Universal chromogenic substrates for lipases and esterases

Article abstract of DOI:10.1016/j.tetasy.2004.07.059

Chromogenic mono- and diacyl-glycerol analogues were prepared and tested as substrates against lipases and esterases in a microtiter plate setup. Thirty-three enzyme samples were analyzed for activity at 100, 10, and 1 μg mL^-1. The reaction rat

Full text of DOI:10.1016/j.tetasy.2004.07.059

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2981–2989
Universal chromogenic substrates for lipases and esterases
Johann Grognux,a Denis Wahler,b Erich Nyfelera and Jean-Louis Reymonda,*
aDepartment of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland
b
´ ´ ˆ
Proteus SA, 70, allee Graham Bell, Parc Georges Besse, 3000 Nımes, France
Received 1 June 2004; accepted 20 July 2004
Available online 11 September 2004
Abstract—Chromogenic mono- and diacyl-glycerol analogues were prepared and tested as substrates against lipases and esterases
in a microtiter plate setup. Thirty-three enzyme samples were analyzed for activity at 100, 10, and 1lgmLꢀ1. The reaction rates
observed were visualized by RGB-color coding of activities, and analyzed by cluster analysis. The C8-, C10-, and C12-monoesters
of the nitrophenol-derived diol 13 reacted very strongly across all enzyme samples tested, and appear as universal substrates for
these enzymes. Such universal chromogenic probes may be used to detect even dilute lipases and esterases in crude cultures.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
lipases and esterases, which are the main class of enzymes
in industrial applications.12 Lipases can be assayed using
fluorescence substrates, which operate by fluorescence
resonance energy transfer (FRET).13 However these
substrates are structurally complex since they require
two chromophores, and are only suited for lipases. In
practice lipase and esterase assays are mostly carried
out using the classical aliphatic esters of nitrophenol,
umbelliferone, resorufin, and fluorescein (as diester).
Unfortunately these substrates are all esters of acidic
phenols, and as such they show high spontaneous and
nonspecific hydrolysis in aqueous buffer. In addition
these substrates do not react very well across all lipases
and esterases. In an attempt to reduce spontaneous reac-
tivity, we have recently investigated acyloxymethyl
ethers14 and 1-acyloxy-1-cyano-3-propyl ethers15 of
umbelliferone as fluorogenic substrates for lipases and
esterases. These substrates show an approximately 10-
fold reduced chemical sensitivity toward spontaneous
hydrolysis. We also developed a chromogenic assay for
lipases and estereases using the adrenaline test for en-
zymes.16 This setup allowed to use triglycerides or 1,2-
diol and carbohydrate acetates as substrates.17 While
the use of such aliphatic esters resulted in a practically
negligible level of nonspecific hydrolysis, the assay was
not suitable to detect trace amounts of lipases.
By virtue of their molecular structure, enzymes are com-
plex catalysts and their detailed reactivity is very difficult
to predict. Most enzymes of industrial interest are cur-
rently isolated from microbial sources or from genetic
libraries.1 In the search for novel enzymes, it is necessary
to apply functional tests for high-throughput screening
that are capable of detecting enzyme activities with high
selectivity and sensitivity.2 Recently we developed en-
zyme substrates that release either umbelliferone as a
blue-fluorescent product or nitrophenol as a yellow
colored product by b-elimination from an intermediate
carbonyl product formed after the enzyme-catalyzed
step (e.g., Scheme 2). This assay methodology practi-
cally suppresses any background reactivity of the sub-
strates in the absence of enzyme, and allows the
detection of various enzyme types with high selectivity,
including alcohol dehydrogenases,3 aldolase catalytic
antibodies,4 acylases and phosphatases,5 proteases,6 lip-
ases and esterases,3,5 epoxide hydrolases,5,7 transaldo-
lases,8 transketolases,9 and Bayer-Villigerases.10 These
assays may also be applied in parallel using arrays of
structurally diverse substrates to produce enzyme-
specific activity fingerprints.11
Many high-throughput enzyme assays are applied as
tools to visualize a particular enzyme class. A number
of assays have been described for the particular case of
In our search for optimal probes for lipases and
esterases, we later found that long chain aliphatic
monoesters of 7-[3,4-dihydroxybut-1-yloxy]coumarin
provide optimal fluorogenic probes for lipases and est-
erases.18 These substrates liberate 7-hydroxycoumarin
(= umbelliferone) as blue fluorescent product by the
*
Corresponding author. Fax: +41 31 631 80 57; e-mail: jean-louis.
reymond@ioc.unibe.ch
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.07.059

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J. Grognux et al. / Tetrahedron: Asymmetry 15 (2004) 2981–2989
indirect mechanism discussed above using sodium perio-
date and bovine serum albumin as secondary reagents,
and show both minimal spontaneous hydrolysis even
under harsh conditions and very high reactivity against
lipases and esterases. An array of such fluorogenic sub-
strates allowed to classify these enzyme according to
their catalytic functionality.19
1. lipase or
esterase
NO2
OH
1 - 12b
HO
O
X
13 (X = H)
14 (X = NO2)
3. BSA, pH > 7
17, 18
Herein we show that the related esters of 4-(nitrophen-
oxy)-1,2-butanediol and 4-(dinitrophenoxy)-1,2-butane-
diol provide excellent chromogenic probes for lipases
and esterases. The chromogenic mono- and diacyl-gly-
cerol analogues 1–12b were synthesized and analyzed
for their reactivity against a series of lipases and
esterases (Scheme 1). Cluster analysis of the reactivity
patterns observed indicate that monoesters of the nitro-
phenol-derived diol 13 with C8-, C10-, and C12-
aliphatic acids behave as highly reactive, universal
chromogenic probes for lipases and esterases.
2. NaIO4
NO2
O
H
O
X
15 (X = H)
16 (X = NO2)
Scheme 2. Principle of chromogenic periodate coupled assay.
cally with proteins,12 substrates 1–12b were triggered
by cleavage of relatively unreactive aliphatic ester
bonds, ensuring that only enzyme-specific activities
would be revealed.
R
NO2
NO2
OH
O
O
O
R
R
O
O
O
Thirty-three enzymes were tested, including lipases, est-
erases, one acylase, and one epoxide hydrolase. Reac-
tions were carried out in 96-well microtiter plates,
recording all substrates (at concentration of 100lM
each) with each enzyme concentration of 100, 10, and
1lgmLꢀ1. The results were represented as 4 · 5 color-
coded grids for each enzyme, with monoesters of
increasing chain length arranged in the first three rows,
and diesters of increasing chain length arranged in the
two lower rows (Fig. 1). The color was coded using each
of the three color channels red (R), green (G), and blue
(B) with the activities observed with 100, 10, and
1lgmLꢀ1 enzyme sample. In this display one expects
to see blue tones only since the reaction rates should in-
crease with increasing enzyme concentration, even with-
in the limitations imposed by the fact that the secondary
oxidation/b-elimination sequence (Scheme 2, steps 2and
3) becomes rate limiting at high enzyme concentration
for very active enzymes, as discussed previously.5 In
practice several enzyme samples, in particular weakly
active preparations, exhibited their highest reactivities
at the lower enzyme concentrations, probably due to
the presence of impurities, or by an enzyme inhibition
effect caused by aggregation.
