highly enantioselective bioreduction of prochiral ketones by stem and germinated plant of ...
TRANSCRIPT
ORIGINAL ARTICLE
Highly enantioselective bioreduction of prochiral ketones by stem and germinated plant of Brassica oleracea variety italica
MEHDI MOHAMMADI , MARYAM YOUSEFI & ZOHREH HABIBI
Department of Chemistry, Faculty of Science, Shahid Beheshti University, G.C., Tehran, Iran
Abstract An eco-friendly and environmentally benign asymmetric reduction of a broad range of prochiral ketones employing Brassica oleracea variety italica (stems and germinated plant) as a novel biocatalyst was developed. It was found that B. oleracea variety italica could be used effectively for enantioselective bioreduction in aqueous medium with moderate to excellent chemical yield and enantiomeric excess (ee). This process is more effi cient and generates less waste than conventional chemical reagents or microorganisms. Both R - and S -confi gurations were obtained by these asymmetric reactions. The best ee were achieved for pyridine derivatives (92 – 99%). The ee in germinated plant reactions were signifi cantly higher than those of stem reactions. The low cost and the easy availability of these biocatalysts suggest their possible use for large scale preparations of important chiral alcohols.
Keywords: bioreduction , prochiral ketones , Brassica oleracea variety italica
Correspondence: Zohreh Habibi, Department of Chemistry, Faculty of Science, Shahid Beheshti University, G.C., Tehran, Iran. Tel: +98-21-29902710.Fax: +98-21-22431663. E-mail: [email protected]
(Received 23 March 2011; revised 26 June 2011; accepted 22 October 2011)
Introduction
Plants represent a unique class of potential biocata-lysts for transformation of important non-natural substrates. Over the past decade, several investiga-tions have been carried out on the biotransformation of foreign substrates (Baladassare et al. 2000). These processes are more effi cient and generate less waste than conventional chemical reagents, enzymes, or microorganisms (Kumaraswamy & Ramesh 2003; Ward & Young 1990; Yousefi et al. 2010). Reactions using chemical methods involve the use of expensive chiral reagents and environmentally hazardous heavy metals, while reactions using isolated enzymes require costly cofactors (NADH, NADPH) which require effi cient regeneration (Noyori 1994). Even with whole cells such as baker ’ s yeast, which is, by far, the most widely used microorganism for biotrans-formation (yielding the corresponding products with fair to excellent enantioselectivity), recovery of the desired product might not be straightforward.
Considerable attention has been paid to the syn-thesis of enantiomerically pure compounds as chiral synthons, which are increasingly in demand for devel-opment of modern drugs and agrochemicals. Chiral alcohols are building blocks for numerous chiral
pharmaceuticals due to their versatile chemical prop-erties (Chartrain et al. 2001; Dau ß mann et al. 2006; Huisman et al. 2010; Strauss et al. 1999). Asymmet-ric reduction of the corresponding prochiral ketones is an effective route to produce chiral alcohols (Kroutil et al. 2004), and biocatalysis, involving either isolated oxido-reductases or living organisms, is one of the most promising methods due to its high enantiose-lectivity, mild reaction conditions and environmental compatibility (Faber 1997; Moore et al. 2007; Schmid et al. 2001). Among different biocatalysts, plants are excellent alternatives to the use of isolated enzymes, since the oxido-reductase, cofactor (NADPH) and its regeneration system are all located inside the plant, and the addition of the expensive cofactor can be avoided (Nakamura et al. 2003). Excellent results have been obtained in the reduction of natural ketones, since they are easily accepted by plant cells (Nagaoka 2004). Over the last decade, various exam-ples of the reduction of prochiral ketones to chiral alcohols have been reported using growing (Villa et al. 1998) or immobilized (Naoshima & Akakabe 1991) plant cell cultures.
Recently, the feasibility of direct use of different parts of plants has been investigated by using both
Biocatalysis and Biotransformation, November–December 2011; 29(6): 328–336
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.DOI: 10.3109/10242422.2011.635302
Bio
cata
l Bio
tran
sfor
mat
ion
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f G
lasg
ow o
n 09
/05/
13Fo
r pe
rson
al u
se o
nly.
Enantioselective bioreduction of ketones by Brassica oleracea 329
freshly cut carrot root (Baladassare et al. 2000; Yadav et al. 2002) and germinated radish sprout (Matsuo et al. 2008) in the reduction of homochiral ketones. In the context of the present study, Sura è z-Franco and co-workers have investigated the ability of B. oleracea variety italica (broccoli) to reduce aromatic aldehydes (Su á rez-Franco et al. 2010).
However, a problem with this approach is the reproducibility of experiments, since the properties of these biocatalysts may depend on their origins. Moreover, even if sourced from the same area, it is diffi cult to obtain biocatalysts with the same proper-ties consistantly throughout a year. To address these problems, we have used both freshly cut stems of B. oleracea and a sprout of vegetable seeds for reduc-tion of different prochiral ketones (Figure 1).
