A novel one-pot multicomponent enzymatic synthesis of 2,4-disubstituted thiazoles

Article abstract of DOI:10.1007/s10562-014-1229-1

A novel one-pot multicomponent synthetic method of some new 2,4-disubstituted thiazoles catalyzed by enzyme was developed. Lipase from porcine pancreas (PPL) displayed good catalytic activity to promote this reaction starting from amines, aldehyde, thioacetic acid and methyl 3-(dimethyl amino)-2-isocyanoacrylate under mild conditions (35 °C) with good yields (up to 93 %). The experimental results revealed PPL showed broad catalytic activity to substrates. The blank and control experiments revealed that PPL played important catalytic role during the process. All the new compounds were confirmed by IR, ¹H NMR, ¹³C NMR, MS and elemental analysis. This PPLcatalyzed one-pot multicomponent synthetic method provided a new strategy to synthesize 2,4-disubstituted thiazoles and expanded the application of enzymes in organic synthesis. Springer Science+Business Media New York 2014.

Full text of DOI:10.1007/s10562-014-1229-1

Catal Lett (2014) 144:928–934
DOI 10.1007/s10562-014-1229-1
A Novel One-Pot Multicomponent Enzymatic Synthesis
of 2,4-Disubstituted Thiazoles
Huiping Zhang
Received: 26 January 2014 / Accepted: 23 February 2014 / Published online: 18 March 2014
Ó Springer Science+Business Media New York 2014
Abstract A novel one-pot multicomponent synthetic
method of some new 2,4-disubstituted thiazoles catalyzed
by enzyme was developed. Lipase from porcine pancreas
(PPL) displayed good catalytic activity to promote this
reaction starting from amines, aldehyde, thioacetic acid and
methyl 3-(dimethyl amino)-2-isocyanoacrylate under mild
conditions (35 °C) with good yields (up to 93 %). The
experimental results revealed PPL showed broad catalytic
activity to substrates. The blank and control experiments
revealed that PPL played important catalytic role during
the process. All the new compounds were confirmed by IR,
1H NMR, 13C NMR, MS and elemental analysis. This PPL-
catalyzed one-pot multicomponent synthetic method pro-
vided a new strategy to synthesize 2,4-disubstituted thia-
zoles and expanded the application of enzymes in organic
synthesis.
exhibit multifarious bioactivities and the synthetic methods
were developed continually [8–10]. Marder et al. reported
the synthesis and applications of some 2,4-disubstituted
thiazole derivatives as small molecule modulators of cel-
lular development [11]. Srinivasan et al. developed an
efficient synthesis of 2,4-disubstituted thiazoles using ionic
liquid [12].
Multicomponent reactions (MCRs) have received much
attention in modern organic chemistry for their high effi-
ciency and greater atom-economy. It can provide new
organic synthetic methods and easier access to get diverse
compounds [13–15]. For the synthesis of thiazole deriva-
tives, Sayyed-Alangi et al. reported the synthesis of func-
tionalized thiazoles using MCRs from isothiocyanates with
high yields and an easy work-up procedure [16]. Domling
et al. found a novel MCRs to synthesize thiazole moiety
from ss-aminothiocarboxylic acids, aldehydes, and
3-dimethylamino-2-isocyanoacylate [17] and they also
reported the new MCRs of 2,4-disubstituted thiazoles
under inert and water-free conditions at 0–10 °C stirred for
1–4 days [18]. This publication prompted us to research a
mild synthetic method via MCRs for the synthesis of 2,4-
disubstituted thiazoles.
Keywords Enzymes Á Multicomponent reactions Á
Heterocycles Á Thiazoles Á Synthesis
1 Introduction
Thiazole derivatives have attracted much attention of
medicinal and organic chemists for their varied biological
activities, such as antitumor, antifungal, antibiotic and
antiviral activities [1–4]. In addition, they are also impor-
tant synthetic intermediates and common substructures in
numerous biological compounds [5–7]. As one of the
important thiazole derivatives, 2,4-disubstituted thiazoles
Enzymatic synthesis, as a novel, green and mild cata-
lytic method, flourished recently [19–21]. Especially for
organic synthesis, enzymes showed enzymatic promiscuity
to form C–C, C–N, C–S bonds through Henry reaction,
Michael addition, Markovnikov addition and Aldol reac-
tions [22–24]. There are some reports about the enzymatic
synthesis of thiazole derivatives [25, 26]. For the impor-
tance of developing new methods, in this paper, the first
chemoenzymatic multicomponent example of synthesizing
2,4-disubstituted thiazoles under mild conditions was
developed. This method was another successful example of
enzyme promiscuity in organic chemistry.
