Laccase-catalyzed oxidation of Hantzsch 1,4 ... · Laccase-catalyzed oxidation of Hantzsch...
Transcript of Laccase-catalyzed oxidation of Hantzsch 1,4 ... · Laccase-catalyzed oxidation of Hantzsch...
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Laccase-catalyzed oxidation of Hantzsch 1,4-dihydropyridines to
pyridines and a new one pot synthesis of pyridines
Heba T. Abdel-Mohsen, Jürgen Conrad and Uwe Beifuss*
Bioorganische Chemie, Institut für Chemie, Universität Hohenheim
Garbenstr. 30, D-70599 Stuttgart, Germany
Fax: (+49) 711 459 22951; Tel: (+49) 711 459 22171
E-mail: [email protected]
Electronic Supplementary Information Table of contents
1. General remarks
2
2. General procedure I for the synthesis of Hantzsch 1,4-dihydropyridines 4a-k
2
3. Synthesis and analytical data of 1,4-dihydropyridines 4a-k
3
4. General procedures II and III for the laccase-catalyzed oxidation of 1,4-
dihydropyridines 4a-k to the corresponding pyridines 5a-k
19
5. General procedures IV and V for the one pot synthesis of pyridines 5a-k
20
6. Synthesis and analytical data of pyridines 5a-k
21
7. Determination of the laccase activity
48
8. References 48
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1. General remarks
All chemicals and the laccase from Trametes versicolor (Fluka) were purchased from
commercial suppliers. Solvents used in extraction and purification were distilled prior to use.
Aldehydes and 1,3-dicarbonyl compounds used in the reactions were freshly distilled before use.
The pH of the buffer was adjusted using a pH 330/SET-1 pH-meter. Reaction temperatures are
reported as bath temperatures. Analytical thin layer chromatography (TLC) was performed on
precoated silica gel 60 F245 aluminium plates (Merck) with visualization under UV light and by
immersion in ethanolic vanillin solution followed by heating. Flash chromatography was carried
out on silica gel MN 60, 0.04-0.053 mm (Macherey & Nagel). Melting points were determined
on a Büchi melting point apparatus B-545 with open capillary tubes and are uncorrected. UV/VIS
spectra were recorded with a Varian Cary 50. IR spectra were measured on a Perkin-Elmer
Spectrum One (FT-IR-spectrometer). 1H and 13C NMR spectra were recorded at 300 (75) MHz
on a Varian UnityInova using CDCl3 or DMSO-d6. The chemical shifts were referenced to the
solvent signals at δH/C 2.49 / 39.50 ppm (DMSO-d6) and 7.26 / 77.00 ppm (CDCl3) relative to
TMS as internal standards. Coupling constants J [Hz] were directly taken from the spectra and
are not averaged. Splitting patterns are designated as s (singlet), d (doublet), t (triplet), q
(quartet), sep (septet) and m (multiplet). Low resolution electron impact mass spectra (EI-LRMS)
and exact electron impact mass spectra (HRMS) were recorded at 70 eV on a Finnigan MAT 95
instrument. The intensities are reported as percentages relative to the base peak (I = 100%).
2. General procedure I for the synthesis of Hantzsch 1,4-dihydropyridines 4a-k 1
An oven-dried 10 mL vial with a magnetic stirrer bar was charged with a mixture of an aldehyde
1 (1 mmol), a 1,3-dicarbonyl compound 2 (2 mmol) and ammonium acetate (3) (1.5 mmol). The
vial was sealed and the reaction mixture was stirred at 80 oC for the time given. After completion
of the reaction, the solid crude product was isolated upon addition of ice-cold water (10 mL)
followed by scratching. The precipitate was filtered and further purified by recrystallization from
methanol to yield the 1,4-dihydropyridines 4a-k.
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3. Synthesis and analytical data of 1,4-dihydropyridines 4a-k
3.1. Synthesis and analytical data of dimethyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4a). According to the general procedure I, paraformaldehyde (1a) (30 mg, 1
mmol), methyl acetoacetate (2a) (232 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5
mmol) were reacted for 10 min. Purification afforded dimethyl-1,4-dihydro-2,6-
dimethylpyridine-3,5-dicarboxylate (4a) (160 mg, 71%) as canary yellow powder; mp 210 - 212 oC (from MeOH) (lit.,2,3 208 - 210 oC); Rf = 0.29 (petroleum ether / EtOAc = 2:1); ṽmax (atr)/cm-1
3350 (NH), 2982, 2962 and 2873 (C_H), 1698 and 1647 (C=O), 1626 (C=C), 1203 and 1073
(C_O); δH (300 MHz; DMSO-d6) 2.10 (6H, s, 1´-H), 3.12 (2H, s, 4-H), 3.57 (6H, s, 2´´-H) and
8.30 (1H, s, 1-H); δC (75 MHz; DMSO-d6) 17.80 (C-1´), 24.78 (C-4), 50.60 and 50.62 (C-2´´),
96.88 (C-3 and C-5), 146.77 (C-2 and C-6) and 167.45 (C-1´´), m/z (EI, 70 eV) 225 (M+, 23%),
224 (50, M+ - 1), 210 (100, M+ - CH3) and 194 (33).
1.000
5.429
2.057
5.678
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
8.3005
3.5700
3.3261
3.1170
2.0991
NH
OO
O O
1
2
34
5
6
1´´1´´ 2´´2´´
1´1´
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PPM 180 160 140 120 100 80 60 40 20 0
167.4506
146.7672
96.8813
50.617850.5995
40.333740.056139.778139.500039.221738.943338.6647
24.7765
17.8019
Fig. 1 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4a in DMSO-d6. 3.2. Synthesis and analytical data of dimethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-
dicarboxylate (4b). According to the general procedure I, acetaldehyde (1b) (66 mg, 1.5 mmol),
methyl acetoacetate (2a) (232 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were
reacted for 30 min. Purification afforded dimethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-
dicarboxylate (4b) (180 mg, 75%) as white crystals; mp 153 - 155 oC (from MeOH) (lit.,2,3 152 -
154 oC); Rf = 0.41 (petroleum ether / EtOAc = 2:1); ṽmax(atr)/cm-1 3340 (NH), 2950 (C_H), 1677
and 1647 (C=O), 1630 (C=C), 1222 and 1054 (C_O); δH (300 MHz; CDCl3) 0.95 (3H, d, 3J 1´´´-H,
4-H 6.6 Hz, 1´´´-H), 2.27 (6H, s, 1´-H), 3.71 (6H, s, 2´´-H), 3.80 (1H, q, 3J 4-H, 1´´´-H 6.6 Hz, 4-H)
and 5.65 (1H, s, 1-H); δC (75 MHz; CDCl3) 19.40 (C-1´), 22.25 (C-1´´´), 28.41 (C-4), 50.94 and
50.95 (C-2´´), 104.41 (C-3 and C-5), 144.63 (C-2 and C-6) and 168.23 (C-1´´); m/z (EI, 70 eV)
239 (M+, 2%), 224 (100, M+ - CH3), 208 (37, M+ - OCH3) and 192 (17).
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1.000
1.1325.653
5.829
3.100
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.2600
5.6542
3.81503.79333.7097
2.2646
0.96110.9394
0.0603
PPM 180 160 140 120 100 80 60 40 20 0
168.2299
144.6245
104.4104
77.423877.000076.5761
50.954450.9407
28.4051
22.253419.3960
0.9877
Fig. 2 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4b in CDCl3.
NH
OO
O O
1
2
34
5
6
1´´1´´ 2´´2´´
1´1´
1´´´
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3.3. Synthesis and analytical data of diethyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4c). According to the general procedure I, paraformaldehyde (1a) (30 mg, 1
mmol), ethyl acetoacetate (2b) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol)
were reacted for 15 min. Purification afforded diethyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4c) (200 mg, 79%) as canary yellow powder; mp 172 - 174 oC (from MeOH)
(lit.,2,3 170 - 172 oC); Rf = 0.39 (petroleum ether / EtOAc = 2:1); ṽmax(atr)/cm-1 3347 (NH), 2988,
2939 and 2897 (C_H), 1691 and 1647 (C=O), 1629 (C=C), 1220 and 1055 (C_O); δH (300 MHz;
CDCl3) 1.28 (6H, t, 3J 3´´-H, 2´´-H 7.2 Hz, 3´´-H), 2.19 (6H, s, 1´-H), 3.26 (2H, s, 4-H), 4.17 (4H, q, 3J 2´´-H, 3´´-H 7.2 Hz, 2´´-H) and 5.20 (1H, s, 1-H); δC (75 MHz; CDCl3) 14.45 (C-3´´), 19.15 (C-
1´), 24.78 (C-4), 59.64 (C-2´´), 99.51 (C-3 and C-5), 144.78 (C-2 and C-6) and 168.05 (C-1´´);
m/z (EI, 70 eV) 253 (M+, 21%), 252 (30, M+ - 1), 224 (100) and 196 (86).
