Urease-catalyzed synthesis of aminocyanopyridines from urea under fully green conditions

Article abstract of DOI:10.1016/j.molcatb.2016.02.015

This is an original work on catalytic performance of urease in organic synthesis which in one-pot dissociation of urea and condensation of the in situ generated ammonia with aldehydes, acetophenones, and malononitrile occurs in water to give 2-amino-3-cyanopyridines. Comparative experiments with ammonium salts supported the enzymatic specify of 0.01 g/mL (50 U/mg) of urease for bio-production of ammonia, while trace amount of heavy metal ions such as Pb²⁺, Hg²⁺, and Ag⁺ inhibit these specific reactions. The scalability and promiscuity of urease facilitate the applicability of the process for biotechnological organic reactions based on ammonia.

Full text of DOI:10.1016/j.molcatb.2016.02.015

Accepted Manuscript
Title: Urease-catalyzed synthesis of aminocyanopyridines
from urea under fully green conditions
Author: Fatemeh Tamaddon Somayeh Ghazi Mohammad
Reza Noorbala
PII:
S1381-1177(16)30029-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.02.015
MOLCAB 3333
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Journal of Molecular Catalysis B: Enzymatic
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Revised date:
Accepted date:
21-10-2015
23-2-2016
24-2-2016
Please cite this article as: Fatemeh Tamaddon, Somayeh Ghazi, Mohammad
Reza Noorbala, Urease-catalyzed synthesis of aminocyanopyridines from urea
under fully green conditions, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2016.02.015
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Urease-catalyzed synthesis of aminocyanopyridines from urea under
fully green conditions
Fatemeh Tamaddon,* Somayeh Ghazi, Mohammad Reza Noorbala
Author 1: Fatemeh Tamaddon, Department of Chemistry, Yazd University, Yazd 89195-741,
Iran, E-mail: ftamaddon@yazd.ac.ir
Author 2: Somayeh Ghazi, Department of Chemistry, Yazd University, Yazd 89195-741, Iran,
E-mail: s.ghazi@stu.yazd.ac.ir
Author 3: Mohammad Reza Noorbala, Department of Chemistry, Yazd University, Yazd 89195-
741, Iran, E-mail: noorbala@yazd.ac.ir
*Corresponding
author: Tel.: 00983531232666; fax: 00983538210644; e‐mail:
ftamaddon@yazd.ac.ir
1

GRAPHICAL ABSTRACT
Highlights



This work supports the catalytic performance of urease in organic synthesis.
This specific urease-catalyzed reaction inhibits by trace amount of Pb2+, Hg2+, or Ag+.
The authority of urease is bio-production of ammonia from urea.
Abstract
This is an original work on catalytic performance of urease in organic synthesis which in one-pot
dissociation of urea and condensation of the in situ generated ammonia with aldehydes,
acetophenones, and malononitrile occurs in water to give 2-amino-3-cyanopyridines.
Comparative experiments with ammonium salts supported the enzymatic specify of 0.01 g/mL
(50 U/mg) of urease for bio-production of ammonia, while trace amount of heavy metal ions
such as Pb2+, Hg2+, and Ag+ inhibit these specific reactions. The scalability and promiscuity of
urease facilitate the applicability of the process for biotechnological organic reactions based on
ammonia.
Keywords: Urease, Enzymatic reactions, In situ generation of ammonia, Biocompatible,
Pyridines
2

1. Introduction
Replacement of hazardous procedures with eco-environmentally benign alternatives is one of
the essential challenges of green chemistry [1,2], though the enzymatic water-based organic
reactions are of the most attractive processes [3-7] due to the biocompatibility, non-toxicity, and
easier workup. Hydrolases are known as substrate specify biocatalysts in organic reactions with
high enzymatic promiscuity [8] that categorized as condition promiscuity, substrate promiscuity,
and catalytic promiscuity. These promiscuities define as preservation of enzymatic activity under
different conditions [9-11], broad range of substrate specify [12,13], and the ability of the active
site of a single enzyme to catalyze various chemical transformations [14]. While immobilization
of enzymes improves their reusability, purity, stability, activity, specificity, selectivity, and
inhibitions [15], hydrolase enzymes illustrated the catalytic promiscuity in water-based
enzymatic organic synthesis [16-18]. Urease is a binuclear Ni-containing hydrolase enzyme with
high substrate specify for dissociation of urea to ammonia which in conjugation with urea can be
considered as a bio source of nitrogen instead of the risky odorous ones.
Pyridine derivatives are among the interest heterocycles which constitute the back bone of
many natural products and bioactive molecules [19] with anti-microbial, cardiotonic, anti-
parkinsonism, anti HIV, antitumor, and anti-inflammatory activities. Multi-functionalized
pyridines with amino- and cyano-substituents (AmCyPys) are of these biologically active and
fluorescent molecules [20] that extra conversion of the cyano and amino into the other functional
groups [21] make them favored for synthesis of bioorganic compounds such as vitamin B3 [22].
There are various malononitrile-based multi-component reactions (MCRs) to synthesis of these
potent pyridines [19, 23-25], although due to the benefits of the MCRs in water [26-30] and
malononitrile-based synthesis of AmCyPys [31-35], their conjugation with advanced enzymatic
3