H. Zhang (&)
Department of Chemistry and Chemical Engineering, Jining
University, Qufu 273100, Shandong, China
e-mail: hpzhangjnu@163.com
123

A Novel One-Pot Multicomponent Enzymatic Synthesis
929
Scheme 1 Model reaction of
preparing 2,4-disubstituted
thiazoles catalyzed by enzyme
This one-pot enzymatic reaction was performed by
employing benzylamine 1a (1 mmol), isobutyraldehyde 2a
(1 mmol), thioacetic acid and methyl 3-(dimethylamino)-2-
(CDCl3, 400 MHz, d, ppm): 0.83 (d, J = 6.8 Hz, 3H,
CH3), 0.95 (d, J = 6.8 Hz, 3H, CH3), 2.06 (s, 3H, CH3-
C=O), 2.84–2.93 (m, 1H, CH), 3.84 (s, 3H, CH3O), 4.59 (d,
J = 17.2 Hz, 1H, CH2N), 4.78 (d, J = 17.2 Hz, 1H,
CH2N), 5.36 (d, J = 7.2 Hz, 1H, CHN), 6.84–7.14 (m, 5H,
Ph-H), 8.01 (s, 1H, CHS). MS(EI): m/z (%) = 346 (M?).
isocyanoacrylate (1 mmol) as
a
model reaction
(Scheme 1). Reaction progress was monitored by TLC and
GC. The reaction conditions including enzyme, solvent,
temperature, reaction time and catalysts amount were
optimized respectively.
2.2 Methyl 2-(2-methyl-1-(N-(prop-2-ynyl)
acetamido)propyl)thiazole-4-carboxylate (3b)
2 Experimental
Yellow solid. M.p. 114–116 °C. IR (KBr): vmax 3306, 3132,
2958, 1728, 1652, 1474, 1440, 1411, 1326, 1255, 1213, 1110,
1
All reagents and solvents were purchased without further
purification, unless otherwise indicated. Reactions were
performed in an end-over-end rotator. NMR was recorded
on a Bruker Avance 400 spectrometer at 400 MHz in
CDCl3 using TMS as internal standard. IR spectra were
recorded on a Bruker Equinox-55 spectrophotometer using
KBr discs in the 4,000–400 cm-1 region. 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 apparatus and were uncorrected.
Elemental analyses were performed on an EA-1110
instrument. All the enzymes were purchased from Aldrich
and used directly.
1075 cm-1. H NMR (CDCl3, 400 MHz, d, ppm): 0.89 (d,
J = 6.4 Hz, 3H, CH3), 1.05 (d, J = 6.4 Hz, 3H, CH3), 2.18 (s,
1H, CH), 2.24 (s, 3H, CH3C=O), 2.72–2.79 (m, 1H, CH), 3.94
(s, 3H, CH3O), 4.01 (d, J = 19.2 Hz, 1H, CH2N), 4.36 (d,
J = 19.2 Hz, 1H, CH2N), 5.51 (d, J = 10.8 Hz, 1H, CHN),
8.14 (s, 1H, CHS). 13C NMR (CDCl3, 100 MHz, d, ppm): 19.2
(CH3), 19.8 (CH3), 22.4 (CH3), 30.5 (CH), 36.8 (CH2), 52.2
(CH3), 67.8 (CH), 72.6 (CH), 79.3 (Cq), 129.6 (CH), 145.3
(Cq), 161.6 (Cq), 169.9 (Cq), 171.3 (Cq). MS(EI): m/
z (%) = 294 (M?). Anal. Calcd. for C14H18N2O3S: C, 57.12;
H, 6.16; N, 9.52; Found: C, 56.99; H, 6.14; N, 9.45 %.