1.000
3.292
1.810
4.957
8.287
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.2600
4.20274.17904.15534.1316
3.2642
2.1896
1.5669
1.30621.28251.2586
0.06710.0551
3´´ 3´´1´´1´´ 2´´2´´
1´1´ 1
2
34
5
6
NH
OO
O O
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PPM 180 160 140 120 100 80 60 40 20 0
168.0457
144.7844
99.5064
77.424377.000076.5758
59.6358
24.7766
19.1503
14.4491
Fig. 3 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4c in CDCl3. 3.4. Synthesis and analytical data of diethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-
dicarboxylate (4d). According to the general procedure I, acetaldehyde (1b) (66 mg, 1.5 mmol),
ethyl acetoacetate (2b) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were
reacted for 15 min. Purification afforded diethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-
dicarboxylate (4d) (188 mg, 70%) as white crystals; mp 128 - 130 oC (from MeOH) (lit.,2,3 128 -
130 oC); Rf = 0.43 (petroleum ether / EtOAc = 2:1); ṽmax(atr)/cm-1 3343 (NH), 2984, 2963 and
2905 (C_H), 1695 (C=O), 1639 (C=C), 1222 and 1056 (C_O); δH (300 MHz; CDCl3) 0.96 (3H, d, 3J 1´´´-H, 4-H 6.6 Hz, 1´´´-H), 1.29 (6H, t, 3J 3´´-H, 2´´-H 7.2 Hz, 3´´-H), 2.26 (6H, s, 1´-H), 3.83 (1H, q, 3J 4-H, 1´´´-H 6.6 Hz, 4-H), 4.15 (2H, dq, 2J 2´´a-H, 2´´b-H 10.9 Hz,
3J 2´´a-H, 3´´H 7.2 Hz, 2´´a-H), 4.21 (2H,
dq, 2J 2´´a-H, 2´´b-H 10.9 Hz, 3J 2´´b-H, 3´´H 7.2 Hz, 2´´b-H) and 5.55 (1H, s, 1-H); δC (75 MHz; CDCl3)
14.41 (C-3´´), 19.48 (C-1´), 22.22 (C-1´´´), 28.50 (C-4), 59.56 and 59.57 (C-2´´), 104.70 (C-3
and C-5), 144.21 (C-2 and C-6) and 167.83 (C-1´´); m/z (EI, 70 eV) 265 (M+ - 2, 5%), 252 (100,
M+ - CH3), 224 (34) and 196 (37).
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1.000
3.908
0.963
5.984
6.139
3.495
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.2600
5.5543
4.24374.23134.21994.20754.18354.18034.15654.14404.13284.12033.83773.8161
2.25812.1655
1.6255
1.31221.28851.26481.24950.97590.9543
0.0630
PPM 180 160 140 120 100 80 60 40 20 0
167.8329
144.2099
104.6986
77.423677.043477.000076.5763
59.571359.5584
28.4992
22.220819.4747
14.4135
0.9919
Fig. 4 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4d in CDCl3.
3´´ 3´´1´´1´´
2´´a
1´1´ 1
2
34
5
6
NH
OO
O O HH2´´b
H H2´´a2´´b 1´´´
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3.5. Synthesis and analytical data of diethyl-1,4-dihydro-2,6-dimethyl-4-phenylpyridine-3,5-
dicarboxylate (4e). According to the general procedure I, benzaldehyde (1c) (106 mg, 1 mmol),
ethyl acetoacetate (2b) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were
reacted for 180 min. Purification afforded diethyl-1,4-dihydro-2,6-dimethyl-4-phenylpyridine-
3,5-dicarboxylate (4e) (148 mg, 45%) as white crystals; mp 160 - 162 oC (from MeOH) (lit.,2,3
158 - 160 oC); Rf = 0.49 (petroleum ether / EtOAc = 2:1); ṽmax(atr)/cm-1 3339 (NH), 3060 and
2982 (C_H), 1686 and 1649 (C=O), 1620 (C=C), 1207 and 1090 (C_O); δH (300 MHz; CDCl3)
1.22 (6H, t, 3J 3´´-H, 2´´-H 7.2 Hz, 3´´-H), 2.33 (6H, s, 1´-H), 4.07 (2H, dq, 2J 2´´a-H, 2´´b-H 10.9 Hz, 3J
2´´a-H, 3´´H 7.0 Hz, 2´´a-H), 4.10 (2H, dq, 2J 2´´a-H, 2´´b-H 10.9 Hz, 3J 2´´b-H, 3´´H 7.1 Hz, 2´´b-H), 4.99
(1H, s, 4-H), 5.63 (1H, s, 1-H), 7.11-7.14 (1H, m, 4´´´-H), 7.18-7.22 (2H, m, 3´´´-H and 5´´´-H)
and 7.26-7.30 (2H, m, 2´´´-H and 6´´´-H); δC (75 MHz; CDCl3) 14.22 (C-3´´), 19.58 (C-1´),
39.62 (C-4), 59.70 (C-2´´), 104.19 (C-3 and C-5), 126.07 (C-4´´´), 127.80 (C-3´´´ and C-5´´´),
127.99 (C-2´´´ and C-6´´´), 143.77 (C-2 and C-6), 147.73 (C-1´´´) and 167.60 (C-1´´); m/z (EI,
70 eV) 329 (M+, 12%), 300 (15, M+ - C2H5), 284 (18, M+ - OC2H5), 252 (100, M+ - C6H5), 224
(36) and 196 (45).
1.000
1.165
4.496
6.948
6.466
1.7832.1011.031
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.29487.28967.28337.26627.26237.26007.22387.21747.20037.19527.17527.14227.13747.13267.1135
5.6248
4.9886
4.13784.12554.11484.10174.09154.07794.06794.05444.0442
2.3265
1.6014
1.23971.21591.1922
3´´ 3´´1´´1´´
2´´a
1´1´ 1
2
34
5
6
NH
OO
O O HH2´´b
H H2´´a2´´b
1´´´
2´´´
5´´´
4´´´
3´´´
6´´´
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PPM 180 160 140 120 100 80 60 40 20 0
167.5962
147.7269143.7665
127.9878127.8043126.0706
104.1934
77.423577.000076.5757
59.6950
39.616139.6062
19.5775
14.2235
Fig. 5 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4e in CDCl3. 3.6. Synthesis and analytical data of di-isopropyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4f). According to the general procedure I, paraformaldehyde (1a) (30 mg, 1
mmol), isopropyl acetoacetate (2c) (288 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5
mmol) were reacted for 30 min. Purification afforded di-isopropyl-1,4-dihydro-2,6-
dimethylpyridine-3,5-dicarboxylate (4f) (196 mg, 70%) as pale yellow crystals; mp 119 - 121 oC
(from MeOH) (lit.,4 123 - 125 oC); Rf = 0.48 (petroleum ether / EtOAc = 2:1); ṽmax(atr)/cm-1 3359
(NH), 2979, 2935 and 2878 (C_H), 1667 and 1644 (C=O), 1630 (C=C), 1232 and 1106 (C_O); δH
(300 MHz; CDCl3) 1.25 (12H, d, 3J 3´´-H, 2´´-H 6.3 Hz, 3´´-H), 2.18 (6H, s, 1´-H), 3.24 (2H, s, 4-H),
5.03 (2H, sep, 3J 2´´-H, 3´´-H 6.3 Hz, 2´´-H) and 5.06 (1H, s, 1-H); δC (75 MHz; CDCl3) 19.23 (C-
1´), 22.08 (C-3´´), 24.84 (C-4), 66.78 and 66.80 (C-2´´), 99.86 (C-3 and C-5), 144.36 (C-2 and C-
6) and 167.63 (C-1´´); m/z (EI, 70 eV) 281 (M+, 4%), 280 (17, M+ - 1), 279 (84, M+ - 2) and 28
(100).
