Chemoenzymatic synthesis of (S)-Pindolol using lipases

Article abstract of DOI:10.1016/j.apcata.2017.08.003

A straightforward chemoenzymatic synthesis of (S)-Pindolol has been developed. The key step involved the enzymatic kinetic resolution of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane with lipase from Pseudomonas fluorescens via hydrolytic process to obtain enantiomerically enriched halohydrin (2S)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (96% ee) and (2R)-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (97% ee). The latter was subjected to a hydrolysis reaction catalyzed by Candida rugosa leading to (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (97% ee), followed by a reaction with isopropylamine, producing (S)-Pindolol (97% ee) in quantitative yield.

Full text of DOI:10.1016/j.apcata.2017.08.003

Accepted Manuscript
Title: Chemoenzymatic synthesis of (S)-Pindolol using lipases
Authors: Gledson Vieira Lima, Marcos Reinaldo da Silva,
Thiago de Sousa Fonseca, Leandro Bezerra de Lima, Maria da
˜
Conceic¸ao Ferreira de Oliveira, Telma Leda Gomes de Lemos,
Davila Zampieri, Jose Cleiton Sousa dos Santos, Nathalia
Saraiva Rios, Luciana Rocha Barros Gonc¸alves, Francesco
Molinari, Marcos Carlos de Mattos
PII:
S0926-860X(17)30373-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.apcata.2017.08.003
APCATA 16358
To appear in:
Applied Catalysis A: General
Received date:
Revised date:
Accepted date:
2-5-2017
24-7-2017
2-8-2017
Please cite this article as: Gledson Vieira Lima, Marcos Reinaldo da Silva, Thiago de
˜
Sousa Fonseca, Leandro Bezerra de Lima, Maria da Conceic¸ao Ferreira de Oliveira,
Telma Leda Gomes de Lemos, Davila Zampieri, Jose Cleiton Sousa dos Santos, Nathalia
Saraiva Rios, Luciana Rocha Barros Gonc¸alves, Francesco Molinari, Marcos Carlos de
Mattos, Chemoenzymatic synthesis of (S)-Pindolol using lipases, Applied Catalysis A,
Generalhttp://dx.doi.org/10.1016/j.apcata.2017.08.003
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Chemoenzymatic synthesis of (S)-Pindolol using lipases
Gledson Vieira Limaa, Marcos Reinaldo da Silvaa, Thiago de Sousa Fonsecaa, Leandro
Bezerra de Limaa, Maria da Conceição Ferreira de Oliveiraa, Telma Leda Gomes de
Lemosa, Davila Zampieria, Jose Cleiton Sousa dos Santosb, Nathalia Saraiva Riosc,
Luciana Rocha Barros Gonçalvesc, Francesco Molinarid and Marcos Carlos de Mattosa *
a
Department of Organic and Inorganic Chemistry, Laboratory of Biotechnology and
Organic Synthesis (LABS), Federal University of Ceará, Campus do Pici, Postal Box
6044, 60455-970 Fortaleza, Ceará, Brazil
b
Institute of Engineering and Sustainable Development, University of International
Integration Lusophone African-Brazilian, 62785-000, Acarape, Ceará,, Brazil
c
Department of Chemical Engineering, Federal University of Ceará, Campus do Pici,
60455-760 Fortaleza, Ceará, Brazil
d
Department of Food, Environmental and Nutritional Sciences (DEFENS), University
of Milan, Via Mangiagalli 25, 20133 Milan, Italy
* Corresponding author. Tel.: +55 8533669144; fax: +55 8533669782
E-mail address: mcdmatto@ufc.br (M.C. de Mattos)
Graphical abstract

Highlights
 A straightforward chemoenzymatic synthesis of (S)-Pindolol has been
developed.
 The key step involved kinetic lipase-mediated resolution of a haloydrin
acetate via hydrolytic process.
 P. fluorescens was a robust biocatalyst and afforded the halohydrin
acetate as chiral intermediate with 97% ee.
 The use of THF as co-solvent was essential for a high enantioselectivity
kinetic resolution process (E > 200).
ABSTRACT
A straightforward chemoenzymatic synthesis of (S)-Pindolol has been developed. The
key step involved the enzymatic kinetic resolution of rac-2-acetoxy-1-(1H-indol-4-
yloxy)-3-chloropropane with lipase from Pseudomonas fluorescens via hydrolytic
process to obtain enantiomerically enriched halohydrin (2S)-1-(1H-indol-4-yloxy)-3-
chloro-2-propanol (96% ee) and (2R)-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane
(97% ee). The latter was subjected to a hydrolysis reaction catalyzed by Candida
rugosa leading to (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (97% ee), followed
by a reaction with isopropylamine, producing (S)-Pindolol (97% ee) in quantitative
yield.
Keywords: Pindolol; lipases; biocatalysis; chemoenzymatic synthesis; enzymatic kinetic
resolution Pseudomonas fluorescens.
;
1. Introduction
Pindolol, 1-(1H-indol-4-yloxy)-3-(isopropylamino)-2-propanol (Fig. 1), is
a
pharmaceutically active substance, commercially marketed under the brand name

