A sensitive and selective high-throughput screening fluorescence assay for lipases and esterases

Article abstract of DOI:10.1002/hlca.200390240

Long-chain fatty acid esters of 7-(3,4-dihydroxybutyloxy)-2H-1-benzopyran-2-one (6) such as octanoate 2a are shown to be exceptionally sensitive and selective fluorogenic substrates for lipases and esterases. Umbelliferone (8) is released upon hydrolysis of the ester function in 2a in the presence of bovine serum albumin and sodium periodate. These substrates are at least by one order of magnitude more sensitive to lipases than the commercial fluorogenic substrate 4-methylumbelliferyl heptanoate. Furthermore, they are stable to a broad range of pH-induced- and thermal-hydrolysis conditions and do not react with non-catalytic proteins such as bovine serum albumin (BSA).

Full text of DOI:10.1002/hlca.200390240

Helvetica Chimica Acta Vol. 86 (2003)
2919
A Sensitive and Selective High-Throughput Screening Fluorescence Assay for
Lipases and Esterases
by Erich Nyfeler, Johann Grognux, Denis Wahler1), and Jean-Louis Reymond*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern
(fax: ‡41316318057; e-mail: jean-louis.reymond@ioc.unibe.ch)
Long-chain fatty acid esters of 7-(3,4-dihydroxybutyloxy)-2H-1-benzopyran-2-one (6) such as octanoate 2a
are shown to be exceptionally sensitive and selective fluorogenic substrates for lipases and esterases.
Umbelliferone (8) is released upon hydrolysis of the ester function in 2a in the presence of bovine serum
albumin and sodium periodate. These substrates are at least by one order of magnitude more sensitive to lipases
than the commercial fluorogenic substrate 4-methylumbelliferyl heptanoate. Furthermore, they are stable to a
broad range of pH-induced- and thermal-hydrolysis conditions and do not react with non-catalytic proteins such
as bovine serum albumin (BSA).
Introduction. Enzyme assays are indispensable tools in enzymology, where they
are used to identify enzymes and to evaluate their purity and activity [1]. When an
enzyme is discovered, one important question is always the identity of its natural
substrate, which is presumably also its best substrate in terms of kinetic behavior. In the
context of enzyme assays, the related problem is to find the optimal method for any
given enzyme, taking into consideration that assays giving spectroscopic signals are
preferred for high-throughput applications, for example, in the context of biodiversity
mining and directed evolution [2]. Herein, we report that C8ÀC14 aliphatic esters of the
fluorogenic diol 6 are exceptionally selective and sensitive probes for lipases
(Scheme 1). These substrates are stable under a variety of conditions, but are rapidly
hydrolyzed whenever only traces of an active lipase are present. The assay is applicable
for high-throughput screening.
Results and Discussion. Lipases and esterases are among the most broadly used
enzymes. The natural function of most of these enzymes is to hydrolyze triglycerides of
fatty acids, which initiates the metabolization of fat. As a consequence, their ideal
substrate should be a triglyceride. Indeed, vegetal oil and glycerol tributyrate
(tributyrin) are well-known substrates of lipases and esterases, respectively. We
recently reported a simple colorimetric enzyme assay with these triglycerides as
substrates [3]. However, despite of using optimal substrates, the assay had a detection
limit in the usual range of 10 100 mg mlÀ1 of enzyme2). We had encountered a similar
1
¬
¬
)
)
Present address: Proteus S.A., 70, allee Graham Bell, Parc Georges Besse, F-30000 Nimes.
Commercial enzyme samples were used in all our studies. The activities observed with vegetal oil [3] or
with the substrates in the present study did not correlate well with the activity units (U ˆ mmol product
minÀ1 mgÀ1) given by the manufacturer, which are most often defined with 4-nitrophenyl butyrate or
palmitate as substrate. This is not surprising considering that enzyme reactivities are generally dependent
on substrate structure.
2

2920
Helvetica Chimica Acta Vol. 86 (2003)
Scheme 1. Fluorogenic Substrates for Lipases and Esterases, and Detection Principle
detection limit when using acetates 1a and 1b as lipase and esterase substrates
(Scheme 1) [4]. These substrates release diol 6 upon reaction with the enzymes, and a
fluorescence signal is produced in the presence of NaIO4 and bovine serum albumin
(BSA) by liberation of umbelliferon (8) through an oxidation/b-elimination sequence
via aldehyde 7 [5]. A first attempt at optimizing the structures of these substrates
towards lipases consisted in modifying diol 6 into a branched analog of glycerol to
produce diglyceride-like aliphatic ester substrates [6]. However, this strategy did not
lead to the expected improvement in sensitivity. We, therefore, went back to the simple
diol 6, which may also be considered as an analog of glycerol, to investigate the effect of
substituting long-chain aliphatic acyl groups for the acetyl group in 1a and 1b.