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1.000
1.320
2.873
5.861
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.2600
5.06055.03965.0188
3.2353
2.1748
1.26461.2438
PPM 180 160 140 120 100 80 60 40 20 0
167.6289
144.3645
99.8563
77.424277.000076.5759
66.796766.7843
24.835622.080819.2299
Fig. 6 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4f in CDCl3.
1
2
34
5
6
NH
OO
O O3´´ 3´´
1´´1´´2´´ 2´´
1´1´
3´´3´´
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3.7. Synthesis and analytical data of di-tert-butyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4g). According to the general procedure I, paraformaldehyde (1a) (30 mg, 1
mmol), tert-butyl acetoacetate (2d) (316 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5
mmol) were reacted for 30 min. Purification afforded di-tert-butyl-1,4-dihydro-2,6-
dimethylpyridine-3,5-dicarboxylate (4g) (246 mg, 80%) as white crystals; mp 151 - 153 oC (from
MeOH) (lit.,4 152 - 154 oC); Rf = 0.64 (petroleum ether / EtOAc = 2:1); ṽmax(atr)/cm-1 3336 (NH),
2958 (C_H), 1694 and 1650 (C=O), 1639 (C=C), 1203 and 1092 (C_O); δH (300 MHz, CDCl3)
1.48 (18H, s, 3´´-H), 2.14 (6H, s, 1´-H), 3.17 (2H, s, 4-H) and 5.02 (1H, s, 1-H); δC (75 MHz,
CDCl3) 19.20 (C-1´), 25.42 (C-4), 28.19 and 28.36 (C-3´´), 79.41 (C-2´´), 100.89 (C-3 and C-5),
143.72 (C-2 and C-6) and 167.57 (C-1´´); m/z (EI, 70 eV) 309 (M+, 68%), 308 (100, M+ - 1), 307
(59, M+ - 2) and 292 (60).
1.000
2.745
8.647
0.479
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.2600
3.1723
2.1433
1.60381.4769
1
2
34
5
6
NH
OO
O O3´´ 3´´
1´´1´´2´´ 2´´
1´1´
3´´3´´
3´´3´´
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PPM 180 160 140 120 100 80 60 40 20 0
167.5719
143.7198
100.8852
79.406577.423877.000076.5758
28.363428.188125.4161
19.1955
1.0016
Fig. 7 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4g in CDCl3.
3.8. Synthesis and analytical data of diallyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4h). According to the general procedure I, paraformaldehyde (1a) (30 mg, 1
mmol), allyl acetoacetate (2e) (284 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol)
were reacted for 10 min. Purification afforded diallyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4h) (200 mg, 72%) as yellow powder; mp 164 - 166 oC (from MeOH); Rf = 0.50
(petroleum ether / EtOAc = 2:1); λmax(MeCN)/nm 366 (log ε, 3.81) and 228 (4.18); ṽmax(atr)/cm-1
3345 (NH), 3085, 2985, 2941 and 2879 (C_H), 1694 and 1655 (C=O), 1627 (C=C), 1211 and
1085 (C_O); δH (300 MHz; CDCl3) 2.20 (6H, s, 1´-H), 3.33 (2H, s, 4-H), 4.62 (4H, dt, 3J 2´´-H, 3´´-H
5.4 Hz, 4J 1.6 Hz, 2´´-H), 5.20 (2H, dq, 2J 1.4 Hz, 3Jcis 4´´a-H, 3´´-H 10.5 Hz, 4J 1.4 Hz, 4´´a-H), 5.31
(2H, dq, 2J 1.7 Hz, 3Jtrans 4´´b-H, 3´´-H 17.4 Hz, 4J 1.7 Hz, 4´´b-H) and 5.96 (2H, ddt, 3J 3´´-H, 2´´-H 5.4
Hz, 3Jcis 3´´-H, 4´´a-H 10.5 Hz, 3Jtrans 3´´-H, 4´´b-H 17.4 Hz, 3´´H); δC (75 MHz; CDCl3) 19.21 (C-1´),
24.73 (C-4), 64.38 (C-2´´), 99.33 (C-3 and C-5), 117.17 (C-4´´), 132.92 (C-3´´), 145.26 (C-2 and
C-6) and 167.50 (C-1´´); ´´); m/z (EI, 70 eV) 277 (M+, 7%), 275 (15, M+ - 2), 236 (44, M+ -
CH2CHCH2), 218 (75), 191 (23), 41 (93, CH2CHCH2) and 28 (100); HRMS (EI, M+) found:
277.1275 calcd. for C15H19NO4: 277.1314 .
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1.000
2.755
2.398
1.321
3.767
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.2600
5.99805.98075.96315.95885.94495.94065.92325.90575.34325.33815.33285.32765.28565.28065.27535.27025.22355.21925.21465.21025.18865.18435.17975.17534.63304.62824.62334.61504.61024.6053
3.3297
2.1975
PPM 180 160 140 120 100 80 60 40 20 0
167.4955
145.2580
132.9191
117.1717
99.3286
77.423977.000076.5760
64.3664
24.7260
19.1885
Fig. 8 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4h in CDCl3.
1
2
34
5
6
3´´ 3´´
1´´1´´ 2´´2´´
1´1´ NH
OO
O O4´´b4´´b
H
H
H
H
H
H4´´a4´´a
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3.9. Synthesis and analytical data of dimethyl-2,6-diethyl-1,4-dihydropyridine-3,5-
dicarboxylate (4i). According to the general procedure I, paraformaldehyde (1a) (30 mg, 1
mmol), methyl propionyl acetate (2f) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5
mmol) were reacted for 15 min. Purification afforded dimethyl-2,6-diethyl-1,4-dihydropyridine-
3,5-dicarboxylate (4i) (227 mg, 90%) as yellow powder; mp 129 - 131 oC (from MeOH); Rf =
0.64 (petroleum ether / EtOAc = 2:1); λmax(MeCN)/nm 363 (log ε, 3.64), 230 (4.14) and 206
(4.14); ṽmax(atr)/cm-1 3350 (NH), 2982, 2962 and 2873 (C_H), 1697 and 1646 (C=O), 1625
(C=C), 1203 and 1072 (C_O); δH (300 MHz; CDCl3) 1.15 (6H, t, 3J 2´H, 1´H 7.5 Hz, 2´-H), 2.61
(4H, q, 3J 1´H, 2´H 7.5 Hz, 1´-H), 3.27 (2H, s, 4-H), 3.70 (6H, s, 2´´-H) and 5.31 (1H, s, 1-H); δC
(75 MHz; CDCl3) 12.54 (C-2´), 24.86 (C-4), 25.62 (C-1´), 50.98 (C-2´´), 98.21 (C-3 and C-5),
150.96 (C-2 and C-6) and 167.86 (C-1´´); m/z (EI, 70 eV) 253 (M+, 24%), 252 (78, M+ - 1), 238
(100, M+ - CH3), 222 (35, M+ - OCH3) and 194 (13, M+ - COOCH3); HRMS (EI, M+) found:
253.1308 calcd. for C13H19NO4: 253.1314.
