Supplementary material for: [https://www.sciencedirect.com/science/article/pii/S0141022919301498?via%3Dihub#bibl0005]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3356

Babu, Ramesh P.

Božić, Nataša

Jeremić, Sanja

Lončar, Nikola L.

Nikodinović-Runić, Jasmina

Opsenica, Igor

Senthamaraikannan, Ramsankar

Simić, Stefan

Vujčić, Zoran

Đokić, Lidija

Enzyme and Microbial Technology

Cherry - Repository of the Faculty of Chemistry;  University of Belgrade

  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Supplementary material for the article: 
 
 
Simić, S.; Jeremic, S.; Djokic, L.; Božić, N.; Vujčić, Z.; Lončar, N.; Senthamaraikannan, R.; 
Babu, R.; Opsenica, I. M.; Nikodinovic-Runic, J. Development of an Efficient Biocatalytic 
System Based on Bacterial Laccase for the Oxidation of Selected 1,4-Dihydropyridines. Enzyme 
and Microbial Technology 2020, 132. https://doi.org/10.1016/j.enzmictec.2019.109411  
1 
 
Supporting information 
Development of an efficient biocatalytic system based on bacterial 
laccase for the oxidation of selected 1,4-dihydropyridines 
Stefan Simić1, Sanja Jeremic2, Lidija Djokic2, Nataša Božić3, Zoran Vujčić1, Nikola Lončar 4,5, 
Ramsankar Senthamaraikannan
6
, Ramesh Babu
 6,7
, Igor M. Opsenica
1,*
, Jasmina Nikodinović-
Runić1,2* 
 
1
University of Belgrade, Faculty of Chemistry, Studentski trg 16, P.O. Box 51, Belgrade, 11158, 
Serbia 
2
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 
444a, Belgrade, 11000, Serbia 
3
Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of 
Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia 
4
Groningen Enzyme and Cofactor Collection (GECCO Biotech), Groningen, The Netherlands 
5
Molecular Enzymology Group, University of Groningen, Groningen, The Netherlands 
6
AMBER Centre, Trinity College Dublin, College Green, Dublin 2, Ireland 
7
BEACON SFI Bioeconomy Research Centre, O'Brien Science Centre, University College 
Dublin, Ireland 
 
 
 
 
 
 
 
 
 
*Corresponding authors: 
Tel. +381 11 3336684; E-mail: igorop@chem.bg.ac.rs 
Tel. +381 11 3976034; E-mail: jasmina.nikodinovic@imgge.bg.ac.rs 
 
 
Supplementary Material
2 
 
Synthesis and characterization of Py1- Py6 
Diethyl 2,6-dimethylpyridine-3,5-dicarboxylate (Py1) [1] 
 
Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, 
diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 26 
mM) was oxidized using TvLacc (final concentration 1.16 U/ml; activity unit determined against 
catechol) during 4 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl 
acetate 9:1 v/v) yielded Py1 as a white powder (28.4 mg, 94%). 
Following the same procedure, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 
(DHPy1) (final concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 
U/ml; activity unit determined against catechol) during 4 h at 50 ˚C. Purification by dry-flash 
chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py1 likewise (28.3 mg, 94%).  
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed BNC-
supported laccase, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final 
concentration 26 mM) was oxidized using TvLacc (5 U) immobilized on BNC (10.7 mg) during 
20 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) 
yielded Py1 (26.3 mg, 87%).  
Following the same procedure, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 
(DHPy1) (final concentration 26 mM) was oxidized using BliLacc (4 U) immobilized on BNC 
(10.7 mg) during 20 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl 
acetate 9:1 v/v) yielded Py1 (20.8 mg, 69%).  
3 
 
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. 
coli whole-cell suspension, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 
(DHPy1) (final concentration 11 mM) was oxidized using E. coli (BliLacc) (0.2 ml, OD600 = 
150) during 7 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane, hexane/ethyl 
acetate 9:1 v/v) yielded Py1 (14.6 mg, 97%). 
 
Following the same procedure, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 
(DHPy1) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.2 ml, OD600 = 150) 
during 24 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane, hexane/ethyl 
acetate 9:1 v/v) yielded Py1 (14.3 mg, 95%).  
 
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the 
BNC-supported E. coli, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) 
(final concentration 11 mM) was oxidized using E. coli (BliLacc) (0.24 ml, OD600 = 150) 
immobilized on BNC (9.7 mg) during 24 h at 50 ˚C. Purification by dry-flash chromatography 
(SiO2; hexane, hexane/ethyl acetate 9:1 v/v) yielded Py1 (13.2 mg, 87%).  
 
Following the same procedure, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 
(DHPy1) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.18 ml, OD600 = 150) 
immobilized on BNC (6.9 mg) during 24 h at 50 ˚C. Purification by dry-flash chromatography 
(SiO2; hexane, hexane/ethyl acetate 9:1 v/v) yielded Py1 (11.4 mg, 75%).  
 
4 
 
The product was obtained as a white solid. FT-IR (ATR): 2980m, 2932m, 2870w, 1722s, 1591m, 
1551m, 1440m, 1369m, 1290m, 1258m, 1224m, 1110m, 1046m; 
1
H NMR (500 MHz, CDCl3): δ 
= 1.42 (t, J = 7.1 Hz, 6H), 2.85 (s, 6H), 4.40 (q, J = 7.1 Hz, 4H), 8.67 (s, 1H) ppm; 
13
C NMR 
(125 MHz, CDCl3): δ =  14.3, 24.9, 61.4, 123.1, 140.9, 162.2, 166.0 ppm.  
 
Diethyl 2,6-dimethyl-4-phenylpyridine-3,5-dicarboxylate (Py2) [1]  
 
 
Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, 
diethyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy2) (final 
concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 U/ml) during 4 h at 
50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py2 
(4.2 mg, 11%).  
 