Materials and methods
General
All ketones were purchased from Merck (Germany). B. oleracea were obtained from a local market. To
increase the contact of the substrate with the bio-catalyst, the external layer was removed, and the rest was carefully cut into small thin pieces (approxi-mately 1 cm long thread-like pieces). B. oleracea seeds were obtained commercially from Falate Iran Co. Tehran, Iran. The reaction products were ana-lyzed by gas chromatography using a Thermoquest-Finnigan (USA) instrument equipped with a HP-CHIRAL-20B column and fl ame ionization detector. Nitrogen was used as the carrier gas at a constant fl ow rate of 1.5 mL/min. The injector and detector (FID) temperature were kept at 250 ° C and 280 ° C, respectively. HPLC analyses of some alco-hols were performed using a Shimadzu LC-10AD instrument. The column was Chiralcel OD, 4.6 � 250 nm. The eluents were hexane/2-propanol (HPLC grade, 85:15) at 0.5 mL per min fl ow rate and mon-itored at 254 nm wavelength. Optical rotations were measured with a Perkin – Elmer 241 polarimeter using chloroform as solvent. Confi guration and enantiomeric excess (ee) of all the compounds was determined by GC and HPLC analyses as well as comparing the optical rotations with those reported
N
O
N
O
N
O
O
4321
O O O
NC
O
F3C
8765
CF3
O O
O2N
O O
Br
O
F
O
BrC6H11
O
ClF
O
BrBr
S
O
O
O O
O
O
PhCl
O
O
O
9 10 11 12
13 14 15 16
17 18 19 20
Figure 1. Chemical structures of the investigated ketones.
Bio
cata
l Bio
tran
sfor
mat
ion
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f G
lasg
ow o
n 09
/05/
13Fo
r pe
rson
al u
se o
nly.
330 M. Mohammadi et al.
in the literature. Authentic standards of racemic alcohols were analysed by GC and HPLC before analysis of the reaction mixtures. The NMR spectra were recorded using a Bruker AVANCE-300 spec-trometer. Control experiments were done wherein only the solvent was added. All other chemicals were analytical grade from various commercial sources. Experiments were performed in duplicate.
Reduction of ketones with B. oleracea stems
The B. oleracea stems were rinsed with 5% sodium hypochlorite solution and sterile distilled H 2 O, and cut into small pieces with a sterile knife. In separate experiments ketones 1 – 20 (50 mg) were individually added to a suspension of the freshly cut B. oleracea stems (10 g) in H 2 O (100 mL). The mixtures were incubated in a shaker (200 rpm) at room tempera-ture for 2 – 4 days, and the reaction process was mon-itored by TLC. Each individual suspension was fi ltered, and the residue washed with water. The aqueous solutions were then extracted with EtOAc (3 � 150 mL), and the organic phases evaporated under reduced pressure. The residues were loaded on a short silica gel column, using n -hexane as elu-ant, to afford the reduced products.
Reduction of ketones with B. oleracea germinated seeds
B. oleracea seeds were obtained commercially and were sterilized in three steps: 1. Rinsing with water and 1% sodium hypochlorite and soaking in water for a while; 2. Sterilizing with ethanol (60 s); 3. Rinsing with H 2 O 2 (10% v/v). Then the seeds were placed on sterilized wet paper fi lters in dark for 3 days at room temperature to be germinated. A grain of germinated seed was cultivated in 10 mL of MS (Murashige & Skoog 1962) medium with 3% sucrose for 8 days to obtain the biocatalyst. The B. oleracea sprout was transferred to a culture tube containing 10 mL of MS medium and 10% (w/v) of each ketone in 20 μ L of DMSO as cosolvent. The reaction was continued under illumination (4000 Lux) for 24 h at room temperature. The fi nal suspensions were then fi ltered off and the fi ltrates extracted with EtOAc (3 � 50 mL).
Preparative scale reduction
B. oleracea stems (200 g), after rinsing with 5% sodium hypochlorite solution and distilled water, were cut into small pieces and placed in a conical fl ask. To the B. oleracea stems in water, 0.5 g of 2-acetylpyridine () was added, and the mixtures incubated in a shaker (200 rpm) for 3 days at room
temperature. Then the broccoli stems were fi ltered off and washed with water. The combined fi ltrate was extracted as previously mentioned. The compound, 1-(pyridin-4-yl) ethanol, was obtained after column chromatography with silica gel.
Recycling of the B. oleracea stems
After completion of the reduction, the reaction mix-ture was fi ltered and the B. oleracea stems washed successively with water and then kept in distilled water at 0 ° C for 2 days. Afterwards, the water was decanted and the B. oleracea stems wiped with soft tissue paper to remove the remaining water. These stems were used further for another reduction reac-tion. It was observed that the activity of the stems was lost completely and no conversion was achieved for 1-(pyridin-4-yl) ethanone after 10 days incuba-tion. So it was concluded that the plant cells lose their activity completely after one round of reduction.
GC and HPLC analysis
1-(Pyridin-4-yl)ethanol . GC analysis, Oven: T1 � 90 ° C for 10 min, T2 � 125 ° C ( D T � 1 ° C/min) for 5 min, T3 � 200 ° C ( D T � 5 ° C/min) for 5 min, Retention time: t R � 14.2 min, t S � 15.02 min. (Nakamura et al. 2002).