Visken (Sandoz) or Barbloc (Alpha) [1]. It is noteworthy that (S)-Pindolol (S-5) is 200
times more active than (R)-Pindolol [2]. This drug is a nonselective β-blocker, which
has a higher intrinsic sympathomimetic activity as compared to any currently available
β-blocker, acting effectively as antianginal, antihypertensive, and antiarrhythmic [3];
interestingly, Pindolol is also used in the treatment of glaucoma [4]. It is an effective
agent for the treatment of hypertension in pregnancy, since it has no effects on fetal
haemodynamics and does not affect fetal cardiac function [5]. Beside these established
uses, (S)-Pindolol can be used in combination with antidepressants selective inhibitors
of serotonin reuptake (SSRI), overcoming the action of these drugs [6,7]. Although an
old-fashioned drug, new applications have been reported for the (S)-Pindolol, such as in
the treatment of fibromyalgia and related fatigue syndromes [8], premenstrual syndrome
and premenstrual dysphoric disorder [9], as well as aiding in the treatment of cachexia
and sarcopenia [10].
Biocatalysis offers an alternative approach to conventional chemical processes for the
production of single-isomer chiral drugs. Lipases are highlighted as being among the
most frequently used enzymes for the production of drugs and their intermediates [11-
14]. The use of lipases is mainly due to the characteristics of the regio-, chemo- and
enantioselectivity in the resolution process of racemates, without the use of cofactors.
Moreover, this class of enzymes has generally excellent stability in the presence of
organic solvents, facilitating the solubility of the organic substrate to be modified [14].
The chemoenzymatic synthesis of pindolol and other β-blockers such as propranolol,
toliprol, moprolol, alprenolol, penbutenol, atenolol, practolol, oxprenolol, acebutolol,
sotalol and nifenalol is a well-documented subject in the literature [15-29].
Syntheses of (S)-Pindolol include the preparation of chiral intermediates via lipase-
mediated kinetic resolution as a key step. An example is the hydrolysis of rac-1,2-
diacetoxy-3-chloropropane, in the presence of lipoprotein lipase, leading to (S)-1,2-
diacetoxy-3-chloropropane which was converted to (S)-3-(4-indolyloxy)-1,2-
propanediol (90% ee and 20% yield). In this case, the authors stated that this latter
chiral intermediate can be used in the synthesis of (S)-Pindolol [28]. Another example is
the hydrolysis of rac-1-acetoxy-2,3-dichloropropane in the presence of pancreatin to
give (S)-1-acetoxy-2,3-dichloropropane (90% ee). The latter was reacted with 4-
hydroxyindole in basic medium, yielding the crude (S)-Pindolol in 45% yield. After
treatment with benzene, the (S)-Pindolol was recovered in 31% yield [[α]D25 = - 2.52 (c

25
1.0, MeOH)]. After recrystallization, the (S)-Pindolol was obtained in 12% yield [[α]D
= - 3.4 (c 5.9, MeOH)] [29]. The syntheses of enantiomerically pure (S)-Pindolol was
also reported from (R)-3-chloro-1,2-propanediol in 65% yield [30,31] as well as from
(S)-glycidyl nosylate in 83% yield [32], but the authors did not informed how these
intermediates were obtained. Another example of synthesis of (S)-Pindolol (88% yield
and 92% ee) include a catalytic asymmetric nitroaldol reaction using Lanthanum-
Lithium-(R)-BINOL, (R)-LLB, as chiral catalyst [33].
In general, a major drawback in the use of epichlorohydrin or glycidyl esters as chiral
intermediates in the preparation of β-blockers is a competition between substitution at
C-1 and ring-opening at C-3 during the nucleophilic attack of the anion aryloxy, leading
to a decrease in the optical purity of the final product [1,30,34]. An effective approach
to circumvent this problem is the enzymatic kinetic resolution of chlorohydrins, rac-1-
aryloxy-3-chloro-2-propanol, to obtain the corresponding chiral compound to be used as
intermediate. This approach has been applied in the synthesis of a series of (S)-β-
blockers as propranolol, toliprol, moprolol, alprenolol, penbutenol, atenolol, practolol
and oxprenolol. In this case, two complementary methods were used, the hydrolysis of
rac-2-acetoxy-1-aryloxy-3-chloropropane and acetylation of rac-1-aryloxy-3-chloro-2-
propanol using vinyl acetate as acyl donor and tert-butyl methyl ether (TBME) as
solvent, both methods in the presence of lipase from Pseudomonas sp. [15]. More
recently, CAL-A (CLEA) [25] and CAL-B [27] mediated the kinetic resolution of
halohydrins, precursors of the β-blocker Atenolol, with E values > 200. It is noteworthy
that various optically pure aromatic chlorohydrins were prepared via dynamic kinetic
resolution (DKR) with the use of lipase from Pseudomonas cepacia (PS-C “Amano” II)
combined with an efficient racemization with Ru complex catalyst [35].
To the best of our knowledge, there are no reports relating to the preparation of chiral
halohydrin 1-(1H-indol-4-yloxy)-3-chloro-2-propanol from lipase-mediated kinetic
resolution to be used as an intermediate for the synthesis of (S)-Pindolol.
Herein, we wish to report the chemoenzymatic synthesis of (S)-Pindolol (S-5) in which
the key step was the preparation of (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (R-
3) (Fig. 1) as intermediate, in the presence of lipases. We have focused especially on the
optimization of the reactional conditions.
2. Experimental

2.1. Enzymes
(i) Immobilized lipases: Candida antarctica lipase type B immobilized on acrylic
resin (CAL-B, Novozym 435, 7,300.0 U/g) and Rhizomucor miehei lipase immobilized
on anionic resin (RML, 150.0 U/g) were purchased from Novozymes®. Thermomyces
lanuginosus lipase immobilized on immobead-150 (TLL, 250.0 U/g), Rhizopus oryzae
lipase immobilized on immobead-150 (ROL, 340.0 U/g) and Amano lipase PS from
Burkholderia cepacia immobilized on diatomaceous earth (PS-IM, ≥ 500 U/g) were
acquired from Sigma-Aldrich® (ii) Crude lipase preparations: Pseudomonas fluorescens
lipase (AK, 22,100.0 U/g), Penicillium camemberti lipase (G, 50.0 U/g), Amano lipase
PS from B. cepacia (PS, ≥ 30,000U/g), lipase from Rhizopus niveus (RNL, ≥ 1.5 U/mg),
Amano lipase from Mucor javanicus (MJL, ≥ 10,000 U/g) and lipase from Aspergillus
niger (ANL, 200 U/g) were acquired from Sigma-Aldrich®. Porcine pancreas lipase
(PPL, 46.0 U/g solid), and C. rugosa lipase (CRL, 1.4 U/g) were obtained from
Sigma®.
2.2. Chemical materials
Chemical reagents were purchased from different commercial sources and used
without further purification. Acetone, hexane and dichloromethane were acquired from
Synth®. Toluene was obtained from Dinamica® and dioxane from Carlo Erba®.
Isopropanol (IPA), acetonitrile and hexane, HPLC grade, were purchased from
TEDIA®. Tetrahydrofuran (THF), heptane, cyclohexane and tert-butylmethylether
(TBME) were acquired from Sigma-Aldrich®. Solvents used in the reaction of
biocatalysis were distilled over an adequate desiccant under nitrogen. Analytical TLC
analyses were performed on aluminum sheets pre-coated with silica gel 60 F254 (0.2
mm thick) from Merck®. Flash chromatographies were performed using silica gel 60
(230-240 mesh).
2.3. Analysis
Melting points of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4) and
(S)-Pindolol (S-5) were determined in open capillary tube Mettler Toledo model FP62
and are uncorrected. IR spectra were recorded on a Perkin - Elmer 1720-X F7 using
1
NaCl plates or KBr in pellets. H and 13C NMR were obtained using Spectrometer
Bruker model Avance DRX-500, operating at frequency of 500 MHz for hydrogen and

