Inhibition of lipoprotein lipase by alkanesulfonyl fluorides

Article abstract of DOI:10.1016/S0960-894X(00)00566-7

A number of alkanesulfonyl halides (chlorides and fluorides) and esters were synthesized and their effect on the activity of lipoprotein lipase (LPL) was studied. Sulfonyl fluorides proved to be efficient inhibitors of LPL when the enzyme was incubated with a 10-fold molar excess of the inhibitors in a buffer containing bile salts (deoxycholate). Hexadecane- and dodecanesulfonyl fluorides caused 50% inhibition of LPL activity at concentrations of 10 to 20 μM. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00566-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2803±2806
Inhibition of Lipoprotein Lipase by Alkanesulfonyl Fluorides
George Kokotos,a,* Stavroula Kotsovolou,a Violetta Constantinou-Kokotou,b
Gengshu Wuc and Gunilla Olivecronac
aLaboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece
bChemical Laboratories, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece
Ê Ê
Department of Medical Biosciences, Medical Biochemistry, Umea University, S-90187 Umea, Sweden
c
Received 15 May 2000; revised 4 October 2000; accepted 4 October 2000
AbstractÐA number of alkanesulfonyl halides (chlorides and ¯uorides) and esters were synthesized and their e€ect on the activity
of lipoprotein lipase (LPL) was studied. Sulfonyl ¯uorides proved to be ecient inhibitors of LPL when the enzyme was incubated
with a 10-fold molar excess of the inhibitors in a bu€er containing bile salts (deoxycholate). Hexadecane- and dodecanesulfonyl
¯uorides caused 50% inhibition of LPL activity at concentrations of 10 to 20 mM. # 2000 Elsevier Science Ltd. All rights reserved.
Plasma lipoproteins are spherical molecular aggregates
composed of a core of insoluble lipids (triacylglycerols
and cholesteryl esters) surrounded by a surface layer of
more polar lipids (phospholipids and cholesterol) and
speci®c lipid-binding proteins (apolipoproteins).1 Chy-
lomicrons are made from dietary lipids in the intestinal
mucosal cells, while the smaller very low density lipo-
proteins are made in the liver from endogenous lipids.
The major step in catabolism of these lipoproteins is the
hydrolysis of their triacylglycerols by lipoprotein lipase
(LPL).2 In addition, LPL has been found to eciently
mediate binding of lipoproteins to cell surfaces, to
extracellular matrix and to the low density lipoprotein
receptor-related protein under cell culture conditions.3,4
This bridging e€ect is not dependent on the catalytic
activity of LPL, but follows from the ability of LPL to
simultaneously interact with proteoglycans/receptors
and with lipoproteins. Experimental studies of the non-
catalytic functions of LPL require inactivated enzyme.5
This may be produced by blocking its active site with
speci®c inhibitors like tetrahydrolipstatin or hex-
adecanesulfonyl ¯uoride.6 8 Tetrahydrolipstatin (Orli-
stat1) was originally developed as a drug to inhibit
digestive lipases (pancreatic and gastric lipases) for the
purpose of reducing fat absorption. It is now a regis-
tered drug for weight reduction. Tetrahydrolipstatin
causes almost complete inhibition of LPL, but it pre-
sents the drawback of slow reversibility since the drug is
slowly turned over by the enzyme.5,6 By having high
concentrations of the inhibitor present during the
experiments, any LPL that regains activity will be
immediately inhibited by a new inhibitor molecule.8
Hexadecanesulfonyl ¯uoride has the advantage of caus-
ing irreversible inhibition of LPL.5,7 It has, however, the
disadvantage of decreasing the solubility of the enzyme,
probably because two long aliphatic chains from the
inhibitor are added per LPL dimer.5 The short-chain
variant, phenylmethanesulfonyl ¯uoride (PMSF) was
previously reported to inhibit LPL,5,9 but the con-
centrations required of the inhibitor are in the mM
range and in our hands the inhibition is incomplete. To
develop more ecient inhibitors, suitable for the study
of the noncatalytic functions of LPL, we have synthe-
sized a number of alkanesulfonyl halides, of short,
medium and long aliphatic chains, and esters, and
studied the inhibition of LPL activity caused by these
compounds.