0.999
6.029
2.195
4.354
5.995
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.2600
5.3113
3.6955
3.2655
2.64942.62422.59912.5740
1.17021.14511.1199
0.0603
1
1´´1´´2´´2´´
1´1´
2
34
5
6
NH
OO
O O
2´ 2´
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PPM 180 160 140 120 100 80 60 40 20 0
167.8558
150.9587
98.2131
77.424377.000076.5762
50.9784
25.614524.8553
12.5421
Fig. 9 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4i in CDCl3. 3.10. Synthesis and analytical data of diethyl-2,6-diethyl-1,4-dihydropyridine-3,5-
dicarboxylate (4j). According to the general procedure I, paraformaldehyde (1a) (30 mg, 1
mmol), ethyl propionyl acetate (2g) (288 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5
mmol) were reacted for 40 min. Purification afforded diethyl-2,6-diethyl-1,4-dihydropyridine-
3,5-dicarboxylate (4j) (196 mg, 70%) as yellow powder; mp 106 - 108 oC (from MeOH); Rf =
0.70 (petroleum ether / EtOAc = 2:1); λmax(MeCN)/nm 363 (log ε, 3.67), 229 (4.13) and 203
(4.05); ṽmax(atr)/cm-1 3344 (NH), 2980, 2935 and 2873 (C_H), 1694 and 1645 (C=O), 1625
(C=C), 1193 and 1071 (C_O); δH (300 MHz; CDCl3) 1.14 (6H, t, 3J 2´H, 1´H 7.5 Hz, 2´-H), 1.27
(6H, t, 3J 3´´H, 2´´H 6.9 Hz, 3´´-H), 2.59 (4H, q, 3J 1´H, 2´H 7.8 Hz, 1´-H), 3.26 (2H, s, 4-H), 4.16 (4H,
q, 3J 2´´H, 3´´H 7.2 Hz, 2´´-H) and 5.33 (1H, s, 1-H); δC (75 MHz; CDCl3) 12.62 (C-2´), 14.39 (C-
3´´), 24.85 (C-4), 25.72 (C-1´), 59.58 (C-2´´), 98.44 (C-3 and C-5), 150.62 (C-2 and C-6) and
167.55 (C-1´´); m/z (EI, 70 eV) 281 (M+, 9%), 238 (48), 220 (28) and 195 (100); HRMS (EI, M+)
found: 281.1581 calcd. for C15H23NO4: 281.1627.
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1.000
3.610
2.048
3.988
5.4245.464
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
5.3276
4.19164.16794.14414.1204
3.2552
2.63152.60642.58132.5562
1.29321.26941.24571.16631.14131.1161
PPM 180 160 140 120 100 80 60 40 20 0
167.5539
150.6212
98.4410
77.424177.000076.5757
59.5844
25.722024.8541
14.392712.6175
Fig. 10 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4j in CDCl3.
1
3´´ 3´´1´´1´´ 2´´2´´
1´1´
2
34
5
6
NH
OO
O O
2´ 2´
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3.11. Synthesis and analytical data of 2,6-dimethyl-3,5-diacetyl-1,4-dihydropyridine (4k).
According to the general procedure I, paraformaldehyde (1a) (30 mg, 1 mmol), acetylacetone
(2h) (200 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for 15 min.
Purification afforded 2,6-dimethyl-3,5-diacetyl-1,4-dihydropyridine (4k) (145 mg, 75%) as
yellow powder; mp 213 - 215 oC (from MeOH) (lit.,5 216 - 220 oC); Rf = 0.25 (petroleum ether /
EtOAc = 1:2); ṽmax(atr)/cm-1 3382 (NH), 2920 (C_H), 1668 and 1643 (C=O) and 1588 (C=C); δH
(300 MHz; DMSO-d6) 2.09 (6H, s, 1´-H), 2.12 (6H, s, 2´´-H), 3.25 (2H, s, 4-H) and 8.27 (1H, s,
1-H); δC (75 MHz; DMSO-d6) 18.67 (C-1´), 26.39 (C-4), 30.10 (C-2´´), 107.62 (C-3 and C-5),
145.24 (C-2 and C-6) and 196.65 (C-1´´); m/z (EI, 70 eV) 193 (M+, 45%), 192 (63, M+ - 1), 178
(50, M+ - CH3), 150 (23, M+ - COCH3), 106 (23) and 43 (38, COCH3).
1.000
5.4302.013
11.45
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
8.2667
3.33203.2493
2.12072.0875
NH
O O
2
34
5
6
1´´1´´2´´2´´
1´1´1
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PPM 180 160 140 120 100 80 60 40 20 0
196.6439
145.2339
107.6176
40.331540.056339.777739.500039.221738.941038.666330.094626.3835
18.6688
Fig. 11 1H (300 MHz) and 13C (75 MHz) NMR spectra of 4k in DMSO-d6.
4. General procedures II and III for the laccase-catalyzed oxidation of 1,4-dihydropyridines
4a-k to the corresponding pyridines 5a-k
4.1. General procedure II for the laccase-catalyzed oxidation of 1,4-dihydropyridines 4a-k
to the corresponding pyridines 5a-k (method A)
A 100 mL round bottomed flask with a magnetic stirrer bar was charged with a solution of 1,4-
dihydropyridine 4 (1 mmol) in methanol (3 - 6 mL). Acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase from Trametes versicolor (168 U, 12 mg) and ABTS diammonium salt (6.9 mg, 0.0125
mmol) were added and the reaction mixture was stirred at 50 oC for the time given. After
extraction with CH2Cl2 (7 × 20 mL), the combined organic phases were dried over anhydrous
Na2SO4, filtered and the solvents were evaporated in vacuo. The crude product was purified by
flash chromatography on SiO2 (petroleum ether / EtOAc = 6:1). It was found that 2-butanone can
be also used for extraction.
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4.2. General procedure III for the laccase-catalyzed oxidation of 1,4-dihydropyridines 4a-k
to the corresponding pyridines 5a-k (method B)
A 100 mL round bottomed flask with a magnetic stirrer bar was charged with a solution of 1,4-
dihydropyridine 4 (1 mmol) in methanol (3 - 6 mL). Acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase from Trametes versicolor (168 U, 12 mg) and ABTS diammonium salt (54.9 mg, 0.1
mmol) were added and the reaction mixture was stirred at 50 oC for the time given. After
extraction with CH2Cl2 (7 × 20 mL), the combined organic phases were dried over anhydrous
Na2SO4, filtered and the solvents were evaporated in vacuo. The crude product was purified by
flash chromatography on SiO2 (petroleum ether / EtOAc = 6:1). It was found that 2-butanone can
be also used for extraction.
5. General procedures IV and V for the one pot synthesis of pyridines 5a-k
5.1. General procedure IV for the one pot synthesis of pyridines 5a-k (method C)
A 100 mL round bottomed flask with a magnetic stirrer bar was charged with an aldehyde 1 (1
mmol), a 1,3-dicarbonyl compound 2 (2 mmol) and ammonium acetate (3) (1.5 mmol). The
mixture was stirred at 80 oC until the starting materials were consumed (TLC). After cooling to
room temperature, the mixture was dissolved in methanol (3 - 6 mL). Acetate buffer (0.2 M, pH
4.37, 30 mL), laccase from Trametes versicolor (168 U, 12 mg) and ABTS diammonium salt (6.9
mg, 0.0125 mmol) were added and the mixture was stirred at 50 oC for the time given. The
reaction mixture was extracted with CH2Cl2 (7 × 20 mL) and the combined organic phases were
dried over anhydrous Na2SO4, filtered and the solvents were evaporated in vacuo. The crude
product was purified by flash chromatography on SiO2 (petroleum ether / EtOAc = 6:1). It was
found that 2-butanone can be also used for extraction.
5.2. General procedure V for the one pot synthesis of pyridines 5a-k (method D)
A 100 mL round bottomed flask with a magnetic stirrer bar was charged with an aldehyde 1 (1
mmol), a 1,3-dicarbonyl compound 2 (2 mmol) and ammonium acetate (3) (1.5 mmol). The
mixture was stirred at 80 oC until the starting materials were consumed (TLC). After cooling to
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room temperature, the mixture was dissolved in methanol (3 - 6 mL). Acetate buffer (0.2 M, pH
4.37, 30 mL), laccase from Trametes versicolor (168 U, 12 mg) and ABTS diammonium salt
(54.9 mg, 0.1 mmol) were added and the mixture was stirred at 50 oC for the time given. The
reaction mixture was extracted with CH2Cl2 (7 × 20 mL) and the combined organic phases were
dried over anhydrous Na2SO4, filtered and the solvents were evaporated in vacuo. The crude
product was purified by flash chromatography on SiO2 (petroleum ether / EtOAc = 6:1). It was
found that 2-butanone can be also used for extraction.
6. Synthesis and analytical data of pyridines 5a-k
6.1. Synthesis and analytical data of dimethyl-2,6-dimethylpyridine-3,5-dicarboxylate (5a)
Synthesis of 5a
According to the general procedure II, dimethyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4a) (225 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 5 h. Purification afforded dimethyl-2,6-dimethylpyridine-3,5-dicarboxylate (5a)
(209 mg, 94%).
