A Green approach towards the synthesis of chiral alcohols using functionalized alginate immobilized Saccharomyces cerevisiae cells

Article abstract of DOI:10.1016/j.molcatb.2016.10.016

The stereochemistry of the drug molecule is gaining greater therapeutic importance and thus much attention was drawn in synthesis of chiral compounds by the pharmaceutical industry. In this study Saccharomyces cerevisiae cells immobilized on functionalize

Full text of DOI:10.1016/j.molcatb.2016.10.016

Accepted Manuscript
Title: A Green approach towards the synthesis of chiral
alcohols using functionalized alginate immobilized
Saccharomyces cerevisiae cells
Author: Narmada Muthineni Manikanta Swamy Arnipally
Sridhar Bojja Harshadas Mitaram Meshram Ajay Kumar
Srivastava Bhaskar Rao Adari
PII:
S1381-1177(16)30211-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.10.016
MOLCAB 3460
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
22-8-2016
21-10-2016
28-10-2016
Please cite this article as: Narmada Muthineni, Manikanta Swamy Arnipally, Sridhar
Bojja, Harshadas Mitaram Meshram, Ajay Kumar Srivastava, Bhaskar Rao Adari,
A Green approach towards the synthesis of chiral alcohols using functionalized
alginate immobilized Saccharomyces cerevisiae cells, Journal of Molecular Catalysis
B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.10.016
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

A Green approach towards the synthesis of chiral
alcohols using functionalized alginate immobilized
Saccharomyces cerevisiae cells
Narmada Muthinenia, Manikanta Swamy Arnipallyb, Sridhar
Bojjac, Harshadas Mitaram Meshrama, Ajay Kumar
Srivastavaa, Bhaskar Rao Adaria*
aMedicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of
Chemical Technology, Hyderabad-500007, India
bAnalytical Chemistry Division, CSIR-Indian Institute of Chemical Technology,
Hyderabad-500007, India
cInorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad-500007, India
*Correspondent: Fax no: +91 40 27160512, Tel. no: +91 40 27193374
e-mail: adarirao2002@yahoo.co.in

Graphical abstract

HIGHLIGHTS
The present study highlights the following;
 Synthesis of chiral alcohols and chiral amino
alcohols by developing simple chemo-enzymatic,
inexpensive, green process
 Functionalized alginate beads entrapped yeast cells
 Reuse of immobilized yeast beads for continuous
synthesis of chiral alcohols
 Hydrogenation of chiral azido alcohols into
respective chiral amino alcohols by incorporating
Palladium (Pd) nanoparticles (<5nm)
 The methodology increase the scope of the
synthesis of pharmaceutically important chiral
precursors by simple fashion to reach industrial
demand.

Abstract
The stereochemistry of the drug molecule is gaining greater
therapeutic importance and thus much attention was drawn in
synthesis of chiral compounds by the pharmaceutical industry. In
this study Saccharomyces cerevisiae cells immobilized on
functionalized alginate beads, catalyze the bio-reduction of
prochiral ketones 1a-12a to their corresponding chiral alcohols 1b-
12b in higher yields of 60-99% and.excellent optical purity 75-
97%. The synthesized chiral azido alcohols 10b-12b were further
subjected to hydrogenation using Palladium(Pd) nanoparticles
(≤5nm), to obtain chiral amino alcohols 10c-12c of therapeutic
importance. Thus, a simple, green and inexpensive continuous
chemo-enzymatic process has been developed in the synthesis of
chiral alcohols/ amino alcohols to enhance the scope of the
methodology towards industrial application.
Key words: Saccharomyces cerevisia cells, functionlized alginate
immobilized beads, Palladium nanoparticles, chiral amino alcohols
1. Introduction
Chiral compounds are important synthons in the synthesis of
numerous pharmaceuticals, agrochemicals, flavors and other value
added fine chemicals [1-2]. Strategies to obtain molecules having a
single stereogenic centre have gained importance in drug discovery

