Parallel synthesis of an ester library for substrate mapping of esterases and lipases

Article abstract of DOI:10.1016/j.tetasy.2004.07.048

Use of solid-supported reagents simplified routine acylation of primary and secondary alcohols because it eliminated traditional purification. Using a parallel synthesizer, eight primary or secondary alcohols reacted with acid chloride in the presence of poly(4-vinylpyridine), which acted as a base and acylation catalyst. Filtration, subsequent addition of amino-functionalized silica gel to remove excess acid chloride and a second filtration affording the corresponding esters in high yield (70-98%), excellent chemical purity (93-99%). Acylation of enantiopure alcohols yielded enantiopure esters. Acylation of two tertiary alcohols gave esters in low yield (27-57%) and variable chemical purity (57-99%).

Full text of DOI:10.1016/j.tetasy.2004.07.048

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3005–3009
Parallel synthesis of an ester library for substrate mapping of
esterases and lipases
à
*
´
Krista L. Morley, Vladimir P. Magloire, Christine Guerard and Romas J. Kazlauskas
´ ´
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6
Received 7 June 2004; accepted 9 July 2004
Available online 11 September 2004
Abstract—Use of solid-supported reagents simplified routine acylation of primary and secondary alcohols because it eliminated tra-
ditional purification. Using a parallel synthesizer, eight primary or secondary alcohols reacted with acid chloride in the presence of
poly(4-vinylpyridine), which acted as a base and acylation catalyst. Filtration, subsequent addition of amino-functionalized silica gel
to remove excess acid chloride and a second filtration affording the corresponding esters in high yield (70–98%), excellent chemical
purity (93–99%). Acylation of enantiopure alcohols yielded enantiopure esters. Acylation of two tertiary alcohols gave esters in low
yield (27–57%) and variable chemical purity (57–99%).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
and solution-phase synthesis,3 where solid-supported
reagents react with soluble compounds.
Advances in genomics and molecular biology have
greatly simplified the preparation of new enzymes. How-
ever, it is still slow and tedious to identify their favored
substrates. This identification is essential to establish an
enzymeÕs likely biochemical role and to identify poten-
tial application in biocatalysis. A key step in identifying
the favored substrates of enzymes is substrate mapping,
which measures the catalytic activity toward a broad
range of substrates. In most cases, these substrates are
not commercially available and must be synthesized.
Herein, we report the application of modern parallel
synthesis methodology to prepare an ester library suita-
ble for substrate mapping of esterases and lipases.
Solid-phase synthesis has the advantages that an excess
of reagents drives reactions to completion and that
washing the support-bound product easily removes
impurities and excess reagents. On the other hand, mon-
itoring reaction progress is often difficult, extra steps are
required for anchoring and removing the substrate from
the solid-support, and synthetic procedures must be
tediously optimized to act on compounds linked to the
solid support. On the other hand, solution-phase synthe-
sis retains the advantages of solid-support chemistry
while eliminating some disadvantages. Traditional puri-
fication is avoided because filtration easily removes
solid-supported reagents and solid-phase scavengers
for impurities. However, one must know the nature of
the impurity to chose a scavenger and avoid products
containing functional groups that might react with the
scavenger.
Parallel synthesis makes individual compounds through
simultaneous parallel reactions.1 The two parallel syn-
thesis methods are: solid-phase synthesis,2 where soluble
reagents act on a compound linked to a solid support,
We required a parallel synthesis of simple esters in high
yield, chemical purity, and conserved enantiomeric pur-
ity to map the substrate range and enantioselectivity of
new hydrolases. For a typical substrate mapping exper-
iment,4 the slowest step is ester synthesis. The subse-
quent screening is fast using colorimetric screening in
96-well plates.5
*
Corresponding author at present address: Department of Biochem-
istry, Molecular Biology & Biophysics and The Biotechnology
Institute, University of Minnesota, 1479 Gortner Ave, St. Paul,
MN 55108, USA. Tel.: +1 612 624 5904; fax: +1 612 625 5780;
e-mail: rjk@umn.edu
´
Present address: 677, rue Frederic Chopin, Montreal, QC, Canada
H1L 6S9.