frequency of 125 MHz for carbon. The chemical shifts are given in delta (δ) values and
the absolute values of coupling constants (J) in Hertz (Hz). Measurement of the optical
rotation was done in a Perkin-Elmer 241 polarimeter. High performance liquid
chromatography (HPLC) analyses were carried out in a Shimadzu chromatograph model
LC solution 20A using a chiral column Chiracel® OD-H (150 m x 4.6 mm I.D.) with
oven set at 35 °C . For the following of the reaction time courses: Flow was 1.0 mL/min
using hexane:IPA (80:20) as eluent and UV detector at 265 nm. Retention times were:
(S)-acetate (S-4) 19.1 min; (R)-acetate (R-4) 20.9 min; (S)-alcohol (S-3) 27.7 min; (R)-
alcohol (R-3) 34.0 min.
2.4 Calculation of enantiomeric excess, conversion and enantioselectivity
The efficiency of the kinetic resolution was evaluated based on the optical purity of
the compounds, expressed in terms of enantiomeric excess of the substrate (e.e.s) and
product (e.e.p), using the following Eq. (1) and (2):
X  Y
e.e.s =
e.e.p =
(1)
(2)
X  Y
X  Y
X  Y
X denote the major enantiomer and Y denote the minor enantiomer represented by their
chromatographic peak areas.
The conversion (c) is calculated using Eq. (3):
e.e.
s
c =
(3)
e.e.
s
 e.e.
p
Enantioselectivity was expressed as enantiomeric ratio (E) and calculated by Eq. (4):
ln[ 1  c (1  e.e.
ln[ 1  c (1  e.e.
p
p
)]
)]
E =
(6):
(4)
The results of the e.e.s and e.e.p are expressed in percentage using the Eq. (5) and
X  Y
e.e.s =
x 100
(5)
X  Y

X  Y
X  Y
e.e.p =
x 100
(6)
Additionally, the conversion (c) is also calculated in percentage, using (5) and (6).
2.5. Synthesis of rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3)
To a suspension of 500 mg (3.75 mmol) of 4-hydroxyindole (1) in 5 mL of aqueous
solution of sodium hydroxide (NaOH 0.75 M), 2.5 mL (32 mmol) of (±)-
epichlorohydrin in 2 mL of dioxane was added. The reaction was stirred for 6 h at room
temperature under N2. The progress of the reaction was monitored by thin layer
chromatography using dichloromethane as eluent. After this time, the solvent was
evaporated under reduced pressure. The crude product was purified by flash
chromatography on silica gel using dichloromethane as eluent, yielding rac-1-(1H-
indol-4-yloxy)-3-chloro-2-propanol (rac-3) (66% yield) and (1H-indol-4-yloxy)(2-
oxiranyl)methane (rac-2) (22% yield). This latter was converted to rac-1-(1H-indol-4-
yloxy)-3-chloro-2-propanol (rac-3) as follows: to a solution of 152.4 mg (0.81 mmol) of
(1H-indol-4-yloxy)(2-oxiranyl)methane (rac-2) in 5 mL of dichloromethane, 5 mL of
concentrated HCl was added. The reaction mixture was stirred for 1 hour. Then, the
product was extracted with dichloromethane (3 x 5 mL) and dried with anhydrous
Na2SO4. After filtration, the solvent was evaporated under reduced pressure. The crude
product was purified by chromatography using silica gel and dichloromethane as eluent.
Finally, the rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3) was obtained with an
overall yield of 85% (718.8 mg).
2.6.
Synthesis of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4)
DMAP (22 mg, 0.19 mmol) and acetic anhydride (360 µL, 3.8 mmol) were added to
a rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3) (50 mg, 0.38 mmol) solution in
dichloromethane (4.0 mL). The reaction was stirred at room temperature during 1 h and
after that time, the solvent was evaporated under reduced pressure. The resulting crude
product was purified by flash chromatography on silica gel , using dichloromethane as
eluent, to afford the desired rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-
4) as a yellow liquid in 93% yield (94.5 mg).
2.7. Synthesis of (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (R-3) via hydrolysis

