Daucus carota root enzyme catalyzed Henry reaction: A green approach

Article abstract of DOI:10.1016/j.tetlet.2018.01.009

Enzyme from Daucus carota root catalyzed Henry reaction of substituted benzaldehydes and nitromethane in phosphate buffer of pH 7 at 28 °C to afford β-nitroalcohols in excellent yields (up to 94%).

Full text of DOI:10.1016/j.tetlet.2018.01.009

Accepted Manuscript
Daucus carota root enzyme catalyzed Henry reaction: A green approach
Chiranjit Acharya, Anushree Achari, Parasuraman Jaisankar
PII:
S0040-4039(18)30021-2
https://doi.org/10.1016/j.tetlet.2018.01.009
TETL 49596
DOI:
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
4 December 2017
30 December 2017
4 January 2018
Please cite this article as: Acharya, C., Achari, A., Jaisankar, P., Daucus carota root enzyme catalyzed Henry
reaction: A green approach, Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.01.009
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Daucus carota root enzyme catalyzed Henry
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reaction: A green approach
Chiranjit Acharya, Anushree Achari and Parasuraman Jaisankar*

1
Tetrahedron Letters
Daucus carota root enzyme catalyzed Henry reaction: A green approach
Chiranjit Acharya, Anushree Achari and Parasuraman Jaisankar*
Laboratory 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
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Enzyme from Daucus carota root catalyzed Henry reaction of substituted benzaldehydes and
nitromethane in phosphate buffer of pH 7 at 28°C to afford -nitroalcohols in excellent yields
(up to 94%).
2009 Elsevier Ltd. All rights reserved.
Available online
Keywords:
Daucus carota
enzyme
Henry reaction
nitromethane
-nitroalcohol
The hidden art of organic synthesis is the carbon  carbon (C
 C) bond forming reactions, the fundamental tool for the
synthesis of complex molecular architecture.1-4 A number of
methodologies have been reported over the years for C  C
bond formation.5-9 Among various other protocols aldol reactions
are the most common and well established methods.10 Besides
this, Henry reaction of aldehydes with nitroalkanes to afford -
nitroalcohols which had been reported in 1895, has attracted the
attention of medicinal chemists due to its applications in the
synthesis of pharmaceutical agents,11 precursors of natural
products11 and bioactive compounds having medicinal
importance (Fig 1).12
The use of base in Henry reactions resulted in side products
along with the desired -nitroalcohol.13 Although, this
shortcoming has been addressed by the use of metal and organo
catalysts in place of conventional bases,14 the problem of toxicity
and high cost is a deterrent. Therefore, further development was
needed to provide green and efficient methodologies.
As a result of this development, enzyme promiscuity has been
introduced.15 But only a few enzymes which are both costly and
substrate specific, were reported to catalyze Henry reactions.16-17
Hence, the search for new enzymes provoked the development of
enzyme catalysis which is an emerging field of modern organic
synthesis.
Fig1. -Aminoalcohols having profound medicinal importance.
 -nitroalcohols from nitrobenzaldehydes and hydrogencyanide
(HCN) or nitromethane with high substrate specificity.15 The
primary role of HNLs is to abstract the acidic proton from HCN
(pKa=9.2) and nitromethane (pKa=10.21). There were few reports
available in the literature on Daucus carota root mediated
reduction of optically active alcohols,18 until our recent report on
its use as catalyst for the asymmetric cross aldol reaction of
nitrobenzaldehyde and acetone.19 In continuation of our prior
work19 we have deliberately used Daucus carota root or its
enzyme in the promiscuous mixture of substituted
Previously, Griengl et. al. reported hydroxynitrile lyases (HNLs,
protease enzyme) catalyzed synthesis of -hydroxynitriles and
*Corresponding author. Tel.: +91-33-24995790; Fax: +91-33-
24735197. E-mail: jaisankar@iicb.res.in