Present address: Universite Pierre et Marie Curie, 4 place Jussieu,
F-75252 Paris cedex 05, France.
à
Although several solid-phase synthesis methods exist for
esters, we wanted to avoid anchoring and removing the
´
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.07.048

3006
K. L. Morley et al. / Tetrahedron: Asymmetry 15 (2004) 3005–3009
product from solid support. For example, carbodiimide
coupling (DMAP/DEC or DIC)6 between resin-bound
alcohol and carboxylic acid (and vice versa) yielded
esters on solid support. Similarly, base-catalyzed addi-
tion of acid chloride to resin-bound alcohol (and vice
versa) afforded resin-bound esters.7 Finally, carboxylic
acids added to trichloroacetimidate-activated Wang
resin to form ester links.8
CHCH2
n
O
C
O
C
O
C
1.
N
R1O
R2
Cl
R2
R1OH
Cl
R2
2. filtration
(excess)
Si
Si
NH2
1.
2. filtration
O
A number of groups have reported solution-phase syn-
thesis of esters, but had not tested them for enantiopure
esters. In the mid 1980Õs, Patchornik et al. used polymer-
supported dimethylaminopyridine (PS-DMAP) as an
acyl transfer agent to make two esters.9 Treating PS-
DMAP with excess acid chloride formed an N-acyl
pyridinium salt. Washing the resin to remove excess acid
chloride followed by addition of alcohol gave an 82–
100% yield of soluble ester. However, our attempts to
extend this method to a broader range of esters were
disappointing.
C
R1O
R2
Scheme 1. Parallel solution-phase synthesis of esters.
method affords enantiopure esters of primary and
secondary alcohols in high yield and chemical purity.
Successful alcohol acylation with five different acid chlo-
rides demonstrates the versatility of this procedure.
Flynn et al.14 acylated amines using a similar method.
Other solution-phase syntheses of esters are extensions
of procedures to make amide links. (Many procedures
to make amides fail when applied to esters.) Carboxylic
acids were linked to N-hydroxysuccinimide (NHS) or to
pentafluorophenol using solid-supported EDAC (ethyl
dimethylaminopropylcarbodiimide).10 Similarly, Dend-
rinos and Kalivretenos made NHS esters via a cap-
ture–release method.11 Carboxylic acids first coupled
to polymer-bound 1-hydroxybenzotriazole using di-
cyclohexylcarbodiimide. Nucleophilic attack by NHS
afforded the corresponding soluble esters. Although var-
ious carboxylic acids could be used, the only suitable
alcohol was NHS. In our laboratory, these methods
worked well to make soluble amides, but no esters
formed using the less nucleophilic aliphatic alcohols or
phenols.
2. Results
First we optimized the scavenger. Amine-functionalized
resins are traditionally used to sequester acid chlorides
in solution-phase acylations.15 However, Kim et al.16
report increased yields for disubstituted guanine
acylations when the scavenger is switched from poly-
mer-bound trisamine to amino-functionalized silica
(Si-Amine, Silicycle). Table 1 compares the efficiency
of PS-Trisamine (commercial scavenger, Argonaut
Technologies) and Si-Amine as acid chloride scavengers
in our alcohol acylations. In these experiments, methyl
(R)-3-hydroxy-2-methylpropionate reacted with excess
4-methylbenzoyl chloride (2.5equiv)17 in the presence
of poly(4-vinylpyridine), followed by the addition of 1,
2, or 2.5equivof scavenger (with respect to excess acid
chloride) upon acylation completion. We observed high
ester yields (98–99%) and enantiomeric purity (99%) for
all reactions, however product purity is significantly
higher for the Si-Amine reactions (Table 1). PS-Tris-
amine matches Si-Amine with respect to acid chloride
scavenging ability, but the presence of dimethylform-
amide (DMF) (presumably left over from scavenger syn-
To make three benzyl esters, Gayo and Suto used the
basic ion-exchange resin Amberlite IRA-68 as both a
reagent and scavenger.12 Acid chloride acylated benzyl
alcohol while the basic resin quenched liberated HCl.
