Lipase-mediated kinetic resolution of (RS)-1-bromo-3-[4-(2-methoxy-ethyl)- phenoxy]-propan-2-ol to (R)-1-bromo-3-(4-(2-methoxyethyl) phenoxy) propan-2-yl acetate

Article abstract of DOI:10.1016/j.tetlet.2011.08.031

A novel biocatalytic method for the enantioselective synthesis of (R)-bromo-3-[4-(2-methoxy-ethyl) phenoxy]-2-propanol [(R)-BMEPP], a precursor for the synthesis of (S)-metoprolol, an anti hypertensive drug is described. We have developed kinetic resolution of rac-BMEPP by transesterification using Candida rugosa lipase and vinyl acetate as the acyl donor affording the product with excellent conversion (49%) and ee (>99%). Various reaction parameters (source of enzyme, reaction media, and concentration of substrate and acylating agent) for the enzymatic kinetic resolution have been reported.

Full text of DOI:10.1016/j.tetlet.2011.08.031

Tetrahedron Letters 52 (2011) 5355–5358
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Tetrahedron Letters
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Lipase-mediated kinetic resolution of (RS)-1-bromo-3-[4-(2-methoxy-ethyl)-p-
henoxy]-propan-2-ol to (R)-1-bromo-3-(4-(2-methoxyethyl) phenoxy) propan-
2-yl acetate
Abhishek Kaler a, Vachan Singh Meena a, Manpreet Singh a, Brahmam Pujala b, Asit K. Chakraborti b,
Uttam Chand Banerjee a,
⇑
a Biocatalysis Laboratory, Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160062, India
b Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel biocatalytic method for the enantioselective synthesis of (R)-bromo-3-[4-(2-methoxy-ethyl)
phenoxy]-2-propanol [(R)-BMEPP], a precursor for the synthesis of (S)-metoprolol, an anti hypertensive
drug is described. We have developed kinetic resolution of rac-BMEPP by transesterification using
Candida rugosa lipase and vinyl acetate as the acyl donor affording the product with excellent conversion
(49%) and ee (>99%). Various reaction parameters (source of enzyme, reaction media, and concentration
of substrate and acylating agent) for the enzymatic kinetic resolution have been reported.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 6 July 2011
Revised 2 August 2011
Accepted 4 August 2011
Available online 11 August 2011
Keywords:
Candida rugosa lipase
Metoprolol
Transesterification
Intermediates
Metoprolol (1-isopropylamino-3-[4-(2-methoxy-ethyl)-phen-
oxy]-propan-2-ol), an antihypertensive drug belongs to the class
of b-blockers having a preferential effect on cardiac b1-adrenore-
ceptors.1–3 Studies indicated on metoprolol have revealed the eud-
ismic ratio (ER) of the order of 270 in favor of the (S)-enantiomer.4
Therefore, it is highly imperative to administer this drug in opti-
cally pure form.
A retro-synthetic approach reveals that optically pure (S)-meto-
prolol can be synthesized by (R)-1-bromo-3-[4-(2-methoxy-ethyl)-
phenoxy]-propan-2-ol (BMEPP) as the chiral drug intermediate.
The existing methods for the chemical synthesis of metoprolol
have been reported in literature.5–9 However, these chemical
methods suffer from disadvantages, such as higher cost, hazardous
reaction conditions, formation of side products, and poor enanti-
oselectivity. Biocatalysts, on the other hand, exhibit better regio-
and stereoselectivity, due to which they are increasingly exploited
to produce complex molecules of industrial interest.10–13 Lipases
are known to bring about a range of biocatalytic conversions by
different reaction mechanisms.14–17 Role of lipase as mediator of
kinetic resolution of number of racemates to enantiopure products
is well established.18–20 Lipase mediated resolution of various
drugs and drug intermediates has been successfully carried out
in our laboratory.21–25 These findings encourage us to use lipase
as a tool for the enantioselective synthesis of metoprolol
intermediate.
The present study demonstrates a novel approach for the
synthesis of enantiopure (R)-BMEPP from 2-[4-(2-methoxy-
ethyl)-phenoxymethyl]-oxirane. The process involves the chemical
synthesis of rac-BMEPP from oxirane and the subsequent kinetic
resolution using lipase-mediated transesterification. The various
parameters for the transesterification reaction have been further
optimized to achieve better enantio-selectivity with higher
conversion.
The synthesis of rac-BMEPP was achieved in a two-step reaction
(Scheme 1). The reaction sequence includes the treatment of
4-(2-methoxyethyl)-phenol with epichlorhydrine under basic con-
ditions to afford the product 2-[4-(2-methoxy-ethyl)-phenoxy-
methyl]-oxirane.26,27 Subsequently epoxide ring was opened by
lithium bromide to obtain rac-BMEPP.28,29 Our next objective was
to achieve the kinetic resolution of rac-BMEPP using different li-
pases and acetylating agent in various organic solvents.
As a model reaction transesterification of rac-BMEPP was tried
with lipase from Pseudomonas aeruginosa in organic solvent
(Scheme 2). Good conversion up to 27% with 97% ee prompted us
to screen lipases from commercial sources such as Candida rugosa
and porcine pancreatic lipase to catalyze the reaction.30
Modified form of the Winkler and Stuckmann method was ap-
plied for the quantification of lipase activity.31 The enzyme activity
of the crude preparation of P. aeruginosa lipase was 854 U mgÀ1
⇑
Corresponding author. Tel.: +91 172 2214682 85; fax: +91 172 2214692
E-mail address: ucbanerjee@niper.ac.in (U.C. Banerjee).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.08.031