1-(Pyridin-3-yl)ethanol. GC analysis, Oven: T1 � 90 ° C for 10 min, T2 � 125 ° C (D T � 1 ° C/min) for 5 min, T3 � 200 ° C ( D T � 5 ° C/min) for 5 min, Retention time: t R � 9.8 min, t S � 10.7 min. (Nakamura et al. 2002).
1-(pyridin-2-yl)ethanol. GC analysis, Oven: T1 � 90 ° C for 10 min, T2 � 125 ° C (D T � 1 ° C/min) for 5 min, T3 � 200 ° C ( D T � 5 ° C/min) for 5 min, Retention time: t R � 11.9 min, t S � 13.1 min. (Yang et al. 2006).
1-Phenylethanol. GC analysis, Oven: T1 � 100 ° C for 10 min, T2 � 170 ° C (D T � 3 ° C/min) for 5 min, Retention time: t R � 13.46 min, t S � 14.31 min. (Nakamura et al. 2002).
1,2,3,4-Tetrahydronaphthalen-1-ol. GC analysis, Oven: T1 � 120 ° C for 5 min, T2 � 130 ° C ( D T � 1 ° C/min) for 10 min, T3 � 200 ° C (D T � 5 ° C/min), Retention time: t R � 19.9 min, t S � 20.8 min. (Matharu et al. 2006).
1-p-Tolylethanol . Retention time: GC analysis, Oven: T1 � 100 ° C for 5 min, T2 � 130 ° C ( D T � 1 ° C/min)
Bio
cata
l Bio
tran
sfor
mat
ion
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f G
lasg
ow o
n 09
/05/
13Fo
r pe
rson
al u
se o
nly.
Enantioselective bioreduction of ketones by Brassica oleracea 331
for 5 min, T3 � 180 ° C ( D T � 3 ° C/min), Retention time: t R � 8.8 min, t S � 10.2 min. (Bandini et al. 2006).
4-(1-Hydroxyethyl)benzonitrile . GC analysis, Oven: T1 � 120 ° C for 5 min, T2 � 130 ° C ( D T � 1 ° C/min) for 10 min, T3 � 200 ° C (DT � 5 ° C/min), Retention time: t R � 26.57 min, t S � 28 min. (Liu & Wolf 2007).
1-(4-(Trifl uoromethyl)phenyl)ethanol . GC analysis, Oven: T1 � 100 ° C for 5 min, T2 � 125 ° C ( D T � 1 ° C/min) for 10 min, T3 � 220 ° C ( D T � 5 ° C/min) for 5 min, Retention time: t R � 11.66 min, t S � 11.87 min. (Savile & Kazlauskas 2005).
2,2,2-Trifl uoro-1-phenylethanol. GC analysis, Oven: T1 � 100 ° C for 5 min, T2 � 130 ° C ( D T � 1 ° C/min) for 5 min, T3 � 180 ° C ( D T � 3 ° C/min), Retention time: t R � 8.9 min, t S � 9.2 min. (Matsuo et al. 2008).
1-(4-Nitrophenyl)ethanol . GC analysis, Oven: T1 � 120 ° C for 5 min, T2 � 130 ° C ( D T � 1 ° C/min) for 10 min, T3 � 200 ° C ( D T � 5 ° C/min), Retention time: t R � 18.2 min, t S � 18.9 min. (Savile & Kazlaus-kas 2005).
1-Mesitylethanol. GC analysis, Oven: T1 � 100 ° C for 5 min, T2 � 130 ° C (D T � 1 ° C/min) for 5 min, T3 � 180 ° C ( D T � 3 ° C/min), Retention time: t R � 11.9 min, t S � 12.4 min. (Savile & Kazlauskas 2005).
1-(4-Bromophenyl)ethanol. GC analysis, Oven: T1 � 100 ° C for 5 min, T2 � 130 ° C ( D T � 1 ° C/min) for 5 min, T3 � 180 ° C ( D T � 3 ° C/min) for 10 min, Retention time: t R � 10.1 min, t S � 10.9 min. (Naka-mura et al. 2002).
1-(4-Fluorophenyl)ethanol. GC analysis, Oven: T1 � 100 ° C for 5 min, T2 � 130 ° C (D T � 1 ° C/min) for 5 min, T3 � 180 ° C (D T � 3 ° C/min), Retention time: t R � 9.2 min, t S � 9.8 min. (Bandini et al. 2006).
1-(2-Bromo-4-cyclohexylphenyl)ethanol. [ α ] 25 D � 25 °
(CHCl 3 , c 0.6) for 90% ee, determined by HPLC analysis, Retention time: t R � 22.1 min, t S � 19.8 min. Relative confi guration of this compound was determined by comparison of the sign of it ' s specifi c
rotation with the reported same molecule (Shimizu et al. 2007).
2-Chloro-1-(4-bromophenyl)ethanol. HPLC analysis, Retention time: t R � 34.8 min, t S � 36.1 min. (Ban-dini et al. 2006).
2-Bromo-1-(4-bromophenyl)ethanol. HPLC analysis, Retention time: t R � 29.9 min, t S � 31.3 min. (Ban-dini et al. 2006).
