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D.-D. Zhao et al. / Journal of Molecular Catalysis B: Enzymatic 95 (2013) 29–35
2.2.3. 2-Amino-5,6-dihydro-4H-cyclopenta[b]
thiophene-3-carbonitrile (3c)
Yellow solid. mp 152–153 ◦C (lit. [20] mp 151 ◦C). IR (KBr): ꢀ.
3438, 3336, 2194 cm−1 1H NMR (400 MHz, CDCl3): ı: 4.64 (bs, 2H),
.
2.78–2.66 (m, 4H), 2.41–2.31 (m, 2H). 13C NMR (100 MHz, CDCl3):
ı: 166.2, 142.9, 125.3, 115.5, 85.6, 29.3, 28.5, 27.4.
Scheme 1. Gewald reaction.
2.2.4. Ethyl 2-amino-5,6-dihydro-4H-cyclopenta[b]
thiophene-3-carboxylate (3d)
(≥30,000 U/g, 1 U is defined as enzyme quantity which produces
1 mol of l-amino acid per 30 min) were purchased from Amano
Enzyme Inc. (Japan). Lipase from Hog pancreas (2.4 U/mg, 1 U is
the amount of immobilized enzyme which forms 1% octyl lau-
rate from 0.5 mmol lauric acid and 1.0 mmol 1-octanol in 10 mL
water-saturated isooctane in 1 h at 20 ◦C), Amano Lipase M from
Mucor javanicus (≥10,000 U/g enzyme activity, pH 7.0, 40 ◦C), Lipase
immobilized on acrylic resin from Candida antarctica (≥10,000 U/g,
recombinant, expressed in A. oryzae) and lipase from Porcine
pancreas (30–90 U/mg protein, 1 U will hydrolyze 1.0 mequiv. of
triacetin in 1 h at pH 7.7 at 37 ◦C) were purchased from Sigma
(Steinheim, Germany). Lipase AY 30 (700–1500 U/mg solid, 1 U will
hydrolyze 1.0 mequiv. of olive oil from a triglyceride in 1 h at pH 7.7
at 37 ◦C) was purchased from Acros (New Jersey, USA). Lipozyme
immobilized from Mucor miehei (MML) was purchased from Fluka.
Bovine serum albumin (BSA) was obtained from Wuxi Enzyme
Co. Ltd., Wuxi, PR China. l-Glutamic acid, l-threonine, l-serine,
l-cysteine and l-lysine were obtained from Sinopharm Chemical
Reagent Co., Ltd. All other chemicals were of the highest purity
commercially available.
Yellow solid. mp 181–182 ◦C (lit. [21] mp 182–183 ◦C). IR (KBr):
ꢀ. 3413, 3297, 1653 cm−1 1H NMR (400 MHz, CDCl3): ı: 5.85 (bs,
.
2H), 4.24 (q, J = 6.8 Hz, 2H), 2.85–2.78 (m, 2H), 2.75–2.67 (m, 2H),
2.36–2.25 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3): ı: 166.2, 165.7, 143.1, 122.1, 102.7, 59.5, 30.8, 28.9, 27.2,
14.4.
2.2.5. 2-Amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carbonitrile (3e)
Yellow solid. mp 125–126 ◦C (lit. [20] mp 126 ◦C). IR (KBr): ꢀ.
3443, 3310, 2203 cm−1 1H NMR (400 MHz, CDCl3): ı: 4.50 (bs, 2H),
;
2.64–2.54 (m, 4H), 1.85–1.77 (m, 2H), 1.68–1.60 (m, 4H). 13C NMR
(100 MHz, CDCl3): ı: 158.1, 136.9, 123.7, 115.9, 91.7, 31.9, 29.9,
29.4, 28.1, 27.2.
2.2.6. Ethyl 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxylate (3f)
Yellow solid. mp 87–88 ◦C (lit. [20] mp 89 ◦C). IR (KBr): ꢀ. 3397,
3301, 1652 cm−1 1H NMR (400 MHz, CDCl3): ı: 5.76 (bs, 2H), 4.28
.
(q, J = 7.2 Hz, 2H), 2.97 (t, J = 5.6 Hz, 2H), 2.57 (t, J = 5.6 Hz, 2H),
1.85–1.76 (m, 2H), 1.68–1.56 (m, 4H), 1.34 (t, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3): ı: 165.9, 159.8, 138.0, 121.3, 107.6, 59.6,
32.1, 29.0, 28.7, 27.8, 26.9, 14.4.
2.2. General procedure for the synthesis of 2-aminothiophenes
A mixture of 1 (1 mmol), 2 (1 mmol), elemental sulfur (1 mmol)
and BSA (20 mg) was added to 1 mL of DMF. The reaction was incu-
bated at 50 ◦C and 200 rpm. After the required time, the BSA was
filtered off to terminate the reaction. For the products with high
yields, the solid crude products precipitated in water, and then fol-
lowed by filtration and drying. For the products with low yields,
the crude residues were purified by flash column chromatography
on silica gel using petroleum/ethyl acetate.