5356
A. Kaler et al. / Tetrahedron Letters 52 (2011) 5355–5358
O
O
OH
Cl
O
K2CO3, MeCN
85 oC, 14 h
O
O
4-(2-Methoxy-ethyl)-phenol
Epichlorohydrin
2-[4-(2-Methoxy-ethyl)-phenoxy methyl]-oxirane
etic acid,
Br
LiBr, ac
THF, 12 h
O
OH
O
1-Bromo-3-[4-(2-methoxy-ethyl)-phenoxy]-propan-2-ol
Scheme 1. Synthesis of 1-bromo-3-[4-(2-methoxy-ethyl)- phenoxy]-propan-2-ol via epoxide ring opening.
Br
Br
Br
O
O
O
O
Lipase, Toluene
OH
O
OH
Vinyl acetate,
30oC, ~1000 rpm,
O
O
O
(S)-1-Bromo-3-(4-(2-methoxyethyl)
(R)-1-Bromo-3-(4-(2-methoxy-ethyl)
1-Bromo-3-[4-(2-methoxy-ethyl)
phenoxy]-propan-2-ol
phenoxy)propan-2-ol
phenoxy)propan-2-yl acetate
Scheme 2. Lipase catalyzed enantioselective transesterification of (RS)-1-bromo-3-[4- (2-methoxy-ethyl)-phenoxy]-propan-2-ol.
much higher than the enzyme activities of C. rugosa (520 U mgÀ1
)
Table 2
and porcine pancreas (325 U mgÀ1). The reactions were carried
out at identical values of the enzyme activity in the reactor. At
the end of the reaction, C. rugosa lipase-catalyzed reaction had at-
tained a rac-BMEPP conversion of nearly 34% (ee ꢀ98%) compared
to the conversion of 27% (ee ꢀ97%) obtained with P. aeruginosa li-
pase (Table 1). The porcine pancreatic lipase did not show any con-
version. During transesterification reaction, vinyl acetate is known
to release acetaldehyde, which in turn deactivates the enzyme.32,33
Lipases are highly stable in organic solvents like n-dodecane, 1-
pentanol, and toluene34 and retention of more than 80% lipase
activity in hexane, toluene, and petroleum ether has already been
reported.35 Hence we tried to study the effect of different organic
solvents such as toluene, hexane, cyclohexane etc. on the reaction
outcome. It is evident from Table 2 that the best results were ob-
tained using toluene as the solvent affording better conversion
and enantiomeric excess as compared to other solvents.
Effect of solvent on the lipase-catalyzed transesterification of rac-BMEPP
Solvents
Conversion (%)
ee (%)
Toluene
Hexane
Cyclohexane
Octane
Tetrahydrofuran
Dioxane
34
5
99
53
48
ND
ND
ND
42
10
ND
ND
ND
7.5
Ethyl acetate
ND: Not detectable.
Further to achieve maximum conversion and enantiomeric ex-
cess of (R)-1-bromo-3-[4-(2-methoxyethyl) phenoxy] propan-2-yl
acetate, the amount of the enzyme required to catalyze the reac-
tion was optimized. The concentration of C. rugosa lipase was var-
ied (5200–26000 U) while keeping the concentrations of the
substrate and vinyl acetate constant (Fig. 1). It was observed that
30 mg C. rugosa lipases (15600 U) in the reaction mixture showed
maximum conversion (40%). The conversion did not increase with
further increase in the enzyme concentration. This may be due to
the increased viscosity of the reaction mixture at higher enzyme
concentration, which reduced the collision between the enzyme
and the substrate.
Figure 1. Effect of enzyme concentration on the transesterification of rac-BMEPP
using C. rugosa lipase.
To study the time course, the reaction was allowed to proceed
for 36 h and the samples were withdrawn every 6 h and subjected
to HPLC analysis.36 It is evident from the Figure 2 that the conver-
sion of alcohol to ester increased as the reaction proceeded, on the
other hand enantiomeric excess decreased with time as commonly
observed in such biotransformation. The best conversion (42%) and
enantiomeric excess (>99%) were achieved after 24 h.
Table 1
Effect of different sources of enzymes on the transesterification reaction
Source
Conversion (%)
ee (%)
C. rugosa
P. aeruginosa
Porcine pancreas
34
27
Nil
98
97
Nil

