Asymmetric reduction of ketones by biocatalysis using medlar (Mespilus germanica L) fruit grown in Algeria

Article abstract of DOI:10.3109/10242422.2014.978305

The biocatalytic reduction of ketones was performed using medlar fruit (Mespilus germanica L), which is grown in large amounts in Algeria. Experiments were performed using aqueous medium with fresh medlar. Prochiral heteroaryl ketones containing furan, thiophene, chroman, and thiochroman moieties were reduced with enantiomeric excess (ee) of up to 98%. High enantioselectivities have been observed especially for the bioreduction of tetralone 4 and thiochromanone 5 with ee of 89% and 98%, respectively. Bioreduction using different parts of medlar indicate that chromanone 6 was reduced with good asymmetric inductions 77% < ee < 85% whichever part of fruit was employed (whole, flesh, or juice) and medlar juice exhibited the best activity.

Full text of DOI:10.3109/10242422.2014.978305

Biocatalysis and Biotransformation, 2014; Early Online: 1–6
ORIGINAL ARTICLE
Asymmetric reduction of ketones by biocatalysis using medlar
(Mespilus germanica L) fruit grown in Algeria
MANHEL BENNAMANE, SAOUSSEN ZEROR & LOUISA ARIBI-ZOUIOUECHE
Ecocompatible Asymmetric Catalysis Laboratory (LCAE), Badji Mokhtar Annaba-University, Annaba, Algeria
Abstract
The biocatalytic reduction of ketones was performed using medlar fruit (Mespilus germanica L), which is grown in large
amounts in Algeria. Experiments were performed using aqueous medium with fresh medlar. Prochiral heteroaryl ketones
containing furan, thiophene, chroman, and thiochroman moieties were reduced with enantiomeric excess (ee) of up
to 98%. High enantioselectivities have been observed especially for the bioreduction of tetralone 4 and thiochromanone
5 with ee of 89% and 98%, respectively. Bioreduction using different parts of medlar indicate that chromanone 6 was
reduced with good asymmetric inductions 77%ꢀeeꢀ85% whichever part of fruit was employed (whole, flesh, or juice)
and medlar juice exhibited the best activity.
Keywords: Biocatalysis, heteroaryl alcohols, Mespilus germanica L
Introduction
alcohols with moderate-to-excellent enantioselec-
tivities (Seebach et al. 1985; Gillois et al. 1988;
Forzato et al. 1997; Howarth et al. 2001; Wei et al.
2001; Patel et al. 2004;Yajima et al. 2004; Silva et al.
2012, 2013). Additionally, several studies report on
the use of parts of fresh plants, vegetables, and fruits
as biocatalysts (Giri et al. 2001; Villa et al. 1998;
Bruni et al. 2002; Borges et al. 2009), for example
freshly cut Daucus carota root has been reported for
the reduction of ketones and aldehydes (Baldassarre
et al. 2000; Comasseto et al. 2004; Andrade et al.
2006; Yadav et al. 2008; Salvano et al. 2011).
Recently, the asymmetric reduction of different kinds
of prochiral ketones to produce the corresponding
chiral alcohols have been realized with Apple (Malus
pumila), carrot (D. carota), cucumber (Cucumis
sativus), onion (Allium cepa), potato (Solanum tubero-
sum), radish (Raphanus sativus), tomato (Lycopersi-
cum esculentum), and sweet potato (Ipomoea batatas)
as biocatalysts. It was found that prochiral ketones
could be reduced by these plant tissues with high
enantioselectivity (Giri et al. 2001;Villa et al. 1998;
Bruni et al. 2002; Borges et al. 2009, Blanchard &
Weghe 2006; Assunção et al. 2008; Utsukihara et al.