1-(Thiophen-2-yl)ethanol. GC analysis, Oven: T1 � 100 ° C for 5 min, T2 � 170 ° C ( D T � 2.5 ° C/min) for 5 min, Retention time: t R � 15.8 min, t S � 16.6 min. (Matharu et al. 2006).
1-(Furan-2-yl)ethanol. GC analysis, Oven: T1 � 100 ° C for 5 min, T2 � 170 ° C (DT � 2.5 ° C/min) for 5 min, Retention time: t R � 14.9 min, t S � 15.6 min. (Matharu et al. 2006).
Benzyl 3-hydroxy butanoate. HPLC analysis, Reten-tion time: t R � 15.1 min, t S � 17.2 min. (Nakamura et al. 2002).
Methyl 4-chloro-3-hydroxy butanoate. GC analysis, Oven: T1 � 120 ° C for 5 min, T2 � 130 ° C ( D T � 1 ° C/min) for 10 min, T3 � 200 ° C ( D T � 5 ° C/min), Retention time: t R � 15.8 min, t S � 16.6 min. (Curt et al. 1992).
Result and discussion
In recent years various whole plant cells such as carrot, apple, cucumber, onion, potato, sweet potato, radish, caulifl ower, pumpkin, artichoke, fennel and banana have been used in plant-mediated reductions for syn-thesis of chiral secondary alcohols (Bruni et al. 2006). For fi nding reductases from new common vegetables, broccoli ( B. oleracea variety italica ) was selected as a novel biocatalyst for asymmetric reductions. Asymmet-ric reduction of various prochiral ketones such as aro-matic ketones and β -ketoesters to their corresponding optically active secondary alcohols with moderate to excellent yields and high ee was achieved. This method is an eco-friendly environmental reduction system with easy available and inexpensive biocatalyst.
In an attempt to avoid potential irreproducibility of reduction reactions due to variations in origin and age of the material, we have also used the sprout prepared from broccoli seed as a novel biocatalyst. Since the seeds are obtainable from any location and
Bio
cata
l Bio
tran
sfor
mat
ion
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f G
lasg
ow o
n 09
/05/
13Fo
r pe
rson
al u
se o
nly.
332 M. Mohammadi et al.
other derivatives (entries 6 – 8,10 – 13,20) have R -confi guration. The enantioselectivities of reduction were increased for all acetophenone derivatives by using germinated seed especially for p -trifl uoromethyl acetophenone (95% ee vs. 70% ee ).
An examination of the experimental results reveals that among the acetophenone derivatives, phenyl trifl uoromethyl ketone, p -trifl uoromethyl acetophenone and p -fl uoroacetophenone with elec-tron-withdrawing substituents were effi ciently reduced with signifi cant enantioselectivities. It seems that the carbonyl group is activated by these sub-stituents. On the other hand steric hindrance caused poor chemical yield such as with mesityl acetophe-none (8 and 11% in germinated seed and stems respectively).
It is noteworthy that for one of the acetophenone derivatives, 2-bromo-1-(4-methoxyphenyl) etha-none, (result not shown) no reduction of the carbo-nyl group was observed (Figure 3), instead the bromo group was replaced by a hydroxyl with high yield (75%) while in analogous compounds, 2-bromo-1-(4-bromophenyl) ethanone and 2-chloro-1-(4-fl uorophenyl) ethanone the carbonyl group was
Table I. 1 H NMR data of the hydroxylated methine of the products (300 MHz, CDCl 3 ).
Products
Chemical shift
(ppm) J (Hz)
1-(pyridin-4-yl)ethanol 4.88 q, 6.451-(pyridin-3-yl)ethanol 4.92 q, 6.421-(pyridin-2-yl)ethanol 4.84 q, 6.541-phenylethanol 4.88 q, 6.161,2,3,4-tetrahydronaphthalen-
1-ol4.71 t, 3.83
1- p -tolylethanol 4.85 q, 6.404-(1-hydroxyethyl)
benzonitrile4.92 q, 5.99
1-(4-(trifl uoromethyl)phenyl)ethanol
4.97 q, 6.25
2,2,2-trifl uoro-1-phenylethanol
5.03 q, 6.75
1-(4-nitrophenyl)ethanol 4.95 q, 6.491-mesitylethanol – –1-(4-bromophenyl)ethanol 4.84 q, 6.431-(4-fl uorophenyl)ethanol 4.87 q, 6.331-(2-bromo-4-
cyclohexylphenyl)ethanol4.98 q, 6.43
2-chloro-1-(4-fl uorophenyl)ethanol
4.91 d, d (8.19, 3.42)
2-bromo-1-(4-bromophenyl)ethanol
4.89–4.92 br
1-(thiophen-2-yl)ethanol 4.99 q, 6.111-(furan-2-yl)ethanol 5.08 q, 6.05benzyl 3-hydroxy butanoate 4.19–4.25 mmethyl 4-chloro-3-hydroxy
butanoate4.22–4.27 m
Figure 2. Asymmetric reduction of prochiral ketones by ger-minated seed.
can be germinated at any time of the year under suitable conditions, this method (Figure 2) should be reproducible regardless of location.