A. Kaler et al. / Tetrahedron Letters 52 (2011) 5355–5358
5357
Figure 4. Effect of vinyl acetate concentration on transesterification of rac-BMEPP
by C. rugosa lipase.
Figure 2. Effect of time-course on the transesterification of rac-BMEPP by C. rugosa
lipase.
Acknowledgments
The effect of substrate concentration was monitored by varying
the amount of rac-BMEPP (5–25 mM) in the transesterification
reaction catalyzed by C. rugosa lipase, keeping the other compo-
nents of the reaction mixture constant. It was observed that high-
est conversion (46%) was obtained when the initial concentration
of rac-BMEPP was 15 mM. Increasing substrate concentration be-
yond 15 mM decreased the conversion (Fig. 3). The enzyme might
have experienced substrate inhibition or this could be due to the
formation of the dead-end inhibition complex between lipase
and rac-BMEPP.
Two acyl donating vinyl esters (vinyl acetate and butyrate)
were compared for the transesterification of rac-BMEPP. The re-
sults showed that vinyl acetate gave excellent conversion (48%)
and enantiomeric excess (>99%) compared to vinyl butyrate where
both the conversion (30%) and enantiomeric excess (96%) were
less. In order to optimize the concentration of acyl donor, reactions
were carried out at various concentrations of vinyl acetate (10–
50 mM) while keeping the other parameters constant. There was
an increase in the conversion (49%) with an increase in vinyl ace-
tate upto 20 mM and then decreased with further increase in its
concentration (Fig. 4).
An efficient and practical synthesis method of 1-bromo-3-[4-(2-
methoxy-ethyl)-phenoxy]-propan-2-ol and their successful enzy-
matic kinetic resolution using lipase has been demonstrated. The
enantiomerically pure isomer may be used for the synthesis of bio-
logically important compounds like metoprolol, a selective b1-
blocker. The reaction conditions have been optimized to provide
an economical and greener method for obtaining the enantiopure
(R)-BMEPP with excellent conversion and enantioselectivity. Stere-
oselective transesterification of rac-BMEPP has been successfully
carried out using lipase from C. rugosa in this study.
A.K. and V.S.M. thank the Department of Biotechnology and B.P.
thanks the Council of Scientific and Industrial Research, Govt. of In-
dia for senior research fellowships.
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(1.56 g, 7.5 mM) with lithium bromide (7.5 mM) in acetic acid (1.35 g,
22.5 mM) and THF (10 ml) at room temperature for12 h. The reaction
mixture was diluted with ethyl acetate (15 ml), washed with water, dried
over Na2SO4, and was concentrated under vacuum to afford (RS)-1-bromo-3-
Figure 3. Effect of substrate concentration on the transesterification of rac-BMEPP
by C. rugosa lipase.

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A. Kaler et al. / Tetrahedron Letters 52 (2011) 5355–5358
[4-(2-methoxy-ethyl)-phenoxy]-propan-2-ol.1H NMR (300 MHz, CDCl3) d: 2.04
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Shimadzu 10AVP instrument equipped with an UV detector on a Chiralcel
(MÀ79)+
30. Enzymatic transesterification of rac-BMEPP. Crude preparation of P. aeruginosa
lipase, commercial lipase from C. rugosa and porcine pancreas were used for
the transesterification of rac-BMEPP. The reaction was carried out in triplicate
using appropriate quantity of lipase in 10 ml stoppard flask containing
substrate rac-BMEPP (15 mM) and vinyl acetate (20 mM) dissolved in
toluene (5 ml). Reaction mixture was magnetically stirred (ꢀ1000 rpm) at
30 °C for 36 h with periodic sampling (6 h). Conversion and enantiomeric
excess of the substrate and product were analyzed by Chiral HPLC. Product (R-
BMEPP-ester) was separated from the reaction mixture and authenticated by
NMR. 1H NMR (CDCl3, 400 MHz): d 2.12 (s, 3H), 2.82 (t, 2H, J = 7 Hz), 3.34 (s,
3H), 3.56 (t, 2H, J = 7 Hz), 3.62–3.70 (m, 2H), 4.10–4.19 (m, 2H), 5.29 (m, 1H),
6.85 (d, 2H, J = 8.6 Hz), 7.14 (d, 2 H, J = 8.6 Hz). 13C NMR (CDCl3, 100 MHz) d
20.9, 30.4, 35.2, 58.6, 66.8, 70.7, 73.7, 114.5, 129.8, 131.9, 156.6, 170.1.
31. Winkler, U. K.; Stuckmann, M. J. Bacteriol. 1979, 138, 663–670.
ODH column (0.46 mm  250 mm, 5
lm, Chiralcel). Elution was done with
hexane-isopropyl alcohol at 90:10 (v/v) and at a flow rate of 0.5 ml/min. The
conversion and enantiomeric excess (ee%) were quantified at 220 nm. The
retention time of (S)-ester and (R)-ester were 12.5 and 15.32 min, respectively
and (S)- and (R)- alcohol enantiomers were eluted at 25.7 and 29.0 min,
respectively. The remaining substrate and the product formed were
authenticated by 1H and 13C NMR spectra recorded on a Bruker Advance DPX
300 MHz NMR spectrometer.