2006; Xu et al. 2010; Liu et al. 2012). The use of
Stereoselective reduction of heteroaryl ketones
containing furan, thiophene, chroman, and thiochro-
man moieties is of utmost importance in organic
synthesis as the resulting chiral alcohols can be used
as antioxidants, or building blocks (Blasig et al. 1998,
Ramadas & Krupadanam 2004; Ellis 1977; Hwang
et al. 2000; Nakamura et al. 2003). Enantiomerically
pure (R)-2-furanyl-2-ethanol was used as a building
block to introduce chirality in the synthesis of the
anthrax tetrasaccharide produced by Bacillus antharacis
(Abrams et al. 2008). Chroman and thiochroman
carbinols are found in many drugs, such as
4-hydroxy tetralol which is employed in the treat-
ment of high blood pressure (Viatcheslav et al. 2009).
Therefore, the synthesis of enantiopure heteroaryl
carbinols is nowadays a large area of research.
Enantioselective reduction of prochiral ketones with
chemical catalysts or biocatalysts is a promising
route for the production of enantioenriched alcohols.
In the context of developing green and sustainable
chemical processes, biotechnology presents attrac-
tive alternatives. Baker’s yeast is the most widely
used microorganism for the chiral reduction of vari-
ous ketones giving the corresponding optically active
Correspondence: Louisa Aribi-Zouioueche, Ecocompatible Asymmetric Catalysis Laboratory (LCAE), Badji Mokhtar Annaba-University, B.P 12, 23000,
Annaba, Algeria. Fax:ꢁ213 (0)38871712. E-mail: louisa.zouioueche@univ-annaba.dz
(Received 26 March 2014; revised 11 July 2014; accepted 15 October 2014)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2014 Informa UK, Ltd.
DOI: 10.3109/10242422.2014.978305

2 M. Bennamane et al.
O
OH
OH
coconut juice as a biocatalyst for selective reduction
of aromatic and aliphatic carbonyl compounds has
been reported and high enantioselectivities were
obtained (Rammohan 2013). Both biological and
organometallic enantioselective catalysts have been
developed as complementary tools for the reduction
of ketones. Asymmetric transfer hydrogenation of
ketones has recently emerged as an alternative
method for the production of chiral alcohols due
to easy workup and availability of the reductants
(Noyori & Hashiguchi 1997; Noyori & Ohkuma
2001; Palmer & Wills 1999; Gladiali & Alberico
2006; Samee et al. 2006; Ikariya et al. 2006). In this
area, we have studied asymmetric transfer hydroge-
nation (ATH) of aromatic ketones in water using
ruthenium-based catalysts (Zeror et al. 2006, 2008;
Boukachabia et al. 2011). In the course of our
previous investigations, the comparison between
enantioselective bio- and metal-catalyzed reactions
has been examined. We have compared the enanti-
oselective reduction of ketoesters by Saccharomyces
cerevisiae- and ruthenium-catalyzed reactions in
water (Zeror et al. 2010).
The use of fruits as biocatalysts has many advan-
tages. First of all, a large range of different fruits is
available at a very low cost.Another important aspect
is that the separation of the product from the reac-
tion mixture can be carried out very easily by filtra-
tion. Moreover, these systems have the advantage of
being environmentally friendly, since the reaction is
carried out in water as solvent and the catalyst is
biodegradable. Medlar grows in various regions
especially in the north of Algeria.This species grows
naturally on our premises, and it is also cultivated in
home gardens. Compared with other fruits, medlar
is available at very low cost (0.5 $/kg) and it has not
previously been investigated as a biocatalyst.We now
report our investigations on the catalytic asymmetric
reduction of ketones by Mespilus germanica L
(medlar) fruit, grown in Algeria (Scheme 1).
Medlar (Mespillus germanica L.)
or
R
R'
R
R'
R
R'
H2O, 30°C
1-8
1a-8a
O
O
O
S
R
X
R
O
8
3
R= H, X= CH2
4
R= H
R= Cl 2
1
R= H, X= S
R= H, X= O
R= Ph, X= O
5
6
7
Scheme 1. Asymmetric reduction of ketones catalyzed by
M. germanica L (medlar).
Thermo Separation Product Pompe P100 with a UV
detector and a chiral stationary-phase column CHI-
RALCEL OD-H and OJ-H. Retention times are
reported in minutes. All of the crude products were
purified by preparative thin-layer chromatography
on Silica Gel 60 PF254 (hexane/ethyl acetate, 2/1).