Addition of water hydrolyzed the excess acid chloride
to the acid, which was also sequestered by the basic
resin. The water-addition step requires additional
work-up steps during product isolation.
Table 1. Evaluation of acid chloride scavengers in the acylation of
alcohol 1 with 4-methylbenzoyl chloride
A more general, but more complicated, method uses
polystyrylsulfonyl chloride resin as a condensation rea-
gent.13 First, alcohol reacts with excess carboxylic acid
in the presence of N-methylimidazole and the sulfonyl
chloride resin. Second, addition of aminomethylated
polystyrene resin and filtration removes excess acid.
Third, addition of acidic ion-exchange resin Amberlyte
15 followed by filtration removes the N-methylimidaz-
ole. This method yielded high purities for a wide range
of esters. Besides the complicated procedure, reaction
times required optimization for different esters.
Scavenger
Equiva
Yield (%)b
Purity (%)b
% Ee
PS-Trisaminec
1
98
98
99
88
91
87
99
99
99
2
2.5
Si-Amined
1
98
98
99
92
97
98
99
99
99
2
2.5
a With respect to excess acid chloride.
b Determined by gas chromatography (flame ionization detector) using
area % of peaks. Si-Amine reactions contained residual acid chloride
as an impurity. PS-Trisamine reactions contained both residual acid
chloride and DMF as impurities.
We report an efficient parallel solution-phase acylation
of alcohols with excess acid chloride, using poly(4-vinyl-
pyridine) as a catalyst and amino-functionalized silica
gel to remove the excess acid chloride (Scheme 1). Our
c Tris-(2-aminoethyl)amine polystyrene.
d Amino-functionalized silica.

K. L. Morley et al. / Tetrahedron: Asymmetry 15 (2004) 3005–3009
3007
thesis18) decreases reaction purity. Based on these obser-
vations, Si-Amine (2.5equiv) is used as the scavenger in
our parallel synthesis. To our knowledge this is the first
application of Si-Amine as an acid chloride scavenger in
alcohol acylation.
sponding alcohols form as byproducts in the acylation
reactions. These byproducts likely arise from octanoyl
chloride ketene formation, dimerization, and subsequent
reaction between the alcohol and resulting diketene
(Scheme 3). Bhushan et al. proposed a similar mechanis-
tic pathway for byproducts observed in the acylation
of tertiary alcohols with equimolar amounts of
4-dimethyl(amino)pyridine (DMAP) and acetic/propi-
onic anhydride.19 Furthermore, Sung and Wu reported
the dimerization of octanoyl chloride with triethyl-
amine, isolation of the resulting-lactone, and subsequent
reaction with methanol to form the corresponding
b-keto methyl ester.20
Next we examined a range of alcohols. Table 2 shows
the acylation results of 10 alcohols (Scheme 2) with octa-
noyl chloride, poly(4-vinylpyridine), and Si-Amine. We
used a dual-sided parallel synthesizer (Quest 210, Argo-
naut Technologies) with 10 reaction vessels on each side.
We used one side of the Quest 210 for alcohol acylation
and then transferred reactions simultaneously via can-
nula to the other side for Si-Amine scavenging. Primary
and secondary alcohols acylate in moderate to excellent
yield (70–98%) with high purity (93–97%). Furthermore,
acylation of all enantiopure alcohols occurs with conser-
vation of enantiomeric purity.
