Using a lipase as a high-throughput screening method for measuring the enantiomeric excess of allylic acetates

Article abstract of DOI:10.1021/jo035067u

This report describes a high-throughput method for measuring the enantiomeric excess of allylic acetates. Such methods are useful tools for screening libraries of potential catalysts for enantioselective reactions. This technique, which is called EMDee fo

Full text of DOI:10.1021/jo035067u

Usin g a Lip a se a s a High -Th r ou gh p u t Scr een in g Meth od for
Mea su r in g th e En a n tiom er ic Excess of Allylic Aceta tes
M. Burak Onaran and Christopher T. Seto*
Department of Chemistry, Brown University, Providence, Rhode Island 02912
christopher_seto@brown.edu
Received J uly 23, 2003
This report describes a high-throughput method for measuring the enantiomeric excess of allylic
acetates. Such methods are useful tools for screening libraries of potential catalysts for enantiose-
lective reactions. This technique, which is called EMDee for an enzymatic method for determining
enantiomeric excess, uses the lipase from Pseudomonas cepacia to hydrolyze the (R) enantiomer of
an allylic acetate, while the (S) enantiomer does not react. The rate of the reaction is monitored by
measuring the acetic acid that is produced during the hydrolysis reaction with a pH indicator.
Using the Michaelis-Menten equation, the rate of the reaction can be correlated with the
concentration of the (R) enantiomer. This method can process 88 samples in less that 30 min.
In tr od u ction
used to screen large libraries of catalysts. These high-
throughput methods fall into four general categories: (1)
kinetic resolution reactions in which an alcohol or amine
that is to be analyzed is coupled with a chiral carboxylic
acid to give a mixture of diastereomers,4 (2) parallel
chromatographic methods that are followed by analysis
with UV, fluorescence or CD spectroscopies,5 (3) com-
parison of the reaction rates of the purified enantiomers
of a chiral starting material,6 and (4) biological methods
that rely on antibodies or enzymes.7
Combinatorial methods have become important tools
in several disciplines including molecular biology, ma-
terials science, and pharmaceutical and synthetic chem-
istry. A number of investigators have been applying these
methods to the discovery of new catalysts for asymmetric
reactions.1,2 Combinatorial techniques are useful in this
area because they have the potential for increasing the
efficiency by which chiral catalysts are discovered and
for optimizing the stereoselectivity of a catalyst for a
particular substrate of interest. Using these techniques,
it is generally straightforward to synthesize thousands
of potential catalysts. However, it is time-consuming to
screen this number of catalysts for enantioselectivity
using standard methods such as chiral HPLC or GC.
With these types of serial analytical methods, the analy-
sis time increases linearly with the size of the library.
In addition, these methods often require that the prod-
ucts from the catalyzed reaction be purified before they
are analyzed.
We have recently developed a new high-throughput
method for screening enantiomeric excess that falls into
the fourth category. This method is called EMDee, for
enzymatic method for determining enantiomeric excess.8
In its most general form, the EMDee screen uses an
enzyme as an analytical tool to measure the % ee of a
sample. The enzyme is introduced into a sample of
unknown stereochemical composition, and it catalyzes a
reaction using only one of the enantiomers as a substrate.
The rate of the enzyme-catalyzed reaction is monitored
by standard methods, and this rate can be correlated to
the % ee of the sample as long as the total concentration
To overcome some of these limitations, new high-
throughput methods for measuring enantiomeric excess
are being developed.3 Many of these new methods rely
on parallel, rather than serial, analyses and thus can be
(3) For reviews, see: (a) Reetz, M. T. Angew. Chem., Int. Ed. 2001,
40, 285. (b) Reetz, M. T. Angew. Chem., Int. Ed. 2002, 41, 1335.
(4) (a) Guo, J .; Wu, J .; Siuzdak, G.; Finn, M. G. Angew. Chem., Int.
Ed. 1999, 38, 1755. (b) Korbel, G. A.; Lalic, G.; Shair, M. D. J . Am.
Chem. Soc. 2001, 123, 361.
