H. Zheng et al.
Yield (%)a
standard. IR spectra were recorded on a Bruker Equinox-55
spectrophotometer in the 4,000–400 cm-1 region. Ele-
mental analyses were performed on an EA-1110 instru-
ment. A Hewlett-Packard model 6890 gas chromatograph
with a capillary column (HP-5) and flame-ionization
detector was used to analyze the yields of products using
tridecane as an internal standard. Melting points were
recorded on an X4-Data microscopic melting point appa-
ratus and were uncorrected. All the enzymes were pur-
chased from Aldrich and used directly. The enzymatic units
of all the enzymes are described below: PPT (2,500 units/mg),
Lipase AY30 (30 units/mg), Lipase from porcine pancreas
(C200 units/mg), Diastase from Aspergillus oryzae
(C3.5 units/mg), a-Amylase from Aspergillus oryzae
(*30 units/mg), a-Amylase from hog pancreas (10 units/mg),
Amano lipase M from Mucor Javanicus (10 units/mg).
General procedure for synthesis of 4-thiazolidinones:
The mixture of 1.0 mmol amines, 1.0 mmol aldehyde,
1.0 mmol mercaptoacetic acid, 20 mg trypsin from porcine
pancreas and 5 mL dichloromethane, was introduced into a
test tube (10 mL), then the mixture was shaken at 160 rpm
end-over-end rotation at 35 °C for 4 h. The reaction mix-
ture was monitored by TLC to end (hexane/AcOEt = 4:1).
The residue was purified on silica gel to afford the target
compounds.
Table 1 Optimization of catalyst
Entry
Catalyst
1
2
3
4
5
6
7
8
9
10
Trypsin from porcine pancreas (PPT)
Lipase AY30
96
85
Lipase from porcine pancreas
Diastase from Aspergillus oryzae
a-Amylase from Aspergillus oryzae
a-Amylase from hog pancreas
Amano lipase M from Mucor javanicus
Bovine serum albumin (BSA)
Trypsin from porcine pancreas (inactivated)b
Blank
82
75
73
64
63
60
Trace
Trace
Reaction conditions: benzylamine (1 mmol), 3-nitrobenzaldehyde
(1 mmol), and mercaptoacetic acid (1 mmol), trypsin from porcine
pancreas (20 mg), dichloromethane (5 mL), shaken at 160 rpm at
35 °C for 4 h
a
GC yields are based on tridecane as an internal standard
b
Trypsin from porcine pancreas denatured with urea at 100 °C for
10 h [25]
The reaction medium has been recognized to be one of
the most important factors influencing the enzymatic
reaction. Several solvents were used to explore the best
solvent for this reaction. The results are shown in Table 2,
which demonstrated that this reaction went smoothly in the
presence of CH2Cl2 with the highest yield (Entry 1,
Table 2) compared to other solvents. From the view of
solvent polarity, non-polar solvents are better than polar
solvents which maybe attributes that polar solvents have a
tendency to strip off constitutive water molecules from the
inner structure of the enzyme, resulting in its unfolding and
reducing catalytic activity.
3 Result and Discussion
In order to find the most suitable enzyme for the envisaged
one-pot synthesis of 4-thiazolinones reaction, we tested the
reaction of 3-nitrobenzaldehyde 1a (1 mmol), benzylamine
2a (1 mmol), and mercaptoacetic acid 3a (1 mmol) as
model substrates. Various enzymes were employed in this
reaction (Scheme 1 and Table 1). It was found that several
enzymes displayed observable catalytic activities for this
reaction. PPT showed an excellent catalytic activity, Lipase
AY30, and Lipase from porcine pancreas also showed good
catalytic activities (Entry 2, 3, Table 1). To determine the
catalytic effects coming from enzyme, according to the
literature [22], the blank control and inactivated enzyme
experiments were performed (Entry 9, 10, Table 1) and
only trace amount of product was detected. The results
demonstrate that PPT played an important catalytic role in
this reaction which maybe attribute to its unique steric and
electronic effects from the catalytic site.
In order to improve the activity of enzyme, other
influencing factors such as temperature, concentration of
catalyst, and reaction time have also been investigated
(Table 3). It was found that the temperatures ranged from
25 to 40 °C, the yield of the products increased when the
enzyme amount is 20 mg. While the enzyme amount
exceeded 30 mg, the yields decrease (Entry 3–7, Table 3)
which maybe attributes to the reunion of enzyme when its
concentration is high in solution. Synthetically, the opti-
mum reaction condition is 20 mg PPT in 5 mL solvent at
35 °C/4 h.
In order to expand the substrates, different amines and
aldehydes were employed (Scheme 2 and Table 4). The
electronic and steric effects are investigated by employing
Scheme 1 The model reaction
of one-pot multicomponent
enzymatic synthesis of
4-thiazolidinones 148 9 25 mm
123
Zheng, Hui