Starting with butane-1,2,4-triol, acetonide formation and tosylation gave the known
tosylate 9 [7]. Substitution with the sodium salt of umbelliferone gave ether 10, which
was deprotected with acid to give the racemic diol 6 (Scheme 2). The four aliphatic acid
monoesters 2a 5a and the four aliphatic acid diester derivatives 2b 5b were prepared

Helvetica Chimica Acta Vol. 86 (2003)
2921
by reaction of this diol with the corresponding acyl chloride and Et3N. These substrates
were conditioned as 1 mm stock solutions in MeCN, and tested with a series of lipases
and esterases. Assays were initiated by addition of substrate to a prediluted solution of
enzyme in a slightly basic buffer (pH 8.8), incorporating NaIO4 as a diol-processing
reagent and BSA as an additive. The latter served both as a catalyst for the b-
elimination of the intermediate aldehyde and as a non-selective protein to mimic the
conditions of high-throughput screening in crude extracts. Fluorescence time profiles
were recorded in microtiter plate assay format by using a series of enzymes, and the
time curves were used to derive apparent reaction rates with the different enzymes
(Table).
Scheme 2. Synthesis of Fluorogenic Lipase Substrates
All enzymes showed a much higher reactivity with the long-chain aliphatic esters
2 5 compared to the acetate derivatives 1a and 1b. While the earlier measurements
with 1a and 1b required 100 mg ¥ mlÀ1 enzyme to obtain a good signal, many enzyme
samples retained activity upon 10-fold, 100-fold, or even 1000-fold dilution. Analysis by
gel electrophoresis (SDS-PAGE) showed that high activity, in fact, correlated with
protein purity. The mono-ester derivatives were generally much more reactive than the
corresponding diesters, which is not surprising if one considers that the diester requires
a double hydrolysis reaction to induce a fluorogenic oxidation sequence. Several
esterases also showed reactivities with the long-chain fatty acid esters largely exceeding
those observed with the acetate derivatives. This is not necessarily surprising
considering the problematic distinction between these two enzyme classes. It should
be noted that the acetates were distinct from the long-chain substrates in that the

2922
Helvetica Chimica Acta Vol. 86 (2003)
Table 1. Reaction Rates Observed with Lipases and Esterasesa)
Fluka No. Enzyme mg mlÀ1 1ab)
2a
3a
4a
5a
9.7
17.8
6.4
25.6
3.5
10.8
25.9
8.2
8.3
12.3
3.7
5.9
7.7
15.2
11.9
7.7
12.7
7.8
10.0
2.3
1.8
1bb)
2b
3b
3.4
4b
3.5
5b
62336
62333
62321
62335
62285
62299
62305
62309
62294
62304
62316
62298
62300
46069
62291
46059
46061
46062
62310
46051
62306
62308
PSBL
CVL
PFL
0.1
0.1
0.1
1
1
10
10
10
10
10
10
(5.2) 17.0
(0.2) 11.9
(4.0)
(1.9) 30.7
(0.2) 4.1
24.2
13.7
13.1
34.4
8.3
23.1
15.6
11.8
33.3
10.5
9.3
20.3
16.2
17.1
19.8
9.8
12.2
29.6
28.6
24.7
18.2
21.7
18.8
9.8
(21)
(5.0)
(14.1)
(7.0) 14.4
(1.3) 3.3
(1.1) 13.8
n.d. 13.3
(1.0) 26.0
(3.1) 15.0
n.d.
(0.5) 14.1
(0.7)
2.4
6.0
0.6
0.3
0.3
0.2
0.4
1.8
n.d.
n.d.
16.2
6.7
3.9
7.2
2.9
1.9
6.8
1.0
7.1
1.5
5.0
2.2
1.9
1.0
1.5
1.8
3.1
2.4
4.1
2.0
3.1
1.1
0.2
1.7
0.0
1.6
2.0
9.4
1.5
9.1
1.1
12.2
0.2
2.4
2.4
4.2
8.8
3.6
5.3
8.2
2.1
3.6
3.4
0.3
0.9
0.1
1.6
2.1
9.5
1.2
10.9
3.2
7.6
4.9
6.2
4.1
3.5
6.6
3.4
3.6
5.1
3.4
4.1
4.2
0.2
1.3
0.1
8.6
PSL
AOL
CAL
RAL
PCL
(4.1) 26.6
n.d. 24.7
(0.4) 13.0
(0.2) 19.2
n.d. 16.4
(0.1) 15.4
(0.1) 16.3
0.3
2.2
0.1
0.1
0.1
0.1
0.1
1.0
n.d.
n.d.