A series of ketones were tested with the freshly cut stems of B. oleracea variety italica in aqueous solution at room temperature for 2 – 3 days. The crude reaction mixtures were initially visualized by TLC, followed by purifi cation by thin layer chromatography. Structures of all alcohols were confi rmed by NMR spectroscopic analysis. The 1 H NMR spectra indicated the presence of one signal characteristic of protons attached to hydroxylated carbon. The oxygenated methine signals were observed at δ H 4 – 5.5 ppm. The downfi eld chem-ical shift of this proton are reported in Table I. The reaction mixtures were also analyzed by chiral GC or HPLC for ee determination.
The results of asymmetric reduction by plant stems and germinated seed are given in Tables II and III. In order to dissolve solid ketones a small quantity of DMSO was used as a co-solvent. Both S -form and R -form alcohols were obtained, but both biocatalysts afforded the same enantiomer for each ketone with variable yields (10 – 100%) and ee (69 – 99%). Results showed that the ee increased for almost all of the alcohols employing germinated plant compared with B. oleracea stems. This increase is highly remarkable for entries 8 ( � 25), 13 ( � 14), entry 15 ( � 18) and entries 4 and 16 ( � 10).
Asymmetric reduction of acetophenones afforded the R -chirality at the carbon atom bearing the hydroxyl group which follows anti-Prelog ' s rule. This result is in accordance with reported confi guration obtained from potato ( Solanum tuberosum ), sweet potato ( Ipo-moea batatas ) and apple ( Malus pumila ) which all fol-lowed anti-Prelog ' s rule (Yang et al. 2008). Compared with the results reported for baker ’ s yeast and other microorganisms, the results are attractive since in the reduction system of plant cell cultures the hydrogen attack takes place preferentially from the re -face of the carbonyl group to give the S -enantiomer of cor-responding alcohol (Hamada et al. 1988; Hirata et al. 1982). As can be seen in Tables II and III four deriv-atives of acetophenone (entries 9, 14 – 16) gave the S -confi guration and follow Prelog ' s rule but all the B
ioca
tal B
iotr
ansf
orm
atio
n D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Gla
sgow
on
09/0
5/13
For
pers
onal
use
onl
y.
Enantioselective bioreduction of ketones by Brassica oleracea 333
Table II. Reduction of ketones with stems of B. oleracea variety italic.
Entry Compounds Products Confi g.a Yield(%)b ee(%)b
Time of conversion
(day)
1 1-(pyridin-4-yl)ethanone 1-(pyridin-4-yl)ethanol R 100 94 32 1-(pyridin-3-yl)ethanone 1-(pyridin-3-yl)ethanol R 97 92 33 1-(pyridin-2-yl)ethanone 1-(pyridin-2-yl)ethanol R 98 92 34 1-phenylethanol 1-phenylethanol R 75 79 45 3,4-dihydronaphthalen-
1(2H)-one 1,2,3,4-tetrahydronaphthalen-1-ol S 76 92 2
6 1-p-tolylethanone 1-p-tolylethanol R 58 82 37 4-acetylbenzonitrile 4-(1-hydroxyethyl)benzonitrile R 97 91 28 1-(4-(trifl uoromethyl)
phenyl)ethanone1-(4-(trifl uoromethyl)phenyl)ethanol R 96 70 2
9 2,2,2-trifl uoro-1-phenylethanone
2,2,2-trifl uoro-1-phenylethanol S 97 94 3
10 1-(4-nitrophenyl)ethanone
1-(4-nitrophenyl)ethanol R 65 85 3
11 1-mesitylethanone 1-mesitylethanol R 11 96 212 1-(4-bromophenyl)
ethanone1-(4-bromophenyl)ethanol R 78 89 3
13 1-(4-fl uorophenyl)ethanone
1-(4-fl uorophenyl)ethanol R 95 78 3
14 1-(2-bromo-4-cyclohexylphenyl)ethanone
1-(2-bromo-4-cyclohexylphenyl)ethanol
S 47 94 2
15 2-chloro-1-(4-fl uorophenyl)ethanone
2-chloro-1-(4-fl uorophenyl)ethanol S 63 69 2
16 2-bromo-1-(4-bromophenyl)ethanone
2-bromo-1-(4-bromophenyl)ethanol S 71 78 2
17 1-(thiophen-2-yl)ethanone
1-(thiophen-2-yl)ethanol R 14 91 3
18 1-(furan-2-yl)ethanone 1-(furan-2-yl)ethanol R 10 95 319 benzyl 3-oxobutanoate benzyl 3-hydroxy butanoate S 69 82 220 methyl 4-chloro-3-
oxobutanoatemethyl 4-chloro-3-hydroxy butanoate R 74 76 3
aThe confi gurations were assigned by comparison of the sign of the specifi c rotation of each alcohol with those of the reported molecules.bConversion and ee were determined by GC and HPLC analysis of crude extract.
O
Br
O
OH
Br
O
O
OH
O
Figure 3. Biotransformation of 2-bromo-1-(4-methoxyphenyl)ethanone by stem and germinated plant of B. oleracea.
reduced and no substitution was observed. At pres-ent, we have no satisfactory explanation for this phe-nomenon.
