Enzyme from Daucus carota root catalyzed asymmetric cross aldol reaction

Article abstract of DOI:10.1016/j.tetlet.2016.08.056

A novel enzyme was isolated from specific variety of Daucus carota root catalyzed asymmetric cross aldol reaction of aromatic aldehydes with acetone to afford cross aldol products with excellent yield (up to 95%) and enantioselectivity (up to 99%). A plau

Full text of DOI:10.1016/j.tetlet.2016.08.056

Accepted Manuscript
Enzyme from Daucus carota root catalyzed asymmetric cross aldol reaction
Chiranjit Acharya, Madhumita Mandal, Trina Dutta, Anil Kumar Ghosh,
Parasuraman Jaisankar
PII:
S0040-4039(16)31083-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.08.056
TETL 48027
Reference:
Tetrahedron Letters
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Received Date:
Revised Date:
Accepted Date:
22 July 2016
13 August 2016
17 August 2016
Please cite this article as: Acharya, C., Mandal, M., Dutta, T., Ghosh, A.K., Jaisankar, P., Enzyme from Daucus
carota root catalyzed asymmetric cross aldol reaction, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/
j.tetlet.2016.08.056
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Tetrahedron Letters
Enzyme from Daucus carota root catalyzed asymmetric cross aldol reaction
Chiranjit Acharya,a Madhumita Mandal,a Trina Dutta,b Anil Kumar Ghoshb and Parasuraman Jaisankara,*
aLaboratory of Catalysis and Chemical Biology, Department of Organic and Medicinal Chemistry, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C.
Mullick Road, Jadavpur, Kolkata 700 032, India.
bDrug Development, Diagnostics & Biotechnology Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032,
India.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel enzyme isolated from specific variety of Daucus carota root catalyzed asymmetric
cross aldol reaction of aromatic aldehydes with acetone to afford cross aldol products with
excellent yield (up to 95%) and enantioselectivity (up to 99%). A plausible mechanism of the
reaction has also been proposed.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Daucus carota,
asymmetric cross aldol reaction,
catalysis,
enantioselectivity,
enzyme
Enantioselective organic transformations remain one of the most exciting and challenging tasks in the realm of organic
chemistry.1 Bio-catalytic approach which mainly focuses on harnessing enzyme catalytic activity to develop novel methodology
and alternative chemical transformations, is a new arena in organic synthesis, attracting much attention of the researchers and
expanding rapidly in recent years.2 Bio-catalytic systems have got advantages of economically viable, ecologically beneficial and
more sustainable than current chemical technologies, due to their inherent advantages of higher selectivity, milder conditions and
comparatively cheaper resources.3 The only limiting factor of enzymes is their specificity towards the substrate.4 Therefore,
searching for new enzymes from easily available natural sources such as edible vegetables is an important task in the field of
asymmetric organic synthesis.
The aldol reaction is one of the most powerful tools for the construction of complex polyol architectures and the best known C–C
bond forming synthetic protocol ever studied in organic synthesis.5 In general, most of the methodologies which are available for
asymmetric aldol reactions fall into one of the following categories: (a) the chiral auxiliary assisted reactions based on the use of
stoichiometric quantities of chiral appendages;6 (b) chiral Lewis acid-catalyzed Mukaiyama type7
and chiral Lewis-base catalyzed reactions;8 and (c) enzymes9 and antibodies10 catalyzed reactions. Recently, there have been reports
demonstrating the utilization of various enzymes in cross aldol
*Corresponding author. Tel.: +91-33-24995790; fax: +91-33-24735197;
E-mail: jaisankar@iicb.res.in
reactions between aldehydes and ketones in aqueous media.11 Notably, Yu et al reported the enzymatic system consisting of either
porcine pancreatic lipase (PPL) or pepsin as catalyst for asymmetric cross aldol reaction of nitrobenzaldehyde and acetone to afford
the corresponding cross aldol product with up to 22% yield and 43.6% ee.12 Other enzymes such as aldolase13 and proteinase14 were
also used as catalysts for asymmetric cross aldol reaction in aqueous media, but the efficacy of these enzymes to induce
enantioselectivity is very poor.
Daucus carota root is a common vegetable with rich nutritious value. There are only a couple of reports available in the literature
on catalytic activity of its root for asymmetric reactions such as reduction of ketones to optically active secondary alcohols with
high yield
and excellent ee (Scheme 1).15, 16
OH
O
Daucus carota root
CH3
CH3
R
R
Yield: up to 95%,
ee: up to 98%
X
X
hydroxyethyl substituted
benzene/pyridine
Substituted
phenyl/pyridyl
methyl ketone
R = OMe, Br, Cl, F,
NO2, OH, H, Me
X = C, N