18.7
14.4
14.5
16.6
11.5
15.1
11.8
28.4
32.0
23.6
28.3
22.3
18.6
15.7
11.0
6.3
ANL
MJL
CCL
MML
HPL
HLE
RML
MME
TBE
BSE
9.2
10
7.4
18.0
8.6
19.3
12.2
3.7
6.1
15.9
2.5
100
100
100
100
100
100
100
100
100
100
35.6
38.8
26.9
31.2
18.5
19.2
17.9
15.5
6.7
RNL
BStE
WGL
PRL
10.6
4.6
3.8
1.0
0.1
4.5
7.3
3.0
no Ec)
0.10
0.23
0.28
0.24
0.22
0.04
0.08
0.02
0.06 0.03
a) Rate of release of umbelliferone (8) in nm sÀ1 from 100 mm substrate, with commercial enzyme samples at the
given concentration. Conditions: aq. 20 mm borate buffer, pH 8.8, 5% (v/v) MeCN, 308, in the presence of 1 mm
NaIO4 and 2 mg ¥ mlÀ1 BSA, 0.1-ml assays were carried out in round-bottom polypropylene 96-well-plates
(Costar) and recorded with a Cytofluor II Fluorescence Plate Reader (Perseptive Biosystems; filters lex ˆ 360 Æ
20, lem ˆ 460 Æ 20 nm). Fluorescence was converted to product concentration with a calibration curve with pure
8, and the steepest linear portion of each curve was used to calculate the reaction rates. b) Data for 1a and 1b
from [4a]. Rates in parentheses are with 100 mg ¥ mlÀ1 enzyme concentration and not the concentration indicated
here. c) Spontaneous reaction in the absence of enzyme under the assay conditions.
diester derivative 1b was consistently more enzyme-reactive than the monoacetate 1a,
indicating that the enzymes require a certain level of lipophilicity in the substrate for
having a good reactivity.
The reactivity of the octanoate 2a with Pseudomonas fluorescens type B lipoprotein
lipase (PSBL) was investigated closer. The substrate gave a signal to background ratio
Vrel ˆ 185 with 10 mg mlÀ1 enzyme (Vrel ˆ (Venz/Vno E) À 1)3). The signal remained very
strong upon dilution of the enzyme, and was still at Vrel ˆ 73 at 0.1 mg mlÀ1 (Table).
Further dilution down to 0.01 mg mlÀ1 enzyme gave a signal-to-background ratio of
V
rel > 10, which is perfectly sufficient for detection (Fig. 1). Both enantiomers (R)-2a
and (S)-2a showed similar reactivities.
3
)
At high enzyme concentration, the rate of release of umbelliferone (8) is limited by the rate of oxidation/
b-elimination of diol 6, so that the apparent reaction rate is not proportional to enzyme concentration
upon dilution of the enzyme.

Helvetica Chimica Acta Vol. 86 (2003)
2923
Fig. 1. Fluorescence signal for the reaction of 100 mm octanoate (R)-2a and (S)-2a in 20 mm aq. borate, pH 8.8,
À1
2 mg mlÀ1 BSA, 1 mm NaIO4, 20% (v/v) DMF, 258. ‡ : No additive, : (S)-2a with 0.1 mg ml BSBL, : (S)-2a
&
&
with 0.01 mg mlÀ1 PSBL, : (R)-2a with 0.1 mg ml PSBL, : (R)-2a with 0.01 mg mlÀ1 PSBL.
À1
~
~
The high reactivity with lipases could be attributed in part to the aliphatic
hydrocarbon chain of the acid. By comparison, we investigated the reactivity of 4-
methylumbelliferyl heptanoate (11), a commercially available fluorogenic lipase
substrate, which is a representative example of aliphatic phenyl ester [8]. 4-
Methylumbelliferyl heptanoate (11) reacted with PSBL within one order of magnitude
of sensitivity of our substrates (Vrel ˆ 4.6 at 0.1 mg mlÀ1 enzyme; Fig. 2). However, the
commercial heptanoate had to be used at exactly neutral pH because it spontaneously
hydrolyzed at pH 8.8, with or without BSA. In addition, its reaction was not selective
for lipases and produced a fluorescence signal in the presence of the noncatalytic
protein BSA even at neutral pH. This nonselective reaction can be attributed to an
acylation of surface lysine residues. By contrast, octanoate 2a showed no reaction in the
presence or absence of BSA at neutral (pH 7) or slightly basic (pH 8.8) conditions. The
reactivity difference between the two ester substrates is a direct consequence of the
difference in leaving-group acidity (pKa ꢀ 7 for the OH group in 4-methylumbellifer-
one and pKa ꢀ 14 for diol 6). The chemical lability problem of 4-methylumbelliferyl
heptanoate (11) also occurs with the resorufin derivative 12, a substrate which is
insoluble in buffer and must be used with detergents for solubilization. Interestingly, it
has been recently shown that substrate 12 undergoes hydrolysis at the activated
resorufin ester bond under the action of lipases, while the glycerol ester bond remains
stable [9]. Probes 11 and 12 must be stored at À188, contain several percent of
hydrolyzed material when purchased, and must be repurified before use.