X
X
O
O
R=
1a (X = H)
2 (X = H)
1b (X = H)
Me
Pr
3 (X = H), 4a (X = NO2)
5a (X = H)
4b (X = NO2)
5b (X = H)
C7H15
C9H19
C11H23
C13H27
C15H31
C17H35
6 (X = H), 7a (X = NO2)
8 (X = H), 9a (X = NO2)
10a (X = H)
7b (X = NO2)
9b (X = NO2)
10b (X = H)
11b (X = H)
12b (X = H)
11a (X = H)
12a (X = H)
7
7
Scheme 1. Chromogenic substrates for lipases and esterases.
2. Results and discussion
Chromogenic substrates are generally more convenient
than fluorogenic substrates. Firstly, their reactions can
be detected visually, implying that they are suitable for
routine activity control without instruments. Secondly,
UV-spectrometers are more broadly available than fluo-
rometers. We therefore decided to investigate chromo-
genic derivatives of our previously developed
fluorogenic substrates as probes for lipases and
esterases.
The highest lipase reactivities were observed with mono-
acyl substrates of intermediate chain length (C8, C10,
and C12). In fact these substrates detect lipase activity
down to 0.01lgmLꢀ1 for the most active samples, which
in our hands is a 100-fold more sensitive than standard
substrates such as nitrophenyl octanoate. The acetate
substrates 1a/b showed their highest reactivity with acetyl
choline esterase (AchE), in agreement with the known
specificity of this enzyme. The C18-derivatives 11a/b
did not show any significant reactivity with any of the
enzymes, probably due to limited solubility. By contrast
the oleate derivatives 12a/b did show the highest reactiv-
ities of the long chain esters. The most active enzyme
samples were those with highest reported specific activ-
The mono- and diacyl-glycerol analogs esters 1–12b of
the glycerol related diols 13 and 14 with aliphatic esters
of varying chain length were prepared by mono- or
diesterification with the acyl chlorides. The chromogenic
reaction followed the oxidation/b-elimination sequence
used in our fluorogenic substrate arrays via the interme-
diate aldehydes 15/16 to produce the colored nitrophe-
nolate 17 and dinitrophenolate 18 as detectable
products (Scheme 2). By contrast to the highly reactive
esters of nitrophenol, which tend to react non-specifi-

J. Grognux et al. / Tetrahedron: Asymmetry 15 (2004) 2981–2989
2983
Figure 1. Enzyme activity patterns against chromogenic lipases substrates. Reaction conditions: 100lM substrate, 5% v/v DMF in aq 20mM borate
buffer pH8.8, 1mM NaIO4, 2mgmLꢀ1 BSA, 26ꢁC. Reactions (0.1mL each) were followed spectrophotometrically at 405nm over 1h in 96-well
polystyrene half-area cell-culture plates (Corning–Costar) using a Spectramax 250 microtiter plate reader (Molecular Devices, Inc.). The steepest
linear portion of each time curve was used to calculate the apparent reaction rate according to a calibration curve with pure p-nitrophenol or 2,4-
dinitrophenol. Enzymes are arranged according to clustering (Fig. 2 and text). The background reaction rates in the absence of enzyme was negligible
(<100pMsꢀ1) for all substrates. The display color for each square in each array is defined by setting the intensities of fundamental colors red (R),
green (G) and blue (B) in proportion to the relative rates observed at 100, 10, and 1lgmLꢀ1 with the given enzyme. Below each array: enzyme code,
activity from manufacturerÕs data, and reaction rate observed for the fastest reaction in the array in pMsꢀ1. The key at bottom left represents six
incremental steps in color intensity. Actual colors are generated proportionally to actual rates, and span the entire color intensity spectrum. Intensity
coefficients F of fundamental colors are calculated from relative rates (Vrel) as FR = 255(1 ꢀ Vrel (100lgmLꢀ1), FG = 255(1 ꢀ Vrel (10lgmLꢀ1)),
FB = 255(1 ꢀ Vrel (1lgmLꢀ1)). Upper left triangles in each 6 · 6 color grid indicate colors generated from the data for Ôwell-behavedÕ systems in
which V(100lgmLꢀ1) > V(10lgmLꢀ1) > V(1lgmLꢀ1). PSBL = Pseudomonas sp. type
B lipoprotein lipase (F62336), PFL = Pseudomonas
fluorescens lipase (F62321), CAL = Candida antarctica lipase (F62299), PCL2 = Pseudomonas cepacia lipase (F62309), PSL1 = Pseudomonas sp.
lipoprotein lipase (SL-9656), PSL2= Pseudomonas sp. lipoprotein lipase (F62335), CVL = Chromobacterium visc. lipoprotein lipase (F62333),
ANL = Aspergillus niger lipase (A39,043-7), PCL1 = Pseudomonas cepacia lipase (F62312), MML = Mucor miehei lipase (F62298), CCL = Candida
cylindracea lipase (F62316), HPL = hog pancreatic lipase (F62300), RAL = Rhizopus arrhizus lipase (F62305), AOL = Aspergillus oryzae lipase
(F62285), RML = Rhizomucor miehei lipase (F62291), MJL = Mucor javanicus lipase (F62304), RNL = Rhizopus niveus lipase (F62310),
PRL = Penicillium roqueforti lipase (F62308), WGL = wheat germ lipase (F62306), CLL = Candida lipolytica lipase (F62303), PLE = pig liver
esterase (F46058), HLE = horse liver esterase (F46069), CLE = Candida lipolytica esterase (F46056), MME = Mucor miehei esterase (F46059),
BTE = Bacillus thermoglucosidasius esterase (F46054), BstE = Bacillus stearothermophilus esterase, (F46051), BSE = Bacillus sp. esterase (F46062),
TBE = Thermoanaerobium brockii esterase (F46061), SCE = Saccharomyces cerevisiae esterase (F46071), AChE = Electrophorus electricus acetyl
choline esterase (F01023), HKA = hog kidney acylase I (F01821), REH = Rhodotorula glutinis epoxide hydrolase.
ity (U/mg). These samples also showed a clear protein
band on SDS-PAGE analysis. Octanoate 3 (position
A3) and decanoate 5a (position B1) appeared as the fast-
est substrates with lipases. This high reactivity also
meant that the activities observed in crude samples of
non-lipase enzymes such as acylase (HKA) and epoxide
hydrolase (REH) most likely indicated the presence of
lipase or esterase impurities.