According to the general procedure III, dimethyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4a) (225 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 5 h. Purification afforded dimethyl-2,6-dimethylpyridine-3,5-dicarboxylate (5a)
(212 mg, 95%).
According to the general procedure IV, paraformaldehyde (1a) (30 mg, 1 mmol), methyl
acetoacetate (2a) (232 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 10 min. The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg,
0.0125 mmol) for 7 h. Purification afforded dimethyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5a) (133 mg, 60%).
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According to the general procedure V, paraformaldehyde (1a) (30 mg, 1 mmol), methyl
acetoacetate (2a) (232 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 10 min. The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9
mg, 0.1 mmol) for 6 h. Purification afforded dimethyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5a) (130 mg, 58 %).
Analytical data of 5a
White powder; mp 98 - 100 oC (lit.,6 101 - 103 oC); Rf = 0.49 (petroleum ether / EtOAc = 2:1);
ṽmax(atr)/cm-1 2970 (C_H), 1721 (C=O), 1595 and 1547 (C=C, C=N), 1227 and 1104 (C_O); δH
(300 MHz; CDCl3) 2.84 (6H, s, 1´-H), 3.92 (6H, s, 2´´-H) and 8.69 (1H, s, 4-H); δC (75 MHz;
CDCl3) 24.92 (C-1´), 52.28 (C-2´´), 122.60 (C-3 and C-5), 141.04 (C-4), 162.61 (C-2 and C-6)
and 166.21 (C-1´´); m/z (EI, 70 eV) 223 (M+, 98%), 192 (100, M+ - OCH3) and 164 (61).
1.000
8.271
8.127
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
8.6920
3.9200
2.8423
N
OO
O O
2
34
5
6
1´´1´´ 2´´2´´
1´1´
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PPM 180 160 140 120 100 80 60 40 20 0
166.2060162.6093
141.0348
122.5999
77.424177.000076.5760
52.2779
24.9179
Fig. 12 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5a in CDCl3.
6.2. Synthesis and analytical data of dimethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate (5b) Synthesis of 5b According to the general procedure II, dimethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-
dicarboxylate (4b) (239 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 4 h. Purification afforded dimethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate
(5b) (213 mg, 90%).
According to the general procedure III, dimethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-
dicarboxylate (4b) (239 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 8 h. Purification afforded dimethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate
(5b) (189 mg, 80%).
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According to the general procedure IV, acetaldehyde (1b) (66 mg, 1.5 mmol), methyl
acetoacetate (2a) (232 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 30 min. The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg,
0.0125 mmol) for 7 h. Purification afforded dimethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate
(5b) (142 mg, 60%).
According to the general procedure V, acetaldehyde (1b) (66 mg, 1.5 mmol), methyl acetoacetate
(2a) (232 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for 30 min.
The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
for 9 h. Purification afforded dimethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate (5b) (118 mg,
50%).
Analytical data of 5b
White powder; mp 78 - 80 oC (lit.,7 78 - 80 oC); Rf = 0.48 (petroleum ether / EtOAc = 2:1);
ṽmax(atr)/cm-1 2980 (C_H), 1713 (C=O), 1558 (C=C, C=N), 1220 and 1043 (C_O); δH (300 MHz;
CDCl3) 2.24 (3H, s, 1´´´-H), 2.50 (6H, s, 1´-H) and 3.92 (6H, s, 2´´-H); δC (75 MHz; CDCl3)
17.11 (C-1´´´), 23.01 (C-1´), 52.40 (C-2´´), 127.27 (C-3 and C-5), 142.35 (C-4), 155.22 (C-2 and
C-6) and 168.84 (C-1´´); m/z (EI, 70 eV) 237 (M+, 30%), 222 (33, M+ - CH3), 206 (56) and 28
(100).
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1.000
2.206
2.189
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.2600
3.9231
2.4990
2.2398
PPM 180 160 140 120 100 80 60 40 20 0
168.8429
155.2195
142.3494
127.2738
77.424177.000076.576576.5351
52.3966
23.0112
17.1054
Fig. 13 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5b in CDCl3.
N
OO
O O
2
34
5
6
1´´1´´ 2´´2´´
1´1´
1´´´
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6.3. Synthesis and analytical data of diethyl-2,6-dimethylpyridine-3,5-dicarboxylate (5c)
Synthesis of 5c
According to the general procedure II, diethyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4c) (253 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 5 h. Purification afforded diethyl-2,6-dimethylpyridine-3,5-dicarboxylate (5c)
(208 mg, 83%).
According to the general procedure III, diethyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4c) (253 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 5 h. Purification afforded diethyl-2,6-dimethylpyridine-3,5-dicarboxylate (5c)
(225 mg, 90%).
According to the general procedure IV, paraformaldehyde (1a) (30 mg, 1 mmol), ethyl
acetoacetate (2b) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 15 min. The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg,
0.0125 mmol) for 5 h. Purification afforded diethyl-2,6-dimethylpyridine-3,5-dicarboxylate (5c)
(133 mg, 53%).
According to the general procedure V, paraformaldehyde (1a) (30 mg, 1 mmol), ethyl
acetoacetate (2b) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 15 min. The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9
mg, 0.1 mmol) for 5 h. Purification afforded diethyl-2,6-dimethylpyridine-3,5-dicarboxylate (5c)
(168 mg, 67%).
Analytical data of 5c
White powder; mp 69 - 71 oC (lit.,6 70 - 71 oC); Rf = 0.63 (petroleum ether / EtOAc = 2:1);
ṽmax(atr)/cm-1 2920 (C_H), 1714 (C=O), 1580 (C=C, C=N), 1221 and 1042 (C_O); δH (300 MHz;
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CDCl3) 1.40 (6H, t, 3J 3´´-H, 2´´-H 7.2 Hz, 3´´-H), 2.83 (6H, s, 1´-H), 4.38 (4H, q, 3J 2´´-H, 3´´-H 7.2 Hz,
2´´-H) and 8.66 (1H, s, 4-H); δC (75 MHz; CDCl3) 14.25 (C-3´´), 24.94 (C-1´), 61.38 (C-2´´),
123.05 (C-3 and C-5), 140.88 (C-4), 162.20 (C-2 and C-6) and 165.95 (C-1´´); m/z (EI, 70 eV)
251 (M+, 82%), 206 (100) and 195 (22).
1.000
5.205
8.124
7.649
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.08.6601
7.2600
4.42094.39714.37334.3495
2.8342
1.7122
1.42751.40371.3799
0.0604
3´´ 3´´1´´1´´ 2´´2´´
1´1´
2
34
5
6
N
OO
O O
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PPM 180 160 140 120 100 80 60 40 20 0
165.9472162.1957
140.8770
123.0463
77.423877.000076.5759
61.3756
24.9353
14.2503
Fig. 14 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5c in CDCl3.
6.4. Synthesis and analytical data of diethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate (5d)
Synthesis of 5d
According to the general procedure II, diethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-
dicarboxylate (4d) (267 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 6 h. Purification afforded diethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate (5d)
(230 mg, 87%).
According to the general procedure III, diethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-
dicarboxylate (4d) (267 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 5 h. Purification afforded diethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate (5d)
(238 mg, 90%).
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According to the general procedure IV, acetaldehyde (1b) (66 mg, 1.5 mmol), ethyl acetoacetate
(2b) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for 15 min.
The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor), ABTS diammonium salt (6.9 mg, 0.0125 mmol) for
6 h. Purification afforded diethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate (5d) (148 mg, 56%).
According to the general procedure V, acetaldehyde (1b) (66 mg, 1.5 mmol), ethyl acetoacetate
(2b) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for 15 min.
The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor), ABTS diammonium salt (54.9 mg, 0.1 mmol) for 6
h. Purification afforded diethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate (5d) (161 mg, 61%).