2924
Helvetica Chimica Acta Vol. 86 (2003)
Fig. 2. Fluorescence signal for the reaction of 100 mm 4-methylumbelliferyl heptanoate in 20 mm aq. phosphate,
À1
À1
À1
~
~
:
&
&
pH 7.0, 20% (v/v) DMF, 258. ‡ : No additive, : 0.5 mg ml BSA, : 2 mg ml BSA, : 0.1 mg ml PSBL,
0.01 mg mlÀ1 PSBL. 4-Methylumbelliferyl heptanoate was repurified before use to remove contaminant 4-
methylumbelliferone. Stock solutions are unstable upon storage.
The resilience of fluorogenic substrate 2a to nonspecific degradation was
remarkable under a variety of conditions such as extreme pH and temperature values.
Thus, there was no significant (< 5%) degradation upon incubation for 60 min in
aqueous buffers containing BSA at pH values as low as pH 1.0 and as high as pH 11, or
by incubation at pH 8.8 at 1008 for 60 min. Clearly, the reaction of 2a with lipases was
not only highly sensitive but also highly selective. Esters 3a 5a and diesters 2b 5b
showed comparable properties in terms of resistance to nonspecific degradation and
sensitivity to lipases and esterases. By contrast to the above mentioned lipase probes,
our substrates also proved shelf-stable as solids and could be handled without any
problems of nonspecific degradation.

Helvetica Chimica Acta Vol. 86 (2003)
2925
Conclusions. In summary, long-chain fatty acid esters of the glycerol analog 6 are
remarkably sensitive and selective fluorogenic substrates for lipases and esterases. The
fluorogenic glyceride analogs reported here are stable under a variety of conditions
including extreme pH and temperature values, yet react selectively with only trace
amounts of lipases. The substrates are also suitable as fluorogenic substrates for
esterases. The coupling of high sensitivity with high selectivity makes these substrates
an optimal choice for the detection of esterolytic activities in high-throughput
screening. The processing reagents NaIO4 and BSA are necessary but do not affect
enzyme activity. These reagents may also be added at the end of the reaction for an
endpoint determination of product formation. It must be mentioned that the
decomposition of the primary diol product 6 by oxidation and b-elimination leading
to the fluorescent signal may become rate-limiting when the enzymatic hydrolysis rate
is very high, implying that our detection system is probably not suited for precise kinetic
studies. The use of the more hydrophobic substrates, in particular, the diesters of the
long-chain esters 3b 5b, in the presence of detergents are under current investigation.
¬
¬
This work was supported by the Swiss National Science Foundation, the Swiss Office Federal de l×Education
¬
et de la Science, and Proteus SA, NÓmes, France.
Experimental Part
General. All reactions were followed by TLC on Alugram SIL G/UV254 silica-gel sheets (Macherey-Nagel)
with detection by UV or with 0.5% phosphomolybdic acid soln. in 95% EtOH. Silica gel 60 (Macherey-Nagel,
230 400 mesh) was used for flash chromatography (FC). M.p.: Kofler apparatus or B¸chi 510 apparatus; IR
Spectra: Perkin-Elmer Spectrum One series FTIR apparatus. 1H- and 13C-NMR spectra: Bruker AC-300
spectrometer.
7-[2-(3,3-Dimethyl-1,2-dioxolan-4-yl)ethoxy]-2H-1-benzopyran-2-one (10). A suspension of umbelliferone
(ˆ 7-hydroxy-2H-1-benzopyran-2-one 8; 270 mg, 1.66 mmol), 18-crown-6 (87 mg, 0.33 mmol), anh. K2CO3
(459 mg, 3.32 mmol), and 2-(3,3-dimethyl-1,3-dioxolan-4-yl)ethyl toluene-4-sulfonate (500 mg, 1.66 mmol) in
3 ml of acetone was heated under refluxfor 16 h. Evaporation, aq. workup (AcOEt, 0.1 n NaOH, then aq. sat.
NaCl) and FC (hexane/AcOEt 2 :1; Rf 0.23) gave 10 (433 mg, 1.49 mmol, 90%). Solid. M.p. 818. IR (KBr):
2988m, 2956m, 2934m, 2890m, 1726s, 1629s. 1H-NMR (300 MHz, CDCl3): 7.63 (d, J ˆ 9.6, 1 H); 7.36 (d, J ˆ 7.7,
1 H); 6.84 (s, 1 H); 6.83 (d, J ˆ 7.7, 1 H); 6.24 (d, J ˆ 9.6, 1 H); 4.31 (m, J ˆ 6.3, 6.6, 1 H); 4.13 (m, J ˆ 6.3, 6.6, 8.1,
3 H); 3.65 (dd, J ˆ 6.6, 8.1, 1 H); 2.07 (m, J ˆ 6.3, 1 H); 1.43 (s, 3 H); 1.37 (s, 3 H). 13C-NMR 75 MHz, CDCl3):
162.7; 144.0; 130.6; 129.5; 128.5; 113.9; 113.5; 113.3; 109.7; 102.2; 73.7; 70.1; 66.0; 34.0; 27.7; 26.4. EI-MS: 290
‡
(M ), 275, 215, 162, 134, 71, 43.
