]oxy}-2-{{[(Z)-1-oxo-8-heptadecenyl]oxy}-
methyl}hexyl ester of 9-octadecenoic acid, (Z)-(9a): Yellowish oil. Yield
4.22 g (69%); 1H NMR: d=5.33 (m, 6H; 3îCH=), 4.35 (dd, 1J=
11.8 Hz, 2J=31.2 Hz, 4H; 2îCH2O), 2.22 2.50 (m, 6H; 3îCH2CO),
1.85 2.10 (m, 14H; 6îCH2CH=, CCH2), 1.55 1.70 (m, 6H; 3îCH2),
1.15 1.45 (m, 74H; 37îCH2), 0.88 ppm (m, 12H; 4îCH3); 13C NMR:
d=173.0 (CH), 172.5 (CO), 130.0 (CH), 81.8 (C), 63.0 (CH2O), 34.2
(CH2), 31.9 (CH2), 29.7 (CH2), 29.5 (CH2), 29.3 (CH2), 29.1 (CH2), 27.2
(CH2), 24.9 (CH2), 24.7 (CH2), 22.7 (CH2), 14.1 ppm (CH3); elemental
1139
Chem. Eur. J. 2004, 10, 1133 1140
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

V. Constantinou-Kokotou et al.
FULL PAPER
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Received: September 25, 2003 [F5573]
1140
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 1133 1140