A suspension of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4) (5
mg, 0.019 mmol) and 10 mg of lipase from Pseudomonas fluorescens in a mixture of
152 μL of phosphate buffer 100 mM (pH 7.0) and 38 μL of THF (80/20 v/v) was shaken
at 40 °C and 250 rpm for 25 h. After this time, the products were extracted with EtOAc
(3 x 1.0 mL) and the organic phases were combined and dried with anhydrous Na2SO4,
filtered and the solvent evaporated under reduced pressure. The crude product was
purified by flash chromatography on silica gel using dichloromethane as eluent,
yielding 2 mg of (2R)-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (R-4) (40.5%
% yield and 97% ee) and 2 mg of (2S)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (S-3)
(46.7% yield and 96% ee). Then, a suspension of (2R)-2-acetoxy-1-(1H-indol-4-yloxy)-
3-chloropropane (R-4) (5 mg, 0.019 mmol) and 5 mg of lipase from Candida rugosa
(ratio 1:1 in weight respect to the R-4) in a mixture of 152 μL of phosphate buffer 100
mM (pH 7.0) and 38 μL of THF (80/20 v/v) was shaken at 40 °C, 250 rpm for 12 h.
After this time, the reaction mixture was centrifuged, the supernatant was removed and
the product was extracted with EtOAc (3 x 1.0 mL). The organic phases were combined
and dried with anhydrous Na2SO4, filtered and the solvent evaporated under reduced
pressure, yielding 4.3 mg of (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (R-3)
(100% yield, [α]D20 = + 2.19 (c 0.9, CHCl3) for 97% e.e. of the (R)-enantiomer).
2.8. Synthesis of (S)-Pindolol (S-5)
A solution of (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (R-3) (100 mg, 0.44
mmol) in excess of isopropylamine (4 mL) was stirred under reflux for 12 h. After this
time, the excess of amine was evaporated under reduced pressure and the product was
extracted with EtOAc (3 x 1.0 mL). The solvent was evaporated under reduced pressure,
yielding 109.5 mg of (S)-Pindolol (S-5) as a white solid in quantitative yield, ([α]D20 = -
20
4.1 (c 0.62, MeOH) for 97% e.e. of the (S)-enantiomer. Lit [α]D = - 3.8 (c 0.62,
MeOH) for 92% e.e. of the (S)-enantiomer) [33].
2.9. General procedure for the lipase-catalyzed hydrolysis of rac-2-acetoxy-1-(1H-
indol-4-yloxy)-3-chloropropane (rac-4) (screening) Table 1
A suspension of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane rac-4 (5 mg,
0.019 mmol), 5 mg of lipase (ratio 1:1 in weight respect to the rac-4) in a mixture of
152 μL of phosphate buffer 100 mM (pH 7.0) and 38 μL of THF (80/20 v/v) was shaken
at 30 °C, 250 rpm for varied times (ranging from 12 to 36 h). After each period, the

products were extracted with EtOAc (3 x 1.0 mL) and the organic phases were
combined and dried with anhydrous Na2SO4, filtered and the solvent evaporated under
reduced pressure. The crude product was purified by flash chromatography on silica gel
using dichloromethane as eluent, yielding (2R)-2-acetoxy-1-(1H-indol-4-yloxy)-3-
chloropropane (R-4) and (2S)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (S-3). Their
enantiomeric excess was determined by HPLC.
2.10. General procedure for the lipase-catalyzed acetylation of rac-1-(1H-indol-4-
yloxy)-3-chloro-2-propanol (rac-3) Table 3
To a suspension of the rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3) (4 mg,
0.018 mmol) and lipase (4 mg) in dry organic solvent (0.18 μL) under nitrogen
atmosphere, vinyl acetate (7.8 µL, 0.085 mmol) was added, and the reaction was shaken
(250 rpm) at temperatures ranging from 30 °C to 40 °C. Aliquots were regularly
analyzed by HPLC and after the adequate time, the reaction was stopped and the
enzyme filtered off and washed with the respective solvent (3 mL). The solvent was
evaporated under reduced pressure and the crude products purified by flash
chromatography on silica gel using dichloromethane as eluent. This procedure yielded
(2S)-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (S-4) and (2R)-1-(1H-indol-4-
yloxy)-3-chloro-2-propanol (R-3), being their enantiomeric excess determined by
HPLC.
rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3): Oil. Rf (100% CH2Cl2): 0.30.
IR max (cm1): 3403, 2924, 1616, 1585, 1502, 1440, 1241, 1087 and 736 cm-1. 1H NMR
(500 MHz, CD3OD) δ (ppm): 2.66 (br s, 1H, OH: exchanges with CD3OD), 3.8 (dd, 1H,
J=11.2 Hz and 5.6 Hz), 3.9 (dd, 1H, J= 11.2 Hz and 5.0 Hz), 4.2-4.3 (m, 3H), 6.6 (d,
1H, J= 7.6 Hz), 6.7 (m, 1H), 7.1-7.2 (m, 3H) and 8.2 (br s, 1H, NH: exchanges with
CD3OD).13C NMR (125 MHz, CD3OD) δ (ppm): 46.4 (CH2), 68.9 (CH2), 70.2 (CH),
99.9 (CH), 101.2 (CH), 105.4 (CH), 118.9(C), 122.9 (CH), 123.2 (CH), 137.6 (C) and
152.1 (C).
rac-(1H-indol-4-yloxy)(2-oxiranyl)methane (rac-2): Oil. Rf (100% CH2Cl2): 0.57. IR

max (cm-1): 3401, 1361, 1240 and 1086 cm-1. 1H NMR (500 MHz, CDCl3) δ (ppm): 2.8
(dd, 1H, J= 5.0 Hz and 2.6 Hz), 2.9 (m, 1H), 3.4-3.5 (m, 1H), 4.2 (dd, 1H, J= 11.1 Hz
and 5.5 Hz), 4.4 (dd, 1H, J= 11.1 Hz and 3.2 Hz), 6.5 (d, 1H, J= 7.6 Hz), 6.7 (m, 1H),
13
7.0-7.1 (m, 3H) and 8.2 (br s, 1H). C NMR (125 MHz, CDCl3) δ (ppm): 45.1 (CH2),