CHCH2
n
CHCH2
n
O
H
C6H13
N
C
O
C6H13
Cl
N
H
Both tertiary alcohols 9 and 10 acylate in low yield (53–
57%) and purity (57–60%). b-Keto esters of the corre-
Cl
H
C6H13
C
O
O
Table 2. Parallel acylation of alcohols 1–10 with octanoyl chloride and
solid-supported reagents
HOCR3
C6H13
O
C6H13
OCR3
Isolated yield (%)a
Purity (%)b
% Ee
O
Alcohol
O
C6H13
1
2
3
4
5
6
7
8
9
10
98
82
83
98
70
94
92
89
57
53
96
93
97
97
94
95
96
96
60
57
>99
98
C6H13
>99
>99
n.a.c
98
Scheme 3. Proposed formation of b-keto tertiary alcohol esters.
Finally, we looked at two alkyl acid chlorides and two
aryl acid chlorides. We observe high ester purity (94–
99%) when we vary the acid chloride in the parallel acyl-
ation of primary 1 and secondary 6 alcohols (Table 3).
Ester yields range from 71% to 96% depending on the
acid chloride. We observed low ester yields (27–31%)
for tert-butanol due to incomplete acylation with the
aryl acid chlorides even after increasing the reaction
time to 24h. Nevertheless, with increased amounts
of scavenger to remove acid chloride, the esters were
95
96
n.a.
n.a.
a Reaction conditions: (a) alcohol, poly(4-vinylpyridine), CH2Cl2, rt,
6h; (b) Si-Amine, rt, 2h.
b Determined by gas chromatography (flame ionization detector) using
area % of peaks. Residual octanoyl chloride identified in reactions
containing alcohols 1–8. b-Keto esters identified as byproducts in
reactions containing 9 and 10.
c n.a. = not applicable because starting alcohol was either racemic or
achiral.
Table 3. Parallel acylation of primary alcohol 1,a secondary alcohol 6,a
and tertiary alcohol 10b with various acid chlorides
Alcohol Chloride
Isolated yield (%) Purity (%)c
1
CH3COCl
75
83
80
94
71
88
87
94
96
95
95
99
94
99
99
99
93
primary alcohols
1
C3H7COCl
C6H5COCl
4-MeC6H4COCl
CH3COCl
O
1
Ph
Ph
Br
OH
OH
MeO
OH
OH
1
4
1
2
3
6
6
C3H7COCl
C6H5COCl
secondary alcohols
6
OH
OH
OH
O
OH
6
4-MeC6H4COClC 96
10
10
C6H5COCl
4-MeC6H4COCl
27
31
OMe
5
7
6
8
a Reaction conditions: (a) acid chloride, poly(4-vinylpyridine), CH2Cl2,
rt, 6h; (b) Si-Amine (1.9mmol, 2.5equivexcess over acid chloride),
2h.
tertiary alcohols
OH
OH
b Reaction conditions: (a) acid chloride, poly(4-vinylpyridine), CH2Cl2,
rt, 24h; (b) Si-Amine (2.8mmol, 2.5equivexcess over acid chloride),
2h.
c Determined by gas chromatography with a flame ionization detector
using area % of peaks. The impurity was residual acid chloride.
9
10
Scheme 2. Primary, secondary, and tertiary alcohols acylated. Yields
and purities were low for the tertiary alcohols.

3008
K. L. Morley et al. / Tetrahedron: Asymmetry 15 (2004) 3005–3009
Table 4. Hydrolase screening with esters containing 0–10% acetyl
chloride
an efficient method for the parallel synthesis of ester
libraries to map the substrate range of lipases and
esterases.
Ester
Hydrolase
Est. Ea
Quick Eb
Menthyl acetate
Menthyl acetate +
PCL
PCL
n.d.
n.d.
1.1 (R)
1.1 (R)
5% acetyl chloride
1-Phenylethyl acetate
1-Phenylethyl acetate +
10% acetyl chloride
1-Phenylethyl acetate
1-Phenylethyl acetate +
10% acetyl chloride
4. Experimental section
PCL
PCL
>50 (R)
>50 (R)
n.d.
n.d.