Ketones were purchased from Sigma or Aldrich.
Fresh medlar fruit (M.germanica) was obtained from
a local market (Scheme 2).
General procedure for enantioselective reduction of
ketones with M. germanica (medlar) as biocatalyst
The ripe medlar fruit was washed with water, then
disinfected with ethanol, and peeled with a sterilized
cutter before cutting carefully into small slices. A
suspension of the medlar (100 g) in water (200 ml)
was stirred in an Erlenmeyer flask at 30°C for
20 min. Then the appropriate ketone (1 mmol) in
dimethylformamide (DMF) (1 mL) was added.After
48 h, the medlar pieces were removed by filtration,
washed several times with ethyl acetate and the
filtrate was extracted with ethyl acetate; the organic
phase was dried over anhydrous MgSO4 and the
Materials and methods
Nuclear magnetic resonance (NMR) spectra were
performed with Bruker spectrometers (300 MHz for
1H, 75 MHz for 13C). Chemical shifts are reported
in δ ppm from tetramethylsilane with the solvent
1
resonance as internal standard for H NMR and
chloroform-d (δ 77.0 ppm) for 13C NMR. The
enantiomeric excess (ee) values were measured by
gas chromatography using aThermo FinniganTrace
gas chromatography (GC), equipped with an
autosampler and using a Chiralsil-DEX CB column
(25 m; 0.25 mm; 0.25 μm), or by a chiral stationary-
phase high-performance liquid chromatography
(HPLC). HPLC analyses were performed on a
Scheme 2. Medlar fruit (M. germanica L).

Reduction of ketone by biocatalysis using medlar (Mespilus germanica L)
3
solvent was evaporated to obtain the product.
Subsequently, each reaction mixture was purified by
column chromatography using ethyl acetate and
n-hexane as eluent to afford the product, and the
percentage yield was calculated based on the weight
of initial and final purified product (Supplementary
Supporting Information, available online at http://
informahealthcare.com/doi/abs/10.3109/10242422.
2014.978305).
was washed, disinfected, and peeled as previously
described and then cut carefully into small slices. A
suspension of the medlar (100 g) in water (200 ml)
was stirred in an Erlenmeyer flask at 30°C for 20 min.
Then chromanone (148 mg, 1 mmol) in DMF (1
mL) was added. After 48 h, the residue was removed
by filtration, washed several times with ethyl acetate,
and the filtrate was extracted with ethyl acetate; the
organic phase was dried over anhydrous MgSO4 and
the solvent was evaporated to yield the product.
Bioreduction of chromanone 6 with flesh or juice of
M. germanica (medlar)
Bioreduction of chromanone 6 with dried flesh of
M. germanica (medlar)
Fresh medlar was washed, disinfected, and peeled as
previously described; 100 g of this material was then
filtered using sterilized compresses as soon as pos-
sible, for the total elimination of the juice from whole
fruit.Then, the juice was filtered through filter paper
to remove solid material and obtain clear juice. Med-
lar flesh (30 g) or juice (40 mL) was used as the bio-
catalytic system for the reduction of chromanone 6.
Fresh medlar was washed with water, disinfected,
and peeled as previously described; 100 g of fruit
was filtered using a sterilized compress as soon as
possible for total elimination of the juice, and the
resulting medlar flesh was then maintained in an
oven at 40°C for 48 h. A suspension of the dried
medlar (mꢂ18 g) in water (200 ml) was stirred in
an Erlenmeyer flask at 30°C for 20 min. Then the
chromanone (1 mmol) in DMF (1 mL) was added.
After 48-h incubation at 30°C, the residue was
removed by filtration, washed several times with
ethyl acetate, and the filtrate was extracted with ethyl
acetate; the organic phase was then dried over anhy-
drous MgSO4 and the solvent was evaporated.