50.6 (CH), 69.0 (CH2), 100.2 (CH), 101.1 (CH), 105.2 (CH), 119.0 (C), 122.9 (CH),
123.0 (CH), 137.6 (C) and 152.4 (C).
rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4): Solid. Mp: 101-106
°C. Rf (100% CH2Cl2): 0.65. IR max (cm-1): 3409, 1736, 1224, and 1047 cm-1. 1H NMR
(500 MHz, CDCl3) δ (ppm): 2.1 (s, 3H), 3.8 (dd, 1H, J= 11.7 Hz and 5.5 Hz), 3.9 (dd,
1H, J= 11.7 Hz and 4.8 Hz), 4.3 (m, 2H), 5.4-5.5 (m, 1H), 6.5 (d, 1H, J= 7.6 Hz), 6.7
(m, 1H), 7.0-7.1 (m, 3H), and 8.2 (br s, 1H). 13C NMR (125 MHz, CDCl3) δ (ppm): 21.1
(CH3), 43.0 (CH2), 66.5 (CH2), 71.5 (CH), 100.0 (CH), 101.2 (CH), 105.4 (CH), 119.0
(C), 122.9 (CH), 123.1 (CH), 137.6 (C), 152.1 (C) and 170.5 (C).
(S)-Pindolol (S-5): Solid. Mp: 168-170 °C. IR max (cm-1): 3303, 2964, 1242, 1086,
and 879 cm-1. 1H NMR (500 MHz, CD3OD) δ (ppm): 1.12 (d, 3H, J= 5.4 Hz), 1.13 (d,
3H, J= 5.2 Hz), 2.7-2.8 (m, 1H), 2.9-3.0 (m, 2H), 4.0-4.2 (m, 3H), 6.5 (dd, 1H, J= 6.7
13
Hz and 1.4 Hz), 6.53-6.54 (m, 1H), 7.0-7.1 (m, 3H). C NMR (125 MHz, CD3OD) δ
(ppm): 22.3 (CH3), 22.4 (CH3), 50.1 (CH), 51.1 (CH2), 69.8 (CH), 72.0 (CH2), 99.7
(CH), 101.3 (CH), 106.3 (CH), 120.2 (C), 123.1 (CH), 124.2 (CH), 139.3 (C) and 153.6
(C). [α]D20 = - 4.1 (c 0.62, MeOH) for 97% e.e. of the (S)-enantiomer. Lit [α]D20 = - 3.8
(c 0.62, MeOH) for 92% e.e. of the (S)-enantiomer [33].
3. Results and Discussion
The chemoenzymatic route for the production of (S)-Pindolol (S-5) is depicted in
Fig. 1. The key step involved the preparation of the enantiomerically enriched
halohydrin (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (R-3) via kinetic resolution
in the presence of a lipase.
INSERT FIGURE 1 HERE
3.1. Synthesis of halohydrin rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3) and
its acetate (rac-4)
Initially, the halohydrin rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3) was
obtained from the reaction between the commercially available reagents 4-
hydroxyindole (1) and epichlorohydrin. In this case, beyond the expected halohydrin,
rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3) (66% yield), the epoxide rac-

(1H-indol-4-yloxy)(2-oxiranyl)methane (rac-2) (22% yield) was obtained. Then, the
epoxide was converted to the desired halohydrin after treatment with concentrated HCl.
Thus, it was possible to obtain the rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-
3) with global yield of 85%. Subsequently, by the use of acetic anhydride and 4-
dimethylaminopyridine (DMAP), the corresponding acetate rac-2-acetoxy-1-(1H-indol-
4-yloxy)-3-chloropropane (rac-4) was prepared with 93% yield. Both compounds, rac-3
and rac-4, were analyzed by chiral HPLC to find the adequate conditions for the
separation of their enantiomers (Fig. 2).
INSERT FIGURE 2A and 2B HERE
3.2. Kinetic resolution of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4)
via hydrolysis reaction
Initially, thirteen commercially available lipases were evaluated in the kinetic resolution
of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4) via hydrolysis reaction,
Fig. 3 (Path a). The reactions were conducted at 30 °C, using 1:1 ratio (w:w) of
substrate/lipase, phosphate buffer (pH 7.0) and THF as a co-solvent (buffer/co-solvent
4:1, v:v). The reactions were monitored by chiral HPLC and stopped within 12-36 h,
when the conversions reached values close to 50%. The experimental data are
summarized in Table 1.
INSERT FIGURE 3 HERE
INSERT TABLE 1 HERE
Lipases from P. camemberti, R. oryzae immobilized on immobead-150, porcine
pancreas, R. miehei immobilized on anionic resin, R. niveus and Amano lipase from M.
javanicus (Table 1, entries 1-6) were inactive in kinetic resolution of rac-2-acetoxy-1-
(1H-indol-4-yloxy)-3-chloropropane (rac-4), under the above conditions. Novozym
435®, Amano PS from B. cepacia immobilized on diatomaceous earth and Amano PS
from B. cepacia were the most active among the evaluated lipases, leading to
conversion values near 50% in only 12 h of reaction, however with low-to-medium E-
values, ranging from 8 to 30 (Table 1, entries 13, 15 and 16). C. rugosa showed an