(5) (a) Reetz, M. T.; Kuhling, K. M.; Deege, A.; Hinrichs, H.; Belder,
D. Angew. Chem., Int. Ed. 2000, 39, 3891. (b) Ding, K.; Ishii, A.;
Mikami, K. Angew. Chem., Int. Ed. 1999, 38, 497. (c) Chen, Y.; Shimizu,
K. D. Org. Lett. 2002, 4, 2937.
(6) (a) Reetz, M. T.; Becker, M. H.; Kuhling, K. M.; Holzwarth, A.
Angew. Chem., Int. Ed. 1998, 37, 2647. (b) J arvo, E. R.; Evans, C. A.;
Copeland, G. T.; Miller, S. J . J . Org. Chem. 2001, 66, 5522. (c) Reetz,
M. T.; Zonta, A.; Schimossek, K.; Liebeton, K.; J aeger, K.-E. Angew.
Chem., Int. Ed. 1997, 36, 2830. (d) Reetz, M. T.; Becker, M. H.; Klein,
H.-W.; Stockigt, D. Angew. Chem., Int. Ed. 1999, 38, 1758.
(7) (a) Taran, F.; Gauchet, C.; Mohar, B.; Meunier, S.; Valleix, A.;
Renard, P. Y.; Creminon, C.; Grassi, J .; Wagner, A.; Mioskowski, C.
Angew. Chem., Int. Ed. 2002, 41, 124. (b) Berkowitz, D. B.; Bose, M.;
Choi, S. Angew. Chem., Int. Ed. 2002, 41, 1603.
* Corresponding author.
(1) For reviews of combinatorial catalyst discovery, see: (a) Hoveyda,
A. H. Chem. Biol. 1998, 5, 187. (b) Shimizu, K. D.; Snapper, M. L.;
Hoveyda, A. H. Chem. Eur. J . 1998, 4, 1885. (c) Kuntz, K. W.; Snapper,
M. L.; Hoveyda, A. H. Curr. Opin. Chem. Biol. 1999, 3, 313. (d)
Snapper, M. L.; Hoveyda, A. H. Comb. Chem. 2000, 433. (e) Gilbertson,
S. R. Progr. Inorg. Chem. 2001, 50, 433. (c) Hoveyda, A. H. Chem. Biol.
1998, 5, 187.
(2) (a) Degrado, S. J .; Mizutani, H.; Hoveyda, A. H. J . Am. Chem.
Soc. 2001, 123, 755. (b) Copeland, G. T.; Miller, S. J . J . Am. Chem.
Soc. 2001, 123, 6496. (c) J acobsen, E. N.; Sigman, M. S. Schiff J . Am.
Chem. Soc. 1998, 120, 4901. (d) Gilbertson, S. R.; Collibee, S. E.;
Agarkov, A. J . Am. Chem. Soc. 2000, 122, 6522. (e) Porte, A. M.;
Reibenspies, J .; Burgess, K. J . Am. Chem. Soc. 1998, 120, 9180. (f)
Hoshino, Y.; Yamamoto, H. J . Am. Chem. Soc. 2000, 122, 10452. (g)
Brouwer, A. J .; van der Linden, H. J .; Liskamp, R. M. J . J . Org. Chem.
2000, 65, 1750. (h) Chataigner, I.; Gennari, C.; Piarulli, U.; Ceccarelli,
S. Angew. Chem., Int. Ed. 2000, 39, 916.
(8) Abato P.; Seto C. T. J . Am. Chem. Soc. 2001, 123, 9206.
10.1021/jo035067u CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/16/2003
8136
J . Org. Chem. 2003, 68, 8136-8141

Measurement of the Enantiomeric Excess of Allylic Acetates
SCHEME 1
dride to give alcohol 1, which was subsequently acylated
with acetic anhydride and catalytic DMAP to give allylic
acetate 2.
Compound 2 was subjected to a stereoselective hy-
drolysis using an immobilized lipase from Candida
antarctica as shown in Scheme 2. After 45% conversion,
the reaction was stopped and the product alcohol was
isolated to give (R)-1 in 99% ee as determined by chiral
HPLC. This alcohol was acylated with acetic anhydride
and catalytic DMAP to give (R)-2, also in 99% ee. The
remaining starting material from the enzymatic reaction,
(S)-2 in 82% ee, was subjected to an additional 10%
conversion with the immobilized lipase. After the product
alcohol was removed by flash chromatography, the
remaining starting material was re-isolated to give (S)-2
in 99% ee. Finally, alcohol (S)-1 was obtained in 99% ee
by base-promoted hydrolysis of (S)-2 as shown in Scheme
3.