2.3 Methyl 2-(1-(N-benzylacetamido)butyl)thiazole-4-
carboxylate (3c)
General procedure for synthesis of 2,4-disubstituted
thiazoles: 1.0 mmol amines, 1.0 mmol aldehyde, 1.0 mmol
thioacetic acid and 1.0 mmol methyl 3-(dimethylamino)-2-
isocyanoacrylate were introduced accordingly into a test
tube (10 ml), then 20 mg lipase from porcine pancreas
(PPL) and 5 ml methanol were added and shaken at
160 rpm end-over-end rotation at 35 °C for 2 h. The
reaction mixture was monitored by TLC to end (hexane/
AcOEt = 3:2). The residue was purified on silica gel to
afford the target compounds. All the new compounds were
Yellow liquid. IR: vmax 2958, 2360, 1735, 1648, 1408, 1323,
1212, 1081 cm-1. 1H NMR (CDCl3, 400 MHz, d, ppm): 0.85
(t, J = 7.2 Hz, 3H, CH3), 1.26–1.32 (m, 2H, CH2), 1.95–2.01
(m, 1H, CH2), 2.09(s, 3H, CH3C=O), 2.25–2.30(m, 1H, CH2),
3.90 (s, 3H, CH3O), 4.63 (s, 2H, CH2N), 5.81 (t, J = 7.2 Hz,
1H, CHN), 7.07–7.27 (m, 5H, Ph-H), 8.08 (s, 1H, CHS). 13
C
NMR (CDCl3, 100 MHz, d, ppm): 13.8 (CH3), 19.6 (CH2),
22.1 (CH3), 33.6 (CH2), 50.6 (CH3), 53.5 (CH2), 61.8 (CH),
126.6 (CH), 127.2 (CH), 128.8 (CH), 129.7 (CH), 136.6 (Cq),
145.9 (Cq), 162.1 (Cq), 168.9 (Cq), 171.9 (Cq). MS(EI): m/
z (%) = 346 (M?). Anal. Calcd. for C18H22N2O3S: C, 62.40;
H, 6.40; N, 8.09; Found: C, 62.39; H, 6.43; N, 8.05 %.
1
informed by IR, H NMR, 13C NMR, MS and elemental
analysis.
2.1 Methyl 2-(1-(N-benzylacetamido)-2-
2.4 Methyl 2-(1-(N-(prop-2-ynyl)acetamido)
butyl)thiazole-4-carboxylate (3d)
methylpropyl)thiazole-4-carboxylate (3a) [13]
Yellow liquid. IR: vmax 2957, 1727, 1648, 1472, 1443,
1410, 1321, 1244, 1210, 1109, 1058 cm-1
Yellow liquid. IR: vmax 3254, 2957, 2872, 1696, 1622,
1431, 1296, 1215, 1094 cm-1
.
1H NMR
.
1H NMR (CDCl3,
123

930
H. Zhang
400 MHz, d, ppm): 0.79 (t, J = 7.2 Hz, 3H, CH3),
1.24–1.28 (m, 2H, CH2), 1.94–1.98 (m, 2H, CH2), 2.22 (s,
3H, CH3C=O), 2.13 (s, 1H, CH), 3.88 (s, 3H, CH3O), 4.04
(d, J = 16.2 Hz, 1H, CH2N), 4.38 (d, J = 16.2 Hz, 1H,
CH2N), 5.51–5.56 (m, 1H, CHN), 8.06 (s, 1H, CHS). 13C
NMR (CDCl3, 100 MHz, d, ppm): 13.1 (CH3), 19.2 (CH2),
22.4 (CH3), 33.2 (CH2), 37.1 (CH2), 52.4 (CH3), 65.8
(CH), 72.8 (CH), 79.9 (Cq), 128.3 (CH), 145.8 (Cq), 161.2
(Cq), 168.8 (Cq), 170.9 (Cq). MS(EI): m/z (%) = 294
(M?). Anal. Calcd. for C14H18N2O3S: C, 57.12; H, 6.16; N,
9.52; Found: C, 57.16; H, 6.21; N, 9.55 %.