intermediate activity, leading to 41% of conversion in 20 h, with low enantioselectivity
(Table 1, entry 14). The best enantioselectivity (E = 79) was presented by lipase from T.
lanuginosus, with 46% conversion after 36 h (Table 1, entry 10). In order to decrease
the reaction time, the temperature was increased from 30 to 40 °C, showing only a slight
acceleration from 36 h to 33 h, keeping the enantioselectivity basically unchanged
(Table 1, entry 11). A further attempt to decrease the reaction time was performed by
increasing the enzyme-substrate ratio from 1:1 to 2:1. Actually, the reaction time
decreased to 24 h, however followed by a decrease in enantioselectivity with E value of
43 (Table 1, entry 12). Lipase from P. fluorescens has provided a conversion value
close to 50% with moderate enantioselectivity (E= 36), but in a very long reaction time,
36 h, (Table 1, entry 7). In order to reduce the reaction time, the temperature was
increased from 30 to 40 °C (Table 1, entry 8). In this case, besides decreasing the
reaction time from 36 to 23 h, there was an increase in selectivity of kinetic resolution
with E values ranging from 36 to 100 (Table 1, entry 8). It is generally assumed that
enantioselectivity in enzyme-catalyzed reactions decreases with increasing temperature.
However, there are several examples in the literature of enzymatic reactions in which
enantioselectivity increases with increasing temperature [36-44]. In these situations,
among the components of the reaction, the degree of flexibility in the active site of the
enzyme is more evident than the stereostatic and electrostatic effects, leading to a
preference of the enantiomer favored by entropy [37,39,44].
This latter result prompted us to verify if the reaction time would be decreased with a
greater amount of enzyme. Thus, the ratio enzyme/substrate was increased from 1:1 to
2:1 (Table 1, entry 9). However, we noticed a slight increase in the reaction time from
23 to 25 h, but with significant increment in the enantioselectivity (E from 100 to 207),
Table 1, entries 8 and 9. It should be noted that, recently, a similar behavior was
observed in the esterification of (R,S)-ibuprofen mediated by lipase from the
thermophilic Geobacillus zalihae, which enantioselectivity increased with increasing
enzyme loading [45]. Concerning the enantiopreference, all the evaluated lipases
showed a marked preference for leading to halohydrin S-3, with the notable exception
of lipase from A. niger, which produces the halohydrin R-3 albeit with low
enantioselectivity (E value of 8; Table 1, entry 17).
Based on the optimized conditions for systems previously studied in our research group
[46,47], hydrolysis reactions of the rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-

chloropropane (rac-4) were performed in the presence of THF as co-solvent. The
enantioselectivity of the reaction underwent a dramatic increase with increasing
temperature and enzyme:substrate ratio. However, it was not clear the influence of the
THF as co-solvent on the activity and selectivity of lipase from P. fluorescens.
Therefore, we decided to investigate the effect of the absence of co-solvent as well as
the presence of other co-solvents on the efficiency of the enzymatic kinetic resolution of
rac-4.
3.3. Co-solvent effect on the enzymatic kinetic resolution of rac-2-acetoxy-1-(1H-indol-
4-yloxy)-3-chloropropane (rac-4) via hydrolytic process
The study of the effect of co-solvent in the hydrolysis of rac-2-acetoxy-1-(1H-indol-4-
yloxy)-3-chloropropane (rac-4) was carried out in the presence of lipase from P.
fluorescens, as it presented the best results of conversion and enantioselectivity (Table
1, entry 9). Apart from THF, which was evaluated in three different ratio with respect to
the buffering medium, hydrolysis reactions were carried out in the presence of acetone,
acetonitrile, dioxane, isopropanol, ethanol, tert-butyl methyl ether (TBME) and hexane
as co-solvents (buffer/co-solvent 4:1, v:v), keeping the temperature at 40 °C, ratio
lipase/substrate 2:1 (w:w) and 25 h of reaction time. It should be noted that the
aforementioned reaction was also performed in the absence of co-solvent. All the
experimental data have been summarized in Table 2.
INSERT TABLE 2 HERE
In the absence of co-solvent, the enantioselectivity of lipase from P. fluorescens was
very low, with E-value of 11 (Table 2, entry 1). All evaluated co-solvents with negative
log P values showed E-values range from low to moderate, 6-39 (Table 2, entries 2-6).
It is noteworthy that protic solvents such as isopropanol and ethanol (Table 2, entries 5
and 6) were the ones that led to the lowest values of enantioselectivity (E). Probably,
these solvents interfere in the active enzymatic site through hydrogen bonds. On the
other hand, when less hydrophilic THF was used as co-solvent (buffer/co-solvent 8:2,
v:v), the enantioselectivity was greatly increased up to an E-value of 207 (Table 2, entry
7). It should be noted that the addition of THF in 9:1 and 7:3 (buffer/co-solvent v:v)
ratios led to lower enantioselectivity (Table 2, entries 8-9). In contrast, solvents with
increasing log P values such as TBME and hexane (Table 2, entries 10 and 11), did not
offer high enantioselectivity, most probably because of a partial loss of efficiency in

dissolving the substrate. As far as we know, there is no general rule that explains the
influence of the co-solvent on the enantioselectivity of enzymatic hydrolysis reaction in
monophasic aqueous systems containing a minor fraction of an organic solvent. A
possible explanation for obtaining a high enantioselectivity value in the kinetic
resolution of rac-4 can be given by evoking the concept of induce-fit motions of the
enzyme [48]. It is known that the two enantiomers of acetates from secondary alcohols
have two different modes of attachment to the active site of a lipase. The large group
(indole moiety) of the most reactive enantiomer (S)-4 points out from the active site
(larger pocket) while the middle group (chlorine atom) is oriented toward the
stereoselective pocket (smaller pocket), constituting a favorable mode. For the less
reactive enantiomer (R)-4, the large group is positioned in the stereoselective pocket,
while the medium group is oriented out of the active site (unfavorable mode). These two
different modes of binding the enantiomers in lipase result in the enantioselectivity
[49,50]. It is evident that the high value of enantioselectivity obtained in the kinetic
resolution of rac-4 in the presence of P. fluorescens is due to a combination of effects,
which includes increasing the reaction temperature and adding an ideal amount of THF
as the co-solvent. These combined effects lead to better accommodation of the (S)-4
enantiomer at the active site and a greater stabilization of the complex between the
substrate and enzyme. The addition of THF strips off an amount of water from the
enzyme, modifying the microenvironment in the active site of the lipase. Probably, the
new microenvironment favors a better accommodation of the indole moiety of the (S)-4
enantiomer. The increase in temperature should be responsible for an increase in the
conformational flexibility of the lipase and this, added to the effect of the addition of
THF, should promote a more effective fitting of the indole moiety of (S)-4 in the large
binding pocket of the enzyme. Thus, the favorable mode becomes even more favorable,
resulting in an increase of the enantioselectivity in the kinetic resolution of rac-4.
Once that the kinetic resolution of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane
(rac-4) was optimized for the production of enantiomerically enriched (2R)-2-acetoxy-
1-(1H-indol-4-yloxy)-3-chloropropane (R-4) (97% ee), we decided to move forward and
explore the enzymatic acetylation of rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol
(rac-3) mediated by P. fluorescens, in the presence of organic solvents.
3.4. Kinetic resolution of rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3) via
acetylation reaction