7-(3,4-Dihydroxybutyloxy)-2H-1-benzopyran-2-one (6). Intermediate 10 (5.47 g, 18.9 mmol) was dissolved
in 60 ml MeOH. 0.1n Aq. HCl (50 ml) was added, and the mixture was stirred at 258 for 17 h. Evaporation and
co-evaporation with toluene, followed by FC (CH2Cl2/acetone 7:3 bis 1 :1), gave 6 (3.13 g, 12.5 mmol, 66%).
Colorless solid. M.p. 988. IR (KBr): 3300s, 2934m, 2876m, 1724s, 1611s. 1H-NMR (300 MHz, CDCl3): 7.63
(d, J ˆ 9.6, 1 H); 7.37 (d, J ˆ 7.7, 1 H); 6.86 (d, J ˆ 7.7, 1 H); 6.84 (d, J ˆ 7.7, 1 H); 6.25 (d, J ˆ 9.6, 1 H); 4.15
(m, 2 H); 3.78 (m, 1 H); 3.46 (m, 2 H); 2.00 (m, 1 H); 1.76 (m, 1 H). 13C-NMR (75 MHz, CDCl3): 163.9; 163.3;
157.0; 145.7; 130.3; 114.1; 113.9; 113.2; 102.2; 70.0; 67.4; 66.5; 33.9. Anal. calc. for C13H14O5: C 62.39, H 5.64;
found: C 62.33, H 5.60.
3-Hydroxy-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butyl Octanoate (2a). A soln. of 6 (100 mg, 0.4 mmol)
and dry Et3N (81 mg, 0.8 mmol) in dry CH2Cl2 (6 ml) and dry DMF (1 ml) at 08 was treated with octanoyl
chloride (69 ml, 0.4 mmol). The mixture was stirred at 08 for 1 h and then at 258 until completion of the reaction
(TLC). Aq. workup (CH2Cl2/aq. sat. NaHCO3 and brine) and FC (hexane/AcOEt 2 :1) gave 2a (91.7 mg,
0.24 mmol, 59%). Colorless solid. M.p. 418. TLC (hexane/AcOEt 1:1): Rf 0.53. IR (film): 3466w (br.), 3008w,
2956m, 2930m, 2858m, 1728s, 1613s. 1H-NMR (300 MHz, CDCl3): 7.63 (d, 1 H); 7.37 (d, 1 H); 6.86 (s, 1 H); 6.84
(d, 1 H); 6.25 (d, 1 H); 4.04 4.25 (m, 5 H); 2.36 (m, 2 H); 1.98 (m, 2 H); 1.63 (m, 2 H); 1.29 (m, 8 H); 0.87

2926
Helvetica Chimica Acta Vol. 86 (2003)
(m, 3 H). 13C-NMR (75 MHz, CDCl3): 174.7; 162.6; 161.9; 156.4; 144.1; 129.4; 113.7; 113.5; 113.3; 102.1; 68.9;
‡
67.7; 65.6; 34.8; 33.3; 32.2; 29.7; 29.5; 25.5; 23.2; 14.7. FAB-MS: 377 (M ), 251, 163.
Compound (R)-2a. The procedure for 2a was carried out starting with (R)-6 [4d] (96% ee; 85 mg,
0.34 mmol) to give the corresponding ester (R)-2a (109 mg, 0.29 mmol, 85%). Colorless waxy solid. [a]2D0 ˆ ‡4.6
(c ˆ 1, CHCl3).
Compound (S)-2a. The procedure for 2a was carried out starting with (S)-6 [4d] (87% ee; 70 mg,
0.28 mmol) to give the corresponding ester (R)-2a (108 mg, 0.29 mmol, 81%). Colorless waxy solid. [a]2D0 ˆ À4.0
(c ˆ 1, CHCl3).