Design of th e Assa y. We have selected the lipase
from Pseudomonas cepacia for the EMDee assays of
allylic acetates. This lipase has a very broad substrate
specificity and generally shows excellent stereoselectivity
with a wide variety of substrates.16 The general scheme
for the assays is shown in Figure 1. A sample of allylic
acetate 2 of unknown stereochemical composition is
treated with the lipase from Pseudomonas cepacia, and
the enzyme catalyzes the hydrolysis of the (R) enantio-
mer. The rate of the enzymatic reaction can be correlated
with the concentration of (R)-2 using the Michaelis-
Menten equation. Since the starting concentration of the
allylic acetate is known, the % ee of the sample can be
calculated.
To monitor the rate of the enzyme-catalyzed reaction
by UV spectroscopy, we used p-nitrophenol as a pH-
sensitive indicator according to the method of Kazlauskas
and co-workers.17 As the enzymatic hydrolysis occurs,
acetic acid is liberated and protonates the indicator. Since
p-nitrophenol and the BES (N,N-bis[2-hydroxyethyl]-2-
aminoethanesulfonic acid) buffer in the reaction have the
same pKa, production of acetic acid is accompanied by a
linear change in absorbance at 405 nm.
Kin etics. We have used this assay system to measure
the kinetics of hydrolysis of (R)-2 by the lipase. Figure 2
shows a plot of the rate of the enzymatic reaction as a
function of the concentration of (R)-2. These data were
used to calculate a KM value for this substrate of 2.6 mM.
We have also determined that (S)-2 is neither a substrate
nor an inhibitor of the lipase up to the limits of solubility.
In addition, further control experiments show that the
corresponding alcohols (R)-1 and (S)-1 do not inhibit the
of the substrate plus its enantiomer is known. Two
important features of the EMDee method are its high
stereoselectivity, since enzymes typically have a strong
specificity for one enantiomer of a substrate, and the
ability to perform the screen in a 96- or 384-well format
using a UV or fluorescence plate reader.
We first demonstrated the EMDee assay using an
alcohol dehydrogenase to oxidize one enantiomer of a
chiral benzylic alcohol.8 This alcohol was obtained through
the catalytic asymmetric addition of diethylzinc to ben-
zaldehyde. In this paper, we broaden the scope of EMDee
by showing that a lipase can be used to measure the
enantiomeric excess of chiral allylic acetates.
Resu lts a n d Discu ssion
Ch oice of th e Su bstr a te. We have chosen chiral
allylic acetates as substrates for this ee screen because
these compounds and their related chiral allylic alcohols
are important intermediates in synthetic chemistry.9
However, this method will be applicable to other esters
and is not limited to allylic acetates in particular. There
are a variety of catalytic asymmetric methods that are
available for synthesizing chiral allylic acetates. These
include palladium-catalyzed allylic substitution reac-
tions10 and the kinetic resolution of racemic alcohols.11
We have selected compound 2 (Scheme 1) as the test
substrate for this lipase-based EMDee assay because it
is a common component of a wide diversity of allylic
substitution reactions,12 it is a useful starting point for
the synthesis medicinally important compounds includ-
ing potential glycosyltransferase inhibitors,13 and it can
be synthesized in chiral form using Fu’s chiral acylation
catalyst14 and via dynamic kinetic resolution.15
Syn th esis of th e Su bstr a te. To perform the enzyme
assays, we needed to synthesize the allylic acetates (R)-2
and (S)-2 and the corresponding alcohols (R)-1 and (S)-1
in enantiomerically pure form. As shown in Scheme 1,
compounds 1 and 2 were prepared in racemic form by
reducing 1-phenyl-1-buten-3-one with sodium borohy-
SCHEME 2
J . Org. Chem, Vol. 68, No. 21, 2003 8137

Onaran and Seto
F IGURE 1. EMDee assay of allylic acetate 2 using the lipase from Pseudomonas cepacia.
F IGURE 3. Analysis of 88 samples of compound 2 that range
in ee from 100% (S) to 100% (R) using the lipase from
Pseudomonas cepacia.