2
nitrobenzaldehyde and nitromethane in aqueous medium to
afford -nitroalcohol in excellent yields (Scheme 1).
Methanol:
14 buffer of pH 7
(1:1)
No
28
24
reaction
See experimental section for general reaction procedure. Reaction was
#
ф
placed without Daucus carota root enzyme. Reaction was placed with
homogenized carrot root (5g). † The reaction was performed at 28 C as the
enzyme was reported to perform best at that temperature.19  Further increase
€
of reaction time has no effect on the yield of the product.
Butylmethylether.
t-
Scheme 1. Daucus carota root catalyzed Henry reaction.
The optimized condition of the Daucus carota root enzyme
catalyzed Henry reaction of 2-nitrobenzaldehyde and
nitromethane to afford -nitroalcohol in excellent yield (93%)
was found to be phosphate buffer of pH 7 at 28 C in 8 hr (Table
1, entry 5). Organic solvents did not have any positive influence
in this reaction, possibly, due to denaturation of the enzyme
occurred in organic solvents. A number of -nitroalcohols have
been synthesized under the optimized conditions and the results
are summarized in table 2. All the synthesized -nitroalcohols,
Initially the reaction was performed by stirring 2-
nitrobenzaldehyde (1a, 1 mmol) and nitromethane (2, 1 mmol)
in a suspension of freshly cut Daucus carota root (5.0 g) in
aqueous medium for 24 hr, which resulted in the formation of -
nitroalcohol (3a) with 55% of isolated yield (Table 1, entry 2).
Encouraged by this initial result we then performed the same
reaction with purified enzyme (10L) from Daucus carota root
(see SI) and it afforded the desired -nitroalcohol with 70% of
isolated yield in 15 hr. Subsequently we went for optimizing the
reaction protocol by changing reaction parameters (Table 1). No
reaction took place in the absence of enzyme.
1
which were racemic in nature have been characterized by H-
NMR, 13C-NMR, ESI-HRMS and FT-IR spectroscopic
techniques (see SI).
Table 2. Substrate scope of Daucus carota root enzyme catalyzed
Henry reactions of substituted benzaldehyde and nitromethane.
Table 1. Optimization of parameters for the Daucus carota root
enzyme catalyzed Henry reaction
R5 OH
R5
OH
Daucus carota root
Daucus carota root
R4
R3
NO2
R4
R3
CHO
NO2
CHO
+
NO2
enzyme
enzyme
+
H3C NO2
H3C NO2
Phosphate buffer
of pH 7, 28 oC,
8-10 h
Solvent, 28 C
R1
R1
NO2
R2
R2
1a
3a
2
2
3a-i
1a-i
Temperature†
(°C)
Isolated
yield (%)
Entry Solvent
Time (h)
Isolated
yield
(%)
Time
(h)
Entry R1
R2
R3
R4 R5
Product
No
reaction
1#
2ф Water
Water
28
24
1
2
3
4
5
6
7
8
9
NO2
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
8
8
3a16
3b16
3c16
3d
3e20
3f21
3g
93
94
92
81
90
89
87
91
81
28
28
24
15
55
70
NO2
H
3
4
Water
H
NO2
H
8
Phosphate
buffer of pH 8
28
8
90
H
OMe OMe
NO2
H
10
9
Phosphate
buffer of pH
7
NO2
H
H
H
H
H
H
H
OMe
CF3
NO2
H
5
28
8
93
H
10
8
NO2
NO2
CF3
H
Phosphate
buffer of pH 6
6
7
8
9
28
28
28
28
28
28
8
82
60
H
8
3h
3i21
Phosphate
buffer of pH 5
H
10
8
It was observed that there was almost no electronic (viz. entry 3
vs. 6) influence of aryl substituents on the yields of the products.
As one would expect, the high specificity of any enzyme in
catalytic activity was corroborated when nitroethane and
nitropropane were used in order to broaden the scope of the
reaction, failed to undergo this reaction. We also checked the
reactivity of aliphatic aldehydes with nitromethane in this
promiscuous reaction but disappointingly, no reaction took place.
The reusability of the Daucus carota root enzyme was
investigated in the reaction of 1a and 2 to yield 3a which was
extracted in diethylether followed by subsequent addition of the
substrates (1a and 2) in the aqueous layer which contains enzyme
to catalyze next batches. It was perceived that the enzyme could
catalyze the Henry reaction of 1a and 2 up to five cycles to afford
3a, though with progressively lower isolated yields (Table 3).
Diethylether
(DEE)
No
reaction
8
DEE: buffer of
pH 7 (1:1)
12
8
50
No
reaction
10 TBME€
TBME: buffer
11
12
60
of pH 7 (1:1)
Acetonitrile:
No
reaction
12 buffer of pH 7
(1:1)
28
28
24
24
Ethanol:
13 buffer of pH 7
(1:1)
No
reaction
Earlier, it was reported that the lysine residue of HNLs acted as
base to abstract a methylene proton from nitromethane to favor

3
the reaction with nitrobenzaldehyde.15b Recently, we disclosed
that the Daucus carota root enzyme also contains Lysine residue
which was proposed to favor the asymmetric cross aldol reaction
of nitrobenzaldehyde and acetone.19 Therefore, we could
visualize that the catalytic site of the enzyme19 which may
contain lysine residue could activate the methylene proton of
nitromethane (2) to favor Henry reaction with 2-
nitrobenzaldehyde (1a), that resulted in the formation of -
nitroalcohol (3a) (Scheme 2). The non-stereospecificity of the
products (3a-i) could be due to lack of binding of the substrate
(2) with the amino acid residues present in the active sites of
enzyme.
nitromethane (2, 35 L, 0.66 mmol) were added to the reaction
mixture and it was stirred at 28 °C for 8 hr. The reaction was
monitored by TLC on silica gel G using 15% ethyl acetate in
petroleum ether as mobile phase. The reaction mass was then
diluted with 50 mL of diethylether. The organic layer was
separated and passed through anhydrous Na2SO4. The solvent
was removed in rotary evaporator to obtain a yellow sticky mass
(152 mg). The product was purified by column chromatography
using 60–120 mesh silica gel and eluting with 10–12% of ethyl
acetate in petroleum ether. A sticky mass obtained which was
washed with diethyl ether to get a yellow solid 3a (130 mg, yield
93%).
Table 3. Investigation of recyclability of Daucus carota root
Acknowledgement:
enzyme in Henry reactionδ
Authors are thankful to the Council for Scientific and Industrial
Research (CSIR), New Delhi, India for financial support (MLP
0116). Thanks are also due to Mr. Satyabrata Samaddar for FT-
IR analysis.
Supplementary Data:
Supplementary data (spectroscopic data of compound 3a–i,
copies of H and 13C NMR spectra) associated with this article
1
No of cycle
Time (h)
% of yield
can
be
found
in
the
online
version,
at
1
2
3
4
5
8
8
8
8
8
93
72
50
35
20
http://dx.doi.org/10.1016/j.tetlet.xxxx.xx.xxx. These data include
MOL files and InChiKeys of the important compounds described
in this article.
References and notes:
δSee experimental section for reaction procedure.
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5
Highlights:
 Daucus carota root enzyme catalyzed
Henry reaction has been reported.
 The products were obtained in excellent
yields (up to 94%).
 This enzymatic C C bond formation
reaction is highly substrate specific.
 A plausible reaction mechanism has been
highlighted in this report.