1.50–1.55 (m, 4H, C5H9), 1.72–1.75 (m, 4H, C5H9), 2.02
(s, 3H, CH3C=O), 2.11–2.18 (m, 2H, CH2), 3.61–3.65 (m,
1H, CHN), 3.88 (s, 3H, CH3O), 5.48 (t, J = 7.2 Hz, 1H,
CHN), 7.98 (s, 1H, CHS). 13C NMR (CDCl3, 100 MHz, d,
ppm): 13.7 (CH3), 21.4 (CH2), 22.5 (CH3), 23.9 (CH2),
24.6 (CH2), 33.9 (CH2), 34.8 (CH2), 52.4 (CH3), 57.6
(CH), 63.6 (CH), 128.4 (CH), 145.4 (Cq), 161.3 (Cq),
169.6 (Cq), 170.8 (Cq). MS(EI): m/z (%) = 338 (M?).
Anal. Calcd. for C17H26N2O3S: C, 60.33; H, 7.74; N, 8.28;
Found: C, 60.38; H, 7.79; N, 8.31 %.
2.8 Methyl 2-(1-(N-butylacetamido)pentyl)thiazole-4-
carboxylate (3h)
2.5 Methyl 2-(1-(N-benzylacetamido)pentyl)thiazole-
4-carboxylate (3e)
Yellow liquid. IR: vmax 2968, 2921, 1740, 1638, 1413, 1321,
1243, 1209, 1076 cm-1. 1H NMR(CDCl3, 400 MHz, d, ppm):
0.88 (t, J = 6.8 Hz, 3H, CH3), 1.22–1.24 (m, 4H, 2CH2), 1.31
(t, J = 5.6 Hz, 3H, CH3), 1.33–1.35 (m, 4H, 2CH2), 2.15 (s,
3H, CH3C=O), 2.31 (q, J = 7.2 Hz, J = 16.2 Hz, 2H, CH2),
3.24 (t, J = 4.8 Hz, 2H, CH2), 3.94 (s, 3H, CH3O), 5.55 (t,
J = 7.2 Hz, 1H, CHN), 8.15 (s, 1H, CHS). 13C NMR (CDCl3,
100 MHz, d, ppm): 13.6 (CH3), 14.0 (CH3), 20.1 (CH2), 21.6
(CH2), 22.4 (CH3), 28.6 (CH2), 32.1 (CH2), 32.1 (CH2), 34.8
(CH2), 52.4 (CH3), 59.3 (CH), 128.8 (CH), 145.5 (Cq), 161.9
(Cq), 169.9 (Cq), 171.3 (Cq). MS(EI): m/z (%) = 326 (M?).
Anal. Calcd. for C16H26N2O3S: C, 58.87; H, 8.03; N, 8.58;
Found: C, 58.91; H, 7.99; N, 8.61 %.
Yellow liquid. IR: vmax 2955, 2869, 1737, 1649, 1408, 1323,
1243, 1213, 1085, 1029 cm-1. 1H NMR (CDCl3, 400 MHz, d,
ppm): 0.74–0.77 (m, 2H, CH2), 0.81 (t, J = 6.4 Hz, 3H, CH3),
1.22–1.25 (m, 2H, CH2), 2.01 (q, J = 7.2 Hz, J = 16.2 Hz,
2H, CH2), 2.10 (s, 3H, CH3), 3.90 (s, 3H, CH3C=O), 4.63 (s,
2H, CH2N), 5.78 (t, J = 7.6 Hz, 1H, CHN), 7.07–7.24 (m, 5H,
Ph-H), 8.08 (s, 1H, CHS). 13C NMR (CDCl3, 100 MHz, d,
ppm): 13.9 (CH3), 20.6 (CH2), 22.6 (CH3), 24.8 (CH2), 33.6
(CH2), 52.8 (CH3), 53.7 (CH2), 62.6 (CH), 126.3 (CH), 127.4
(CH), 128.7 (CH), 129.8 (CH), 136.8 (Cq), 145.4 (Cq), 161.8
(Cq), 169.5 (Cq), 171.8 (Cq). MS(EI): m/z (%) = 360 (M?).
Anal. Calcd. for C19H24N2O3S: C, 63.31; H, 6.71; N, 7.77;
Found: C, 63.28; H, 6.69; N, 7.79 %.