Bioreduction of chromanone 6 with medlar juice
Chromanone 6 (148 mg, 1mmol) in DMF was
added to the freshly prepared juice (40 mL) in water
(200 ml). The reaction mixture was stirred at room
temperature for a maximum of 48 h.Then the reac-
tion mixture was filtered, washed several times with
ethyl acetate, and the filtrate was extracted with ethyl
acetate; the organic phase was dried with anhydrous
MgSO4. Chroman-4-ol (104 mg) was obtained after
purification by thin-layer chromatography with
hexane/ethyl acetate mixtures.
Chiral GC analysis and/or chiral HPLC analysis
The absolute configuration of major enantiomer was
assigned by comparison with our previous work
(Zeror et al. 2006, 2008). The conditions for the
analysis of all alcohols are reported below:
(S)-(-)-1-phenyl-1-ethanol 1a: GC CHIRAL-
CEL Dex β-PM: tR ꢂ4.43 min, tS ꢂ4.62 min
(Tcolumn ꢂ140°C) (Zeror et al. 2006).
(S)-(1)-1-(4-chlorophenyl)-1-ethanol 2a: GC
CHIRALCEL Dex β-PM: tR ꢂ11.32 min, tS ꢂ12.63
min (Tcolumn ꢂ140°C) (Zeror et al. 2008).
(S)-(1)-1-indanol 3a: HPLC (CHIRALCEL
OD-H, Isohexane/i-PrOH: 98/2, flow 0.5 mL/min):
tS ꢂ25.99 min, tR ꢂ30.49 min (Zeror et al. 2008).
Bioreduction of chromanone 6 with medlar flesh
A suspension of the medlar flesh (mꢂ30 g) in water
(200 ml) was stirred in an Erlenmeyer flask at 30°C
for 20 min. Then chromanone 6 (148 mg, 1mmol)
in DMF (1 mL) was added and the mixture stirred
at room temperature for 48 h. The flesh was then
removed by filtration, washed several times with
ethyl acetate, and the filtrate was extracted with ethyl
acetate; the organic phase was dried over anhydrous
MgSO4 and the solvent was evaporated to get the
product. Additionally, the reaction product was puri-
fied by column chromatography on silica gel as men-
tioned above to give the chroman-4-ol (38 mg).
(R)-(-)-1,2,3,4-tetrahydronaphtalen-1-ol
4a:
HPLC (CHIRALCEL OJ-H, Isohexane/i-PrOH:
90/10, flow 0.5 mL/min): tR ꢂ14.05 min, tS ꢂ17.14
min (Zeror et al. 2008) or HPLC (CHIRALCEL
OJ-H, Isohexane/i-PrOH: 90/10, flow 1 mL/min):
tR ꢂ10.78 min, tS ꢂ14.54 min.
(R)-(1)-thiochroman-4-ol 5a: HPLC (CHIRAL-
CEL OJ-H, Isohexane/i-PrOH: 90/10, flow 1.0 mL/
min): tSꢂ7.68 min, tRꢂ9.55 min (Zeror et al. 2008) or
HPLC (CHIRALCEL OJ-H,Isohexane/i-PrOH:90/10,
flow 0.5 mL/min): tSꢂ23.12 min, tRꢂ24.52 min.
Bioreduction of chromanone 6 with rotten medlar
Medlar fruits were left unrefrigerated for 30 days.
Browning of the skin and residue of moisture were
observed and it gained a rotting odor. Rotten medlar

4 M. Bennamane et al.
Table I. Enantioselective reduction of aromatic ketones catalyzed
by M. germanica L.
(S)-(-)-chroman-4-ol 6a: HPLC (CHIRALCEL
OJ-H, Isohexane/i-PrOH: 90/10, flow 1.0 mL/
min): tS ꢂ 6.35 min, tR ꢂ 7.29 min (Zeror et al.
2008) or HPLC (CHIRALCEL OJ-H, Isohexane/i-
PrOH: 90/10, flow 0.5 mL/min): tS ꢂ 12.57 min,
tR ꢂ 14.20 min.