because one of the enantiomer having an effective therapeutic
action, while the other isomer exhibits undesirable therapeutic
effect or toxic effect [3-4]. A number of chemical and bio-catalytic
methodologies are known in the synthesis of value added chiral
compounds. Biocatalytic reactions have proven to be a promising
green methodology in the synthesis of chiral compounds. Mostly,
the conventional chemical synthesis use expensive chemical
catalyst or transition metals and requires multiple steps to get the
desired product, whereas using biocatalyst (whole cells or by
isolated enzymes) the reactions are carried out in single step under
mild reaction conditions [5-7]. The biocatalytic reactions show
substrate specificity and the products obtained are in good yields
with high enantio- selectivity/ purity.
Though, it is believed that biocatalytic processes are superior to
chemical methodologies in the synthesis of chiral molecules, the
use of the expensive co-factors (NADH, NADPH) has limited their
scope for commercial applications. An alternative approach to
improve the biocatalytic efficiency in the synthesis of chiral
molecules is by immobilizing biocatalysts (cells/ enzymes); that
enable the re-use of biocatalyst, resulting in enhancing their ability
to catalyze a wide range of stereo selective reactions [8]. Several
chiral alcohols/ amino alcohols are known potential precursors in
synthesis of biologically and pharmaceutically active molecules. In

continuation to our interest in synthesis of novel chiral molecules
of medicinal importance using biocatalysts [9-11], the present
study is focused on extending our methodology for a continuous
process of chiral alcohols/ amino alcohols which are known
building blocks for various pharmaceuticals and agrochemical
products.
There has been an increasing awareness in the use of biocatalysts
in the biotransformation of pro-chiral compounds to their
corresponding chiral products with high chemo-, regio- and
enantio-selectivity [12]. Bio-reduction of pro-chiral ketones to
their corresponding chiral alcohols transformation using whole
microbial cell system are advantageous over the use of isolated
pure enzymes dehydrogenases (ADH) as they do not require
expensive co-enzyme regeneration system (NADH, NADPH) [13].
Immobilization of whole cells/enzymes demonstrate several
advantages like easy separation of cells from the reaction mixture,
repeated or continuous use of biocatalyst with enhanced stability
and enantio-selective properties. Keeping in the view the pros and
cons of yeast immobilization, we here in report the synthesis of
chiral alcohols using Saccharomyces cerevisiae (Baker’s yeast)
immobilized over functionalized alginate beads to give optimum
bead stability and no loss of enzyme activity with a potential for
continuous process.

2. Experimental
2.1. Materials
Baker's yeast from Saccharomyces cerevisiae (Type II) was
purchased from Sigma-Aldrich (USA). Substituted acetophenones,
1-(2-hydroxyphenyl) propan-1-one, 1-(3-(trifluoromethyl) phenyl)
propan-2-one, azido ketones, sodium alginate, succinic anhydride
and Palladium chloride (II) bought from Sigma-Aldrich (USA).
Calcium chloride purchased from Hi-Media Pvt. Ltd, Mumbai. All
the analytical and HPLC grade chemicals and solvents used in the
study were purchased from Merck, India.
2.2. Instruments
1H and 13C NMR spectra were recorded on Bruker Avance-
400MHz spectrometer. IR spectra were recorded on Thermo
Nicolet FT/IR-5700 spectrometer. Mass spectra were recorded
using Waters mass spectrometer. Polari meter-Perkin Elmer model
341, HPLC Gilson solution 321, UK, SHIMADZU- GC-2010 were
used to record the HPLC and GC spectra respectively.
2.3. Synthesis of chiral alcohols using functionalized alginate
immobilized yeast cells
The fermentation medium, in a total volume of 1L, consists of 20 g
L-1 glucose, 20 g L-1 peptone and 10 g L-1 yeast extract, to this
yeast cells were inoculated, followed by incubation at 28-30 °C for
48 h. After optimal growth, the medium was centrifuged to isolate