Analytical data of 5d
Colourless oil (lit.,6 oil); Rf = 0.66 (petroleum ether / EtOAc = 2:1); ṽmax(atr)/cm-1 2981 and 2962
(C_H), 1721 (C=O), 1565 (C=C, C=N), 1214 and 1038 (C_O); δH (300 MHz; CDCl3) 1.36 (6H, t, 3J 3´´-H, 2´´-H 7.2 Hz, 3´´-H), 2.24 (3H, s, 1´´´-H), 2.49 (6H, s, 1´-H) and 4.40 (4H, q, 3J 2´´-H, 3´´-H
7.2 Hz, 2´´-H); δC (75 MHz; CDCl3) 14.11 (C-3´´), 16.88 (C-1´´´), 22.86 (C-1´), 61.50 (C-2´´),
127.51 (C-3 and C-5), 141.97 (C-4), 154.86 (C-2 and C-6) and 168.33 (C-1´´); m/z (EI, 70 eV)
265 (M+, 35%), 220 (74) and 28 (100).
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1.000
1.301
1.974
1.990
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
4.41854.39464.3708
2.4884
2.2424
1.38581.36201.3382
PPM 180 160 140 120 100 80 60 40 20 0
168.3292
154.8588
141.9704
127.5073
77.423977.000076.5754
61.5035
22.8621
16.879714.1094
Fig. 15 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5d in CDCl3.
N
OO
O O
2
34
5
6
1´´1´´ 2´´2´´
1´1´
1´´´
3´´ 3´´
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6.5. Synthesis and analytical data of diethyl-2,6-dimethyl-4-phenylpyridine-3,5-
dicarboxylate (5e)
Synthesis of 5e
According to the general procedure II, diethyl-1,4-dihydro-2,6-dimethyl-4-phenylpyridine-3,5-
dicarboxylate (4e) (329 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 24 h. Purification afforded diethyl-2,6-dimethyl-4-phenylpyridine-3,5-
dicarboxylate (5e) (88 mg, 27%).
According to the general procedure III, diethyl-1,4-dihydro-2,6-dimethyl-4-phenylpyridine-3,5-
dicarboxylate (4e) (329 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 9 h. Purification afforded diethyl-2,6-dimethyl-4-phenylpyridine-3,5-
dicarboxylate (5e) (261 mg, 80%).
According to the general procedure V, benzaldehyde (1c) (106 mg, 1 mmol), ethyl acetoacetate
(2b) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for 180 min.
The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
for 24 h. Purification afforded diethyl-2,6-dimethyl-4-phenylpyridine-3,5-dicarboxylate (5e) (65
mg, 20%).
Analytical data of 5e
White powder; mp 63 - 65 oC (lit.,6 60 - 62 oC); Rf = 0.63 (petroleum ether / EtOAc = 2:1);
ṽmax(atr)/cm-1 2980 (C_H), 1714 (C=O), 1555 (C=C, C=N), 1226 and 1039 (C_O); δH (300 MHz;
CDCl3) 0.90 (6H, t, 3J 3´´-H, 2´´-H 7.2 Hz, 3´´-H), 2.60 (6H, s, 1´-H), 3.99 (4H, q, 3J 2´´-H, 3´´-H 7.2 Hz,
2´´-H), 7.23-7.25 (3H, m, 3´´´-H, 4´´´-H and 5´´´-H) and 7.35-7.37 (2H, m, 2´´´-H and 6´´´-H); δC
(75 MHz; CDCl3) 13.50 (C-3´´), 22.88 (C-1´), 61.29 (C-2´´), 126.89 (C-4´´´), 128.04 and 128.05
(C-3´´´ and C-5´´´), 128.08 and 128.37 (C-2´´´ and C-6´´´), 136.55 (C-1´´´), 146.08 (C-4), 155.38
(C-2 and C-6) and 167.84 (C-1´´); m/z (EI, 70 eV) 327 (M+, 100 %), 282 (56, M+ - OC2H5), 236
(79) and 209 (24).
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0.9991.324
1.863
2.943
2.748
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
7.36707.35667.34537.26007.25497.25137.24547.23927.2278
4.02954.00573.98193.9581
2.5986
0.91990.89610.8723
PPM 180 160 140 120 100 80 60 40 20 0
167.8415
155.3831
146.0805
136.5486128.3718128.0808128.0518128.0389126.8851
77.423977.000076.5757
61.2901
22.8813
13.5044
0.9790
Fig. 16 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5e in CDCl3.
3´´ 3´´1´´1´´ 2´´2´´
1´1´
2
34
5
6
3´´´
2´´´
1´´´
4´´´
N
OO
O O6´´´
5´´´
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6.6. Synthesis and analytical data of di-isopropyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5f)
Synthesis of 5f
According to the general procedure II, di-isopropyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4f) (281 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 20 h. Purification afforded di-isopropyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5f) (226 mg, 81%).
According to the general procedure III, di-isopropyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4f) (281 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 8 h. Purification afforded di-isopropyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5f) (260 mg, 93%).
According to the general procedure IV, paraformaldehyde (1a) (30 mg, 1 mmol), isopropyl
acetoacetate (2c) (288 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 30 min. The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg,
0.0125 mmol) for 21 h. Purification afforded di-isopropyl-2,6-dimethylpyridine-3,5-
dicarboxylate (5f) (159 mg, 57%).
According to the general procedure V, paraformaldehyde (1a) (30 mg, 1 mmol), isopropyl
acetoacetate (2c) (288 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 30 min. The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9
mg, 0.1 mmol) for 9 h. Purification afforded di-isopropyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5f) (189 mg, 68%).
Analytical data of 5f
White powder; mp 63 - 65 oC (lit.,4 64 - 66 oC); Rf = 0.71 (petroleum ether / EtOAc = 2:1);
ṽmax(atr)/cm-1 3020, 2984 and 2936 (C_H), 1712 (C=O), 1591 (C=C, C=N), 1221 and 1100
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(C_O); δH (300 MHz; CDCl3) 1.38 (12H, d, 3J 3´´-H, 2´´-H 6.0 Hz, 3´´-H), 2.82 (6H, s, 1´-H), 5.25
(2H, sep, 3J 2´´-H, 3´´-H 6.0 Hz, 2´´-H) and 8.60 (1H, s, 4-H); δC (75 MHz; CDCl3) 21.88 (C-3´´),
24.96 (C-1´), 69.10 (C-2´´), 123.55 (C-3 and C-5), 140.77 (C-4), 161.74 (C-2 and C-6) and
165.65 (C-1´´); m/z (EI, 70 eV) 279 (M+, 10 %), 250 (22, M+ - C2H5), 220 (21), 195 (55) and 31
(100); HRMS (EI, M+) found: 279.1451 calcd. for C15H21NO4: 279.1471.
1.000
2.298
8.586
17.43
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.08.5982
5.27375.25285.2319
2.8200
1.39131.3704
0.0621
2
34
5
6
N
OO
O O3´´ 3´´
1´´1´´2´´ 2´´
1´1´
3´´3´´
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PPM 180 160 140 120 100 80 60 40 20 0
165.6498161.7389
140.7670
123.5539
77.423877.000076.5762
69.0976
24.956421.8793
Fig. 17 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5f in CDCl3.
6.7. Synthesis and analytical data of di-tert-butyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5g)
Synthesis of 5g
According to the general procedure II, di-tert-butyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4g) (309 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 15 h. Purification afforded di-tert-butyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5g) (185 mg, 60%).
According to the general procedure III, di-tert-butyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4g) (309 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 7 h. Purification afforded di-tert-butyl-2,6-dimethylpyridine-3,5-dicarboxylate
(5g) (275 mg, 90%).
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According to the general procedure IV, paraformaldehyde (1a) (30 mg, 1 mmol), tert-butyl
acetoacetate (2d) (316 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 30 min. The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg,
0.0125 mmol) for 24 h. Purification afforded di-tert-butyl-2,6-dimethylpyridine-3,5-
dicarboxylate (5g) (93 mg, 30%).
According to the general procedure V, paraformaldehyde (1a) (30 mg, 1 mmol), tert-butyl
acetoacetate (2d) (316 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 30 min. The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9
mg, 0.1 mmol) for 24 h. Purification afforded di-tert-butyl-2,6-dimethylpyridine-3,5-
dicarboxylate (5g) (190 mg, 62%).