3-Hydroxy-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butyl Decanoate (3a). The procedure for 2a was applied
with decanoyl chloride (83 ml, 0.4 mmol). FC gave 3a (80 mg, 0.2 mmol, 49%). Colorless solid. M.p. 458. TLC
(hexane/AcOEt 1:1): Rf 0.57. IR (film): 3492w (br.), 3013w, 2928m, 2856w, 1728s, 1613s. 1H-NMR (300 MHz,
CDCl3): 7.63 (d, 1 H); 7.36 (d, 1 H); 6.86 (s, 1 H); 6.83 (d, 1 H); 6.25 (d, 1 H); 4.04 4.24 (m, 5 H); 2.35
(m, 2 H); 1.97 (m, 2 H); 1.61 (m, 2 H); 1.26 (m, 12 H); 0.86 (m, 3 H). 13C-NMR (75 MHz, CDCl3): 174.7; 162.6;
161.8; 156.6; 144.0; 129.5; 113.9; 113.5; 113.4; 102.23; 69.0; 67.9; 65.7; 34.9; 33.3; 32.5; 30.1; 29.9; 29.8; 25.6; 23.3;
‡
14.8. FAB-MS: (M ), 251, 163, 13. Anal. calc. for C32H32O6: C 68.29, H 7.97; found: C 68.19, H 8.00.
3-Hydroxy-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butyl Dodecanoate (4a). The procedure for 2a was
applied with dodecanoyl chloride (96 ml, 0.4 mmol). FC gave 4a (130 mg, 0.3 mmol, 75%). Colorless solid.
M.p. 538. TLC (hexane/AcOEt 1:1): Rf 0.69. IR (film): 3466w (br.), 3009w, 2927s, 2855m, 1728s, 1613s.
1H-NMR (300 MHz, CDCl3): 7.63 (d, 1 H); 7.37 (d, 1 H); 6.86 (s, 1 H); 6.84 (d, 1 H); 6.25 (d, 1 H); 4.01 4.28
(m, 5 H); 2.34 (m, 2 H); 1.97 (m, 2 H); 1.52 (m, 2 H); 1.25 (m, 16 H); 0.87 (m, 3 H). 13C-NMR (75 MHz,
CDCl3): 174.6; 162.6; 161.8; 156.5; 144.0; 129.4; 113.8; 113.5; 113.3; 102.2; 69.0; 67.7; 65.7; 34.8; 33.3; 32.5; 30.2;
‡
‡
30.1; 29.9; 29.9; 29.8; 25.6; 23.3; 14.7. FAB-MS: 433 (M ), 251, 228, 163. HR-ESI-MS: 455.2425 ([M ‡ Na ],
‡
C25H36O6 ; calc. 455.2409).
3-Hydroxy-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butyl Tetradecanoate (5a). The procedure for 2a was
applied with tetradecanoyl chloride (108 ml, 0.4 mmol). FC gave 5a (142 mg, 0.31 mmol, 77%). Colorless solid.
M.p. 608. TLC (hexane/AcOEt 1:1): Rf 0.67. IR (film): 3469w (br.), 3011w, 2927s, 2855m, 1728s, 1613s. 1H-NMR
(300 MHz, CDCl3): 7.63 (d, 1 H); 7.37 (d, 1 H); 6.86 (s, 1 H); 6.84 (d, 1 H); 6.25 (d, 1 H); 4.04 4.28 (m, 5 H);
2.36 (m, 2 H); 1.98 (m, 2 H); 1.62 (m, 2 H); 1.25 (m, 20 H); 0.88 (m, 3 H). 13C-NMR (75 MHz, CDCl3): 174.7;
162.6; 161.9; 156.6; 144.1; 129.5; 113.9; 113.5; 113.4; 102.2; 69.1; 67.9; 65.7; 34.9; 33.3; 32.6; 30.4; 30.3; 30.3; 30.1;
‡
‡
30.0; 29.9; 29.8; 25.6; 23.4; 14.8. FAB-MS: 461 (M ), 256, 251, 163. HR-ESI-MS: 461.2914 ([M ‡ H] ;
‡
C27H40O6 ; calc. 461.2903).
4-[(2-Oxo-2H-1-benzopyran-7-yl)oxy]butane-1,2-diyl Dioctanoate (2b). A soln. of diol 6 (100 mg,
0.4 mmol) and 4-(dimethylamino)pyridine (134 mg, 1.1 mmol) in dry CH2Cl2 (7 ml) was treated with octanoyl
chloride (171 ml, 1.1 mmol) and stirred at 258 until completion of the reaction (18 h, TLC). Aq. workup (CH2Cl2/
aq. sat. NaHCO3 sat. and brine) and FC (hexane/AcOEt 5 :1) gave 2b (60 mg, 0.12 mmol, 30%). Colorless solid.
M.p. 388. TLC (hexane/AcOEt 2 :1): Rf 0.56. IR (film): 2955m, 2928m, 2857m, 1738s, 1614s. 1H-NMR
(300 MHz, CDCl3): 7.63 (d, 1 H); 7.35 (d, 1 H); 6.84 (s, 1 H); 6.80 (d, 1 H); 6.25 (d, 1 H); 5.33 (m, 1 H); 4.35
(m, 1 H); 4.04 4.16 (m, 3 H); 2.31 (m, 4 H); 2.13 (m, 2 H); 1.59 (m, 4 H); 1.27 (m, 16 H); 0.86 (m, 6 H).