Chemistry
Hexane-, dodecane- and hexadecanesulfonyl chlorides
and ¯uorides were used in this study. Hexane- and
dodecanesulfonyl chlorides (3, 4) were prepared in good
yield by the treatment of commercially available sodium
sulfonates (1, 2) with N-chlorosuccinimide in the pres-
ence of triphenylphosphine under mild conditions
(room temperature, 1 h), whereas hexadecanesulfonyl
chloride (5) is commercially available. This mild chlor-
ination method is applicable even to complex substrates
containing other sensitive functional groups. Alkane-
sulfonyl ¯uorides 6±8 were obtained from the
*Corresponding author. Fax: +301-7274761; e-mail: gkokotos@
cc.uoa.gr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00566-7

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G. Kokotos et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2803±2806
corresponding chlorides 3±5 by re¯uxing in anhydrous
acetone for 3 h, in the presence of a 10-fold molar excess
of anhydrous sodium ¯uoride.10
As an adaptation to the linear range of the assay, each
sample had to be immediately diluted 400-fold (in 2 mL
cold 5 mM deoxycholate, 0.1% sodium dodecyl sulfate,
10 mM Tris±HCl, pH 8.5) before assay, and 5 mL of the
diluted material was then used for the incubation with
the lipid substrate. The assay system contained in a total
volume of 0.2 mL (pH 8.5): 5 mg triglycerides/mL (from
radiolabelled Intralipid), 0.1 M NaCl, 0.15 M Tris±HCl,
0.1 mg heparin/mL, 60 mg bovine serum albumin/mL
and 5% (v/v) rat serum.18 The incubations were carried
out in duplicate for 15 min at 25 ꢀC.
Two esters of hexanesulfonic acid have been chosen to
be prepared and tested for their ability to react with the
active site of LPL. p-Nitrophenyl hexanesulfonate (9) is
an analogue of p-nitrophenyl phosphonates, which have
been proven potent inhibitors of digestive lipases11 and
microbial lipases.12,13 N-[(Hexanesulfonyl)oxy]succini-
mide (10) was chosen because derivatives of N-
(sulfonyloxy)succinimide have been reported to inhibit
the serine proteases human leukocyte elastase14 and
chymotrypsin.15 LPL is also a serine hydrolase with a
catalytic triad consisting of Ser-132, Asp-156 and His-
241.5 Compound 9 was prepared by addition of hex-
anesulfonyl chloride (3) to a solution of p-nitrophenol
and NaH in THF at 30 ꢀC, followed by stirring for
three days at room temperature. For the synthesis of
Porcine pancreatic lipase (PPL, Sigma, type VI-S) in
1 mM Tris±HCl, pH 8.0, containing 0.1 M NaCl and
5 mM CaCl2, was preincubated with the inhibitor (dis-
solved in DMSO) at a ratio calculated on active enzyme.
The preincubation solution contained 3% DMSO.
Residual PPL activity was measured with the pH-stat
method at pH 8, in a mixture containing: 10.5 mL 1 mM
Tris±HCl with 0.1 M NaCl and 5 mM CaCl2, 4 mL
15 mM deoxycholate and 0.5 mL tributyrin.19 The pre-
incubations and measurements of catalytic activity were
carried out at 37 ꢀC. The results are mean values from
three identical experiments.
compound 10
a
solution containing equimolar
amounts of N-hydroxysuccinimide and compound 3,
and a few drops of pyridine in CH2Cl2 was re¯uxed
for 1 h.