Analytical data of 5g
White powder; mp 107 - 109 oC (lit.,4 108 - 109 oC); Rf = 0.80 (petroleum ether / EtOAc = 2:1);
ṽmax(atr)/cm-1 3003, 2980 and 2937 (C_H), 1709 (C=O), 1595 (C=N, C=C), 1150 and 1105
(C_O); δH (300 MHz; CDCl3) 1.59 (18H, s, 3´´-H), 2.79 (6H, s, 1´-H) and 8.51 (1H, s, 4-H); δC
(75 MHz; CDCl3) 24.95 (C-1´), 28.18 (C-3´´), 82.08 (C-2´´), 124.61 (C-3 and C-5), 140.74 (C-
4), 161.11 (C-2 and C-6) and 165.44 (C-1´´); m/z (EI, 70 eV) 279 (M+- C2H4, 23%), 264 (100)
and 251 (77).
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1.000
8.080
25.24
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
8.5078
2.7885
1.5937
0.0596
PPM 180 160 140 120 100 80 60 40 20 0
165.4401
161.1124
140.7424
124.6132
82.083677.423077.000076.5757
28.183724.9476
0.9957
Fig. 18 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5g in CDCl3.
2
34
5
6
N
OO
O O3´´ 3´´
1´´1´´2´´ 2´´
1´1´
3´´3´´
3´´3´´
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6.8. Synthesis and analytical data of diallyl-2,6-dimethylpyridine-3,5-dicarboxylate (5h)
Synthesis of 5h
According to the general procedure II, diallyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4h) (277 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 7 h. Purification afforded diallyl-2,6-dimethylpyridine-3,5-dicarboxylate (5h)
(236 mg, 86%).
According to the general procedure III, diallyl-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (4h) (277 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 8 h. Purification afforded diallyl-2,6-dimethylpyridine-3,5-dicarboxylate (5h)
(220 mg, 80%).
According to the general procedure IV, paraformaldehyde (1a) (30 mg, 1 mmol), allyl
acetoacetate (2e) (284 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 10 min. The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg,
0.0125 mmol) for 24 h. Purification afforded diallyl-2,6-dimethylpyridine-3,5-dicarboxylate (5h)
(167 mg, 61%).
According to the general procedure V, paraformaldehyde (1a) (30 mg, 1 mmol), allyl
acetoacetate (2e) (284 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted
for 10 min. The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH
4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9
mg, 0.1 mmol) for 9 h. Purification afforded diallyl-2,6-dimethylpyridine-3,5-dicarboxylate (5h)
(136 mg, 49 %).
Analytical data of 5h
White powder; mp 64 - 66 oC; Rf = 0.80 (petroleum ether / EtOAc = 2:1); λmax(MeCN)/nm 272
(log ε, 3.60), 235 (4.07) and 207 (4.54); ṽmax(atr)/cm-1 3091 and 2928 (C_H), 1716 (C=O), 1591
(C=N, C=C), 1219 and 1106 (C_O); δH (300 MHz; CDCl3) 2.85 (6H, s, 1´-H), 4.83 (4H, dt, 3J 2´´-
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H, 3´´-H 5.6 Hz, 4J 1.3 Hz, 2´´-H), 5.32 (2H, dq, 2J 1.3 Hz, 3Jcis 4´´a-H, 3´´-H 10.5 Hz, 4J 1.3 Hz, 4´´a-H),
5.40 (2H, dq, 2J 1.4 Hz, 3Jtrans 4´´b-H, 3´´-H 17.2 Hz, 4J 1.4 Hz, 4´´b-H), 6.03 (2H, ddt, 3J 3´´-H, 2´´-H 5.7
Hz, 3Jcis 3´´-H, 4´´a-H 10.4 Hz, 3Jtrans 3´´-H, 4´´b-H 17.3 Hz, 3´´-H) and 8.73 (1H, s, 4-H); δC (75 MHz;
CDCl3) 25.00 (C-1´), 65.99 (C-2´´), 118.85 (C-4´´), 122.73 (C-3 and C-5), 131.79 and 131.81 (C-
3´´), 141.02 (C-4), 162.60 (C-2 and C-6) and 165.47 (C-1´´); m/z (EI, 70 eV) 275 (M+, 5%), 234
(15), 218 (33) and 41 (100); HRMS (EI, M+) found: 275.1146 calcd. for C15H17NO4: 275.1158.
1.000
2.015
4.491
5.363
8.333
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
8.7302
7.2600
6.08386.04906.02666.00755.99185.44385.43915.43465.38645.38175.37735.33195.32805.29725.29334.84124.83704.83284.82214.81784.8137
2.8496
0.0639
2
34
5
6
3´´ 3´´
1´´1´´ 2´´2´´
1´1´ N
OO
O O4´´b4´´b
H
H
H
H
H
H4´´a4´´a
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PPM 180 160 140 120 100 80 60 40 20 0
165.4684162.6043
141.0227
131.8115131.7885
122.7249118.8486
77.424277.000076.5762
65.9881
24.9955
0.9942
Fig. 19 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5h in CDCl3.
6.9. Synthesis and analytical data of dimethyl-2,6-diethylpyridine-3,5-dicarboxylate (5i)
Synthesis of 5i
According to the general procedure II, dimethyl-2,6-diethyl-1,4-dihydropyridine-3,5-
dicarboxylate (4i) (253 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
were reacted for 20 h. Purification afforded dimethyl-2,6-diethylpyridine-3,5-dicarboxylate (5i)
(200 mg, 80%).
According to the general procedure III, dimethyl-2,6-diethyl-1,4-dihydropyridine-3,5-
dicarboxylate (4i) (253 mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 14 h. Purification afforded dimethyl-2,6-diethylpyridine-3,5-dicarboxylate (5i)
(179 mg, 71%).
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According to the general procedure IV, paraformaldehyde (1a) (30 mg, 1 mmol), methyl
propionyl acetate (2f) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were
reacted for 15 min. The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2
M, pH 4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt
(6.9 mg, 0.0125 mmol) for 26 h. Purification afforded dimethyl-2,6-diethylpyridine-3,5-
dicarboxylate (5i) (186 mg, 74%).
According to the general procedure V, paraformaldehyde (1a) (30 mg, 1 mmol), methyl
propionyl acetate (2f) (260 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were
reacted for 15 min. The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2
M, pH 4.37, 30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt
(54.9 mg, 0.1 mmol) for 20 h. Purification afforded dimethyl-2,6-diethylpyridine-3,5-
dicarboxylate (5i) (167 mg, 67%).
Analytica data of 5i
White powder; mp 50 - 52 oC; Rf = 0.84 (petroleum ether / EtOAc = 2:1); λmax(MeCN)/nm 272
(log ε, 3.52), 235 (3.99) and 207 (4.47); ṽmax(atr)/cm-1 2976, 2958 and 2938 (C_H), 1716 (C=O),
1593 (C=N, C=C), 1229 and 1048 (C_O); δH (300 MHz; CDCl3) 1.30 (6H, t, 3J 2´-H, 1´-H 7.5 Hz,
2´-H), 3.20 (4H, q, 3J 1´-H, 2´-H 7.5 Hz, 1´-H), 3.92 (6H, s, 2´´-H) and 8.63 (1H, s, 4-H); δC (75
MHz, CDCl3) 13.69 (C-2´), 30.36 (C-1´), 52.29 (C-2´´), 122.03 (C-3 and C-5), 141.22 (C-4),
166.35 (C-1´´) and 167.23 (C-2 and C-6); m/z (EI, 70 eV) 251 (M+, 83%), 236 (100, M+ - CH3)
and 220 (25, M+ - OCH3); HRMS (EI, M+) found: 251.1157 calcd. for C13H17NO4: 251.1158.
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1.000
8.148
5.708
8.858
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
8.6297
7.2600
3.9199
3.23193.20693.18193.1570
1.32261.29771.2727
0.0646
PPM 180 160 140 120 100 80 60 40 20 0
167.2279166.3453
141.2213
122.0306
77.423877.000076.5760
52.2909
30.3636
13.6921
Fig. 20 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5i in CDCl3.