enzyme activity fingerprint was then decomposed in a
series of relative activities for each substrate, such that
the total reactivity for each enzyme would total 100%
across all substrates measured. The analysis of relative
rather than absolute reaction rates circumvents the
problem of determining the absolute amount of active
enzyme in the sample, as well as that of finding a sub-
strate that reacts with all enzymes to assign absolute
activity units to each enzyme.19
Cluster analysis was performed to gain a systematic in-
sight into the measured reactivity profiles. In order to
circumvent the observed problem of enzyme concentra-
tion dependent enzyme activities, each enzyme/substrate
pair was assigned a total reactivity corresponding to the
sum of reaction rates observed with the substrate at the
three different enzyme concentrations measured. Each
The dataset was processed using the multivariate ana-
lysis softwares WINIDAMS and VISTA.20 Clustering was
carried out using WardÕs method on the basis of stand-
ardized euclidean distances. The 20 substrates were
defined as variables and the 32enzymes tested as
observations. Cluster analysis is based on algorithms,

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J. Grognux et al. / Tetrahedron: Asymmetry 15 (2004) 2981–2989
which group observations on the basis of their spatial
proximity in an N-dimensional space defined by N var-
iables.21 The enzyme series thus ordered was represented
as a color-coded representation of the symmetrical dis-
similarity matrix of relative distances (Fig. 2).
ple showing an enhanced reactivity with the C16- and
oleic acid monoesters 10a and 12a.
3. Conclusion
Analysis of the clustered enzyme set according to the
dissimilarity matrix showed a relatively large cluster of
14 similar enzymes at the center (AOL to CCL in Fig.
2). The reactivity pattern in this cluster corresponded
to that of the four most reactive lipase samples in the
series (PFL, CVL, and two samples of PSL), which
was characterized by a very strong reactivity with the
4-nitrophenoxy-derived monoesters 3 (C8), 5a (C10), 6
(C12), 8 (C14), 10a (C16), and 12a (oleate). Two addi-
tional clusters appeared at upper left (HKA to RgEH)
and at lower right (BTE to CLL) containing mostly
esterases. The first cluster was characterized by a very
strong reactivity with the nitrophenyl derived C8- and
C10-monoesters 3 and 5a over the other substrates,
while the second cluster contained enzymes with gener-
ally very low and distributed reactivities across most
substrates. The other enzymes appeared singular in their
reactivity pattern, including acetyl choline esterase
(AchE) reacting more strongly with the mono- and diac-
etate esters 1a and 1b, and the weakly active RNL sam-
The experiments above demonstrate that aliphatic esters
of the periodate-activated chromogenic diols 13 and 14
are sensitive probes for lipase and esterase activities
releasing nitrophenol and dinitrophenol, respectively.
The mono- and diacetate derivatives exhibit a similarly
selective reactivity for acetyl choline esterase (AchE).
The monoesters with longer chain aliphatic acids show
remarkable reactivities with both lipases and esterases.
The most useful lipase probes are the C8-, C10-, and
C12-monoesters of the nitrophenol-derived diol 13.
The corresponding dinitrophenol derived esters of diol
14 are generally much less reactive against lipases, which
is probably caused by steric hindrance limiting access to
the enzyme active sites. These reactivity differences are
largely conserved across all enzymes tested.
The nitrophenol-derived substrates 3 (C8), 5a (C10), and
6 (C12) are optimal to detect lipase and esterase activi-
ties with high sensitivity in unknown samples, and it is
reasonable to assume that these substrates will react
Figure 2. Color-coded representation of the dissimilarity matrix of the enzyme series according to their relative activity fingerprints across the
substrates series 1a–12b. Black = identity; white = maximum distance. The actual highest level (dissimilarity) is 0.376, and the agglomerative
coefficient is 0.75.

J. Grognux et al. / Tetrahedron: Asymmetry 15 (2004) 2981–2989
2985
with any lipase or esterase. Such a universal chromo-
genic lipase and esterase assay can be extremely useful
for enzyme discovery during screening. For example,
we have used the C10-ester 5a to discover microbial
thermophilic lipases by direct screening.22 Evidently, it
must then be coupled with a more detailed assay proto-
col to test the enzyme for reactivity and selectivity,
including stereo- and enantioselectivity, against particu-
lar substrates of synthetic interest.
1333 (s), 1303 (m), 1265 (s), 1243 (m), 1176 (m), 1124
(m), 1105 (m), 1067 (m), 1046 (m) ; Elemental analysis:
calcd for C10H13NO5: C, 52.86%, H, 5.77%, N, 6.16%.
Found: C, 52.55%, H, 5.91%, N, 5.90%
4.4. ( )-1,2-O-Isopropylidene-4-(2,4-dinitrophenyloxy)-
1,2-butanediol 20
1,2-O-isopropylidene-1,2,4-butanetriol (0.5g, 3.4mmol)
was treated with 2,4-dinitro fluorobenzene (636mg,
3.42mmol) and triethylamine (0.53mL, 3.76mmol).
The dark brown solution was stirred for 6h at 25ꢁC.
Aqueous workup (diethyl ether/satd aq NaHCO3, then
brine) and FC (hexane/AcOEt 3/1–1/1) gave 20 (0.66g,
2.00mmol, 58%) as yellow oil; TLC (Hex/EtOAc 1/1)
4. Experimental
4.1. General
1
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 solution in
95% EtOH. Silica gel 60 (Macherey–Nagel 230–
400mesh) was used for flash chromatography (FC).
Melting points were determined on a Kofler apparatus
Rf 0.61; H NMR (300MHz, CDCl3) d (ppm): 8.74 (d,
1H), 8.43 (dd, 1H), 7.24 (d, 1H), 4.36 (m, 3H), 4.14
(m, 1H), 3.67 (m, 1H), 2.12 (m, 2H), 1.41 (s, 3H), 1.35
(s, 3H). MS (EI) m/z: 313 (M++1), 297, 167.
4.5. ( )-4-(2,4-Dinitrophenyloxy)-1,2-butanediol 14
or with a Buchi 510 apparatus and are uncorrected.
¨
1H NMR and 13C NMR spectra were recorded with a
Bruker AC-300 or a BRUKER DMX 400 spectrometer.
The procedure for 13 was applied with 19 (0.6g,
1.92mmol) using MeOH (6mL) and 0.1N aq HCl
(6mL). FC (acetone/hexane 2/1, Rf 0.68) gave 14
(520mg, 1.91mmol, 99%) as pale yellow solid,
4.2. ( )-1,2-Isopropylidene-4-(4-nitrophenyloxy)-1,2-
butanediol 19
1
mp 74ꢁC; H NMR (300MHz, CDCl3) d (ppm): 8.78
(d, 1H, J = 2.9Hz), 8.44 (dd, 1H, J = 2.6 and 9.2Hz),
7.26 (d, 1H, J = 9.5Hz), 4.45 (m, 2H), 4.05 (m, 1H),
3.76 (dd, 1H, J = 11.0 and 3.3Hz), 3.57 (dd, 1H,
J = 11.0 and 6.6Hz), 2.05 (m, 2H); 13C NMR
(75MHz, CDCl3) d (ppm): 157.91, 141.32, 140.24,
130.15, 122.35, 116.22, 69.62, 68.80, 67.32, 33.61; IR
(KBr) cmꢀ1: 3384 (b, s), 3116 (m), 2932 (m), 2889 (m),
1610 (s), 1529 (s), 1348 (s), 1289 (s); MS (EI) m/z: 241,
184, 168, 154.