fermented yeast cells, which were then washed with sterile distilled
water. These fermented yeast cells were immobilized different
alginate matrix i.e. a) Calcium alginate immobilized yeast beads
and b) Succinyl modified alginate immobilized beads as described
in our earlier studies [14]. The degree of succinylation was
determined by the titration method as described by Wurzburg [15].
To the 4% sodium alginate/succinylated alginate solution, 2% (v/v)
of fermented S.cerevisiae cells were added and the resultant cell
suspension (107cfu/cm3) was extruded through a needle with
diameter-0.5 mm injector added as droplets into 2% calcium
chloride solution under continuous stirring to get the calcium
alginate/succinylated alginate-immobilized beads. Beads having a
diameter in the range of 1.5 to 2.5 mm were selected for
subsequent fermentation experiments. Cell viability was
determined by plate counts. The stability of yeast entrapped in the
succinyl alginate matrix (beads) was measured as described in
[14]. The binding efficiency of yeast cells in the alginate/
functionalized alginate matrix has been confirmed by SEM studies
(Fig: 1).
To the above immobilized yeast cells (10 gm in 250 mL of
phosphate buffer, pH 7.5), the pro-chiral ketones 1a-9a or azido
ketones 10a-12a, (100 mg/ mL) each were added and incubated at
°
30-37 C. The progress of the reactions at different time intervals

was monitored by TLC and HPLC. After optimal
biotransformation of ketones to respective chiral alcohols the
immobilized beads containing yeast cells were separated and
reused up to 7 cycles without much loss enzyme activity. Thus
collected reaction medium containing products was extracted with
equal amounts of ethyl acetate and the products obtained were
isolated and purified by column chromatography. The spectral data
of all the chiral products synthesized has confirmed by literature
[9, 10, 17-23]. Further the chiral azido alcohols 10b-12b obtained
were subjected to hydrogenation by using Pd nanoparticles to
obtain chiral amino alcohols.
2.4. Synthesis of chiral amino alcohols by using Palladium (Pd)
nanoparticles
The chloride salt of palladium used for preparation of palladium
nanoparticles according to the reported method [16], In brief
palladium nanoparticles were prepared by adding 50 mg of PdCl2,
6 mL of 0.02M HCl and make up the volume to 250 mL with
water to get 1.2 Mm H2PdCl4 stock solution. The prepared solution
was sonicated for 3h and incubated at room temperature for 24 h
before use. Add 10 mL of 1.2 mM H2PdCl4 to the 10 mL of de-
ionized water maintaining pH 2.5, by adding 0.1M HCl. To this
added 30 mg of PVP, the solution was heated upto 100 °C for 1 h
by adding ethanol drop wise (10 mL) the solution was refluxed for

3 h, which resulted in a dark brown colloidal Pd solution. Thus
obtained Pd nanoparticles were separated and incorporated into the
hydrogenation reaction of chiral azido alcohols 10b-12b to give
chiral amino alcohols 10c-12c as described in the literature [13].
2.5. HPLC and GC Analysis:
The progress of product formation was monitored by TLC and
HPLC analysis. Waters C18 column (250× 4.6 mm) using mobile
phase Actonitrile:Water (80:20) at 254 nm and the enantiomeric
excess (ee) was determined by HPLC using Diacel-chiracel OD-H
(50 x 4.6mm x 3mm) and OJ-H using mobile phase n-Hexane:
isopropanol (95:5) and PDA Multi detector at 265 nm, with
1ml/min flow rate.
GC analysis: For determining the enantiomeric excess of products
6b and 8b, (SHIMADZU, GC-2010) equipped with a flame
ionization detector and a HP Chiral 10% cyclodextrin
chromatography column (30 m×0.25 mm×0.25 μm). N2 was used
as carrier gas and a sample volume of 0.1μl was injected by using a
split ratio of 20︰1. The column temperature was held at 120 °C
for 1 min and was increased to 220 °C at a rate of 2 °C·min−1, and
then kept constant for 10 min. The temperature of the injector and
the detector were 240 °C and 250 °C, respectively [26].
3. Results and discussion