All reactions were carried out under nitrogen in anhy-
drous dichloromethane in a Quest 210 parallel synthe-
sizer (Argonaut Technologies, Foster City CA, USA
http://www.argotech.com). Poly(4-vinylpyridine) (Sig-
ma-Aldrich Co., Oakville, ON) was dried under vacuum
at room temperature for 24h before use. Silica-sup-
ported amine (Si-Amine, Silicycle (Quebec City QC,
http://www.silicycle.com) and polymer-supported amine
(Trisamine, Argonaut Technologies) were used as re-
ceived. Reactions were monitored by thin-layer chroma-
tography using UV light as a visualizing agent or
phosphomolybdic acid solution and heat as a develop-
ing agent. Gas chromatography was carried out with
Chromopak Chiralsil-Dex CB column (25m · 0.25mm,
PFE
PFE
30 (R)
29 (R)
n.d.
n.d.
a The ratio of the initial rates of hydrolysis of the two enantiomers
without competition.
b Calculated from the initial rates of hydrolysis of the two enantiomers
in the presence of resorufin acetate. n.d. = not determined. Accurate
measurement of high Quick E values requires a slower reacting ref-
erence compound to accurate compare to the slow reaction of the
slow reacting enantiomer.
1
isolated in high purity (93–99%). Unreacted tert-butanol
evaporated during solvent removal. Acylation with
acetyl and butyryl chloride was not examined, but we
expect significant b-keto ester byproducts for these
reactions.
Raritan, NJ). H NMR spectra were acquired at 500,
400, or 300MHz in CDCl3.
4.1. Parallel acylation procedure
Ten 5-mL vessels (side A) of the Quest 210 parallel syn-
thesizer were each loaded with poly(4-vinylpyridine)
(0.250g) and purged with nitrogen for 1h. Dry dichloro-
methane (4mL), alcohol (0.50mmol), and acylating
agent (1.25mmol, 2.5equiv) were added to each reaction
vessel and the reaction mixtures were agitated for 6h at
room temperature. All reactions were simultaneously fil-
tered and transferred via cannula to individual 5-mL
reaction vessels of the Quest 210 (side B) containing
Si-Amine (1.15g, 1.9mmol, 2.5equivexcess over acid
chloride). Side A reaction vessels were rinsed with
dichloromethane (1mL) and the rinse solutions were
added to the reaction mixtures in Side B. After 2h agi-
tation at room temperature, each reaction was filtered
through a 30lm frit into a 25-mL test tube. The
Si-Amine scavenger was rinsed with dichloromethane
(3 · 5mL) and rinse solutions were added to the
25-mL test tubes. The dichloromethane was removed
by rotary evaporation yielding oils.
Although the purity of esters from primary and second-
ary alcohols is high (93–99%), the impurity, residual
acid chloride, is a potential problem for using these
products directly for screening. However, we found no
detectable interference from this impurity and suspect
that it hydrolyzed rapidly during preparation of the
stock solution. As a test, we compared enantioselectivi-
ties measured using pure menthyl acetate with menthyl
acetate containing 5% deliberately added acetyl chlo-
ride. In both cases, hydrolysis catalyzed by lipase from
Pseudomonas cepacia (PCL) showed the same low enan-
tioselectivity—E = 1.1 in favor of the (R)-enantiomer
(Table 4). Similarly, we did not detect any interference
from added acid chloride for reactions with higher E
values. Using our method, we synthesized 1-phenylethyl
acetate (99% purity) and compared it with 1-phenylethyl
acetate containing 10% deliberately added acetyl
chloride. Both measurements showed the same high esti-
mated E values consistent with previous measure-
ments.21,22 The PCL-catalyzed hydrolysis showed
E >50 in favor of the (R)-enantiomer, while esterase
from Pseudomonas fluorescens (PFE) showed E values
of 30 and 29. Thus, in spite of the small amount of acid
chloride impurities, the esters produced by this parallel
synthesis method are suitable for use in enzyme screen-
ing without further purification.