A solution of ( )-1,2-isopropylidene-4-(4-toluene-sulfo-
nate)-1,2,4-butanetriol23 (5g, 16.6mmol), 4-nitrophenol
17 (2.780g, 20mmol), K2CO3 (4.600g, 33.2mmol), and
18-C-6 (0.300g, 1.1mmol) in 100mL acetone is stirred
under reflux for 18h. Acetone is then removed in vacuo,
and the residue taken in EtOAc and water. The organic
phase is washed with 0.1N NaOH and brine, dried over
MgSO4 and concentrated to dryness. Crude 19 (5.440g)
is used without further purification for the next step.
TLC (Hex/EtOAc 75/25, UV 254nm): Rf 0.49; 1H
NMR (300MHz, CDCl3) d (ppm): 8.20 (d, J = 9.2Hz,
2H), 6.95 (d, J = 9.2Hz, 2H), 4.35–4.27 (m, 1H), 4.22–
4.11 (m, 3H), 3.68–3.63 (m, 1H), 2.11–2.04 (m, 2H),
1.43 (s, 3H), 1.36 (s, 3H).
4.6. ( )-(4-(p-Nitrophenyloxy-2-hydroxybut-1-yl)-buty-
rate 2
A solution of diol 13 (100mg, 0.44mmol) and dry trieth-
ylamine (89.0mg, 0.88mmol) in dry CH2Cl2 (6mL) and
dry DMF (1mL) at 0ꢁC was treated with the butyroyl
chloride (46lL, 0.44mmol). The reaction mixture was
stirred at 0ꢁC for 1h and then at 25ꢁC until completion
of the reaction (TLC). Aqueous workup (CH2Cl2/satd
aq NaHCO3 and brine), and FC (hexane/AcOEt 3/1–1/
1) gave 2 (73mg, 0.25mmol, 56%) as pale yellow oil;
TLC (Hex/EtOAc 1/1) Rf 0.65; 1H NMR (300MHz,
CDCl3) d (ppm): 8.20 (d, 2H), 6.96 (d, 2H), 4.04–4.29
(m, 5H), 2.34 (m, 2H), 1.99 (m, 2H), 1.68 (m, 2H),
0.97 (m, 3H); 13C NMR (75MHz, CDCl3) d (ppm):
174.50, 164.43, 142.25, 126.57, 115.12, 68.97, 67.69,
65.83, 36.67, 33.30, 19.06, 14.28; IR (film) cmꢀ1: 3525
(b, w), 2957 (s), 2927 (s), 2851 (m), 1732 (m), 1593
(m), 1510 (s), 1342(s), 1263 (s); MS (FAB) m/z: 2 98
(M+), 281, 210, 155, 119.
4.3. ( )-4-(4-Nitrophenyloxy)-1,2-butanediol 13
A solution of 19 (5.440g) in 60mL MeOH is degassed
with nitrogen for 1h. A solution of 0.1N HCl (45mL)
is added, and the mixture is further degassed for
30min. After 18h stirring at rt under N2, the solvent is
removed in vacuo and the residue taken in EtOAc,
washed with satd aq NaHCO3 and brine, dried over
MgSO4, and concentrated. The crude residue is recrys-
tallized in EtOAc/Hex to give 3.480g (15.3mmol, 92%
from 19) of ( )-4-(4-nitrophenoxy)-1,2-butanediol 13
as a yellow solid, mp 98–100ꢁC; TLC (EtOAc/Hex, 65/
1
35, UV 254nm): Rf 0.16; H NMR (200MHz, CD3OD)
d (ppm): 8.2(d, J = 9.3Hz, 2H), 7.1 (d, J = 9.3Hz, 2H),
4.37–4.14 (m, 2H), 3.93–3.76 (m, 1H), 3.53 (d,
J = 5.9Hz, 2H), 2.19–1.97 (m, 1H), 1.93–1.72 (m, 1H);
13C NMR (75MHz, CD3OD) d (ppm): 165.7, 142.7,
126.8, 115.8, 69.9, 67.4, 66.8, 33.9; IR (KBr) m (cmꢀ1):
3380 (s), 3275 (s), 3081 (w), 2964 (w), 2926 (w), 2880
(w), 1610 (s), 1597 (s), 1513 (s), 1499 (s), 1389 (m),
4.7. ( )-(4-(4-p-Nitrophenyloxy-2-hydroxybut-1-yl)-
octanoate 3
The procedure for 2 was applied using octanoyl chloride
(76lL, 0.44mmol). FC gave 3 (97mg, 0.27mmol, 62%)

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J. Grognux et al. / Tetrahedron: Asymmetry 15 (2004) 2981–2989
as pale yellow solid, mp 38ꢁC; TLC (Hex/EtOAc 1/1) Rf
0.68; 1H NMR (300MHz, CDCl3) d (ppm): 8.20 (d, 2H),
6.96 (d, 2H), 4.04–4.32 (m, 5H), 2.36 (m, 2H), 1.89–2.08
(m, 2H), 1.64 (m, 2H), 1.28 (m, 8H), 0.88 (m, 3H); 13C
NMR (75MHz, CDCl3): 174.67, 164.43, 142.23,
126.54, 115.10, 68.97, 67.65, 65.83, 34.80, 33.30, 32.26,
29.71, 29.52, 25.56, 23.20, 14.66; IR (film) cmꢀ1: 3505
(b, w), 2957 (m), 2930 (m), 2858 (m), 1731 (m), 1594
50) to give compounds 5a (1.503g, 83%) and 5b
(0.117g, 5%).
Compound 5a: yellow solid, mp 41ꢁC; TLC (Hex/
EtOAc 50/50, UV 254nm): Rf 0.80; 1H NMR
(300MHz, CDCl3) d (ppm): 8.19 (d, J = 8.5Hz, 2H),
6.95 (d, J = 8.8Hz, 2H), 4.31–4.04 (m, 5H), 2.38–2.33
(m, 2H), 2.09–1.89 (m, 2H), 1.66–1.61 (m, 2H), 1.28–
1.27 (m, 12H), 0.89–0.85 (m, 3H); 13C NMR (75MHz,
CDCl3) d (ppm): 174.7, 164.4, 142.3, 126.6, 115.1,
69.0, 67.7, 65.8, 34.8, 33.3, 32.5, 30.1, 29.9, 25.6, 23.3,
14.8; IR (neat) m (cmꢀ1): 3479 (w), 2928 (s), 2856 (s),
1737 (s), 1609 (m), 1595 (s), 1515 (s), 1499 (s), 1468
(m), 1343 (s), 1299 (m), 1264 (s), 1174 (s), 1112 (s);
HR-ESI-MS: calcd for C10H32NO6+: 382.2229. Found:
382.2221.