In this study the optimum reaction conditions in the synthesis of
chiral alcohols 1a-12a by fermented yeast cells immobilized on
derivatized alginate beads found to be pH 7.5 and incubation
temperature was 30-37 °C (Fig: S1 and S2). The results show that
maximum conversion of pro-chiral ketones 1a-12a to respective
chiral alcohols 1b-12b was observed after 24 h by fermented yeast
cells immobilized on to the alginate and functionalized alginate
matrices (Scheme 1, Scheme 2, Scheme 3). When compared to
other immobilized matrices the succinyl alginate beads
immobilized yeast cells has shown promising reproducible yields
of 60-99% and high enantiomeric excess of 93-97% ee (Table: 1).
The ortho substituted compound 1-(2-hydroxyphenyl) propan-1-
one 9b has shown lower yields (50%) and enantiomeric excess
(>20%) when compared to para substituted acetophenones 1b-6b,
azido ketones 10b-11b and meta substituted compounds 8b [1].
The progress of bio-reduction of keto compounds to respective
chiral alcohols by functional alginate immobilized yeast cells was
summarized (Table: S1 and S2). The absolute configuration of
chiral alcohols 1b-9b and 12b was found to be ‘S’ (Prelog’s rule)
[9], [17-19], whereas 10b, 11b has shown ‘R’ configuration (anti-
Prelog’s rule) [10]. The functionalized alginate beads shown
maximum bead stability without functionalized alginate bead
degeneration and thus improving the enzyme activity and enantio-

selectivity (ee) even after repeated use of beads in a continuous
process up to 7 cycles, (Fig: 2). Where as in calcium alginate
immobilized yeast cells, diminution of enzymatic activity which is
associated with loss of cell viability or decline in intracellular co-
factor regeneration (NADH, NADPH) cycle resulting in loss of
stability of alginate beads, resulting in decrease in enzyme activity
after 3rd cycle. The improved enzyme activity in functionalized
alginate immobilized beads containing yeast cells, may be due to
high amount of water/ medium around the cavities of beads, which
helps in maintaining cell viability and enzyme activity (ADH),
and also takes part in the internal cofactor (NADH, NADPH)
regeneration system via, glucose present in the medium [20]. Thus
the results establishes that the succinic anhydride derivatized
alginate beads provide better integrity and intact support to the
immobilized yeast cells without altering the extracellular (ADH)
enzyme activity, in comparison to conventional calcium alginate
beads. Several immobilized methods have been reported for
immobilization of baker’s yeast cells on different matrices,
resulted in low yields of 70-80% and enantiomeric purity 50-90%
when compared to present study [21-22]. The main drawbacks of
all these methods were decreased in the enzyme activity and
stability of immobilized beads after few cycles due to dissolution
of the matrix support. Therefore, all these known methods have

limited scope in scale up and continuous production of chiral
intermediates. Chiral compounds 1b-5b, 7b are the important
intermediates in development of various drugs and agrochemicals
[9]. Compound 6b used as chiral intermediate in the synthesis of
anti viral CCR5 antagonists [17]. The compound 8b has its
importance in the synthesis of drug (S) - (-) - fenfluramine [18],
10b-12b are the intermediates for synthesis of drugs (R) - (-) -
tembamide, β-adrenergic blocker, (S) - (+) - fluoxetine
respectively [23-25].
This study was further extended in hydrogenation of chiral azido
alcohols 10b-12b to obtain respective chiral amino alcohols 10c-
12c in good yields (85-92%), by implementing the Pd
nanoparticles ≤ 5nm, prepared by ethanol reduction and the
particle size along with distribution was confirmed by TEM (Fig: 3
and 4), The absolute configuration of the synthesized chiral amino
alcohols 10c, 11c found to be (R) configuration and 12c was (S) ,
these values were compared with literature values [23-25].
The chiral amino alcohols synthesized (Scheme 3), are the known
intermediates/ precursors of the anti-hyperglycemic drug R - (-) -
tembamide, β-adrenergic blocker, anti-depressant drug (S) - (+) –
fluoxetine, respectively.
Thus the study highlights the use of yeast cell bound functionalized
alginate immobilized beads, which are stable and can be reused for