F IGURE 2. Plot of the rate of the lipase-catalyzed reaction
as a function of the concentration of (R)-2.
SCHEME 3
were analyzed in a 96-well formal using a UV plate
reader. The total concentration of compound 2 in each
well was 1 mM. The assay solutions also contained 10%
acetonitrile to improve solubility of the substrate. Data
were collected over 25 min and are shown in Figure 3.
The solid line in the plot in Figure 3 represents the
theoretical curve that is generated using the Michaelis-
Menten equation with the constraints that the total
concentration of compound 2 remains constant at 1 mM,
that (R)-2 is the substrate, and that (S)-2 is neither a
substrate nor an inhibitor for the lipase. The good
correlation between the experimental data and the
theoretical curve demonstrates that the rate of the lipase-
catalyzed reaction can be used to estimate the stereo-
chemical composition of samples of compound 2. In
addition, this is a fast, parallel analysis in which 88
samples can be analyzed in less than 30 min.
lipase and that there is no significant background hy-
drolysis of compound 2 under the assay conditions in the
absence of enzyme.
To demonstrate that the lipase can be used to measure
the % ee of compound 2, we prepared 88 samples of allylic
acetate 2 that varied in ee from 100% (S) to 100% (R).
The ee of these samples were confirmed by chiral HPLC
analysis prior to the assays. These samples, along with
eight control reactions that did not contain substrate,
(9) (a) Charette, A. B.; Beauchemin, A. Org. React. 2001, 58, 1. (b)
Hanson, R. M. Org. React. 2002, 60, 1. (c) Katsuki, T.; Martin, V. S.
Org. React. 1996, 48, 1.
An a lysis of Cr u d e Acyla tion P r od u cts. We have
also investigated the possibility of using this lipase-based
EMDee assay on crude reaction products. Fu and co-
workers have used a planar-chiral DMAP derivative to
(10) (a) J acobsen, E. N., Pfaltz, A., Yamamoto, H., Eds. Compre-
hensive Asymmetric Catalysis I-III; Springer-Verlag: Berlin, 1999;
Chapter 24. (b) Trost, B. M.; Organ, M. G. J . Am. Chem. Soc. 1994,
116, 10320. (c) Longmire, J . M.; Wang, B.; Zhang, X. Tetrahedron Lett.
2000, 41, 5435. (d) Lussem, B. J .; Gais, H.-J . J . Am. Chem. Soc. 2003,
125, 6066. (e) Gilbertson, S. R.; Lan, P. Org. Lett. 2001, 3, 2237.
(11) (a) Fu, G. Acc. Chem. Res. 2000, 33, 412. (b) Copeland, G. T.;
Miller, S. J . J . Am. Chem. Soc. 2001, 123, 6496. (c) Clapham, B.; Cho,
C.-W.; J anda, K. D. J . Org. Chem. 2001, 66, 868. (d) For a review, see:
Long, Y. O.; Paquette, L. A. Chemtracts 2000, 13, 1 and references
therein.
(12) For several examples, see: (a) Makkov, A. V.; Baxendale, I. R.;
Mansfield, D. J .; Kocovsky, P. J . Chem. Soc., Perkin Trans. 1 2001,
1234. (b) Wang, Y.; Ding, K. J . Org. Chem. 2001, 66, 3238. (c)
Kobayashi, Y.; Tokoro, Y.; Watatani, K. Eur. J . Org. Chem. 2000, 3825.
(d) Blacker, A. J .; Clarke, M. L.; Loft, M. S.; Mahon, M. F.; Humphries,
M. E.; Williams, J . M. J . Chem. Eur. J . 2000, 6, 353. (e) Uozumi, Y.;
Dango, H.; Hayashi, T. J . Org. Chem. 1999, 64, 3384.
(15) (a) Lee, D.; Huh, E. A.; Kim, M.-J .; J ung, H. M.; Koh, J . H.;
Park, J . Org. Lett. 2000, 2, 2377. (b) Choi, Y. K.; Suh, J . H.; Lee, D.;
Lim, I. T.; J ung, J . Y.; Kim, M.-J . J . Org, Chem. 1999, 64, 8423.
(16) (a) Pchelka, B. K.; Loupy, A.; Plenkiewicz, J .; Blanco, L.