(s), 1512(s), 1342(s), 1624 (s); MS (FAB)
m/z: 354
(M+), 337, 215, 210, 127.
4.8. ( )-(4-(2,4-Dinitrophenyloxy)-2-hydroxybut-1-yl)-
octanoate 4a and ( )-(4-(2,4-dinitrophenyloxy)-2-octa-
noyloxybut-1-yl)-octanoate 4b
A solution of diol 14 (100mg, 0.37mmol), triethylamine
(153lL, 1.1mmol) and 4-dimethylamino-pyridine
(2.5mg, 0.02mmol) in CH2Cl2 (6mL) was treated at
0ꢁC with octanoyl chloride (95lL, 0.55mmol). The
reaction mixture was stirred for 1h at 0ꢁC, then at
25ꢁC until completion of the reaction (5h, TLC). Aque-
ous workup (CH2Cl2/satd aq NaHCO3, then brine) and
separation by FC (hexane/AcOEt 3/1–1/1) gave first 4b
(36mg, 0.07mmol, 18%) as pale yellow oil, then 4a
(74mg, 0.19mmol, 50%) as pale yellow oil. Compound
4a: TLC (Hex/EtOAc 1/1) Rf 0.50; 1H NMR
(300MHz, CDCl3) d (ppm): 8.77 (d, 1H), 8.44 (dd,
1H), 7.25 (d, 1H), 4.06–4.52 (m, 5H), 2.36 (m, 2H),
2.06 (m, 2H), 1.64 (m, 2H), 1.28 (m, 8H), 0.87 (m,
3H); 13C NMR (75MHz, CDCl3) d (ppm): 174.81,
157.29, 140.80, 139.35, 129.94, 122.69, 115.07, 68.85,
67.99, 67.37, 34.79, 32.95, 32.28, 29.73, 29.55, 25.55,
23.23, 14.70; IR (film) cmꢀ1: 3565 (b, m), 3094 (w),
2956 (s), 2930 (s), 2858 (s), 1732 (s), 1610 (s), 1538 (s),
1344 (s), 1288 (s); MS (FAB) m/z: 399 (M++1), 381,
369, 215, 127.
Compound 5b: yellow oil; TLC (Hex/EtOAc 90/10, UV
254nm): Rf 0.30; H NMR (300MHz, CDCl3) d (ppm):
1
8.19 (d, J = 7.0Hz, 2H), 6.93 (d, J = 7.3Hz, 2H), 5.32
(m, 1H), 4.36–4.31 (m, 1H), 4.17–4.08 (m, 3H), 2.35–
2.27 (m, 4H), 2.15–2.13 (m, 2H), 1.64–1.56 (m, 6H),
1.26–1.24 (m, 24H), 0.89–0.85 (m, 6H); 13C NMR
(75MHz, CDCl3) d (ppm): 174.1, 173.8, 164.2, 142.4,
126.6, 115.1, 69.2, 65.4, 65.3, 35.0, 34.8, 32.5, 31.2,
30.1, 29.9, 29.8, 25.6, 23.3, 14.7; IR (neat) m (cmꢀ1):
2928 (s), 2856 (s), 1741 (s), 1610 (m), 1595 (s), 1518
(s), 1499 (m), 1468 (m), 1343 (s), 1299 (m), 1263 (s),
1173 (s), 1112(s); HR-EI-MS: calcd for C 30H49NO7:
535.3509. Found: 535.3526.
4.10. ( )-(4-(4-Nitrophenyloxy-2-hydroxybut-1-yl)-dode-
canoate 6
The procedure for 2 was applied using dodecanoyl chlo-
ride (104lL, 0.44mmol). FC gave 6 (105mg, 0.26mmol,
58%) as pale yellow solid, mp 48ꢁC; TLC (Hex/EtOAc
1/1) Rf 0.70; 1H NMR (300MHz, CDCl3) d (ppm):
8.20 (d, 2H), 6.96 (d, 2H), 4.04–4.30 (m, 5H), 2.34 (m,
2H), 1.99 (m, 2H), 1.64 (m, 2H), 1.25 (m, 16H), 0.85
(t, 3H); 13C NMR (75MHz, CDCl3) d (ppm): 174.72,
164.41, 142.26, 126.60, 115.11, 69.01, 67.72, 65.81,
34.83, 33.28, 32.56, 30.25, 30.10, 29.98, 29.91, 29.80,
25.59, 23.34, 14.77; IR (film) cmꢀ1: 3505 (b, w), 3028
(w), 2927 (s), 2855 (m), 1731 (s), 1594 (s), 1512 (s),
1342(s), 1263 (s); MS (FAB) m/z: 410 (M+), 393, 271,
210, 183, 119.
Compound 4b: TLC (Hex/EtOAc 1/1) Rf 0.83; 1H NMR
0
(300MHz, CDCl3) d (ppm): 8.76 (d, 1H, H3 ), 8.42(dd,
1H), 7.17 (d, 1H), 5.30 (m, 1H), 4.08–4.36 (m, 4H), 2.30
(m, 4H), 2.20 (m, 2H), 1.60 (m, 4H), 1.26 (m, 16H), 0.87
(m, 6H); 13C NMR (75MHz, CDCl3) ppm: 174.04,
173.86, 157.03, 141.01, 139.65, 129.74, 122.69, 114.93,
69.14, 67.74, 65.17, 34.95, 34.77, 32.33, 31.22, 29.74,
29.58, 25.61, 25.56, 23.26, 14.71; IR (film) cmꢀ1: 3031
(w), 2957 (m), 2930 (s), 2858 (m), 1736 (s), 1609 (s),
+
1534 (s), 1345 (s), 1288 (s); MS (FAB) m/z: 52 5 (M),
495, 381, 341, 127.
4.11. ( )-(4-(2,4-Dinitrophenyl)-2-hydroxybut-1-yl)-
dodecanoate 7a and ( )-(4-(2,4-dinitrophenyl)-2-dodeca-
noyloxy-but-1-yl)-dodecanoate 7b
4.9. ( )-(4-(4-Nitrophenyloxy)-2-hydroxybut-1-yl)-decan-
oate 5a and ( )-(4-(4-nitrophenyloxy)-2-decanoyloxybut-
1-yl)-decanoate 5b
The procedure for 5a/b was applied using dodecanoyl
chloride (133lL, 0.56mmol). FC gave first 7b (70.3
mg, 0.11mmol, 30%) as yellow solid, mp 35ꢁC, then
7a (91mg, 0.2mmol, 54%) as yellow solid, mp 42 ꢁC.