reactions is highly desirable. To the best of our knowledge this is the first report on the
application of urease in organic synthesis that due to the benefits of the water-based enzymatic
reactions have been designed to improve the four-component synthesis of AmCyPys under fully
biocompatible conditions (Scheme 1).
<< Scheme 1 >>
2. Material and methods
2.1. Materials and analytical methods
All materials and urease with art no from jack beans were purchased from Merck. All
reagents were obtained from commercial suppliers and were used without further purification
unless otherwise noted. The NMR spectra were recorded on a Bruker 500 MHz instrument using
DMSO-d6 or CDCl3 as solvents. Chemical shifts (δ) were expressed in ppm with TMS as internal
standard, and coupling constants (J) were reported in Hz.
2.2. General procedure for the synthesis of 2-amino-3-cyanopyridines
A mixture of an aldehyde (1 mmol), substituted acetophenone (1 mmol), malononitrile (1
mmol), urea (1.5 mmol) and urease (0.01 g (50 U/mg)) in 0.25 mL water was stirred at 70 oC for
appropriate times (Table 2). The progress of the reaction was monitored by TLC (70:30, n-
hexane/acetone). The reaction mixture was cooled after completion and 95% cold EtOH (2 mL)
was added. The precipitate was filtered off, washed with cold ethanol, and dried to give the pure
product.
3. Results and discussion
4

Initially, to optimize the reaction conditions for access to the maximum activity and specify
of enzyme, the catalytic performance of the commercially available urease was investigated in
the reaction of benzaldehyde (1), acetophenone (2), malononitrile (3), and urea (4) screened at
various temperatures and loading of ureases (Table 1).
<< Table 1 >>
As results show, the maximum yield of 2-amino-4,6-diphenylnicotinonitrile 5 was obtained
in water using 0.01 g of commercially available urease after 1.5 h at 70 C (Table 1, entry 13). A
control experiment without urease under similar conditions did not give the product 5 even after
24 h and confirmed the catalytic role of urease for dissociation of urea to ammonia.
To distinct the catalytic role of urease for the in situ generation of ammonia or further
transformations, the model reaction was run with ammonium acetate instead of urea in the
presence and absence of 0.01 g of urease. The similar yields and reaction times of both of
reactions support the substrate specify of urease for the only in situ generation of ammonia from
urea in the first step of the reaction. Another control experiment with thiourea supported the
promiscuity of urease for exclusive dissociation of urea (entry 16).
To determine the details of this enzymatic reaction, the influences of enzyme loading (Fig
1.), molar ratio of starting materials (Fig 2.), and reaction temperature (Fig 3.) were finely
investigated. In respect of the reaction time and yield, the optimal reaction temperature, enzyme
loading, and molar ratios of components 1-4 were 70 °C, 0.01 g/mL of enzyme, and mole ratio of
1:1:1:4 for (1), (2), (3), and (4), respectively.
<< Fig 1 >>
5

<< Fig 2 >>
<< Fig 3 >>
In order to investigate the influence of pH, the urease-catalyzed model reaction was
performed in phosphate buffers with pH 6-8. As Table 2 shows, pH 7 is optimum for superior
activity of urease in this enzymatic reaction, presumably due to the changes in active site of
urease or denaturation of peptide residual of enzyme in acid or base conditions.
<< Table 2 >>
Owing to the importance of the enzymatic methods in large scale, the model reaction was run
with 50 mmol scale of substrates under optimized conditions and fortunately the product 5 was
obtained in 85% yield. The reusability tests for this large scale reaction show only 3% decrease
in the reaction yield with reused reaction medium after three consequence runs (Fig. 4).
<< Fig 4 >>
Heavy metal ions are non-competitive inhibitors for urease, thus to check the enzyme
inhibition, the model reaction was run with 0.5 mL of aqueous solution of 10-6 molar of heavy
metal ions such as Pb2+, Hg2+, and Ag+ that resulted in the dramatic reduction of the reaction
yield in all cases, even in the longer reaction times (Fig. 5).
<< Fig 5 >>
A plausible mechanism for the urease-catalyzed model reaction is proposed in Scheme 2,
which begins with urease-catalyzed hydrolysis of urea to ammonia.
<< Scheme 2 >>
6