13C-NMR (75 MHz, CDCl3): 174.1; 173.83; 162.4; 159.4; 158.8; 144.0; 129.5; 114.0; 113.7; 113.4; 102.0; 69.2; 65.4;
‡
65.1; 35.0; 34.8; 32.3; 31.2; 29.7; 29.6; 25.7; 25.6; 23.3; 14.7. FAB-MS: 503 (M ), 377, 341, 251, 233, 215, 127. HR-
‡
ESI-MS: 525.2827 ([M ‡ H], C29H42O7 ; calc. 525.2828).
4-[(2-Oxo-2H-1-benzopyran-7-yl)oxy]butane-1,2-diyl Didecanoate (3b). The procedure for 2b was applied
with decanoyl chloride (204 ml, 1.1 mmol). FC gave 3b (166 mg, 0.29 mmol, 73%). Colorless solid. M.p. 558. TLC
(hexane/AcOEt 2 :1): Rf 0.67. IR (film): 3024w, 2955m, 2927s, 2855m, 1740s, 1613s. 1H-NMR (300 MHz,
CDCl3): 7.63 (d, 1 H); 7.36 (d, 1 H); 6.84 (s, 1 H); 6.80 (d, 1 H); 6.26 (d, 1 H); 5.32 (m, 1 H); 4.34 (m, 1 H);
4.04 4.16 (m, 3 H); 2.31 (m, 4 H); 2.14 (m, 2 H); 1.57 (m, 4 H); 1.24 (m, 24 H); 0.86 (m, 6 H). 13C-NMR
(75 MHz, CDCl3): 174.1; 173.8; 166.6; 162.44; 161.7; 144.0; 129.5; 114.0; 113.7; 113.4; 102.0; 69.2; 65.4; 65.1; 35.1;
‡
34.8; 32.5; 31.2; 30.1; 30.0; 29.8; 25.7; 25.6; 23.4; 14.8. FAB-MS: 559 (M ), 405, 251, 233, 215, 155. HR-ESI-MS:
‡
‡
559.3640 ([M ‡ H] , C33H50O7 ; calc. 559.3635).
4-[(2-Oxo-2H-1-benzopyran-7-yl)oxy]butane-1,2-diyl Didodecanoate (4b). The procedure for 2b was
applied with dodecanoyl chloride (237 ml, 1.1 mmol). FC gave 4b (184 mg, 0.3 mmol, 75%). Colorless solid. M.p.
648. TLC (hexane/AcOEt 2 :1): Rf 0.58. IR (film): 3020m, 2925s, 2854s, 1735s, 1612s. 1H-NMR (300 MHz,
CDCl3): 7.63 (d, 1 H); 7.36 (d, 1 H); 6.84 (s, 1 H); 6.80 (d, 1 H); 6.26 (d, 1 H); 5.34 (m, 1 H); 4.34 (m, 1 H);
4.04 4.16 (m, 3 H); 2.30 (m, 4 H); 2.13 (m, 2 H); 1.58 (m, 4 H); 1.24 (m, 32 H); 0.87 (m, 6 H). 13C-NMR
(75 MHz, CDCl3): 174.1; 173.8; 162.4; 161.7; 156.6; 144.0; 129.5; 114.0; 113.7; 113.4; 102.0; 69.2; 65.4; 65.1; 35.0;

Helvetica Chimica Acta Vol. 86 (2003)
2927
‡
34.8; 32.6; 31.2; 30.3; 30.2; 30.1; 30.0; 29.8; 29.7; 25.6; 25.6; 23.4; 14.8. FAB-MS: 615 (M ), 433, 251, 233, 215.
‡
‡
HR-ESI-MS: 615.4242 ([M ‡ H] , C37H58O7 ; calc. 615.4261).
4-[(2-Oxo-2H-1-benzopyran-7-yl)oxy]butane-1,2-diyl Ditetradecanoate (5b). The procedure for 2b was
applied with tetradecanoyl chloride (297 ml, 1.1 mmol). FC gave 5b (261 mg, 0.39 mmol, 88%). Colorless solid.