2.9 Methyl 2-(cyclohexyl(N-cyclopentylacetamido)
methyl)thiazole-4-carboxylate (3i)
2.6 Methyl 2-(1-(N-(prop-2-ynyl)acetamido)
pentyl)thiazole-4-carboxylate (3f)
Yellow liquid. IR: vmax 2965, 1731, 1657, 1411, 1325, 1235,
1
Yellow liquid. IR: vmax 3242, 2956, 1733, 1655, 1408, 1320,
1244, 1216, 1086 cm-1. 1H NMR(CDCl3, 400 MHz, d, ppm):
0.79 (t, J = 6.4 Hz, 3H, CH3), 0.81–0.85 (m, 2H, CH2),
1.20–1.23 (m, 2H, CH2), 2.05 (q, J = 7.2 Hz, J = 16.2 Hz,
1H, CH2), 2.20 (s, 3H, CH3C=O), 2.5 (s, 1H, CH), 3.94 (s, 3H,
CH3O), 4.05 (d, J = 16.2 Hz, 1H, CH2N), 4.39 (d,
J = 16.2 Hz, 1H, CH2N), 5.97 (t, J = 7.6 Hz, 1H, CHN),
8.14 (s, 1H, CHS). 13C NMR (CDCl3, 100 MHz, d, ppm): 13.8
(CH3), 21.1 (CH2), 22.8 (CH3), 24.7 (CH2), 33.7 (CH2), 36.6
(CH2), 52.4 (CH3), 64.7 (CH), 72.9 (CH), 79.8 (Cq), 128.9
(CH), 145.3 (Cq), 161.6 (Cq), 169.4 (Cq), 171.2 (Cq). MS(EI):
m/z (%) = 308 (M?). Anal. Calcd. for C15H20N2O3S: C,
58.42; H, 6.54; N, 9.08; Found: C, 58.39; H, 6.59; N, 9.11 %.
1221, 1088 cm-1. H NMR (CDCl3, 400 MHz, d, ppm):
1.40–1.44 (m, 10H, C6H11), 1.51–1.53 (m, 4H, C5H9),
1.73–1.75 (m, 4H, C5H9), 2.03 (s, 3H, CH3C=O), 2.41–2.43
(m, 1H, CH), 3.86 (s, 3H, CH3O), 4.95 (d, J = 7.2 Hz, 1H,
CHN), 7.82 (s, 1H, CHS). 13C NMR (CDCl3, 100 MHz, d,
ppm): 22.4 (CH3), 23.9 (CH2), 24.6 (CH2), 25.9 (CH2), 29.8
(CH2), 30.1 (CH2), 32.3 (CH2), 32.7 (CH2), 39.6 (CH), 52.4
(CH3), 55.4 (CH), 61.3 (CH), 128.1 (CH), 145.2 (Cq), 161.2
(Cq), 169.3 (Cq), 170.1 (Cq). MS(EI): m/z (%) = 364 (M?).
Anal. Calcd. for C19H28N2O3S: C, 62.61; H, 7.74; N, 7.69;
Found: C, 62.58; H, 7.79; N, 7.72 %.
2.10 Methyl 2-((N-butylacetamido)(cyclohexyl)
methyl)thiazole-4-carboxylate (3j)
2.7 Methyl 2-(1-(N-cyclopentylacetamido)pentyl)
thiazole-4-carboxylate (3g)
Yellow liquid. IR: vmax 2958, 1712, 1645, 1413, 1322,
1
1243, 1220, 1084 cm-1. H NMR (CDCl3, 400 MHz, d,
Yellow liquid. IR: vmax 2958, 2925, 1730, 1649, 1418,
1315, 1265, 1220, 1077 cm-1
ppm): 0.33 (t, J = 5.6 Hz, 3H, CH3), 1.35–1.37 (m, 4H,
2CH2), 1.40–1.44 (m, 10H, C6H11), 2.11 (s, 3H, CH3C=O),
2.20 (t, J = 4.8 Hz, 2H, CH2), 2.44–2.46 (m, 1H, CH),
3.94 (s, 3H, CH3O), 5.52 (d, J = 7.2 Hz, 1H, CH), 8.17 (s,
.
