Biotransformations with Rhizopus arrhizus and Geotrichum candidum for the preparation of (S)-atenolol and (S)-propranolol

Article abstract of DOI:10.1016/S0968-0896(00)00131-0

(±)-Atenolol/(±)-propranolol and their acetates were incubated with the fungus Rhizopus arrhizus and Geotrichum candidum separately for different time intervals to afford (S)-atenolol/(S)-propranolol in good optical yield. The time and pH for this biotransformation was optimised. The present biodegradations using Rhizopus arrhizus and Geotrichum candidum provides a simple and useful method to obtain (S)-atenolol and (S)-propranolol which are active enantiomers of the β-adrenergic blockers. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00131-0

Bioorganic & Medicinal Chemistry 8 (2000) 2067±2070
Biotransformations with Rhizopus arrhizus and Geotrichum
candidum for the Preparation of (S)-Atenolol and (S)-Propranolol
Subhash V. Damle, Prashant N. Patil and Manikrao M. Salunkhe*
Department of Chemistry, The Institute of Science, 15, Madam Cama Road, Mumbai-400 032, India
Received 13 March 2000; accepted 9 May 2000
AbstractÐ(Æ)-Atenolol/(Æ)-propranolol and their acetates were incubated with the fungus Rhizopus arrhizus and Geotrichum can-
didum separately for di€erent time intervals to a€ord (S)-atenolol/(S)-propranolol in good optical yield. The time and pH for this
biotransformation was optimised. The present biodegradations using Rhizopus arrhizus and Geotrichum candidum provides a simple
and useful method to obtain (S)-atenolol and (S)-propranolol which are active enantiomers of the b-adrenergic blockers. # 2000
Elsevier Science Ltd. All rights reserved.
Introduction
preparation of (S)-atenolol and (S)-propranolol using
above whole cells.
The biological activity exhibited by the drug molecule is
directly related to its absolute stereochemistry.1 Atenolol
and propranolol, the b-adrenergic blockers used to treat
cardiovascular disorders are characterised by the aryl-
oxypropanolamine structure with one chiral centre and
the activity resides in the (S)-enantiomer.2,3 On the con-
trary the opposite (R)-enantiomer may be responsible for
the undesirable e€ects.4
Results and Discussions
Optimisation of microbial transformations of (Æ)-atenolol
and (Æ)-propranolol with respect to optical purity and
chemical yield have been carried out. (Æ)-Atenolol (1a)
or (Æ)-propranolol (1b) were incubated with R. arrhizus
or G. candidum separately at di€erent pH conditions
and varying time intervals, 2±8 days. It was interesting to
observe that the yield of recovered substrate was found
to decrease during the course of biotransformation.
Therefore optical rotation of the recovered substrate
was taken and found to be optically active. The best
results were obtained at pH=7.0 (Fig. 2a±d) and after 6
days (Fig. 1a±b). The absolute con®guration ((S)-ate-
nolol (2a) and (S)-propranolol (2b)) was assigned after
correlating with literature data. So it is clear that (R)-
alcohol gets preferentially metabolised.
The synthesis of (R)- and (S)-atenolol has been achieved
by lipase catalysed hydrolysis5 of the intermediate O-
acetyl esters. The synthesis of (R)- and (S)-propranolol
has been achieved by using lipase catalysed hydrolysis6
of the intermediate O-acetyl esters and also by using
lipase catalysed acylation7 of the intermediate secondary
alcohols. R. arrhizus has been utilised in the reduction
of prochiral ketones,8 hydrolysis of racemic acetates9 and
in the biosorption studies of radionuclides.10 G. candidum
has been utilised in the reduction of various prochiral
ketones.11 Excellent catalytic asymmetric synthesis of the
optically active b-blockers by using chiral catalysts12a j
and industrial scale synthesis by utilising above-men-
tioned methods have been reported. The processes con-
sisting of whole cells being economical and operationally
simple and recent use in biosorption studies prompted
us to develop this methodology. To our knowledge, this
is the ®rst report on the biodegradation studies for the
In another approach similar experiments were carried out
with (Æ)-atenolol acetate (3a) and (Æ)-propranolol acetate
(3b). It was interesting to note that substrate (R)-atenolol
acetate and (R)-propranolol acetate were metabolised
maybe after hydrolysis as per earlier observations. How-
ever (S)-atenolol acetate and (S)-propranolol acetate can
be recovered. The recovered acetates were chemically
hydrolysed (with K2CO3±MeOH) to give (S)-atenolol
and (S)-propranolol, respectively. In this case also, the
maximum enantiopurities were obtained at pH=7.0
and after 6 days (Fig. 1a±b). The absolute con®guration
*Corresponding author. Tel.:+91-22-2816750; Fax: +91-22-2816750;
e-mail: mmsalunkhe@hotmail.com
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00131-0

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S. V. Damle et al. / Bioorg. Med. Chem. 8 (2000) 2067±2070
Figure 1. (a) Biotransformation of atenolol and atenolol acetate; 1 Atenolol; R. arrhizus; 2 Atenolol; G. candidum; 3 Atenolol acetate; R. arrhizus; 4
Atenolol acetate; G. candidum. (b) Biotransformation of propranolol and propranolol acetate; 1 propranolol; R. arrhizus; 2 Propranolol; G. candi-
dum; 3 Propranolol acetate; R. arrhizus; 4 Propranolol acetate; G. candidum.
Figure 2. (a) Optimization of pH during the biotransformations (atenolol; R. arrhizus); (b) Optimization of pH during the biotransformations (ate-
nolol; G. candidum); (c) Optimization of pH during the biotransformations (propranolol; R. arrhizus). (d) Optimization of pH during the bio-
transformations (propranolol; G. candidum).
((S)-atenolol (2a) and (S)-propranolol (2b)) was
assigned after correlating with literature data. It could
thus be inferred that both the fungus (R. arrhizus and G.
candidum) preferentially metabolised the (R)-acetates.
From the literature it appears that the lipase catalysed
resolution of the intermediate chlorohydrin is the ®rst
report on the synthesis of chiral atenolol.5 The method
reports the optical yield >95% with the multi-step