more number of cycles without degeneration of beads and leakage
of yeast cells. The developed methodology has shown greater
advantage in synthesis of chiral compounds over conventional
methods in demonstrating the recyclability, easiness and the use of
yeast cell bound functionalized alginate immobilized beads
without degeneration of beads and leakage of yeast cells, followed
by the use of palladium nanoparticles in selective hydrogenation to
obtain chiral amino alcohols. Thus the process enlightens the scope
in the synthesis of chiral alcohols/ amino alcohols of
pharmaceutical importance.
4. Conclusions
In summary, a continuous chemo-enzymatic synthesis of
chiral alcohols followed by amino alcohols was achieved
successfully by using functionalized alginate immobilized
beads containing yeast cells with increased efficiency and
high enantio selectivity by improving cell viability and co-
enzyme regeneration system. This green process developed
also overcome the drawbacks like waste management and
product loss and has the advantage in continuous use of yeast
cells immobilized in alginate beads. The yeast cells show
broad substrate specificity and it has been observed that
electronic effects were not influencing the stereo chemical
features and enantio-selectivity during the process of bio-

reduction the keto compounds. Thus, the method developed
provides a green approach to ameliorate the efficiency of
biocatalyst in the synthesis of optically pure chiral alcohols
and provides tremendous opportunity in developing simple,
easy, inexpensive and scalable route in synthesis of chiral
compounds of biological interest.
Conflict of interest
The authors do not declare any conflict of interest,
whatsoever, either financially or otherwise.
Acknowledgements
Authors thanks DST Government of India for financial
support to the project no. SB/S5/GC-16/2013 (GAP-0489). N.
M. would like to thank Director CSIR- IICT, for awarding
project fellowship and providing necessary facilities. We are
thankful to Dr. G. Sheelu, for guiding in GC analysis.

References
[1] J.A.R. Rodrigues, P.J.S. Moran, J.A.G. Conceicao, L.C.
Fardelone, Food Technol. Biotech. 42 (2004) 295-303.
[2] N.J. Turner, Nat. Chem. Biol. 5 (2009) 567-573.
[3] L.A. Nguyen, H. He, C.P. Huy, Int. J. Biol. Sci. 2 (2006) 85-
100.
[4] P. Borowiecki, D. Paprocki, A. Dudzik, J. Plenkiewicz, J. Org.
Chem. 81 (2016) 380-395.
[5] M. Hall, A. S. Bommarius, Chem. Rev. 111 (2011) 4088-4110.
[6] K. Nakamura, R. Yamanaka, T. Matsuda, T. Harada,
Tetrahedron: Asymmetry. 14 (2003) 2659-2681.
[7] Nicolas Blanchard, Pierre van de Weghea, Org. Biomol. Chem.
14 (2006) 2348-2353.
[8] S.E. Milnera, A.R. Maguire, Arkivoc. 1 (2012) 321-382.
[9] J.S. Yadav, S. Nanda, P. Thirupathi Reddy, A. Bhaskar Rao,
J. Org. Chem. 67 (2002) 3900-3903.
[10] J.S. Yadav, P. Thirupathi Reddy, S. Nanda, A. Bhaskar Rao,
Tetrahedron: Asymmetry. 12 (2001) 63-67.
[11] S.S. Deepthi, E. Prasad, B.V. Subba Reddy, B. Sreedhar, A.
Bhaskar Rao, Green and Sustainable Chemistry. 4 (2014) 15-19.

[12] R.N. Patel, Biomolecules. 3 (2013) 741-777.
[13] J.H. Schrittwieser, F. Coccia, S. Kara, B. Grischek, W.
Kroutil, N. Alessandrod, F. Hollmann, Green. Chem. 15 (2013)
3279-3490.
[14] Ch. Subba Rao, R.S. Prakasham, A. Bhaskar Rao, J.S.
Yadav, Appl. Biochem. Biotechnol. 149 (2008) 219-228.
[15] O.B. Wurzburg, Methods in Carbohydrate Chemistry,
fourth ed., Academic Press, New York, 2000.
[16] N.V. Long, T. Hayakawa, T. Matsubara, N.D. Chien, M.
Ohtaki and M. Nogami,
Nanoscienc. (2012) 426–439.
Journal of Experimental
7
[17] M.J. Homann, R.B. Vail, Edward Previte, Maria Tamarez,
Brian Morgan, D.R. Dodds, Aleksey Zaks, Tetrahedron. 60 (2004)
789–797.
[18] Marco Fogagnolo, P.P Giovannini, Alessandra Guerrini,
Alessandro Medici, Paola Pedrini, Nicola Colombi, Tetrahedron:
Asymmetry. 9 (1998) 2317–2327.
[19] Andrea Kišić, Michel Stephan, Barbara Mohar, Adv. Synth.
Catal. 357 (2015) 2540–2546.
[20] A. Wolfson, N. Haddad, C. Dlugy, D. Tavor, Y. Shotland,
Org. Commun. 1 (2008) 9-16.