All products gave satisfactory analytical and spectro-
scopic data in full accord with their assigned structures.16
Results and Discussion
Figure 1 shows the time courses for inhibition of LPL
by alkanesulfonyl ¯uorides 6±8 and esters 9 and 10 at a
10-fold molar excess of the inhibitors. All sulfonyl
¯uorides proved to be ecient inhibitors of LPL. After
24 h the remaining enzymatic activity was zero (com-
pounds 7, 8) or almost zero (compound 6). The potency
of the inhibitors decreased as the chain length
decreased. At 3 h compound 8 (hexadecanesulfonyl
¯uoride) had inhibited more than 90% of the LPL
activity, while compound 6 (hexanesulfonyl ¯ouride) had
inhibited only 60% of the activity. On the contrary, the
sulfonyl esters (9 and 10) were almost inecient, causing
signi®cant inhibition under the conditions used.
Inhibition Studies
LPL puri®ed from bovine milk17 (500 mg/mL in 5 mM
deoxycholate, 10 mM Tris±HCl, pH 8.5)5 was pre-
incubated at room temperature with the inhibitor at a
ratio calculated on the concentration of LPL monomer
(50 kDa). LPL alone was virtually stable under the
conditions used. The inhibitors were dissolved and
diluted in DMSO. The ®nal concentration of DMSO in
the mixtures with LPL was 1% (v/v). Control experi-
ments showed that this concentration per se did not
a€ect the activity of LPL. Samples (5 mL) were taken at
the indicated times for assay of remaining LPL activity.

G. Kokotos et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2803±2806
2805
The inhibitory e€ect of alkanesulfonyl chlorides was
compared to that of the corresponding ¯uorides, as shown
in Figure 2. A 25-fold molar excess of the inhibitors 3, 4, 6,
and 7 was used. It is clear that sulfonylchlorides were quite
inecient as inhibitors in comparison with the ¯uorides.
After 90 min of incubation, dodecanesulfonyl ¯uoride
completely inhibited LPL, while the corresponding chlo-
ride caused less than 10% inhibition.
explain the structural determinants of LPL, which are
responsible for heparin binding, dimer formation and
phospholipase activity.20 The alkanesulfonyl halides 3±8
and esters 9 and 10, studied with LPL, were also studied
for their ability to inhibit PPL using the pH-stat
method. Among the compounds tested in this study,
only dodecanesulfonyl and hexadecanesulfonyl chlor-
ides 4 and 5 inhibited PPL activity at a high enzy-
me:inhibitor molar ratio (1:250). Fig. 4 shows the
dependency of the remaining PPL activity as a function
of time for compounds 4 and 5. These results are in
agreement with the ®ndings that short chain alkane-
sulfonyl ¯uorides did not inhibit PPL, while aromatic
sulfonyl chlorides may inhibit PPL if they contain elec-
tronegative substituents in the ring.22,23
The dose-dependent inhibition of LPL catalytic activity
by alkanesulfonyl ¯uorides was studied. Plots of
remaining LPL activity versus concentration of inhibi-
tors 6±8 are shown in Figure 3. In this experiment the
concentration of LPL monomer was about 5-fold lower
than in the previous experiments (2 mM as compared to
10 mM).
Taking into consideration that the common serine
hydrolase inhibitor phenylmethanesulfonyl ¯uoride
inhibits LPL only at high concentrations (mM) and the
inhibition is usually not complete,5 it seems that for
ecient inhibition of LPL linear alkanesulfonyl ¯uo-
rides with at least medium chain lengths (12 carbon
atoms) are needed. Hexanesulfonyl ¯uoride is also pos-
sible to use, but a higher ratio of inhibitor to LPL is
needed for ecient inhibition. In all experiments with
LPL a detergent-containing bu€er (5 mM deoxycholate)
was necessary for ecient inhibition. It is not known
whether the detergent is required for solubilization of
the inhibitors, or whether it a€ects the conformation of
LPL so that its active site is accessible to the inhibitors.
Hexadecane- and the dodecanesulfonyl ¯uorides caused
50% inhibition of LPL at concentrations of 10 to
20 mM. The decrease of the chain length from 16 to 12
carbon atoms caused a slight decrease in the inhibitory
power. A decreased chain length to six carbon atoms
(compound 6) resulted, however, in that about a 10-fold
higher concentration of the inhibitor was required for
50% inhibition of LPL.