1H NMR (CDCl3,
400 MHz, d, ppm): 0.96 (t, J = 6.8 Hz, 3H, CH3),
1.26–1.29 (m, 2H, CH2), 1.31–1.34 (m, 2H, CH2),
123

A Novel One-Pot Multicomponent Enzymatic Synthesis
931
1H, CHS). 13C NMR (CDCl3, 100 MHz, d, ppm): 13.8
(CH3), 20.4 (CH2), 22.4 (CH3), 24.6 (CH2), 26.9 (CH2),
28.6 (CH2), 28.9 (CH2), 32.1 (CH2), 39.6 (CH), 47.9
(CH2), 52.4 (CH3), 61.3 (CH), 128.4 (CH), 145.6 (Cq),
Table 1 Optimization of catalyst
Entry
Enzymes
Yield (%)b
1
2
Lipase from porcine pancreas
a-Amylase from hog pancreas
Typsin from porcine pancreas
Bovine serum albumin (BSA)
Diastase from Aspergillus oryzae
Amano lipase M from Mucor javanicus
a-Amylase from Aspergillus oryzae
a-Amylase from Bacillus subtilis
Blank
88
71
161.6 (Cq), 169.8 (Cq), 171.1 (Cq). MS(EI):
m/
z (%) = 352 (M?). Anal. Calcd. for C18H28N2O3S: C,
61.33; H, 8.01; N, 7.95; Found: C, 61.38; H, 7.98; N,
8.00 %.
3
52
4
32
5
51
6
32
2.11 Methyl 2-((N-cyclopentylacetamido)(cyclopropyl)
methyl)thiazole-4-carboxylate (3k)
7
33
8
22
9
Trace
Trace
10
Denatured lipase from porcine pancreasa
Yellow liquid. IR: vmax 2944, 1738, 1650, 1415, 1323, 1248,
1219, 1083 cm-1. 1H NMR (CDCl3, 400 MHz, d, ppm): 0.43
(q, J = 4.4 Hz, J = 8.8 Hz, 2H, CH2), 0.64 (q, J = 4.4 Hz,
J = 8.8 Hz, 2H, CH2), 1.25–1.27 (m, 1H, CH), 1.67–1.69 (m,
4H, C5H9), 1.75–1.77 (m, 4H, C5H9), 2.20 (s, 3H, CH3C=O),
3.92 (s, 3H, CH3O), 4.04 (d, J = 7.6 Hz, 1H, CHN), 8.14 (s,
1H, CHS). 13C NMR (CDCl3, 100 MHz, d, ppm): 6.2 (CH2),
6.3 (CH2), 15.8 (CH), 22.5 (CH3), 23.3(CH2), 34.1 (CH2), 34.3
(CH2), 52.3 (CH3), 56.4 (CH), 65.3 (CH), 128.2 (CH), 145.3
(Cq), 161.7 (Cq), 169.5 (Cq), 170.6 (Cq). MS(EI): m/
z (%) = 322 (M?). Anal. Calcd. for C16H22N2O3S: C, 59.60;
H, 6.88; N, 8.69; Found: C, 59.58; H, 6.90; N, 8.71 %.
Reaction conditions benzylamine (1 mmol), thioacetic acid (1 mmol),
isobutyraldehyde (1 mmol), and methyl 3-(dimethylamino)-2-isocy-
anoacrylate (1 mmol), enzyme (20 mg), methanol (5 ml), shaken at
160 rpm at 35 °C for 2 h
a
Lipase from porcine pancreas was refluxed in urea for 24 h at
100 °C [27]
b
GC yields are based on tridecane as an internal standard
which maybe attribute to the different catalytic sites in
their protein structures. To determine the catalytic effects
coming from enzyme, the control experiments were per-
formed (Entries 9, 10; Table 1) according to the literature
[27]. The results revealed that only trace product was
detected in the blank and denatured enzyme experiments.
So we conclude that PPL played an important catalytic role
in this reaction.