3, 4-dihydro-2-phenyl-2H-chroman-4-ol 7a:
HPLC (CHIRALCEL OD-H, Isohexane/i-PrOH:
90/10, flow 0.5 mL/min): t(ꢁ) ꢂ20.38 min, t(ꢃ) ꢂ
26.26 min.
Entry
Substratea
Yeildb (%)
eec(%)
Confd
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
39
34
38
34
35
31
41
51
62
85
25
89
98
83
32
51
(S)
(S)
(S)
(R)
(R)
(S)
(-)
(S)
(S)-(-)-1-thiophen-2-yl-ethanol 8a: GC [CHI-
RALCEL Dex β-PM, at 90°C during 10 min (3°C/
min), heated to 141°C during 1 min (20°C/min) and
maintained at 180°C. tR ꢂ15.05, tS ꢂ15.59 (Zeror
et al. 2008) or HPLC (CHIRALCEL OD-H,
hexane/i-PrOH: 98/2, flow 0.5 mL/min): t(ꢁ) ꢂ17.72
min, t(ꢃ) ꢂ19.21 min.
aReactions were performed with 100 g medlar and 200 mL water
for 1 mmol substrate in water at 30°C during 48 h.
bIsolated yield.
cee determined by chiral GC or HPLC, dAbsolute configuration
of the major enantiomer was assigned by comparison with our
previous paper.
1 and 2). The presence of an electron-withdrawing
group on the phenyl ring led to an increase in the ee
values. The isolated alcohol 2a had the same con-
figuration (S) as that of 1a. As part of our interest
was in the enantioselective synthesis of biologically
active precursors, we decided to apply our reduction
with medlar to the asymmetric reduction of het-
eroaryl compounds. The reduction of indanone 3
catalyzed by medlar furnished a product with low ee
values (25%, entry 3). Interestingly, high enantiose-
lectivity was observed for the reduction of tetralone
4 which includes a six-membered ring (eeꢂ89%,
entry 4), whereas only ee of 39% was reported when
Lens culinaris was used as biocatalyst for this sub-
strate (Daniele et al. 2012).The influence of heteroa-
tomsinthecyclewasfurtherinvestigated.Remarkable
enantioselectivity was achieved for thiochromanone
5 reduction, providing the (R)-thiochroman-4-ol
5a with ee of 98% (entry 5). Extending this to
the synthesis of oxygen-containing heteroaryl alco-
hols, prochiral chromanone 6 was successfully
reduced to its corresponding alcohol with high
enantioselectivity (eeꢂ83%, entry 6).The reduction
with thiochromanone 5 and chromanone 6 gave
interesting results with similar or slightly higher ee
values than those for reduction of tetralone 4 (com-
pare entries 4, 5, and 6). However, while the alcohols
formed by the reduction of 4 and 5 had the
same configuration, this was opposite to that of the
alcohol 6a produced. These chiral benzylic alcohols
are used as key synthons for many drugs (Bauer
et al. 1997; Macor 2007). In order to evaluate the
influence of a phenyl substituent on chromanone,
2-phenyl-chroman-4-one 7 was also reduced by
medlar. However, this alcohol gave a lower ee than
6a (entries 6 and 7). In order to extend the scope of
this biocatalyst, we explored the reduction of various
five-membered heterocycles. Medlar was unable to
Results and discussion
With the joint aims of developing green chemistry
methods and using endemic flora, the biocatalytic
reduction of ketones was studied using fresh ripe
medlar fruit purchased from a local market. Medlar
(M. germanica L) has not previously been investi-
gated as a biocatalyst. In order to examine activity
and enantioselectivity, we selected a variety of
substrates 1–8 including acetophenone derivatives
and prochiral heteroaryl ketones containing furan,
thiophene, chroman, and thiochroman moieties.