2.2.7. 2-Amino-4-methylthiophene-3-carbonitrile (3g)
Yellow solid. mp 118 ◦C (lit. [22] mp 118–119 ◦C). IR (KBr): ꢀ.
3419, 3325, 2204 cm−1 1H NMR (400 MHz, CDCl3): ı: 5.96 (s, 1H),
.
4.75 (bs, 2H), 2.19 (s, 3H). 13C NMR (100 MHz, CDCl3): ı: 162.3,
135.8, 115.4, 105.2, 90.8, 15.3.
The structures of the products were confirmed by IR, 1H NMR
and 13C NMR. 1H (400 MHz) and 13C NMR (100 MHz) spectra were
recorded on a Bruker Avance 400 spectrometer in CDCl3 using TMS
(tetramethylsilane) as internal reference. IR spectra were obtained
with a Nicolet Nexus 470 FT-IR spectrophotometer. HPLC was car-
ried out using an Agilent 1100 series with an Agilent TC-C18 column
(3a, 3c, 3e, 3g, 3i, 3k: methanol/water ratio = 60/40, 1.0 mL/min
and 220 nm; 3b, 3d, 3f, 3h, 3g, 3l: methanol/water ratio = 60/40,
1.0 mL/min and 229 nm).
2.2.8. Ethyl 2-amino-4-methylthiophene-3-carboxylate (3h)
Yellow solid. mp 78 ◦C (lit. [23] mp 76–78 ◦C). IR (KBr): ꢀ. 3411,
3301, 1651 cm−1 1H NMR (400 MHz, CDCl3): ı: 6.03 (bs, 2H), 5.83
.
(s, 1H), 4.28 (q, J = 7.2 Hz, 2H), 2.28 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H). 13
C
NMR (100 MHz, CDCl3): ı: 172.7, 160.9, 127.3, 114.7, 104.0, 60.6,
20.1, 13.1.
2.2.9. 2-Amino-4,5-dimethylthiophene-3-carbonitrile (3i)
Yellow solid. mp 142 ◦C (lit. [24] mp 141–142 ◦C). IR (KBr): ꢀ.
For all reactions, solvents for column chromatography were dis-
tilled before use.
3432, 3335, 2200 cm−1 1H NMR (400 MHz, CDCl3): ı: 4.58 (bs, 2H),
.
2.15 (s, 3H), 2.06 (s, 3H). 13C NMR (100 MHz, CDCl3): ı: 158.9, 129.7,
2.2.1. 2-Amino-4,5,6,7-tetrahydrobenzo[b]
thiophene-3-carbonitrile (3a)
117.3, 115.3, 90.9, 22.71, 13.1.
Yellow solid, mp 140–142 ◦C (lit. [19] mp 144–146 ◦C). IR (KBr):
2.2.10. Ethyl 2-amino-4,5-dimethylthiophene-3-carboxylate (3j)
Yellow solid. mp 94 ◦C (lit. [24] mp 91–92 ◦C). IR (KBr): ꢀ. 3403,
ꢀ. 3447, 3329, 2198 cm−1 1H NMR (400 MHz, CDCl3): ı: 4.62 (bs,
.
2H), 2.53–2.46 (m, 4H), 1.85–1.73 (m, 4H). 13C NMR (100 MHz,
3303, 1648 cm−1 1H NMR (400 MHz, CDCl3): ı: 5.89 (bs, 2H), 4.27
;
(q, J = 7.2 Hz, 2H), 2.16 (s, 3H), 2.15 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H). 13
C
CDCl3): ı: 159.9, 132.4, 120.7, 115.5, 88.8, 24.5, 24.1, 23.4, 22.1.
NMR (100 MHz, CDCl3): ı: 166.1, 161.0, 130.4, 113.9, 107.7, 59.5,
14.8, 14.4, 12.3.
2.2.2. Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]
thiophene-3-carboxylate (3b)
Yellow solid, mp 114–115 ◦C (lit. [19] mp 117–118 ◦C). IR (KBr):
2.2.11. 2-Amino-4-ethyl-5-methylthiophene-3-carbonitrile (3k)
Yellow solid. mp 154 ◦C (lit. [25] mp 155–159 ◦C). IR (KBr): ꢀ.
ꢀ. 3403, 3299, 1647 cm−1 1H NMR (400 MHz, CDCl3): ı: 5.93
;
3430, 3321, 2199 cm−1 1H NMR (400 MHz, CDCl3): ı: 4.60 (bs, 2H),
.
(bs, 2H), 4.25 (q, J = 7.2 Hz, 2H), 2.72–2.68 (m, 2H), 2.52–2.46 (m,
2H), 1.82–1.70 (m, 4H), 1.33 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3): ı: 166.1, 161.2, 132.5, 118.0, 106.1, 59.4, 26.9, 24.6, 23.3,
22.8, 14.5.
2.47 (q, J = 7.6 Hz, 2H), 2.16 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H). 13C NMR
(100 MHz, CDCl3): ı: 159.6, 136.4, 118.4, 115.7, 89.1, 21.0, 14.3,
12.2.