Tetrahedron: Asymmetry 2000, 11, 2719. (b) Ghanem, Ashraf, Schurig,
V. Tetrahedron: Asymmetry 2001, 12, 2761. (c) Ghanem, Ashraf,
Schurig, V. Chirality 2001, 13, 118. (d) Adam, W.; Diaz, M. T.; Fell, R.
T.; Saha-Moller, C. R. Tetrahedron: Asymmetry 1996, 7, 2207. (e)
Nicolosi, G.; Spatafora, C.; Tringali, C. J . Mol. Catal. B: Enzymatic
2002, 16, 223. (f) Athawale, V.; Manjrekar, N.; Athawale, M. J . Mol.
Catal. B: Enzymatic 2001, 16, 169. (g) Guieysse, D.; Salagnad, C.;
Monsan, P.; Remaud-Simeon, M. Tetrahedron: Asymmetry 2001, 12,
2473. (h) Pchelka, B. K.; Loupy, A.; Plenkiewicz, J .; Petit, A.; Blanco,
L. Tetrahedron: Asymmetry 2001, 12, 2109. (i) Park, S.; Kazlauskas,
R. J . J . Org. Chem. 2001, 66, 8395. (j) Zanoni, G.; Agnelli, F.; Meriggi,
A.; Vidari, G. Tetrahedron: Asymmetry 2001, 12, 1779.
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42, 2289.
(14) Bellemin-Laponnaz, S.; Tweddell, J .; Ruble, J . C.; Breitling, F.
M.; Fu, G. C. Chem. Commun. 2000, 1009.
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8138 J . Org. Chem., Vol. 68, No. 21, 2003

Measurement of the Enantiomeric Excess of Allylic Acetates
F IGURE 4. Preparation and EMDee analysis of 88 samples of crude allylic acetate 2.
F IGURE 6. Indene derivative (R)-4 is a substrate for the
lipase.
F IGURE 5. Lipase-based screen of 88 samples of compound
2 that range in ee from 100% (S) to 100% (R). These samples
are crude products from the acylation reaction shown in Figure
4 and were analyzed without isolation or purification.
selectively acylate one enantiomer of a racemic mixture
of a variety of allylic alcohols.14 To model the products
from these types of enantioselective acylation reactions,
we prepared 88 samples of allylic alcohol 1 that varied
in ee from 100% (S) to 100% (R). These samples were
acylated with 1.2 equiv of acetic anhydride and catalytic
DMAP in acetonitrile as shown in Figure 4. The crude
allylic acetates were then directly subjected to the EMDee
assay without prior removal of the solvent, workup, or
purification. The buffer concentration in the enzyme
assays was high enough to ensure that the excess acetic
acid that is generated during the acylation reactions did
not interfere with the functioning of the pH indicator.
F IGURE 7. Structures of several substrates for the lipase
from Pseudomonas cepacia.
while its enantiomer (S)-4 is not a substrate or an
inhibitor of the enzyme.
To further probe the potential scope of this ee screen,
we have found other alcohols and esters reported in the
literature that are substrates for the lipase from Pseu-
domonas cepacia. Seven of these structures are shown
in Figure 7. These particular compounds were chosen to
highlight the ability of this enzyme to accept substrates
with a diversity of structures.16 The lipase catalyzes
either the esterification or hydrolysis of all of these
substrates with high stereoselectivity. Thus, our own
experiments with compounds (R)-2 and (R)-4, along with
previous studies of this enzyme suggest that the lipase-
based EMDee screen should be applicable to a wide
variety of allylic acetates and other esters that are of
interest to the synthetic community.
Figure 5 shows the data from the assay of these crude
samples. There is a strong correlation between the % ee
of the allylic alcohol starting material and the rate of the
enzymatic reaction with the crude allylic acetate prod-
ucts. These results demonstrate that the lipase-based
EMDee screen is a robust method that can reliably
measure the enantiomeric excess of products from an
acylation reaction without the need for isolation or
purification of the allylic acetates.
(18) (a) Lyle, T. A.; Wiscount, C. M.; Guare, J . P.; Thompson, W. J .;
Anderson, P. S.; Darke, P. L.; Zugay, J . A.; Emini, E. A.; Schleif, W.