Due to the high values of conversion and enantioselectivity obtained in the hydrolysis of
rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4) (Table 1, entry 7),
catalysed by lipase from P. fluorescens, we selected this enzyme to mediate the kinetic
resolution of rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3) via acetylation (Fig
3, Path b). Based on previously reported studies, we choose the vinyl acetate as acyl
donor [46,47]. Various solvents were evaluated at temperatures ranging between 30 and
40 °C, (Table 3).
INSERT TABLE 3 HERE
As THF was successfully used as co-solvent in the hydrolysis of rac-2-acetoxy-1-(1H-
indol-4-yloxy)-3-chloropropane (rac-4), it was the first solvent evaluated in acetylation
of rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3), in the presence of lipase from
P. fluorescens. However, THF decreases the enzymatic activity leading to a conversion
of only 2%, while the selectivity remained high, E-value > 200 (Table 3, entry 1). With
increasing temperature of 30 to 40 °C, the enzyme activity showed only a slight increase
to 4% of conversion, with a decrease of E-value to 22, (Table 3, entry 2). In more
hydrophobic solvents (i.e., heptane, toluene, cyclohexane and hexane), conversion
values range from low to moderate (13-31%), but with quite low enantioselectivity
(Table 3, entries 3-6). When the acetylation was carried out in the presence of TBME as
solvent, the corresponding acetate (S-4) was obtained with e.e. of 91%, but the
conversion value was only 19% (Table 3, entry 7). This result encouraged us to provide
conditions to achieve a conversion close to 50% with still high enantioselectivity.
Firstly, temperature was increased from 30 to 40 °C, leading a desired conversion value
(51%). However, the selectivity decreased dramatically, with E-value of only 11 (Table
3, entry 8). More attempts to optimize reaction conditions in the presence of TBME
were performed; the amount of vinyl acetate was doubled from 5 to 10 equivalents and
the reaction time prolonged from 24 to 68 h, but the selectivity of the reaction remained
low, with E-value of only 14, (Table 3, entries 9-10). In acetonitrile and reaction time of
24 h, the acetylated product (S-4) was obtained with a high value e.e. (94%), but low
conversion value, only 9% (Table 3, entry 11). By increasing the reaction time from 24
to 68 h, conversion increased from 9 to 20% while maintaining high e.e. (94%) of the
product, (Table 3, entry 12). To decrease reaction time, we decided to increase the
temperature from 30 to 35 °C. Thus, in 24 h, the acetylated product (S-4) was obtained

with high selectivity (99% e.e.) and E-value > 200, but with conversion of 10% (Table
3, entry 13). In order to achieve 50% conversion, reaction was monitored up to 120 h,
but the conversion remained practically unaltered (Table 3, entries 14-16). An increase
of 5 °C in the temperature (40 °C) led to only a slight increase in the conversion of 10 to
13%, while maintaining the high e.e. (>99%) to (S-4) (Table 3, entry 17). A higher
reaction time (48 h) afforded a higher conversion value (22%), but with a decrease in
e.e. (94%) to (S-4) (Table 3, entry 18).
These results were not so promising when compared with the hydrolytic process. This
prompted us to move our attention to further optimize the performance of the hydrolysis
process. In the kinetic resolution of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-
chloropropane (rac-4), the compound with the desired configuration is the remaining
substrate (2R)-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (R-4). Then, the latter
should be subjected to a hydrolysis reaction to get the key intermediate in the synthesis
of (S)-Pindolol (S-5), the haloydrin (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (R-
3). To catalyze this hydrolysis, we choose the lipase from C. rugosa, which proved to be
active, but not selective (Table 1, entry 14).
3.5. Synthesis of the key intermediate haloydrin (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-
propanol (R-3)
Once obtained the (2R)-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (R-4)
enantiomerically enriched (97% e.e.), we turn our attention to the preparation of the
halohydrin (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (R-3), the key intermediate
in the synthesis of (S)-Pindolol (S-5). The hydrolysis of (2R)-2-acetoxy-1-(1H-indol-4-
yloxy)-3-chloropropane (R-4) (97% e.e.), mediated by lipase from C. rugosa in
phosphate buffer (pH 7)/THF (4:1, v:v) at 40 °C for 12 h, led to (2R)-1-(1H-indol-4-
yloxy)-3-chloro-2-propanol (R-3) (97% e.e.) in quantitative yield.
3.6. Synthesis of (S)-Pindolol

Finally, the haloydrin (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (R-3) was
subjected to reaction with excess of isopropylamine, under reflux, leading to (S)-
Pindolol (S-5) (97% e.e.), in a quantitative yield.
4. Conclusion
In summary, we have prepared the (S)-Pindolol based on a chemoenzymatic process
using lipases. Lipase from P. fluorescens proved to be a robust catalyst in the kinetic
resolution of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4), via
hydrolysis reaction, leading to the intermediate (2R)-2-acetoxy-1-(1H-indol-4-yloxy)-3-
chloropropane (R-4) with 97% ee. It is noteworthy that the kinetic resolution was
efficient when an ideal amount (buffer/co-solvent 8:2, v:v) of THF was used as co-
solvent and when the reaction temperature was increased from 30 to 40 °C, resulting in
the higher enantioselectivity (E >200). This result could be explained through a model
in which the accommodation of (S)-4 enantiomer at the active site was improved with
the great conformational flexibility of the enzyme. Therefore, parameters such as
temperature and addition of co-solvent are ways of tuning the enantioselectivity of an
enzyme. Lipase from C. rugosa was efficient in the hydrolysis of the chiral acetate
yielding as key intermediate the halohydrin (2R)-1-(1H-indol-4-yloxy)-3-chloro-2-
propanol (R-3) with 97% ee in quantitative yield. The kinetic resolution was more
efficient in the hydrolytic process rather than acylation process in organic solvent.
Acknowledgements
The authors thank to the Fundação Cearense de Apoio ao Desenvolvimento
Científico e Tecnológico (FUNCAP), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Ensino
Superior (CAPES) for fellowships and financial support. The authors thank the
Brazilian funding agency Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) for providing the Special Visiting Researcher fellowship (process
400171/2014-7) under the Brazilian Scientific Program “Ciência sem Fronteira” and the
research sponsorships of M. C. de Mattos (Process: 308034/2015-5) and M. C. F. de
Oliveira (Process: 303365/2014-5).