For investigating the reduction of halogen-containing aromatic ketones, 4 ¢ -bromo and 4 ¢ -fl uoroacetophenones were chosen as model sub-strates. As mentioned above, the confi guration of the resulting alcohols were similar to that from
acetophenone. In stems of broccoli, yields were higher than that of acetophenone, while ee for 4 ¢ -fl uoroacetophenone (78%) were similar to acetophe-none (79%) but higher for 4 ¢ -bromoacetophenone (89%). The ee obtained using the germinated seed (Table III) were notably higher for 4¢ -bromo and 4¢ -fl uoroacetophenones (94%, 92% respectively) than acetophenone (89%). This phenomenon was
Bio
cata
l Bio
tran
sfor
mat
ion
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f G
lasg
ow o
n 09
/05/
13Fo
r pe
rson
al u
se o
nly.
334 M. Mohammadi et al.
also reported for 4 ¢ -chloroacetophenone reduction by different plant tissues (Bruni et al. 2006) indicat-ing that halogenated aromatic ketones are better substrates in plant cells than simple aromatic ketones. The halogen (here bromide and fl uoride) also enlarges the difference between the size of two sub-stituents of the carbonyl, and therefore, improves the enantioselectivity of this reaction. Also, the increase in ee values observed by using germinated seeds was greater for 4 ¢ -fl uoroacetophenone ( � 14% increase) than for 4 ¢ -bromoacetophenone ( � 5% increase).
Reduction of β -ketoesters to chiral alcohols had been studied extensively using several plant cell biocatalysts (carrot, apple, cucumber, onion, potato and radish) and baker’ s yeast (Yang & Yao 2004). In this current study, methyl-4-chloro-3-oxobu-tanoate and benzyl-3-oxobutanoate were chosen as model substrates for examining β -ketoester asym-metric reduction. Results are presented in Tables II and III. An anti-Prelog selectivity was observed for
methyl-4-chloro-3-oxobutanoate both with plant stems and germinated seed (76%, 81% ee respec-tively). In comparison benzyl-3-oxobutanoate afforded a Prelog-like selectivity (82%, 81% ee respectively). The yields of reactions were higher with plant stems (74%, 69%) compared with germinated plant (56%, 52%). Yang and co-workers reported an S -confi gura-tion for the reduced product of ethyl 4-chloroacetoac-etate using several plant tissues among which the best results were obtained with carrot (45.5% yield, 91.0% ee ) (Yang et al. 2008) Blanchard and Van De Weghe (2006) also investigated reduction of 4-chloro and 4-trifl uoromethyl acetoacetate. The Prelog rule was obeyed by 4-chloroacetoacetate, as seen with carrot and baker ' s yeast as biocatalyst which resulted in the S -form of the alcohol (90%, 55% ee respectively) but 4-trifl uoromethyl acetoacetate showed an anti-Prelog selectivity ( R -confi guration) with 78%, 62% ee respec-tively. With these substrates, germinated seeds did not improve the ee values signifi cantly.
Table III. Reduction of ketones with germinated seed of B. oleracea variety italic.
Entry Compounds Products Confi g.a Yield(%)b
ee%(compared with brassica
stems)b
Time of conversion
(day)
1 1-(pyridin-4-yl)ethanone 1-(pyridin-4-yl)ethanol R 78 99(� 5) 12 1-(pyridin-3-yl)ethanone 1-(pyridin-3-yl)ethanol R 94 98(� 6) 1
3 1-(pyridin-2-yl)ethanone 1-(pyridin-2-yl)ethanol R 88 97(� 5) 14 1-phenylethanol 1-phenylethanol R 71 89(� 10) 15 3,4- dihydronaphthalen-
1(2H)-one1,2,3,4-tetrahydronaphthalen-1-ol S 75 91(�1) 1
6 1-p-tolylethanone 1-p-tolylethanol R 57 90(� 8) 17 4-acetylbenzonitrile 4-(1-hydroxyethyl)benzonitrile R 81 97(� 6) 18 1-(4-(trifl uoromethyl)
phenyl)ethanone1-(4-(trifl uoromethyl)phenyl)
ethanolR 78 95(� 25) 1
9 2,2,2-trifl uoro-1-phenylethanone
2,2,2-trifl uoro-1-phenylethanol S 89 93(�1) 1
10 1-(4-nitrophenyl)ethanone
1-(4-nitrophenyl)ethanol R 64 89(� 4) 1
11 1-mesitylethanone 1-mesitylethanol R 8 99(� 3) 112 1-(4-bromophenyl)
ethanone1-(4-bromophenyl)ethanol R 60 94(� 5) 1
13 1-(4-fl uorophenyl)ethanone
1-(4-fl uorophenyl)ethanol R 85 92(� 14) 1
14 1-(2-bromo-4-cyclohexylphenyl)ethanone
1-(2-bromo-4-cyclohexylphenyl)ethanol
S 37 90(�4) 1
15 2-chloro-1-(4-fl uorophenyl)ethanone
2-chloro-1-(4-fl uorophenyl)ethanol
S 61 87(� 18) 1
16 2-bromo-1-(4-bromophenyl)ethanone
2-bromo-1-(4-bromophenyl)ethanol
S 72 88(� 10) 1
17 1-(thiophen-2-yl)ethanone
1-(thiophen-2-yl)ethanol R 11 95(� 4) 1
18 1-(furan-2-yl)ethanone 1-(furan-2-yl)ethanol R 12 93(�2) 119 benzyl 3-oxobutanoate benzyl 3-hydroxy butanoate S 52 81(�1) 120 methyl 4-chloro-3-
oxobutanoatemethyl 4-chloro-3-hydroxy
butanoateR 56 81(�5) 1
aThe confi gurations were assigned by comparison of the sign of the specifi c rotation of each alcohol with those of the reported molecules.bConversion and ee were determined by GC and HPLC analysis of crude extract.