Encouraged by results of the model reaction under optimal conditions, the generality of the
urease-catalyzed one-pot synthesis of AmCyPys was investigated for substituted aldehydes and
acetophenones in water that led to the high yield synthesis of the desired products under these
biocompatible conditions (Table 3).
<< Table 3 >>
Table 4 shows the merit of this enzymatic reaction and catalytic performances of urease
versus the previously reported methods for the model reaction with ammonium acetate [32,34].
<< Table 4 >>
4. Conclusion
In conclusion, an extremely efficient and green enzymatic process has been developed to
synthesis of 2-amino-3-cyanopyridines via the one-pot condensation of aldehydes,
acetophenones, and malononitrile with the in situ released ammonia from the urea without using
any chemical catalyst, solvent, or additive. This method represents merits such as no use of any
base, metal, or Lewis acid catalyst, simple isolation/purification of products by aqueous work-up,
high selectivity, low cost, simplicity of handling, and eco-environmentally benign of the process.
Scalability of this novel example of enzyme promiscuity provides the possible applications of
urease in biotechnological processes.
Acknowledgements
We gratefully acknowledge the financial support of the Yazd University.
References
7

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10

Fig 1. Effect of enzyme loading on the reaction time and yield.
Fig 2. Effect of urea loading on the yield of 5.
11

Fig 3. Effect of temperature on the rate of urease-catalyzed reaction.
Fig 4. Reusability of the reaction medium.
12

Fig 5. Inhibition of the urease-catalyzed reaction.
13

Scheme 1. Biocompatible synthesis of AmCyPys in water
Scheme 2. Plausible mechanism for urease-catalyzed synthesis of AmCyPy.
14

Table 1
Optimization of urease-catalyzed reactiona
Entry
Conditions
Urea
(mmol)
1.2
1.2
1.2
1.2
1.2
2
3
4
4
4
Yield b
(%)
Temp.
(C)
60
70
70
70
80
70
70
60
65
70
70
70
70
70
80
Urease
(g)
Time
(h)
8
1
2
3
4
5
6
7
8
0.01
0.005
0.01
0.02
0.01
0.01
0.01
0.01
0.01
0.005
0.01
0.015
0.02
0.02
0.01
0.01
50
40
68
70
50
75
80
60
70
60
85
85
87
85
60
-
8
4.5
4.5
6
3.5
2.5
3
9
2
5
10
11
12
13
14
15
16
4
4
4
5
1.5
1.5
1.4
1.5
3
4
4c
70
12
a Reaction was performed with benzaldehyde, acetophenone, malononitrile, and urea (in ratio of 1:1:1:1.2-4 mmol) in 0.5 mL of water and 0.005-
0.02 g of urease.
b Isolated yield based on 3.
c Reaction was run with thiourea.
15

Table 2
Effect of pH
Entry pH Time a Isolated yield
(min) (%)
1
2
3
4
6
7
8
7
140
100
80
58
86
83
90
87 (This work)
16

Table 3
Urease catalyzed synthesis of 2-amino-3-cyanopyridines.
Entry
R
R1
Time Yieldb Mp
(min) (%)
Ref.
(C)
1
H
H
H
H
H
H
H
H
H
H
H
90
85
80
70
75
80
75
88
85
70
80
89
86
80
78
80
85
87
(187-189) [25]
(175-177) [31]
2
4-CH3
3-OMe
4-OMe
4-NO2
2-Cl
100
90
3
(201-204)
-
4
85
(189-192) [24]
(173-177) [31]
(180-183) [31]
(234-236) [31]
(217-218) [34]
(206-210) [35]
(190-194) [35]
(123-125) [25]
(182-183) [25]
(201-203) [33]
5
150
60
6
7
4-Cl
20
8
4-F
20
9
3-pyridyl
2-furyl
H
60
10
11
12
13
14
15
16
17
110
4-NO2 50
4-CH3 90
4-CH3 95
4-CH3 120
H
4-NO2
2-furyl
H
(150-153)
-
4-Cl
4-Cl
4-Cl
90
70
80
(238-241) [25]
(245-247) [33]
(205-207) [25]
4-Cl
4-OMe
a Reaction was performed with, malononitrile (1 mmol), aldehyde (1 mmol), acetophenone (1 mmol) and urea (4 mmol) in 0.5 mL of water using
0.01 g (50 U/mg) of urease.
b Isolated yield.
17

Table 4
Catalytic performance of urease in synthesis of 2-amino-3-cyanopyridines
Entry Catalyst/Nitrogen source/Solvent/Temp./Time/Yield Ref.
(mol% or g)/-/-/(°C)/(h)/(% Isolated)
1
2
3
SnO2/SiO2 (0.1 g)/NH4OAc/EtOH/Reflux/24/91
Yb(PFO)3 (1 mol%)/NH4OAc/EtOH/r.t/24/90
Urease (0.01 g)/Urea/H2O/70/1.5/85
[34]
[32]
This work
18