3
Scheme 1 Reported Daucus carota root catalyzed asymmetric reduction of ketones to chiral alcohols.
However, the use of enzymes isolated from Daucus carota root for asymmetry aldol condensation is unexplored. Thus, we wish
to disclose for the first time the direct asymmetric cross aldol reaction of aromatic aldehydes with acetone to afford -hydroxy
ketones (Scheme 2). Details of the study is presented in this communication.
O
H
HO
enzyme /
Phosphate buffer
R4
R3
R4
R3
CHO
O
S
CH3
+
H3C
CH3
R1
R1
of pH 7.5,
28 oC, 8-10 h
R2
R2
3a-i
R1 = H, NO2, CF3; R2 = NO2, H, CN
R3 = H, NO2, CF3, OMe; R4 = H, Cl, OMe
1a-i
2
Scheme 2 Present work on asymmetric cross aldol reaction catalyzed by
enzyme isolated from Daucus carota root.
Initially, the cross aldol reaction was carried out by stirring 3-nitro benzaldehyde (1a, 1.0 mmol) and acetone (2, 73 µL, 1 mmol)
with freshly cut Daucus carota root (5.0 g) in water for 72 hr and the desired product, (-)-(S)-4-hydroxy-4-(3-nitrophenyl)butan-2-
one (3a) was obtained in good yield (60%) with moderate enantioselectivity (62%). Encouraged by this initial result, we thought
that the isolation of pure enzyme from Daucus carota root could improve the yield and enatiopurity of the corresponding cross aldol
adduct. The isolation, purification and characterization of enzyme from Daucus carota roots of variety 1 (Fig 1) and its activity
towards cross aldol reaction is described in detail in the supplementary information. Surprisingly roots of variety 2 (Fig 1) failed to
catalyze this reaction.
Variety 1(active enzyme)
Variety 2
Fig. 1 Available variants of Daucus carota roots.
Optimization of cross aldol reaction catalyzed by purified enzyme
Purified enzyme was used as catalyst for asymmetric cross aldol reactions under various reaction conditions. In order to optimize
the reaction condition, first we tested the effect of pH on catalytic activity of enzyme catalyst in asymmetric cross aldol reaction
between 3-nitrobenzaldehyde (1a) and acetone (2) in phosphate
buffer of varying pH from mild basic (pH 8.0) to mild acidic (pH
6.0) condition (Table 1). Eventually we found the optimum pH
for this reaction is 7.5 which afforded the product 3a in 95%
yield and S-selective enantiomeric excess of 98% (Table 1, entry
2). However, moving from basic to mild acidic condition reduces
the yield as well as enantioselectivity of 3a.
After achieving an optimum pH for the asymmetric cross aldol
reaction, we focused our attention on the effect of temperature on
catalytic activity of the enzyme. There was a strong correlation
of enzyme activity with variation of temperature on the reaction.
Increase of temperature from 28 ⁰C to 50 ⁰C and above resulted in a reduced yield of 3a, which could probably be due to
denaturation of enzyme at higher temperatures, although deterioration of enantioselectivity at elevated temperatures was negligible.
The optimum temperature for this enzyme catalysis was found to be 28 °C (Fig. 2).
Table 1 Effect of pH on catalytic activity of enzyme on the cross aldol reaction of substrate 1a.
Time
(h)
% Isolated
Yield
%
eec
Entrya pH