Triethylamine (1.4mL, 9.5mmol) is added under N2 to a
solution of ( )-4-(4-nitrophenoxy)-1,2-butanediol 13
(1.080g, 4.75mmol) in dry DCM (40mL) and DMF
(10mL), and the mixture is cooled to 0ꢁC. Capric acid
chloride (1.200g, 6.3mmol) is then added, and the mix-
ture stirred at rt for 6h. After concentration to dryness
the crude product is taken in EtOAc and water, washed
with satd aq NaHCO3 and brine, dried over MgSO4 and
concentrated. The residue is finally purified by flash
chromatography on silica gel (Hex/EtOAc 75/25–50/
Compound 7a: TLC (Hex/EtOAc 1/1) Rf 0.44; 1H NMR
(300MHz, CDCl3) d (ppm): 8.78 (d, 1H), 8.44 (dd, 1H),
7.24 (d, 1H), 4.06–4.52 (m, 5H), 2.36 (m, 2H), 2.05 (m,
2H), 1.62 (m, 2H), 1.25 (m, 16H), 0.86 (m, 3H); 13C
NMR (75MHz, CDCl3) ppm: 174.78, 157.28, 140.86,
139.44, 129.87, 122.66, 115.09, 66.85, 68.03, 67.42,

J. Grognux et al. / Tetrahedron: Asymmetry 15 (2004) 2981–2989
2987
34.81, 33.01, 32.54, 30.24, 30.09, 29.96, 29.89, 29.79,
25.57, 23.32, 14.74; IR (film) cmꢀ1: 3566 (b, w), 2927
(s), 2855 (m), 1734 (m), 1610 (s), 1538 (s), 1344 (s),
1288 (s); MS (FAB) m/z: 455 (M+), 437, 425, 271, 183,
109.
69.16, 67.75, 65.17, 34.98, 34.78, 32.61, 31.23, 30.34,
30.18, 30.04, 29.97, 29.82, 25.64, 25.57, 23.37, 14.79;
IR (film) cmꢀ1: 3031 (w), 2925 (s), 2854 (s), 1737 (s),
1609 (s), 1534 (s), 1344 (s), 1288 (s); MS (FAB) m/z:
693 (M+), 663, 607, 509, 465, 284.
Compound 7b: TLC (Hex/EtOAc 1/1) Rf 0.88; 1H NMR
(300MHz, CDCl3) d (ppm): 8.76 (d, 1H), 8.42(dd, 1H),
7.17 (d, 1H), 5.30 (m, 1H), 4.12–4.36 (m, 4H), 2.31 (m,
4H), 2.21 (m, 2H), 1.59 (m, 4H), 1.24 (m, 32H), 0.87
(m, 6H); 13C NMR (75MHz, CDCl3) d (ppm): 174.02,
173.84, 157.02, 140.99, 139.41, 129.72, 122.66, 114.94,
69.14, 67.73, 65.15, 34.95, 34.87, 34.76, 32.57, 31.20,
30.28, 30.15, 30.00, 29.94, 29.89, 29.81, 29.79, 25.61,
25.55, 23.34, 14.76; IR (film) cmꢀ1: 3094 (w), 2927 (s),
2855 (s), 1736 (s), 1609 (s), 1534 (s), 1345 (s), 1287 (s);
MS (FAB) m/z: 636 (M+), 618, 606, 453, 183.
4.14. ( )-(4-(4-Nitrophenyloxy)-2-hydroxybut-1-yl)-hexa-
decanoate 10a and ( )-(4-(4-nitrophenyloxy)-2-hexa-
decanoyloxybut-1-yl)-hexadecanoate 10b
Triethylamine (0.7mL, 4.5mmol) and DMAP (0.0025g,
0.02mmol) are added under N2 to a solution of ( )-4-(4-
nitrophenoxy)-1,2-butanediol 13 (0.500g, 2.2mmol) in
dry DCM (20mL) and DMF (5mL), and the mixture
is cooled to 0ꢁC. Palmitoyl chloride (0.910g, 3.3mmol)
is then added, and the mixture stirred at rt for 18h.
After concentration to dryness the crude product is ta-
ken in EtOAc and water, washed with satd aq NaHCO3,
and brine, dried over MgSO4 and concentrated. The res-
idue is finally purified by flash chromatography on silica
gel (Hex/EtOAc 100/0–50/50) to give compounds 10a
(0.808g, 79%) and 10b (0.032g, 2%).
4.12. ( )-(4-(4-Nitrophenyloxy-2-hydroxybut-1-yl)-tet-
radecanoate 8
The procedure for 2 was applied using tetradecanoyl
chloride (119lL, 0.44mmol). FC gave 8 (126mg,
0.29mmol, 65%) as pale yellow solid, mp 81ꢁC; TLC
Compound 10a: yellow oil; TLC (Hex/EtOAc 50/50, UV
1
1
(Hex/EtOAc 1/1) Rf 0.77; H NMR (300MHz, CDCl3)
254nm): Rf 0.80; H NMR (300MHz, CDCl3) d (ppm):
d (ppm): 8.20 (d, 2H), 6.96 (d, 2H), 4.04–4.29 (m, 5H),
2.36 (t, 2H), 1.98 (m, 2H), 1.64 (m, 2H), 1.25 (m,
20H), 0.88 (t, 3H); 13C NMR (75MHz, CDCl3) ppm:
174.70, 164.42, 142.33, 126.61, 115.13, 69.03, 67.79,
65.85, 34.85, 33.32, 32.58, 30.31, 30.13, 30.01, 29.92,
29.82, 25.61, 23.35, 14.77; IR (film) cmꢀ1: 3506 (b, w),
2926 (s), 2854 (m), 1735 (m), 1594 (m), 1516 (s), 1342
(s), 1263 (s); MS (FAB) m/z: 438 (M+), 421, 299, 284,
211, 124.
8.18 (d, J = 9.2Hz, 2H), 6.95 (d, J = 9.2Hz, 2H), 4.28–
4.03 (m, 5H), 2.37–2.32 (t, J = 7.5Hz, 2H), 2.03–1.93
(m, 2H), 1.65–1.61 (m, 2H), 1.25–1.23 (m, 24H), 0.89–
0.85 (t, J = 6.6Hz, 3H); 13C NMR (75MHz, CDCl3) d
(ppm): 174.7, 164.4, 142.3, 126.6, 115.1, 69.0, 67.7,
65.8, 34.8, 33.3, 32.6, 30.3, 30.2, 30.2, 30.0, 29.9, 29.8,
25.6, 23.3, 14.8; IR (neat) m (cmꢀ1): 3479 (m), 2926 (s),
2855 (s), 1736 (s), 1609 (m), 1595 (s), 1515 (s), 1499
(s), 1468 (m), 1343 (s), 1264 (s), 1174 (s), 1112 (s).