1´´1´´2´´2´´
1´1´
2
34
5
6
N
OO
O O
2´ 2´
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6.10. Synthesis and analytical data of diethyl-2,6-diethylpyridine-3,5-dicarboxylate (5j)
Synthesis of 5j
According to the general procedure II, diethyl-2,6-diethyl-1,4-dihydropyridine-3,5-dicarboxylate
(4j) (281 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL), laccase (168
U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol) were
reacted for 24 h. Purification afforded diethyl-2,6-diethylpyridine-3,5-dicarboxylate (5j) (200
mg, 72%).
According to the general procedure III, diethyl-2,6-diethyl-1,4-dihydropyridine-3,5-
dicarboxylate (4j) (281 mg, 1 mmol), methanol (6 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
were reacted for 9 h. Purification afforded diethyl-2,6-diethylpyridine-3,5-dicarboxylate (5j)
(237 mg, 85%).
According to the general procedure IV, paraformaldehyde (1a) (30 mg, 1 mmol), ethyl propionyl
acetate (2g) (288 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for
40 min. The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH 4.37,
30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg,
0.0125 mmol) for 24 h. Purification afforded diethyl-2,6-diethylpyridine-3,5-dicarboxylate (5j)
(162 mg, 58%).
According to the general procedure V, paraformaldehyde (1a) (30 mg, 1 mmol), ethyl propionyl
acetate (2g) (288 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for
40 min. The reaction mixture was treated with methanol (6 mL), acetate buffer (0.2 M, pH 4.37,
30 mL), laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1
mmol) for 8 h. Purification afforded diethyl-2,6-diethylpyridine-3,5-dicarboxylate (5j) (155 mg,
56%).
Analytical data of 5j
Colourless solid; mp 35 - 37 oC; Rf = 0.83 (petroleum ether / EtOAc = 2:1); λmax(MeCN)/nm 272
(log ε, 3.58), 235 (4.05) and 206 (4.52); ṽmax(atr)/cm-1 2977, 2936 and 2874 (C_H), 1717 (C=O),
1590 (C=N or C=C), 1224 and 1044 (C_O); δH (300 MHz; CDCl3) 1.30 (6H, t, 3J 2´-H, 1´-H 7.5 Hz,
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2´-H), 1.40 (6H, t, 3J 3´´-H, 2´´-H 7.2 Hz, 3´´-H), 3.18 (4H, q, 3J 1´-H, 2´-H 7.5 Hz, 1´-H), 4.39 (4H, q, 3J 2´´-H, 3´´-H 7.2 Hz, 2´´-H) and 8.59 (1H, s, 4-H); δC (75 MHz; CDCl3) 13.80 (C-2´), 14.22 (C-
3´´), 30.42 (C-1´), 61.35 (C-2´´), 122.55 (C-3 and C-5), 141.02 (C-4), 166.13 (C-1´´) and 166.78
(C-2 and C-6); m/z (EI, 70 eV) 279 (M+, 19%), 250 (100, M+ - C2H5) and 222 (43); HRMS (EI,
M+) found: 279.1446 calcd. for C15H21NO4: 279.1471.
1.000
0.199
5.364
5.821
7.8467.899
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
8.5869
7.2600
4.42064.39694.37314.3494
3.21673.19183.16693.1420
1.42551.40171.37791.31961.29481.2698
3´´ 3´´1´´1´´ 2´´2´´
1´1´
2
34
5
6
N
OO
O O
2´ 2´
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PPM 180 160 140 120 100 80 60 40 20 0
166.7820166.1288
141.0229
122.5495
77.423677.000076.5756
61.3461
30.4192
14.222713.7998
Fig. 21 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5j in CDCl3.
6.11. Synthesis and analytical data of 2,6-dimethyl-3,5-diacetylpyridine (5k)
Synthesis of 5k
According to the general procedure II, 2,6-dimethyl-3,5-diacetyl-1,4-dihydropyridine (4k) (193
mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL), laccase (168 U, 12 mg,
Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol) were reacted for 10 h.
Purification by flash chromatography on SiO2 (petroleum ether / EtOAc = 2:1) afforded 2,6-
dimethyl-3,5-diacetylpyridine (5k) (165 mg, 86%).
According to the general procedure III, 2,6-dimethyl-3,5-diacetyl-1,4-dihydropyridine (4k) (193
mg, 1 mmol), methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL), laccase (168 U, 12 mg,
Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol) were reacted for 10 h.
Purification by flash chromatography on SiO2 (petroleum ether / EtOAc = 2:1) afforded 2,6-
dimethyl-3,5-diacetylpyridine (5k) (172 mg, 90%).
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According to the general procedure IV, paraformaldehyde (1a) (30 mg, 1 mmol), acetylacetone
(2h) (200 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for 15 min.
The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (6.9 mg, 0.0125 mmol)
for 14 h. Purification by flash chromatography on SiO2 (petroleum ether / EtOAc = 2:1) afforded
2,6-dimethyl-3,5-diacetylpyridine (5k) (112 mg, 59%).
According to the general procedure V, paraformaldehyde (1a) (30 mg, 1 mmol), acetylacetone
(2h) (200 mg, 2 mmol) and ammonium acetate (3) (116 mg, 1.5 mmol) were reacted for 15 min.
The reaction mixture was treated with methanol (3 mL), acetate buffer (0.2 M, pH 4.37, 30 mL),
laccase (168 U, 12 mg, Trametes versicolor) and ABTS diammonium salt (54.9 mg, 0.1 mmol)
for 10 h. Purification by flash chromatography on SiO2 (petroleum ether / EtOAc = 2:1) afforded
2,6-dimethyl-3,5-diacetylpyridine (5k) (115 mg, 60%).
Analytical data of 5k
White crystals; mp 65 - 67 oC (lit.,6 68 - 70 oC); Rf = 0.29 (petroleum ether / EtOAc = 2:1);
ṽmax(atr)/cm-1 2980 (C-H), 1673 (C=O) and 1578 (C=N, C=C); δH (300 MHz; CDCl3) 2.61 (6H, s,
2´´-H), 2.75 (6H, s, 1´-H) and 8.22 (1H, s, 4-H); δC (75 MHz; CDCl3) 24.95 (C-1´), 29.32 (C-
2´´), 130.11 (C-4), 137.73 (C-3 and C-5), 160.23 (C-2 and C-6) and 199.19 (C-1´´); m/z (EI, 70
eV) 191 (M+, 37 %), 176 (77, M+ - CH3), 106 (39) and 28 (100).
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1.000
6.6626.034
PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
8.2155
2.74842.6095
PPM 180 160 140 120 100 80 60 40 20 0
199.1881
160.2342
137.7302
130.1092
77.481177.423977.000076.5762
29.3237
24.9484
Fig. 22 1H (300 MHz) and 13C (75 MHz) NMR spectra of 5k in CDCl3.
N
O O
2
34
5
6
1´´1´´2´´2´´
1´1´
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7. Determination of the laccase activity8
A 0.1 M solution of ABTS (0.3 mL) in 0.2 M acetate buffer (pH 4.37) was diluted with 0.2 M
acetate buffer (2.6 mL, pH 4.37) and treated with a solution of laccase in the same buffer (0.1
mL). The change in absorption was followed via UV spectroscopy (λ = 414 nm). One unit was
defined as the amount of laccase (Trametes versicolor, Fluka) that converts 1 mmol of ABTS per
minute at pH 4.37 at rt.
8. References
[1] M. A. Zolfigol and M. Safaiee, Synlett, 2004, 827.
[2] Suresh, D. Kumar and J. S. Sandhu, Synth. Commun., 2009, 39, 1957.
[3] H. Salehi and Q.-X. Guo, Synth. Commun., 2004, 34, 4349.
[4] J. Uldrikjis, G. Duburs, I. Dipans, B. Cekavicius, Khimiya Geterotsiklicheskikh Soedinenii,
1975, 9, 1230.
[5] K. Kikugawa, T. Nakahara and K. Sakurai, Chem. Pharm. Bull., 1987, 35, 4656.
[6] J.-J. Xia and G.W. Wang, Synthesis, 2005, 2379.
[7] A. Ghorbani-Choghamarani and J. Zeinivand, Synth. Commun., 2010, 40, 2457.
[8] S. Nicotra, A. Intra, G. Ottolina, S. Riva and B. Danieli, Tetrahedron: Asymmetry,
2004, 15, 2927.
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