4
1
8.0
8
95
98
2
3
4
7.5
7.0
6.5
8
95
75
60
98
95
72
8b
8 b
% of ee‡
(Absolute
configuration)
8 b
40
65
% of
Yield†
(Product)
5
6.0
Time
(h)
Entry R1
R2
R3
R4
1
H
NO2
H
H
8
95 (3a)
98(S)17
a Reaction condition: A mixture of 1a (1 mmol), acetone (2, 73 µL, 1 mmol) and
enzyme (10 µL of concentration 0.07 mg/mL) was stirred at 28 ⁰C in buffer
solution of different pH. b Further increase of reaction time has no effect on yield
and ee of the products. c The ee was determined by chiral HPLC analysis using
DAICEL CHIRALPAK® AS-H column.
Fig. 2 Effect of temperature on catalytic activity of enzyme.
Reaction conditions: 1a (1 mmol), acetone (2, 73 µL, 1 mmol) and 10 µL of pre
incubated enzyme at different temperatures in buffer solution stirred for 8 hr.
Yield was measured by column chromatography. ee is the value for S-isomer
determined by DAICEL CHIRALPAK® AS-H column (5m, 4.6 mm x 250
mm).
Various other substrates have been subjected to cross aldol reactions
under optimized reaction conditions and the results are summarized
in Table 2. Some of nitro substituted benzaldehydes responded to
these enzymatic cross aldol reactions with significant enantiomeric excess and yield, whereas other substituted benzaldehydes
(Table 2, entries e-i) were unable to produce any enantiomeric excess although the products were formed in good yields.
Similarly, we checked the catalytic activity of this enzyme in the cross aldol reaction of different heterocyclic aldehydes (thiophene-
2-carboxaldehyde, indole-3-carboxaldehyde, pyridine-3-carboxaldehyde and furan-2-carboxaldehyde) with acetone (2) and 3-
nitrobenzaldehyde (1a) with other ketone and active methylene compounds (acetophenone, acetyl acetone and diethylmalonate)
instead of acetone under the optimized reaction conditions. But none of these responded to this reaction to yield the product. This
may be due to the lack of binding of substrates with the amino acids present in the active site of enzyme. These results indicate that
the enzyme isolated from Daucus carota root is highly substrate specific towards cross aldol reaction.
O
H
Table 2 Substrate scope for asymmetric cross aldol reactions
to afford of 4-hydroxy-4-aryl-butan-2-one derivatives 3a-i.
HO
enzyme /
Phosphate buffer
R4
CHO
R4
O
CH3
S
+
H3C
CH3
R3
R1
R3
R1
of pH 7.5,
28 oC, 8-10 h
R2
R2
3a-i
1a-i
2