S. V. Damle et al. / Bioorg. Med. Chem. 8 (2000) 2067±2070
2069
synthesis but with the use of expensive biocatalysts. On
the contrary our present method describes the synthesis
of enantiomerically pure b-blockers by microbial asym-
metric destruction, with 88±99% ee. Our present
approach is simple, single-step and totally new from the
classical method of synthesis of chiral b-blockers.
The lipase catalysed (chemoenzymatic) preparation of
(S)-propranolol via resolution of intermediates, cynohy-
drin and chlorohydrin were recently reported. Matsuo et
al.6 described the preparation of (S)-propranolol with
71% ee while Bevinakatti et al.16 report good optical
purity, 95% ee and overall 30% yield. The methods are
multi-step synthesis and use expensive biocatalysts.
Therefore our present method is simple, environmentally
friendly, single-step producing (S)-propranolol with 40±
88% ee, but su€ers from the disadvantage like that of
low atom eciency. As far as our knowledge goes this is
the ®rst report on the synthesis of (S)-atenolol and (S)-
propranolol employing the biodegradation approach.
From the experimental results (Scheme 1, Table 1), it
clearly demonstrates that the incubation of the (Æ)-ate-
nolol and (Æ)-propranolol itself with the fungus is a
better method for the biodegradation of drugs men-
tioned above as compared with the method involving
biodegradation of corresponding esters (Scheme 2,
Table 1).
Conclusion
Scheme 1.
Biodegradation using R. arrhizus and G. candidum pro-
vides a simple and useful method to obtain (S)-atenolol
and (S)-propranolol.
Experimental
Materials and methods
Chemicals. All reactions were monitored by TLC (thin
layer chromatography) on precoated silica gel sheet 60
F 254 (Merck). Compounds were visualised by iodine
vapours or in UV light. Preparative TLC was carried
out with silica gel (Merck). The substrate (Æ)-atenolol,13
(Æ)-propranolol14 and their corresponding O-acetyl
esters were prepared by literature procedure.15
Micro-organisms. Rhizopus arrhizus (NCIM 997), Geo-
trichum candidum (NCIM 980) were obtained from
National Collection of Industrial Micro-organisms,
National Chemical Laboratory, Pune, India.
Instrumentation. IR spectra were recorded on Perkin
Elmer 170-X Infrared Fourier Transform Spectro-
Scheme 2.
Table 1. Results of biotransformation after 6 days at pH=7.0a
Compound
Fungus
[a]d (c, solvent)
ee (%)
Recovered
yield (%)
Con®guration
atenolol
atenolol acetate
propranolol
propranolol acetate
Atenolol
Atenolol acetate
propranolol
propranolol acetate
R. arrhizus
R. arrhizus
R. arrhizus
R. arrhizus
G. candidum
G. candidum
G. candidum
G. candidum
13.6 (1%, ethanol)5
12.7 (0.25%, ethanol)5
9.0 (0.5%, ethanol)16
6.9 (1%, ethanol)16
12.0 (0.5%, ethanol)5
11.6 (0.5%, ethanol)5
8.0 (1%, ethanol)16
4.0 (2%, ethanol)16
>99
96
88
68
91
88
78
39
75
69
74
70
74
67
73
68
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
aLit.5 [a]d= 13.10 (0.9%, ethanol, >99%ee); Lit.16[a]d= 10.2 (1.02%, ethanol).

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S. V. Damle et al. / Bioorg. Med. Chem. 8 (2000) 2067±2070
1
photometer. H NMR spectra were recorded on (VRX)
300 MHz Spectrometer, using CDCl3 or DMSO-d6 as
solvents (TMS as internal reference). Optical rotation
measurements were carried out on Jasco-360 polarimeter.
Acknowledgements
We thank C. S. I. R. New Delhi and Rameshwardas Birla
Smarak Kosh, Mumbai (India) for the ®nancial support.
Determination of absolute con®guration and enantio-
meric excess. The absolute con®guration [a]d and
enantiomeric excess (ee%) of microbial products were
determined by correlating the data with those reported
in the literature.5,16
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Enzymatic (whole cell) resolution. At the end of the fer-
mentation, the cells were ®ltered o€. The ®ltrate was
extracted with n-butanol (atenolol)/CHCl3 (propranolol/
acetates). The cells were also washed with the respective
organic solvent and the combined organic solvent was
washed with water, dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure and the resi-
due was subjected to preparative TLC. (Solvent system :
[for atenolol]=CHCl3:MeOH 8:2/[for propranolol]=
CHCl3:MeOH 9.8:0.2). Recovered yields being 75 mg
with R. arrhizus and 74 mg when G. candidum was used.
In case of O-acetyl esters, the residue obtained was
subjected to preparative TLC. The isolated pure O-ace-
tyl ester was hydrolysed (with K2CO3±MeOH) to yield
optically pure atenolol/propranolol. The compounds
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1
obtained were characterised by IR and H NMR.