4.13. ( )-(4-(2,4-Dinitrophenyl)-2-hydroxybut-1-yl)-tet-
radecanoate 9a and ( )-(4-(2,4-dinitrophenyl)-2-tetra-
decanoyloxybut-1-yl)-tetradecanoate 9b
Compound 10b: yellow solid, mp 50–51ꢁC; TLC (Hex/
EtOAc 90/10, UV 254nm): Rf 0.38; 1H NMR
(300MHz, CDCl3) d (ppm): 8.19 (d, J = 9.5Hz, 2H),
6.93 (d, J = 9.5Hz, 2H), 5.36–5.31 (m, 1H), 4.32 (dd,
J = 3.7, 12.1Hz, 1H), 4.16–4.08 (m, 3H), 2.33–2.27 (m,
4H), 2.17–2.10 (m, 2H), 1.63–1.57 (m, 4H), 1.25–1.23
(m, 48H), 0.90–0.85 (t, J = 6.6Hz, 6H); 13C NMR
(75MHz, CDCl3) d (ppm): 173.8, 164.2, 145.6, 126.6,
115.1, 69.2, 65.4, 65.3, 35.0, 34.8, 32.6, 31.2, 30.4, 30.2,
30.0, 29.8, 25.7, 25.6, 23.4, 14.8; IR (Chl) m (cmꢀ1):
3025 (w), 2926 (s), 2855 (s), 1741 (s), 1610 (m), 1595
(m), 1518 (m), 1499 (m), 1468 (m), 1343 (s), 1263 (s),
The procedure for 5a/b was applied using diol 14 (60mg,
0.24mmol), triethylamine (100lL, 0.72mmol), DMAP
(1.5mg, 0.01mmol) in CH2Cl2 (4mL). FC gave first 9b
(46mg, 0.07mmol, 27%) as yellow solid, mp 44ꢁC, then
9a (49mg, 0.1mmol, 42%) as yellow solid, mp 53ꢁC.
Compound 9a: TLC (Hex/EtOAc 1/3) Rf 0.82; 1H NMR
(300MHz, CDCl3) d (ppm): 8.78 (d, 1H), 8.45 (dd, 1H),
7.24 (d, 1H), 4.06–4.52 (m, 5H), 2.36 (m, 2H), 2.05 (m,
2H), 1.63 (m, 2H), 1.26 (m, 20H), 0.87 (m, 3H); 13C
NMR (75MHz, CDCl3) ppm: 174.80, 157.29, 140.91,
136.74, 129.88, 122.69, 115.08, 68.89, 68.03, 67.46,
34.82, 33.03, 32.58, 30.34, 30.30, 30.26, 30.11, 30.01,
29.92, 29.82, 25.59, 23.35, 14.76; IR (film) cmꢀ1: 3467
(b, w), 3090 (w), 3026 (w), 2926 (s), 2854 (s), 1732 (s),
1610 (s), 1538 (s), 1345 (s), 1289 (s); MS (FAB) m/z:
465(M++1ꢀOH), 453, 299, 211, 167, 149, 113.
Compound 9b: TLC (Hex/EtOAc 1/3) Rf 0.97; 1H NMR
(300MHz, CDCl3) d (ppm): 8.76 (d, 1H), 8.42(dd, 1H),
7.17 (d, 1H), 5.30 (m, 1H), 4.08–4.36 (m, 4H), 2.31 (m,
4H), 2.19 (m, 2H), 1.58 (m, 4H), 1.25 (m, 40H), 0.88
(m, 6H); 13C NMR (75MHz, CDCl3) d (ppm): 174.05,
173.87, 157.03, 140.44, 139.67, 129.74, 122.70, 114.93,
+
1173 (m), 1112(m) ; EI-MS: 704 [M] .
4.15. ( )-(4-(4-Nitrophenyloxy)-2-hydroxybut-1-yl)-octa-
decanoate 11a and ( )-(4-(4-nitrophenyloxy)-2-octadeca-
noyloxybut-1-yl)-octadecanoate 11b
Triethylamine (1.4mL, 9.5mmol) and DMAP (0.0025g,
0.02mmol) are added under N2 to a solution of ( )-4-(4-
nitrophenoxy)-1,2-butanediol 13 (0.500g, 2.2mmol) in
dry DCM (20mL) and DMF (5mL), and the mixture
is cooled to 0ꢁC. Stearoyl chloride (1g, 3.3mmol) is then
added, and the mixture stirred at rt for 18h. After con-
centration to dryness the crude product is taken in
EtOAc and water, washed with satd aq NaHCO3 and
brine, dried over MgSO4, and concentrated. The residue
is finally purified by flash chromatography on silica gel

2988
J. Grognux et al. / Tetrahedron: Asymmetry 15 (2004) 2981–2989
(Hex/EtOAc 90/10–50/50) to give compounds 11a
(0.680g, 63%) and 11b (0.206g, 12%).
Compound 12b: yellow oil; TLC (Hex/EtOAc 90/10, UV
254nm): Rf 0.38; H NMR (300MHz, CDCl3) d (ppm):
1
8.19 (d, J = 9.2Hz, 2H), 6.93 (d, J = 9.2Hz, 2H), 5.37–
5.32(m, 5H), 4.33 (dd, J = 3.7, 12.1Hz, 1H), 4.17–4.08
(m, 3H), 2.34–2.27 (m, 4H), 2.17–2.10 (m, 2H), 2.01–
1.99 (m, 8H), 1.61–1.57 (m, 4H), 1.30–1.26 (m, 40H),
0.90–0.85 (t, J = 6.6Hz); 13C NMR (75MHz, CDCl3) d
(ppm): 173.8, 164.2, 142.1, 130.8, 130.4, 130.3, 126.6,
115.1, 69.2, 65.3, 35.0, 34.8, 32.6, 30.4, 30.2, 30.0, 29.9,
29.8, 27.9, 25.6, 23.4, 14.8; IR (neat) m (cmꢀ1): 2928 (s),
2857 (m), 1737 (m), 1646 (s), 1518 (w), 1499 (w), 1467
(w), 1343 (w), 1263 (w), 1216 (w), 1174 (w), 1112 (w).
Compound 11a: yellow oil; TLC (Hex/EtOAc 50/50, UV
254nm): Rf 0.80; H NMR (300MHz, CDCl3) d (ppm):
1
8.17 (d, J = 9.2Hz, 2H), 6.94 (d, J = 9.2Hz, 2H), 4.27–
4.05 (m, 5H), 2.36–2.31 (m, 2H), 2.00 (m, 2H) 1.64–
1.59 (m, 2H), 1.25–1.21 (m, 28H), 0.87–0.83 (m, 3H);
13C NMR (75MHz, CDCl3) d (ppm): 174.7, 164.4,
126.6, 115.1, 69.0, 67.8, 65.8, 65.3, 35.1, 34.9, 33.3,
32.6, 30.9, 30.4, 30.3, 30.1, 30.0, 29.9, 29.8, 25.7, 25.6,
23.4, 14.8; IR (neat) m (cmꢀ1): 3492(w), 302(w),
2927 (s), 2855 (s), 1736 (s), 1609 (m), 1595 (s), 1515
(s), 1499 (s), 1468 (m), 1343 (s), 1264 (s), 1174 (s),
1113 (s); EI-MS: 493 [M]+; HR-EI-MS: calcd for
C28H47NO6: 493.3403. Found: 493.3403.