4.2. Octanoic acid (S)-2-methoxycarbonyl-propyl ester
1H NMR (400MHz, CDCl3) d 0.92 (t, 3H), 1.28 (d, 3H),
1.36 (m, 8H), 1.64 (t, 2H), 2.34 (t, 2H), 2.84 (m, 1H),
3.76 (s, 3H), 4.28 (m, 2H).
4.3. (S)-3-Acetoxy-2-methyl-propionic acid methyl ester
1H NMR (500MHz, CDCl3) d 1.22 (d, 3H), 2.00 (s, 3H),
2.80 (m, 1H), 3.66 (s, 3H), 4.14 (dd, 1H), 4.20 (dd, 1H).
3. Discussion
In summary, we report a convenient method for the par-
allel acylation of primary and secondary alcohols using
commercially available poly(4-vinyl)pyridine and Si-
Amine. The procedure works well with alkyl and aryl
acid chlorides and affords esters in high yield, chemical
purity, and enantiomeric purity. We believe this will be
4.4. (S)-Butyric acid 2-methoxycarbonyl-propyl ester
1H NMR (500MHz, CDCl3) d 0.91 (t, 3H), 1.22 (d, 3H),
1.60 (m, 2H), 2.28 (t, 2H), 2.80 (m, 1H), 3.66 (s, 3H),
4.14 (dd, 1H), 4.20 (dd, 1H).

K. L. Morley et al. / Tetrahedron: Asymmetry 15 (2004) 3005–3009
3009
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(2H), 7.55 (1H), 8.00 (2H).
4.6. (S)-4-Methyl-benzoic acid 2-methoxycarbonyl-propyl
ester
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2.93 (m, 1H), 3.73 (s, 3H), 4.40 (dd, 1H), 4.48 (dd, 1H),
7.24 (d, 2H), 7.88 (d, 2H).
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using hexane/ethyl acetate (500:1) eluent: 1H NMR
(300MHz, CDCl3) d 0.89 (t, 3H), 0.90 (t, 3H), 1.28
(m, 16H), 1.46 (s, 9H), 1.59 (m, 2H), 1.80 (m, 2H),
2.50 (m, 2H), 3.32 (t, 1H); 13 C NMR (300MHz, CDCl3)
d 14.41, 14.49, 22.91, 22.97, 23.03, 23.91, 27.72, 28.28,
28.49, 29.43, 29.47, 31.90, 32.02, 42.06, 60.57, 81.80,
169.31, 205.98; MS (FAB) m/z 327 (7, [M+H]+), 271
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16H), 1.46 (s, 6H), 1.59 (m, 2H), 1.80 (m, 4H), 2.50
(m, 2H), 3.32 (t, 1H). The 1H NMR spectrum was nearly
identical to that of the tert-butyl b-keto ester. Differ-
ences in integration values correspond to the dimethyl-
propyl moiety of ester.
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Acknowledgements
15. Parlow, J. J.; Devraj, R. V.; South, M. S. Curr. Opin.
Chem. Biol. 1999, 3, 320–336.
16. McComas, W.; Chen, L.; Kim, K. Tetrahedron Lett. 2000,
41, 3573–3576.
We thank NSERC (Canada) for financial support and a
postgraduate scholarship to K.L.M., Dr. Robert Zam-
´
boni (Merck Frosst, Montreal) for the loan of Argonaut
Quest 210 parallel synthesizer and Sarah McDermott
and Candice Suter for preliminary experiments.
17. Using less acid chloride resulted in incomplete reactions.
For example, 10% unreacted alcohol remained when using
2equivof acid chloride.
18. Booth, R. J.; Hodges, J. C. J. Am. Chem. Soc. 1997, 119,
4882–4886.
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