LPL and PL have about 30% sequence identity, sug-
gesting a similar tertiary fold.20 Despite the considerable
sequence homology, they display some important func-
tional di€erences, for example substrate speci®city2 and
anity to heparin-like molecules and lipoproteins.21
Three-dimensional models have been proposed to
A
similar experience was previously made with
tetrahydrolipstatin as an inhibitor for LPL.5,6
Figure 1. Time courses for inhibition of LPL by alkanesulfonyl ¯uo-
rides and esters (-^- 8, -&- 7, -~- 6, -x- 10, -*- 9). LPL and inhibi-
tors were incubated at a molar ratio 1:10 (calculated on LPL
monomer) in 10 mM Tris±HCl, 5 mM deoxycholate, pH 8.5 at room
temperature.
Figure 3. Dose-dependent inhibition of LPL catalytic activity (mono-
mer concentration 2 mM) by increasing concentrations of alkanesulfonyl
¯uorides (-^- 8, -&- 7, -~- 6). In this experiment the incubation time
with the inhibitors was 60 min at room temperature.
Figure 2. Time courses for inhibition of LPL activity by alkanesulfo-
nyl ¯uorides and chlorides (-^- 7, -&- 6, -~- 4, -*- 3). LPL and
inhibitors were incubated at a molar ratio 1:25 in 10 mM Tris±HCl,
5 mM deoxycholate, pH 8.5 at room temperature.
Figure 4. Time courses for inhibition of PPL by alkanesulfonyl chlor-
ides. PPL was preincubated with inhibitor 4 at molar ratios 1:250 (-&-),
1:100 (-^-) and with inhibitor 5 at a molar ratio 1:250 (-~-), pH 8 at
37 ꢀC.

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G. Kokotos et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2803±2806
14. Groutas, W. C.; Brubaker, M. J.; Stanga, M. A.; Cas-
Acknowledgements
trisos, J. C.; Crowley, J. P.; Schatz, E. J. J. Med. Chem. 1989,
32, 1607.
15. Groutas, W. C.; Stanga, M. A.; Brubaker, M. J. J. Am.
Chem. Soc. 1989, 111, 1931.
16. For example: Compound 7: H NMR (300 MHz, CDCl3)
This research has been supported in part by the EU
(BIO2-CT94-3041) and by grants from the Swedish
Medical Research Council (03X-727 and 13X-12203).
1
d ppm: 3.4 (m, 2H, CH2SO2F), 1.95 (m, 2H, CH2CH2SO2F),
1.5 (m, 2H, CH2CH2CH2SO2F), 1.3 (m, 16H, 8ÂCH2), 0.95 (t,
3H, CH3, J=6.8 Hz). Analysis for C12H25SO2F (252.39): calcd
C 57.11, H 9.98; found C 57.21, H 10.09. Compound 9: 1H
NMR (300 MHz, CDCl3) d ppm: 8.35 (d, 2H, J=10 Hz, 2-H,
6-H), 7.45 (d, 2H, J=10 Hz, 3-H, 5-H), 3.4 (m, 2H, CH2SO2),
2.05 (m, 2H, CH2CH2SO2), 1.5 (m, 2H, CH2CH2CH2SO2), 1.4
(m, 4H, CH3CH2CH2), 0.9 (t, 3H, CH3, J=9 Hz). FAB MS:
m/z 310 (M+Na, 5%), 288 (M+H, 100), 272 (20), 257 (30),
242 (5), 166 (16). Compound 10: mp 70±72 ꢀC; 1H NMR
(300 MHz, CDCl3) d ppm: 3.55 (m, 2H, CH2SO2), 2.9 (s, 4H,
OCCH2CH2CO), 2.05 (m, 2H, CH2CH2SO2), 1.55 (m, 2H,
CH2CH2CH2SO2), 1.35 (m, 4H, CH3CH2CH2), 0.9 (t, 3H,
CH3, J=6.3 Hz). FAB MS: m/z 286 (M+Na, 3%), 264
(M+H, 40), 165 (2), 116 (100). Analysis for C10H17NO5S
(263.32): calcd C 45.61, H 9.22, N 3.46%; found C 45.71, H
9.00, N 3.47%.
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