Asymmetric reduction of ketones catalyzed by
M. germanica L (medlar)
It is known that whole cells have their own cofactor
regeneration system that can be used without any
additives or by adding cosubstrates or reducing sugar
(Goldberg et al. 2007).Thus, in this work there was
no external addition of a source of reducing power
or cofactor, but there are a large amount of endog-
enous reducing sugars in medlar. The reaction with
medlar was first optimized (reaction time, tempera-
ture, and the amount of medlar) for the asymmetric
reduction of acetophenone. The mixture was stirred
at 30°C for 48 h, after which the reaction yield was
constant, indicating that the reaction had reached
equilibrium. Medlar was able to reduce the ketone
to its corresponding alcohol, with modest yields
(31–51%). The results are shown in Table I.
The (S)-1-phenyl-1-ethanol 1a was obtained
with good ee (62%), but low isolated yield (39%).
The effect of a substituent in the aromatic moiety on
the biotransformation was studied using p- chloro-
acetophenone 2, which was reduced with high
enantioselectivity compared with that of 1a (entries

Reduction of ketone by biocatalysis using medlar (Mespilus germanica L)
5
reduce 2-acetylfuran and 2-acetylpyrrole but, sur-
prisingly, bioreduction of 2-acetylthiophene 8 gave
the corresponding alcohol with moderate yield and
ee (entry 8). The presence of a sulfur atom in the
ring seems to provide an improvement in activity and
enantioselectivity (substrates 5 and 8). In summary,
high enantioselectivities were observed for substrates
1, 2, 4, 5, and 6, and in particular for the reduction
of heterocyclic ketones including a six-membered
ring (eeꢂ83–98%). The best results obtained for
reduction with medlar were recorded for ketones 2,
4–6.
activity, therefore, depends upon the concentration
of juice in each component of the medlar.
Conclusion
Medlar fruits contain an enzyme system, which
appears to be predominantly contained in the juice,
with the ability to enantioselectively reduce ketones.
The enzyme in the fruit appears to be relatively
stable when stored without refrigeration for a
number of days, making it simple to handle. High
enantioselectivities have been observed for some
substrates especially for the bioreduction of hetero-
cyclic ketones including a six-membered ring
(83ꢀeeꢀ98%). This biotechnological process is
eco-friendly and cheap, the isolation of the final
product is easy, and the fruits are readily available.
Indeed, fruits represent a new source of enzymes for
use as catalysts in organic synthesis.
Reductions of chromanone 6 by various parts of
M. germanica L
In order to valorize the effect of not using fresh mate-
rial, medlar fruits were left outside the refrigerator
for 30 days and then we compared the reduction of
chromanone 6 as a model substrate with the results
obtained with fresh fruit (Table II (entries 1and 2)).
Interestingly, the flesh of rotten medlar was still able
to reduce chromanone 6 with a high ee (75%) which
suggests that the use of fresh or refrigerated material
is not critical to obtain good enzyme activity.
In an attempt to determine which component of
medlar contains the main biocatalytic activity, differ-
ent parts of the medlar were tested for chromanone
6 bioreduction (Table II (entries 1, 3–5)). Using
compressed fruit flesh the resulting alcohol was
obtained with very poor yield (25%), but there was
no decrease in the ee compared with that observed
with whole fruit (entries 1 and 4). Fruit juice was
examined and gave the alcohol with an increased
yield (69%) and an ee close to that obtained with
whole fruit and medlar flesh (entries 1, 4, and 5).
Thus, the use of medlar juice as a biocatalyst
improved the yield of the reaction. Consistent with
this, no bioreduction was observed using dried fruit
as a catalyst (entry 3). The differences in yield
observed were probably due to the presence of flesh
in the whole fruit, hindering the reaction by partially
binding the substrate. The observed bioreduction
Acknowledgments
Algerian Ministry of education and scientific research
(FNR 2000 and PNR) is gratefully acknowledged
for financial support of this work. The authors wish
to thank Professor Olivier Riant (MOST/INCM)
Catholic University of Louvain for welcoming
Manhel Bennamane to carry out chiral HPLC and
GC measurements.
Declaration of interest: The authors report no
declarations of interest. The authors alone are
responsible for the content and writing of the
paper.
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Supplementary material available online
Supplementary Supporting Information, at http://
informahealthcare.com/doi/abs/10.3109/10242422.
2014.978305.