2.12 Methyl 2-((N-butylacetamido)(cyclopropyl)
methyl)thiazole-4-carboxylate (3l)
Yellow liquid. IR: vmax 2945, 1734, 1657, 1410, 1328, 1245,
1
1214, 1089 cm-1. H NMR (CDCl3, 400 MHz, d, ppm):
The reaction medium was one of the important influ-
encing factors. Especially for the enzyme promiscuity, the
solvents will affect the configuration of the enzyme. Sev-
eral solvents were screened in this reaction. The experi-
mental results demonstrated this reaction went smoothly in
the presence of methanol with the highest yield (Entry 1;
Table 2) compared to other organic solvents. Interestingly,
as the similar polar solvents, ethanol and water seemed to
be inefficient in this reaction (Entries 3, 9; Table 2). It
maybe revealed that methanol was the most suitable can-
didate in term of polarity and structure. So we concluded
that PPL had higher ‘‘promiscuous’’ activity than its
‘‘natural’’ activity in organic solvents. Based on the
experimental results, methanol was selected as the opti-
mum solvent in this reaction.
0.46 (q, J = 4.8 Hz, J = 9.6 Hz, 2H, CH2), 0.65 (q,
J = 4.8 Hz, J = 9.6 Hz, 2H, CH2), 0.90 (t, J = 7.2 Hz, 3H,
CH3), 1.27–1.29 (m, 1H, CH), 1.30–1.33 (m, 4H, 2CH2),
2.15 (s, 3H, CH3C=O), 3.71 (t, J = 7.2 Hz, 2H, CH2), 3.93
(s, 3H, CH3O), 4.71 (d, J = 10.4 Hz, 1H, CH), 8.14 (s, 1H,
CHS). 13C NMR (CDCl3, 100 MHz, d, ppm): 6.7 (CH2), 6.8
(CH2), 13.6 (CH3), 15.8 (CH), 20.1 (CH2), 22.5 (CH3), 34.1
(CH2), 47.1 (CH2), 52.4 (CH3), 67.3 (CH), 128.1 (CH), 145.1
(Cq), 161.3 (Cq), 169.2 (Cq), 170.1 (Cq). MS(EI): m/
z (%) = 310 (M?). Anal. Calcd. for C15H22N2O3S: C, 58.04;
H, 7.14; N, 9.02; Found: C, 58.11; H, 7.11; N, 8.99 %.
3 Results and Discussion
According to above conditions, we applied PPL to
optimize other conditions. A GC yield was selected as the
criterion to optimize the reaction temperature, time and
concentration of PPL in this reaction. The experimental
results were listed in Table 3. For the temperature, five
experiments were performed. The results showed that the
yields went up from 54–84 % at the region 25–35 °C.
When the temperature continues to rise to 45 °C the yield
decreased to 78 %. This maybe attributed to the partly
We initially screened the enzymes and the results were
summarized in Table 1. Several enzymes displayed
observable catalytic activities for this reaction. Especially,
PPL showed better catalytic activity (Entry 1; Table 1)
compared to other enzymes. Interestingly, a-amylase from
hog pancreas had higher catalytic ability (Entry 2; Table 1)
than that of a-amylase from Aspergillus oryzae and a-
Amylase from Bacillus subtilis (Entries 7, 8; Table 1),
123

932
H. Zhang
Table 2 Optimization of solvents
Table 4 Synthesis of different 2,4-disubstituted thiazoles catalyzed
by PPL
Entry
Solvent
T (°C)
Yielda (%)
Entry
Compound
R1
R2
Yield (%)a
1
2
3
4
5
6
7
8
9
Methanol
35
35
35
35
35
35
35
35
35
88
1
3a
3b
3c
3d
3e
3f
Isopropyl
Isopropyl
n-Propyl
n-Propyl
n-Butyl
Benzyl
88 (79)b
93 (86)
90 (84)
89 (82)
81 (72)
78 (67)
67 (56)
78 (65)
75 (66)
68 (57)
71 (60)
72 (61)
Acetonitrile
Ethanol
52
2
2-Propynyl
Benzyl
50
3
n-Hexane
41
4
2-Propynyl
Benzyl
Dichloromethane
1,4-Dioxane
Acetone
39
5
37
6
n-Butyl
2-Propynyl
Cyclopentyl
n-Butyl
22
7
3g
3h
3i
n-Butyl
Tetrahydrofuran
Water
20
8
n-Butyl
Trace
9
Cyclohexyl
Cyclohexyl
Cyclopropyl
Cyclopropyl
Cyclopentyl
n-Butyl
Reaction conditions benzylamine (1 mmol), thioacetic acid (1 mmol),
isobutyraldehyde (1 mmol), and methyl 3-(dimethylamino)-2-isocy-
anoacrylate (1 mmol), lipase from porcine pancreas (20 mg), solution
(5 ml), shaken at 160 rpm at 35 °C
10
11
12
3j
3k
3l
Cyclopentyl
n-Butyl
a
GC yields are based on tridecane as an internal standard
Reaction conditions amines (1 mmol), aldehyde (1 mmol), thioacetic
acid (1 mmol) and methyl 3-(dimethylamino)-2-isocyanoacrylate
(1 mmol), PPL (20 mg), methanol (5 ml), shaken at 160 rpm at 35 °C
Table 3 Optimization of the reaction conditions
a
GC yields are based on tridecane as an internal standard
Yield (%)a
Isolated yields
b
Entry
Amount (mg)
T (°C)
Time (h)
1
20
20
20
20
20
10
20
30
40
50
20
20
20
25
30
35
40
45
35
35
35
35
35
35
35
35
2
2
2
2
2
2
2
2
2
2
1
2
3
54
73
88
80
78
62
88
81
75
74
56
88
83
cycloalkanes to check the application scope of this reaction.