A.; Quintero, J . C.; Dixon, R. A. F.; Sigal, I. S.; Huff, J . R. J . Med.
Chem. 1991, 34, 1228. (b) Askin, D.; Wallace, M., A.; Vacca, J . P.;
Reamer, R. A.; Volante, R. P.; Shinkai, I. J . Org. Chem. 1992, 57, 2771.
(c) Askin, D.; Eng, K. K.; Rossen, K.; Purick, R. M.; Wells, K. M.;
Volante, R. P.; Reider, P. J . Tetrahedron Lett. 1994, 35, 673.
(19) Hoppe, I.; Marsch, M.; Harms, K.; Boche, G.; Hoppe, D. Angew.
Chem., Int. Ed. Engl. 1995, 34, 2158. WARNING: This procedure for
the synthesis of compound 3 (inden-1-ol) involves the preparation of
indene 1-peroxide as an intermediate. We have performed this
procedure a number of times, using appropriate safety precautions
including the use of a blast shield, without mishap. However, on one
occasion while the reaction mixture was being quenched with a solution
of NaI, a serious explosion occurred that resulted in a small fire and
injury from flying glass. We have not determined the exact cause of
this explosion, but recommend that all appropriate safety precautions
be used when carrying out this procedure.
Ap p lica tion to Oth er Ester s. One potential limita-
tion of the EMDee method is that many enzymes are
highly specific for a particular substrate and do not
tolerate significant variations in substrate structure. To
address this issue, we have examined a second allylic
acetate, indene derivative (R)-4 (Figure 6), as a potential
substrate for the lipase. Chiral derivatives of indene are
useful substructures in medicinal chemistry, and have
been used in the synthesis of a number of inhibitors for
HIV protease.18 Compound (R)-4 was synthesized in 99%
ee from racemic inden-1-o119 using a procedure similar
to that shown in Scheme 2.20 We have found that (R)-4
is a substrate for the lipase with a KM value of 5.2 mM,
(20) For the details of the synthesis of racemic 4, (R)-3, (R)-4, and
(S)-4 see the Supporting Information.
J . Org. Chem, Vol. 68, No. 21, 2003 8139

Onaran and Seto
no workup or purification of the products. The reactions and
subsequent enzymatic analyses were performed multiple times
with similar results.
In our current studies, we have analyzed samples in
which the total concentration of allylic acetate is known.
However, if this technique is used to analyze a combi-
natorial library of asymmetric catalysts, there are two
factors that will influence the rate that is observed in
the EMDee assay; the % ee of the sample, and the extent
of conversion of the catalyzed reaction. We believe that
the EMDee assay will be most useful as a preliminary
screening technique to identify promising catalysts from
large libraries that give both high yields and that show
high stereoselectivity. These catalysts will give the
highest rates in the EMDee assay. Conversely, catalysts
that give low conversion or that have low stereoselectivity
will give low rates. If it becomes necessary to identify
catalysts that have low conversion but good stereoselec-
tivity, these compounds could be identified using a second
EMDee assay, as we have shown previously.8 This
requires two enzymes with opposite stereoselectivity that
are used to quantitate each of the two enantiomers of
the product.
Ra cem ic Alcoh ol 1. (E)-3-Hydroxy-1-phenyl-1-butene 1
was prepared by reducing (E)-4-phenyl-3-buten-2-one (25 g,
171 mmol) with NaBH4 (6.47 g, 171 mmol) in ethanol (250 mL).
The reaction mixture was stirred at 0 °C for 30 min and then
at room temperature for 3 h. The reaction was quenched with
1 N HCl, and the ethanol was removed by rotary evaporation.
The aqueous phase was extracted with ethyl acetate, and the
organic phase was washed with water and brine and dried over
MgSO4. Purification by flash column chromatography (EtOAc/
hexanes 1:4) gave a clear oil (22.8 g, 154 mmol, 90%): 1H NMR
(CDCl3, 400 MHz) δ 1.40 (d, J ) 6.4 Hz, 3H), 1.64 (s, 1H),
4.51-4.54 (m, 1H), 6.29 (dd, J ) 15.9, 6.4 Hz, 1H), 6.60 (d, J
) 15.9 Hz, 1H), 7.26 (t, J ) 7.6 Hz, 1H), 7.34 (dd, J ) 7.0, 1.7
Hz, 2H), 7.41 (d, J ) 3.3 Hz, 2H); 13 C NMR (CDCl3, 100 MHz)
δ 137.1, 134.0, 129.8, 129.0, 128.1, 126.9, 69.4, 23.8; HRMS
(ESI) calcd for C10H12O 148.0888, found 148.0892.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
2 a n d 4. Racemic 2, racemic 4, (R)-2, and (R)-4 were prepared
from the corresponding alcohols by DMAP-catalyzed acylation.