5. Enzyme measurements
All substrates were diluted from 1mM stock solutions in
50% aq DMF. The solutions could be stored over
months at +4ꢁC without noticeable decomposition of
the substrates. Commercial enzyme samples were freshly
prepared by weighting the solid sample and diluting it to
a stock solution of 1mg/mL in PBS (aq 10mM phos-
phate, 160mM NaCl, pH7.4). BSA (bovine serum albu-
min) was diluted from a 40mg/mL stock solution in
20mM aqueous borate buffer pH8.8. Sodium periodate
(NaIO4) was freshly prepared as a 10mM stock solution
in water. Measurements were carried out in 96-well half-
area clear-bottom polystyrene microtiter plates. Sub-
strate solutions (10lL) were first placed in the wells. A
prediluted solution of 2.5mL containing the enzyme at
the indicated concentration, BSA (2mg/mL) and sodium
periodate (1mM) in aq 20mM borate buffer pH8.8 was
prepared, and 90lL were added to each substrate-con-
taining well. The microtiter plates were then placed in
Compound 11b: yellow solid, mp 58–59ꢁC; TLC (Hex/
EtOAc 90/10, UV 254nm): Rf 0.41; 1H NMR
(300MHz, CDCl3) d (ppm): 8.19 (d, J = 9.2Hz, 2H),
6.93 (d, J = 9.2Hz, 2H), 5.35–5.30 (m, 1H), 4.36–4.31
(m, 1H), 4.16–4.08 (m, 3H), 2.53–2.46 (m, 2H), 2.34–
2.27 (m, 2H), 2.15–2.13 (m, 2H), 1.63–1.56 (m, 6H),
1.26–1.24 (m, 56H), 0.90–0.85 (m, 6H); 13C NMR
(75MHz, CDCl3) d (ppm): 174.0, 170.0, 164.1, 126.6,
115.2, 70.3, 70.1, 65.3, 65.2, 65.1, 60.1, 59.6, 42.8, 42.6,
34.8, 32.6, 31.1, 30.4, 30.2, 30.1, 30.0, 29.9, 29.8, 29.0,
28.3, 25.5, 24.2, 23.4, 14.8; IR (CHCl3) m (cmꢀ1): 3025
(w), 2926 (s), 2855 (s), 1739 (s), 1610 (m), 1595 (s),
1518 (s), 1499 (m), 1468 (m), 1344 (s), 1263 (s), 1216
(s), 1173 (s), 1113 (s).
4.16. ( )-(4-(4-Nitrophenyloxy)-2-hydroxybut-1-yl)-cis-9-
octadecenoate 12a and ( )-(4-(4-nitrophenyloxy)-2-(cis-9-
octadecenoyl)oxybut-1-yl)-cis-9-octadecenoate 12b
a
Spectramax 250 Microplate Spectrophotometer
(Molecular Devices) and the reaction followed at
405nm over 1h. The primary OD-versus-time data were
first converted to product-versus-time using a calibra-
tion curve with pure nitrophenol or dinitrophenol under
the same conditions. The steepest linear portion of each
curve was then used to calculate the reaction rate in each
well. There was no significant reaction with buffer,
NaIO4, and BSA under the reaction conditions in the
absence of enzyme.
Triethylamine (0.7mL, 4.5mmol) and DMAP (0.0025g,
0.02mmol) are added under N2 to a solution of ( )-4-(4-
nitrophenoxy)-1,2-butanediol 13 (0.500g, 2.2mmol) in
dry DCM (20mL) and DMF (5mL), and the mixture
is cooled to 0ꢁC. Oleoyl chloride (1g, 3.3mmol) is then
added, and the mixture stirred at rt for 18h. After con-
centration to dryness the crude product is taken in
EtOAc and water, washed with satd aq NaHCO3 and
brine, dried over MgSO4 and concentrated. The residue
is finally purified by flash chromatography on silica gel
(Hex/EtOAc 100/0–50/50) to give compounds 12a
(0.644g, 60%) and 12b (0.025g, 2%).
6. Data treatment
All data were processed in MS-Excel. A table with four
columns and five rows containing the observed reaction
rates with the different substrates following the layout in
Figure 1 was assembled. For each enzyme, a second
table was then assembled with 12columns and 5 rows.
Starting at the first row and the first column, each group
of three entries on the same rows was filled with the rate
data measured with the enzyme at 0.1, 0.01, and
0.001mg/mL, again following the layout of substrates.
All rate data in the table were then divided by the max-
imum rate in the table to give relative reaction rates Vrel.
The color intensity factors F were then computed
according to the formula F = 255(1ꢀVrel). The resulting
numbers were rounded to 0 decimal, and saved in the
Ôcomma separated valueÕ file format. The file was then
Compound 12a: yellow oil; TLC (Hex/EtOAc 50/50, UV
254nm): Rf 0.80; H NMR (300MHz, CDCl3) d (ppm):
1
8.19 (d, J = 9.2Hz, 2H), 6.96 (d, J = 9.2Hz, 2H), 5.36–
5.32 (m, 2H), 4.32–4.04 (m, 5H), 2.38–2.33 (m, 2H),
2.05–1.95 (m, 6H), 1.64–1.61 (m, 2H), 1.30–1.26 (m,
20H), 0.88–0.85 (m, 3H); 13C NMR (75MHz, CDCl3)
d (ppm): 174.7, 164.4, 142.2, 130.6, 130.3, 126.5, 115.1,
72.7, 68.9, 67.6, 65.8, 65.6, 65.1, 35.0, 34.8, 33.2, 32.5,
30.8, 30.4, 30.3, 30.1, 29.9, 29.8, 29.7, 27.8, 25.5, 23.3,
14.7; IR (neat) m (cmꢀ1): 3481 (m), 3006 (m), 2927 (s),
2856 (s), 1739 (s), 1609 (s), 1595 (s), 1516 (s), 1499 (s),
1467 (s), 1343 (s), 1264 (s), 1174 (s), 1112 (s); EI-MS:
:
+
491 [M]+; HR-ESI-MS: calcd for C28H46NO6
492.3325. Found: 492.3321.

J. Grognux et al. / Tetrahedron: Asymmetry 15 (2004) 2981–2989
2989
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re-opened in a text editor and the following four lines
were inserted at the top:
line 1: P3
line 2: # (optional line with identification)
line 3: 4 5
line 4: 255
The file was then saved in the Ôportable pixel mapÕ for-
mat by adding Ô.ppmÕ to the file name. This new file is
then opened in a graphical program such as COREL-
DRAW, PHOTOSHOP, or PAINTSHOP, and resized to
approximately 100pixel broad for visualization.
´
9. Sevestre, A.; Helaine, V.; Guyot, G.; Martin, C.; Hecquet,
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Acknowledgements
This work was supported by the Swiss National Science
´ ´
foundation, the Office Federal Suisse de lÕEducation et
´
de la Science, the COST Action D16, and Proteus SA,
ˆ
Nımes, France.
`
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