The yields were relatively higher when the substituent
groups R1 were isopropyl and n-propyl (Entries 1–4;
Table 4). When R1 was n-butyl in entries 5–8 (Table 4), the
yields kept moderate to good (Entries 5–8; Table 4). When
the substituent groups R1 were cycloalkanes, the yields were
a little lower (Entries 9–12; Table 4). It seemed that the
higher yields could be obtained when the shorter chain
aldehydes were employed. Therefore, two conclusions can
be drawn from the above experimental results. One, the steric
effect in aldehydes have some influence on yield, namely
bulkier groups provide lower yields. The other one, the
amines and aldehydes with different groups can react
smoothly to afford 2,4-disubstituted thiazoles with moderate
to high yields, so the enzyme PPL have a wide tolerance
range towards amines and aldehydes in this reaction.
2
3
4
5
6
7
8
9
10
11
12
13
Reaction conditions benzylamine (1 mmol), thioacetic acid (1 mmol),
isobutyraldehyde (1 mmol), and methyl 3-(dimethylamino)-2-isocy-
anoacrylate (1 mmol), PPL, methanol (5 ml), shaken at 160 rpm
a
GC yields are based on tridecane as an internal standard
A plausible reaction mechanism for this process is
proposed as depicted in Scheme 3. It is likely that the
presence of the enzyme activates the relevant substrates via
the hydrogen bond effects in the whole process [28]. It
seems that the carbonyl group in substrates 1 accelerates
dehydration via hydrogen bond to produce imine 5, which
generates ion pair and reacts with methyl 3-(dimethyl-
amino)-2-isocyano-acrylate to obtain the intermediate 6.
Then, the carbonyl group in 6 is activated via hydrogen
bond of enzyme and attacked by secondary amine group to
give intermediate 7, which transform to 8 via hydrogen
bond of enzyme. After that, the C=S bond is activated by
enzyme to form 9. Subsequently, the intermediate 10 was
obtained by intramolecular cyclization from 9 and the
dimethylamine leaves from 10 to give product 3. This
inactivated enzyme at higher temperature. So the optimum
temperature was 35 °C. The optimal enzyme amount was
screened from 10–50 mg. The results showed that the
highest yield was achieved with 20 mg of enzyme, which
maybe attributed to the reunion of enzyme when its con-
centration was high in solution. As to reaction time, the
highest yield was obtained at 2 h. Synthetically, the opti-
mum reaction condition is 20 mg PPL in 5 ml solvent and
2 h.
Based on these optimized conditions, we employed this
reaction with various different amines and aldehydes in order
to extend the substrate (Scheme 2; Table 4). We chose dif-
ferent amines and aldehydes bearing alkanes and
123

A Novel One-Pot Multicomponent Enzymatic Synthesis
933
Scheme 2 One-pot
multicomponent enzymatic
synthesis of 2,4-disubstituted
thiazoles
Scheme 3 A plausible
mechanism for the
enzymatic synthesis
of 2,4-disubstituted thiazoles
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