In a typical procedure, the alcohol (100 mmol) was combined
with THF (70 mL), acetic anhydride (120 mmol), triethylamine
(180 mmol), and DMAP (0.5 mmol) at 0 °C. The reaction
mixture was maintained at 0 °C for 1 h and then was allowed
to stir at room temperature overnight. The solvent was
removed by rotary evaporation, and the resulting material was
washed with water, brine and dried over MgSO4. Purification
by flash column chromatography (EtOAc/hexanes 1:4) gave the
desired product.
Con clu sion s
In summary, we have developed an EMDee screen that
uses the lipase from Pseudomonas cepacia to measure
the enantiomeric excess of allylic acetates in a high-
throughput manner. This method can accommodate
samples that range in stereochemistry from 100% (S) to
100% (R), and is able to process 88 samples in ap-
proximately 25 min. Because the lipase has a very broad
substrate specificity, this assay procedure should be
useful for screening potential enantioselective catalysts
for a variety of synthetic transformations.
Ra cem ic 2. Racemic 2 was prepared as described above
from alcohol 1 (14.0 g, 95 mmol). Purification gave a clear oil
(18.0 g, 95 mmol, 100%): 1H NMR (CDCl3, 400 MHz) δ 1.45
(d, J ) 6.5 Hz, 3H), 2.11 (s, 3H), 5.55-5.58 (m, 1H), 6.22 (dd,
J ) 16.0, 6.8 Hz, 1H), 6.64 (d, J ) 16.0 Hz, 1H), 7.29 (t, J )
4.3 Hz, 1H), 7.35 (m, 2H), 7.42 (d, J ) 9.6 Hz, 2H); 13 C NMR
(CDCl3, 100 MHz) δ 170.7, 136.8, 132.0, 129.2, 129.0, 128.3,
127.0, 71.4, 21.8, 20.8; HRMS (ESI) calcd for C12H14O2 190.0994,
found 190.0986.
Exp er im en ta l Section
Alcoh ol (R)-1. (R)-1 was synthesized from racemic acetate
2 by enantioselective hydrolysis using an immobilized lipase
from Candida antarctica. To the racemic acetate 2 (60.3 mg,
0.317 mmol) were added acetonitrile (3 mL), water (3 mL), and
the immobilized lipase (120.6 mg), and the reaction mixture
was gently shaken at room temperature for 24 h under
ambient atmosphere. After the reaction had reached 45%
completion as determined by the 1H NMR spectrum of an
aliquot of the reaction mixture, the immobilized enzyme was
removed by filtration and the acetonitrile was removed by
rotary evaporation. Water (4 mL) was added, the product was
extracted into ethyl acetate, and the organic phase was dried
over MgSO4. Purification by flash column chromatography
(EtOAc/hexanes 1:4) yielded alcohol (R)-1 (19.2 mg, 0.13 mmol,
91% of the theoretical yield) in 99% ee as judged by HPLC
analysis (Chiralcel OD-H column, 90:10 hexane/2-propanol, 1
mL/min, 254 nm UV detection), tR ) 8.3 min for (R)-1 and tR
) 12.5 min for (S)-1. The chromatographic purification also
yielded (S)-2 in 82% ee.