5
†Isolated yield. ‡ee was determined by HPLC analysis using DAICEL
CHIRALPAK® AS-H column (5m, 4.6 mm x 250 mm) and absolute
2
3
4
5
6
7
8
9
NO2
H
H
H
H
NO2
H
H
H
8
8
92 (3b)
90 (3c)
89 (3d)
85 (3e)
88 (3f)
84 (3g)
79 (3h)
81 (3i)
91(S)17
95(S)17
configuration was assigned by comparison with literature reports 17, 18
.
O
HO
H
NO2
NO2
NO2
CF3
NO2
H
H
Cl 10
99(S)18
NO2
O
O
CH3
n
s
A
H
O
1a
H
H
CF3
NO2
H
H
H
H
8
8
Racemic
Racemic
Racemic
Racemic
Racemic
NO2 3a
H
CH3
s
y
O
2
NH2
H2O
L
CH3
H
10
H
OMe OMe 10
10
Lys
H3C
Asn
B
A
Lys
NH
CH3
Asn
CN
H
H
O
NH
H
O
O
H
O
H3C
O
H
O
H3C
H2C
L
H
N
y
s
H
NO2
NO2
H
A
O
H
O
s
n
O
O2N
Scheme 3 Plausible mechanism of cross aldol reaction catalyzed by the enzyme isolated from Daucus carota roots.
A partial sequence of thirty seven amino acid residue (MERVYIRMCKNINCDSGICEIANEAPYPVKVPQKNEL) with
molecular weight 4.3 KDa was derived from MALDI TOF MS/MS analysis of isolated enzyme which was digested with trypsin.
This sequence was matched with NCBI database using tBLASTn (Protein-nucleotide Basic Local Alignment Tool). It revealed
similarity with CSCP mRNA of proteases obtained from other plant sources including Daucus carota. The amino acid analysis
revealed that the enzyme is rich with cysteine (18.8%) among other amino acids such as tryptophan, phenylalanine, tyrosine etc.
(see SI).
It is known that Lysine and Asparagine (in the active site of an enzyme) act as ketone donor and aldehyde acceptor respectively.11, 19
Therefore, a plausible mechanism of this reaction has been depicted in scheme 3. Accordingly, Lysine (Lys) covalently bound with
acetone (2) to form Schiff base intermediate A which reacted with 3-nitrobenzaldehyde (1a) activated by Asparagine (Asn) through
H-bonding interaction to form an enzyme-product complex B; subsequent hydrolysis affords the cross aldol product 3a (Scheme 3).
1
All the synthesized compounds have been characterised by H-NMR, 13C-NMR, ESI-HRMS and FT-IR spectroscopic analysis
(see SI).
In conclusion, we have successfully developed a simple eco-friendly protocol to carry out asymmetric cross aldol reaction with
excellent yield and ee by using an enzyme isolated from specific variety of Daucus carota root as biocatalyst. The specificity of
enzyme towards substrates may be exploited for making important natural product precursors. Using this enzyme catalyst for novel
organic transformations is being investigated in this laboratory.
Acknowledgement
This project was funded by Council of Scientific and Industrial Research (CSIR), New Delhi, India in the form of Network Project
(ORIGIN, CSC-0108). Thanks are also due to Mr. S. Samaddar for IR analyses.
Typical procedure for the synthesis of chiral 4-hydroxy-4-(substituted phenyl)-butan-2-one:
3-Nitrobenzaldehyde (1a; 100 mg, 0.66 mmol) was taken in 1 mL of phosphate buffer of pH 7.5 and treated with 73 µL (1
mmol) of acetone (2) and 10 µL (0.7 µg) of enzyme (isolated from Daucus carota roots) of concentration 0.07 mg/mL. Then the
whole reaction mixture was stirred at 28 ⁰C for 8 hr. The reaction was monitored by TLC using 20% ethyl acetate in petroleum
ether as mobile phase. The reaction mass was diluted with 25 mL of ethyl acetate and washed with 25 mL of water (twice) followed
by brine (25 mL). The organic layer was passed through anhyd. Na2SO4 and the solvent was removed in rotary evaporator to obtain
a yellow sticky mass (154 mg). The product was purified by column chromatography using 60-120 mesh silica gel. It was eluted
with 10-12 % ethyl acetate in petroleum ether. The sticky mass was washed with diethyl ether to get an yellow solid (131 mg, yield
95%). Enantiomeric excess was determined by Chiral HPLC using DAICEL CHIRALPAK® AS-H column (5m, 4.6 mm x 250
mm) with the solvent system Hexane: Isopropanol = 90:10 with 1 mL/min flow rate at 20 ⁰C.

6
Supplementary data
Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tetlet.2016. xxxxx.
These data include spectroscopic data of compound 3a-i, copies of 1H, 13C NMR spectra, MALDI-TOF MS of the isolated enzyme
from Daucus carota root and HPLC spectra of compounds 3a-d, HPGFLC and native PAGE electrophoresis of the enzyme.
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Graphical Abstract
Enzyme from Daucus carota root catalyzed
Leave this area blank for abstract info.
asymmetric cross aldol reaction
Chiranjit Acharya,a Madhumita Mandal,a Trina Dutta,b Anil Kumar Ghoshb and Parasuraman Jaisankara,*

Tetrahedron Letters
 Asymmetric cross aldol reaction by an
8
Highlights
enzyme from Daucus carota root is
reported.
 Variation of temperature, pH and
substrates are covered.
 Active enzyme is present in specific
variety of Daucus carota.
 Covered a plausible mechanism of this
enzyme catalysis.