Bio
cata
l Bio
tran
sfor
mat
ion
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f G
lasg
ow o
n 09
/05/
13Fo
r pe
rson
al u
se o
nly.
Enantioselective bioreduction of ketones by Brassica oleracea 335
As part of a wider evaluation process, other aro-matic ketones (pyridine, furan and thiofen derivatives) were investigated. All of these compounds afforded R -alcohols. According to the results pyridine deriva-tives yielded excellent ee for brassica stems (93 – 96%) and germinated seed (97 – 99%). Chemical yields for furan (10 – 12%) and thiofen (11 – 14%) were lower than pyridine derivatives (88 – 100%) but ee were com-parable with those of pyridine derivatives (91 – 95%).
To demonstrate the potential for large scale prep-aration using this bioreduction protocol we con-ducted a scaled-up reduction for the best substrate with 100% chemical yield i.e. 2-acetylpyridine (0.5 g) using stems of B. oleracea (200 g). The ee and isolated yield of the product were 90% and 76% respectively.
Conclusion
We have reported a straightforward and mild eco-friendly process for the synthesis of chiral alcohols using B. oleracea variety italica as biocatalyst with excellent yields and high optical purity. The key features of the new process are the use of a low-cost enzymatic system, the ease of work-up, high enantioselectivity and mild conditions (water as reaction media and room temperature). Aromatic, halogen-containing aromatic ketones and β -ke-toesters were effi ciently reduced to their corre-sponding alcohols with the best results being obtained for pyridine derivatives, especially 3-acetylpyridine. Both R -and S -alcohols could be obtained with these asymmetric reactions. Germi-nated seed gave faster reactions and higher enanti-oselectivities than B. oleracea ’ s stems with the majority of substrates examined.
Acknowledgment
The authors are thankful to Iran National Science Foundation (INSF), Tehran, Iran.
Declaration of interests: This work was supported by Iran National Science Foundation (INSF), Tehran, Iran. The authors report no confl icts of interest. The authors alone are responsible for the content and writ-ing of the paper.
References
Baladassare F, Bertoni G, Chiappe C, Marioni F. 2000. Prepara-tive synthesis of chiral alcohols by enantioselective reduction with Daucus carota root as biocatalyst. J Mol Catal B: Enzym 11:55 – 56.
Bandini M, Bottoni A, Cozzi PG, Miscione GP, Monari M, Pierciaccante R, Umani-Ronchi A. 2006. Chiral C 2-Boron-
Bis(oxazolines) in Asymmetric Catalysis – A Theoretical Study of the Catalyzed Enantioselective Reduction of Ketones Pro-moted by Catecholborane. Eur J Org Chem 4596 – 4608.
Blanchard N, Van De Weghe P. 2006. Daucus carota L. mediated bioreduction of prochiral ketones. Org Biomol Chem 4:2348 – 2353.
Bruni R, Fantin G, Maietti S, Medici A, Pedrini P, Sacchetti G. 2006. Plants-mediated reduction in the synthesis of homochiral secondary alcohols. Tetrahed Asymm 17:2287 – 2291.
Chartrain M, Greasham R, Moore J, Reider P, Robinson D, Buckland B. 2001. Asymmetric bioreductions: application to the synthesis of pharmaceuticals. J Mol Catal B Enzym 11:503 – 512.
Curt W, Bradshaw HF, Gwo-Jenn S, Chi-Huey W. 1992. A Pseudomonas sp. alcohol dehydrogenase with broad substrate specifi city and unusual stereospecifi city for organic synthesis. J Org Chem 57:1526 – 1532.
Dau ß mann T, Rosen TC, Dunkelmann P. 2006. Oxidoreductases and hydroxynitrilase lyases: complementary enzymatic tech-nologies for chiral alcohols. Eng Life Sci 6:125 – 129.
Faber K. 1997. Biotranformations in organic chemistry. Berlin: Springer.
Hamada H, Nakamura N, Ito S, Kawabe S, Funamoto T. 1988. Enantioselectivity in the reduction of α -dicarvelone by cell sus-pension cultures of Nicotiana tabacum . Phytochemistry 27:3807 – 3808.
Hirata T, Hamada T, Aoki T, Suga T. 1982. Stereoselectivity of the reduction of carvone and dihydrocarvone by suspension cells of Nicotiana tabacum . Phytochem 21:2209 – 2212.
Huisman GW, Liang J, Krebber A. 2010. Practical chiral alcohol manufacture using ketoreductases. Curr Opin Chem Biol 14:122 – 129.