M.p. 728. TLC (hexane/AcOEt 2 :1): Rf 0.73. IR (film): 3022w, 2927s, 2855m, 1732s, 1614s. 1H-NMR (300 MHz,
CDCl3): 7.63 (d, 1 H); 7.36 (d, 1 H); 6.84 (s, 1 H); 6.80 (d, 1 H); 6.26 (d, 1 H); 5.34 (m, 1 H); 4.34 (m, 1 H);
4.04 4.16 (m, 3 H); 2.31 (m, 4 H); 2.13 (m, 2 H); 1.58 (m, 4 H); 1.25 (m, 40 H); 0.87 (m, 6 H). 13C-NMR
(75 MHz, CDCl3): 174.1; 173.8; 162.4; 161.8; 156.5; 144.0; 129.5; 113.9; 113.7; 113.4; 101.9; 69.2; 65.3; 65.1; 35.0;
34.8; 32.6; 31.1; 30.33; 30.30; 30.27; 30.14; 30.12; 30.03; 29.9; 29.79; 29.76; 25.6; 25.5; 23.3; 14.8. FAB-MS: 671
‡
(M ), 461, 443, 251, 233, 215.
Kinetic Measurements. All substrates were diluted from stock solns. in 50% aq. MeCN (for 1a 5a) or pure
MeCN (for 1b 5b), and stored at À208. 4-Methylumbelliferyl heptanoate (11) was purchased from Fluka
(No. 75230), purified by flash chromatography (AcOEt/hexane 1:3) and recrystallization from MeOH, and
used as a freshly prepared 10 mm stock soln. in MeCN/H2O 1 :1. Enzymes were diluted from 1 mg mlÀ1 stock
solns. of the supplied solid in PBS (10 mm phosphate, 150 mm NaCl, pH 7.4). Assays (0.1 ml) were followed in
individual wells of round-bottom polypropylene 96-well-plates (Costar) with a Cytofluor II Fluorescence Plate
Reader (Perseptive Biosystems, filters lex ˆ 360 Æ 20, lem ˆ 460 Æ 20 nm). Fluorescence data were converted to
umbelliferone concentration by means of a calibration curve. The rates indicated in the Table are derived from
the linear portion in each curve.
REFERENCES
[1] a) M. T. Reetz, Angew. Chem. 2001, 113, 292; Angew. Chem., Int. Ed. 2001, 40, 284; b) D. Wahler, J.-L.
¡
Reymond, Curr. Opin. Chem. Biol. 2001, 5, 152; c) F. Beisson, A. Tiss, C. Riviere, R. Verger, Eur. J. Lipid
Sci. Technol. 2000, 133; d) D. Wahler, J.-L. Reymond, Curr. Opin. Biotechnol. 2001, 12, 535.
[2] a) B. Marrs, S. Delagrave, D. Murphy, Curr. Opin. Microbiol. 1999, 2, 241; b) W. P. C. Stemmer, Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 10747; c) W. P. C. Stemmer, Nature 1994, 370, 389; d) L. You, F. H. Arnold,
Protein. Eng. 1996, 9, 77; e) M. T. Reetz, K.-E. Jaeger, Chem. Eur. J. 2000, 6, 407; f) S. V. Taylor, P. Kast, D.
Hilvert, Angew. Chem. 2001, 113, 3408; Angew. Chem., Int. Ed. 2001, 40, 3310.
[3] D. Wahler, J.-L. Reymond, Angew. Chem., Int. Ed. 2002, 41, 1229.
[4] a) F. Badalassi, D. Wahler, G. Klein, P. Crotti, J.-L. Reymond, Angew. Chem., Int. Ed. 2000, 39, 4067; b) D.
Wahler, F. Badalassi, P. Crotti, J.-L. Reymond, Angew. Chem., Int. Ed. 2001, 40, 4457; c) D. Wahler, F.
Badalassi, P. Crotti, J.-L. Reymond, Chem. Eur. J. 2002, 8, 3211; d) J.-L. Reymond, D. Wahler, F. Badalassi,
H. K. Nguyen, Patent WO0192563, 7.12.2001.
[5] a) G. Klein, J.-L. Reymond, Bioorg. Med. Chem. Lett. 1998, 8, 1113; b) G. Klein, J.-L. Reymond, Helv.
¬
¡
Chim. Acta 1999, 82, 400; c) R. Perez Carlon, N. Jourdain, J.-L. Reymond, Chem. Eur. J. 2000, 6, 4154;
d) E. M. Gonzalez-Garcia, V. Helaine, G. Klein, M. Schuermann, G. Sprenger, W.-D. Fessner, J.-L.
Reymond, Chem. Eur. J. 2003, 9, 893.
[6] E. M. Gonzalez-Garcia, J. Grognux, D. Wahler, J.-L. Reymond, Helv. Chim. Acta 2003, 86, 2458.
[7] J. L. Cage, B. P. Branchaud, J. Org. Chem. 1996, 61, 831.
[8] G. G. Guilbault, J. Hieserman, Anal. Chem. 1969, 41, 2006 2009.
[9] B. Bothner, R. Chavez, J. Wei, C. Strupp, Q. Phung, A. Schneemann, G. Siuzdak, J. Biol. Chem. 2000, 275,
13455.
Received April 10, 2003