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Fig.
1.
Chemoenzymatic synthesis of (S)-Pindolol.
Fig.
2A. HPLC chromatogram of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-
4: (S) tR 19.1 min.; (R) tR 20.9 min.)

Fi
g. 2B. HPLC chromatogram of rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3:
(S) tR 27.7 min.; (R) tR 34.0 min.).
Fig. 3. (Path
a) Enzymatic kinetic resolution of rac-4 via hydrolysis reaction. (Path b) Enzymatic
kinetic resolution of rac-3 via acetylation reaction.

Table 1. Kinetic resolution of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane
(rac-4) via hydrolysis using lipases.a
Entry Lipase
Reaction
time (h)
e.e.s
(%)b
e.e.p
(%)b
c
Ed
(%)c
1
2
P. camemberti
R. oryzae
24
-
-
-
-
0
0
-
-
immobilized
on 24
immobead-150
3
4
Porcine pancreas
24
-
-
-
-
0
0
-
-
R. miehei immobilized on anionic 24
resin
5
R. niveus
24
24
36
23
25
-
-
0
-
6
Amano lipase from M. javanicus
P. fluorescens
-
-
0
-
7
81
90
97
80
87
94
96
94
48
49
50
46
36
100
207
79
8e
9f
10
P. fluorescens
P. fluorescens
T. lanuginosus immobilized on 36
immobead-150
11e
12f
T. lanuginosus immobilized on 33
87
81
93
89
48
48
78
43
immobead-150
T. lanuginosus immobilized on 24
immobead-150
13
14
15
Novozym 435®
12
20
91
37
63
81
54
78
53
41
45
30
5
C. rugosa
Amano lipase PS from B. cepacia 12
15
immobilized on diatomaceous

earth
16
17
Amano lipase PS from B. cepacia
12
24
55
54
63
64
47
46
8
8
A. niger g
a Conditions: 30 ºC, lipase:rac-4 (1:1) at 250 r.p.m.
b Determined by HPLC.
c Conversion, c = e.e.s/(e.e.s+e.e.p).
d Enantiomeric ratio, E = ln[1-c(1+e.e.p)]/ln[1-c(1-e.e.p)]
e Conditions: 40 ºC, lipase:rac-4 (1:1) at 250 r.p.m.
f Conditions: 40 ºC, lipase:rac-4 (2:1) at 250 r.p.m.
g Inverse configurations

Table 2. Co-solvent effect on the lipase from P. fluorescens-mediated enzymatic kinetic
resolution of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropropane (rac-4)a
Entry Co-solvent logP e.e.s (%)b e.e.p(%)b c(%)c Ed
1
-
None
55
85
75
62
50
9
73
84
89
88
68
68
96
72
95
82
12
43
50
46
41
42
12
50
53
51
50
14
11
31
39
29
8
2
-0.24
-0.34
-0.27
-0.16
-0.10
0.46
0.46
0.46
0.94
3.90
Acetone
Acetonitrile
Dioxane
Isopropanol
Ethanol
THFe
3
4
5
6
6
7
97
80
98
83
2
207
15
180
26
2
THFf
8
THFg
9
10
11
TBME
Hexane
a Conditions: 40 ºC, 25 h and lipase from P. fluorescens:rac-4 (2:1) at 250 r.p.m.
b Determined by HPLC.
c Conversion, c = e.e.s/(e.e.s+e.e.p).
d Enantiomeric ratio, E = ln[1-c(1+e.e.p)]/ln[1-c(1-e.e.p)]
e (buffer/THF 8:2, v:v)
f (buffer/THF 9:1, v:v)
g (buffer/THF 7:3, v:v)
Table 3. Kinetic resolution of rac-1-(1H-indol-4-yloxy)-3-chloro-2-propanol (rac-3)
via acetylation using lipase from P. fluorescens.a

Entry Solvent
Reaction
time (h)
Temperature
(°C)
e.e.s
(%)b
e.e.p
(%)b
c
Ed
(%)c
1
THF
24
24
24
24
30
40
30
30
30
30
30
40
30
30
30
30
35
35
35
35
40
40
2
>99
91
2
>200
22
2
THF
4
4
3
Heptane
Toluene
13
15
20
35
21
72
24
47
9
88
13
17
27
31
19
51
22
37
9
18
4
75
8
5
Cyclohexane 24
53
4
6
Hexane
TBME
TBME
TBMEe
TBMEe
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
24
24
24
24
68
24
68
24
48
72
120
24
48
79
12
7
91
26
8
69
11
9
84
14
10
11
12
13
14
15
16
17
18
80
14
94
35
23
11
13
14
14
15
26
94
20
10
11
12
12
13
22
40
>99
>99
>99
>99
>99
94
>200
>200
>200
>200
>200
42
aConditions: lipase from P. fluorescens:rac-3 (1:1), vinyl acetate as acyl donor (5 eq.) in organic solvent,
at 250 r.p.m.
b Determined by HPLC.
c Conversion, c = e.e.s/(e.e.s+e.e.p).
d Enantiomeric ratio, E = ln[1-c(1+e.e.p)]/ln[1-c(1-e.e.p)]
e (10 eq. of vinyl acetate)