[21] T.R. Gervais, G. Carta, J.L. Gainer, Biotechnol. Prog. 19
(2003) 389-395.
[22] K. Kato, H. Nakamura, K. Nakanishi, Appl. Surf. Sci. 293
(2014) 312-317.
[23] A. Kamal, A.A. Shaik, S. Mahendra, M.S. Malik,
Tetrahedron: Asymmetry, 15 (2004) 3939-3944.
[24] Taeko Izumi, Katsumi Fukaya, Bull. Chem. Soc. Jpn. 66
(1993) 1216-1221.
[25] J. Whittall, Regio- and stereo- controlled oxidation and
reductions, in: Stanley M. Roberts , John Whittall (Eds.), Catalysts
for Fine Chemical Synthesis, John Wiley & Sons, Ltd., UK, 2009,
pp. 153.
[26] Wang Pu, Su Huizhen, Sun Liming, He Junyao, Lu Yaping,
Chinese Journal of Chemical Engineering. 19 (2011) 1028-1032.

List of Figures and Tables:
Fig: 1. Scanning Electronic Microscopic (SEM) image of
functionalized alginate immobilized alginate beads entrapped yeast
cells
Fig: 2. Demonstration reuse cycles of functionalized alginate
immobilized yeast beads
Fig: 3. Transmission electron microscopy (TEM) image to
represents Palladium (Pd) nanoparticles
Fig: 4. Particle Size distribution of Palladium (Pd) nanoparticles
(≤5nm)

Figures and Tables
Fig: 1. Scanning Electronic Microscopic (SEM) image of
functionalized alginate immobilized alginate beads entrapped yeast
cells

Fig: 2. Represents number of reuse cycles of functionalized
alginate immobilized yeast beads

Fig: 3. Transmission electron microscopy (TEM) image to
represents Palladium (Pd) nanoparticles

Fig: 4. Particle size distribution of Palladium (Pd) nanoparticles
(≤5nm)

Scheme 1: Asymmetric reduction of para- substituted
acetophenones 1a-6a and 2-acetyl naphthone 7a by functionalized
alginate immobilized baker’s yeast
Scheme 2: Asymmetric reduction of substituted propiophenones
8a, 9a by functionalized alginate immobilized baker’s yeast

Scheme 3: Chiral Reduction Azido ketones 10a-12a by
functionalized alginate immobilized Baker’s yeast followed by
hydrogenation with Pd nanoparticles.

Table: 1. Bio-reduction of pro-chiral ketones 1a-12a with
S .cerevisiae cells
Table 1: Bio-reduction of pro-chiral ketones 1a-12a by
by fermented S. cerevisiae cells
Entry Substrate Product
Fermented yeast
cells
Alginate immobilized Succinylated alginate Confi
gurati
on
yeast cells
Yeast cells
Yield [%]a ee [%]b Yield [%]a
ee [%]b Yield [%]a ee [%]b
1.
2.
1a
2a
1b
2b
S
S
S
S
S
S
S
S
S
R
R
S
77
70
56
62
56
40
66
52
20
55
50
60
43
64
52
55
60
34
45
25
-
78
67
77
78
77
65
71
76
40
70
80
67
55
40
53
62
70
59
50
60
<5
84
78
77
88
91
89
99
94
92
96
80
55
99
91
98
97
93
95
94
89
95
97
96
20
95
85
93
3.
3a
3b
4.
4a
4b
5.
5a
5b
6.
6a
6b
7.
7a
7b
8.
8a
8b
9.
9a
9b
10.
11.
12.
10a
11a
12a
10b
11b
12b
50
64
54
a Isolated yield of products 1b-12b after 24h by column
chromatography
b
Enantiomeric excess determined indepenendently by
HPLC and/or GC