Kroutil W, Mang H, Edegger K, Faber K. 2004. Stereoselective bioreduction of bulky-bulky ketones by a novel ADH from Ralstonia sp. Curr Opin Chem Biol 8:120 – 126.
Kumaraswamy G, Ramesh S. 2003. Soaked Phaseolus aureus L: an effi cient biocatalyst for asymmetric reduction of prochiral aromatic ketones. Green Chem 5:306 – 308.
Liu S, Wolf C. 2007. Chiral amplifi cation based on enantioselec-tive dual-phase distribution of a scalemic bisoxazolidine catalyst. Org Lett 9:2965 – 2968.
Matharu DS, Morris DJ, Clarkson GJ, Wills M. 2006. An out-standing catalyst for asymmetric transfer hydrogenation in aqueous solution and formicacid/triethylamine. Chem Com-mun 30:3232 – 3234.
Matsuo K, Kawabe SI, Tokuda Y, Eguchi T, Yamanaka R, Nakamura K. 2008. Asymmetric reduction of ketones with a germinated plant. Tetrahed Asymm 19:157 – 159.
Moore JC, Pollard DJ, Kosjek B, Devine PN. 2007. Advances in the enzymatic reduction of ketones. Acc Chem Res 40:1412 – 1419.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473 – 497.
Nagaoka H. 2004. Ability of different biomaterials to enantiose-lectively catalyze oxidation and reduction reactions. Biotechnol Prog 20:128 – 133.
Nakamura K, Takenaka K, Fujii M, Ida Y. 2002. Asymmetric syn-thesis of both enantiomers of secondary alcohols by reduction with a single microbe. Tetrahed Lett 43:3629 – 3631.
Nakamura K, Yamanaka R, Matsuda T. 2003. Recent develop-ments in asymmetric reduction of ketones with biocatalysts. Tetrahed Asymm 14:2659 – 2681.
Naoshima Y, Akakabe Y. 1991. Biotransformation of aromatic ketones with cell cultures of carrot, tobacco and Gardenia. Phytochem 30:3595 – 3597.
Noyori R. 1994. Asymmetric catalysis in organic synthesis. New York: Wiley.
Bio
cata
l Bio
tran
sfor
mat
ion
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f G
lasg
ow o
n 09
/05/
13Fo
r pe
rson
al u
se o
nly.
336 M. Mohammadi et al.
Savile CK, Kazlauskas RJ. 2005. How substrate solvation contrib-utes to the enantioselectivity of subtilisin toward secondary alcohols. J Am Chem Soc 127:12228 – 12229.
Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B. 2001. Industrial biocatalysis today and tomorrow. Nature 409:258 – 268.
Shimizu H, Igarashi D, Kuriyama W, Yusa Y, Sayo N, Saito T. 2007. Asymmetric hydrogenation of aryl ketones mediated by a cop-per catalyst. Org Lett 9:1655 – 1657.
Strauss UT, Felfer U, Faber K. 1999. Biocatalytic transforma-tion of racemates into chiral building blocks in 100% chemi-cal yield and 100% enantiomeric excess. Tetrahed Asymm 10:107 – 117.
Su á rez-Franco G, Hern á ndez-Quiroz T, Navarro-Ocana A, Oliart-Ros RM, Valerio-Alfaro G. 2010. Plants as a green alter-native for alcohol preparation from aromatic aldehydes. Biotech Bioproc Eng 15:441 – 445.
Villa R, Molinari F, Levati M, Aragozzini F. 1998. Stereoselective reduction of ketones by plant cell cultures. Biotechnol Lett 20:1105 – 1108.
Ward OP, Young CS. 1990. Reductive biotransformations of organic compounds by cells or enzymes of yeast. Enzym Micro-biol Technol 12:482 – 493.
Yadav JS, Nanda S, Thirupathi Reddy P, Bhaskar Rao A. 2002. Effi cient enantioselective reduction of ketones with Daucus carota root. J Org Chem 67:3900 – 3903.
Yang W, Xu JH, Xie Y, Xu Y, Zhao G, Lin GQ. 2006. Asymmetric reduction of ketones by employing Rhodotorula sp. AS2.2241 and synthesis of the b-blocker (R)-nifenalol. Tetrahed Asymm 17:1769 – 1774.
Yang Z, Yao S. 2004. Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl 4-chloro-3-hydroxybutanoate by yeast cells in aqueous phase. Chin J Catal 25:434 – 438.
Yang ZH, Zeng R, Yang G, Wang Y, Li LZ, Lv ZS, Yao M, Lai B. 2008. Asymmetric reduction of prochiral ketones to chiral alco-hols catalyzed by plants tissue. J Ind Microbiol Biotechnol 35:1047 – 105.
Yousefi M, Mohammadi M, Habibi Z, Shafi ee A. 2010. Biotrans-formation of progesterone by Acremonium chrysogenum and Absidia griseolla var. igachii. Biocatal Biotransform 28:254 – 258.
Bio
cata
l Bio
tran
sfor
mat
ion
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f G
lasg
ow o
n 09
/05/
13Fo
r pe
rson
al u
se o
nly.