P r oced u r e for Lip a se Assa ys. Samples of compound 2
were used as substrates for Amano lipase PS from Pseudomo-
nas cepacia. Protonation of the indicator p-nitrophenoxide by
the acetic acid that is formed during the enzymatic reaction
was monitored by observing the decrease in absorbance at 404
nm. Each well of the 96-well plate contained 300 µL of a
solution of 1 mM substrate, 6.67 mM BES buffer (N,N-bis(2-
hydroxyethyl)-2-aminoethanesulfonic acid) at pH 7.20, 0.22
mM p-nitrophenol, 60 µL of enzyme stock solution, and 10%
acetonitrile as a cosolvent. Samples of compound 2 varying in
% ee composition were prepared using enantiomerically pure
(R)-2 and (S)-2. Enzyme stock solutions were prepared by
dissolving the lipase (1 mg/mL) and BSA (bovine serum
albumin, 1 mg/mL) in BES buffer. This solution was agitated
with a shaker at room temperature for 30 min, then centri-
fuged twice to remove the insoluble particles. The reactions
were initiated by the addition of enzyme stock solution, and
absorbance data was collected over 25 min. These assays were
performed under ambient atmosphere.
Acet a t e (R)-2. (R)-2 was prepared from alcohol (R)-1 as
described above in the general procedure. 99% ee by HPLC
analysis (Chiralcel OD-H column, hexane, 1 mL/min, 254 nm
UV detection), tR ) 22.7 min for (R)-2 and tR ) 26.5 min for
(S)-2.
Aceta te (S)-2. The (S)-2 (82% ee) that was obtained during
the synthesis of alcohol (R)-1 was subjected to an additional
10% conversion with the immobilized lipase from Candida
antarctica. After the desired conversion was attained as
determined by the 1H NMR spectrum of an aliquot of the
reaction mixture, the immobilized enzyme was filtered and the
acetonitrile was removed by rotary evaporation. Water (4 mL)
was added, the product was extracted into ethyl acetate, and
P r oced u r e for Acyla tion of 11 Sa m p les of Com p ou n d
1 a n d An a lysis of th e Resu ltin g Cr u d e Allylic Aceta tes
Usin g th e Lip a se. Eleven samples of compound 1 ranging
in % ee from 100% (R)-1 to 100% (S)-1 were prepared using
stock solutions of pure (R)-1 and (S)-1 in acetonitrile. These
samples were acylated using acetic anhydride (1.2 equiv), NEt3
(1.2 equiv), and DMAP (3 mol %) in acetonitrile solvent at room
temperature. The reactions were monitored by HPLC and
shown to be complete in less than 1 h. Samples were adjusted
to a final concentration of 10 mM in the allylic acetate product
using acetonitrile, and eight aliquots of each reaction was
subjected to analysis using the lipase as described above. The
enzymatic analyses were performed on the crude samples, with
8140 J . Org. Chem., Vol. 68, No. 21, 2003

Measurement of the Enantiomeric Excess of Allylic Acetates
the organic phase was dried over MgSO4. Purification by flash
column chromatography (EtOAc/hexanes 1:4) yielded acetate
(S)-2 (23 mg, 0.12 mmol, 85% of theoretical): 99% ee as judged
by HPLC analysis (Chiralcel OD-H column, hexane, 1 mL/min,
254 nm UV detection), tR) 22.7 min for (R)-2 and tR) 26.5
min for (S)-2.
Purification by flash column chromatography (EtOAc/hexanes
1:4) yielded (S)-1 (491 mg, 3.32 mmol, 90%): 99% ee as judged
by HPLC analysis (Chiralcel OD-H column, 90:10 hexane/2-
propanol, 1 mL/min, 254 nm UV detection), tR) 8.3 min for
(R)-1 and tR) 12.5 min for (S)-1.
Alcoh ol (S)-1. Alcohol (S)-1 was synthesized from acetate
(S)-2 (99% ee) by basic hydrolysis. To the acetate (S)-2 (700
mg, 3.68 mmol) were added methanol (30 mL), water (60 mL),
and sodium carbonate (1.12 g), and the reaction mixture was
stirred at room temperature for 10 h. The methanol was
removed by rotary evaporation, the product was extracted into
ethyl acetate, and the organic phase was dried over MgSO4.
Su p p or tin g In for m a tion Ava ila ble: Procedures for the
synthesis of racemic 4, (R)-3, (R)-4, and (S)-4; 1H and 13C NMR
spectra for compounds 1, 2, and 4; HPLC traces for (R)-1, (S)-
1, (R)-2, (S)-2, (R)-3, (R)-4, and (S)-4. This material is available
free of charge via the Internet at http://pubs.acs.org.
J O035067U
J . Org. Chem, Vol. 68, No. 21, 2003 8141