The product was obtained as a white solid. FT-IR (ATR): 3058w, 2981m, 2959m, 2927m, 2854w, 
1727s, 1559m, 1495w, 1447w, 1410w, 1376w, 1292m, 1235s, 1211m, 1105m, 1043m; 
1
H NMR 
(500 MHz, CDCl3): δ = 0.90 (t, J = 7.1 Hz, 6H), 2.60 (s, 6H), 4.00 (q, J = 7.1 Hz, 4H), 7.23-7.26 
(m, 2H), 7.34-7.37 (m, 3H) ppm; 
13
C NMR (125 MHz, CDCl3): δ = 13.5, 22.9, 61.3, 126.9, 
128.0, 128.1, 128.3, 136.5, 155.4, 167.8 ppm.  
 
 
 
 
5 
 
Diethyl 2,6-dimethyl-4-propylpyridine-3,5-dicarboxylate (Py3) [2] 
 
Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, 
diethyl 2,6-dimethyl-4-propyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy3) (final 
concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 U/ml) during 4 h at 
50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py3 
(5.4 mg, 15%).  
 
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. 
coli whole-cell suspension, 2,6-dimethyl-4-propyl-1,4-dihydropyridine-3,5-dicarboxylate 
(DHPy3) (final concentration 11 mM) was oxidized using E. coli (BliLacc) (0.2 ml, OD600 = 
150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be 11% by 
NMR spectroscopy.  
 
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. 
coli whole-cell suspension, 2,6-dimethyl-4-propyl-1,4-dihydropyridine-3,5-dicarboxylate 
(DHPy3) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.2 ml, OD600 = 150) 
during 24 h at 50 ˚C. The conversion of the starting material was determined to be 7% by NMR 
spectroscopy. 
 
6 
 
The product was obtained as a colorless solid. FT-IR (ATR): 2965m, 2932m, 2875w, 1728s, 
1568m, 1449m, 1412w, 1380w, 1283m, 1236s, 1200m, 1104m, 1040m; 
1
H NMR (500 MHz, 
CDCl3): δ = 0.93 (t, J = 7.3 Hz, 3H), 1.39 (t, J = 7.1 Hz, 6H), 1.58 (m, 2H), 2.51 (s, 6H), 2.55 
(m, 2H), 4.41 (q, J = 7.1 Hz, 4H) ppm; 
13
C NMR (125 MHz, CDCl3): δ = 14.1, 14.4, 22.9, 24.2, 
33.4, 61.5, 127.2, 146.3, 155.0, 168.5 ppm. 
 
1,1'-(2,6-dimethylpyridine-3,5-diyl)diethanone (Py4) [1] 
 
Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, 
1,1'-(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)diethanone (DHPy4) (final concentration 26 
mM) was oxidized using BliLacc (final concentration 0.87 U/ ml) during 4 h at 50 ˚C. 
Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 8:2 v/v, hexane/ethyl 
acetate 7:3 v/v) yielded Py4 (21.7 mg, 94%).  
 
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. 
coli whole-cell suspension, 1,1'-(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)diethanone (DHPy4) 
(final concentration 11 mM) was oxidized using E.coli (BliLacc) (0.2 ml, OD600 = 150) during 24 
h at 50 ˚C. The conversion of the starting material was determined to be >99% by NMR 
spectroscopy.  
 
7 
 
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. 
coli whole-cell suspension, 1,1'-(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)diethanone (DHPy4) 
(final concentration 11 mM) was oxidized using E. coli TOP10 (0.2 ml, OD600 = 150) during 24 
h at 50 ˚C. The conversion of the starting material was determined to be >99% by NMR 
spectroscopy.  
 
The product was obtained as a white solid. FT-IR (ATR): 2970w, 2925w, 1682s, 1593m, 1531m, 
1434m, 1358m, 1266s, 1206m, 1072m, 1025m, 952m, 928m; 
1
H NMR (500 MHz, CDCl3): δ = 
2.63 (s, 6H), 2.77 (s, 6H), 8.23 (s, 1H) ppm; 
13
C NMR (125 MHz, CDCl3): δ = 24.9, 29.3, 130.1, 
137.7, 160.2, 199.2 ppm. 
 
3,3,6,6-tetramethyl-3,4,6,7-tetrahydroacridine-1,8(2H,5H)-dione (Py5) [3] 
 
Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, 
3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (DHPy5) (final 
concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 U/ml) during 4 h at 
50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v, 
hexane/ethyl acetate 7:3 v/v) yielded Py5 (5.1 mg, 16%).  
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. 
coli whole-cell suspension, 3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-
dione (DHPy5) (final concentration 11 mM) was oxidized using E. coli (BliLacc) (0.2 ml, 
8 
 
OD600 = 150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be 
9% by NMR spectroscopy.  
 
Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. 
coli whole-cell suspension, 3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-
dione (DHPy5) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.2 ml, OD600 
= 150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be 7% by 
NMR spectroscopy. 
The product was obtained as a white solid. FT-IR (ATR): 2957s, 2928s, 2871m, 1699s, 1588s, 
1563w, 1465m, 1417m, 1389w, 1371w, 1335w, 1296w, 1260w, 1234m, 1120w; 
1
H NMR (500 
MHz, CDCl3): δ = 1.12 (s, 12H), 2.57 (s, 4H), 3.05 (s, 4H), 8.81 (s, 1H) ppm. 
 
Fig. S1. Recycling of the E. coli (BliLacc)/BNC catalyst in the oxidation of 1a. The reactions 
were run with 0.03 mmol of the substrate, according to general procedure. Substrate 
amount/CDW = 8.55.  
 
>99 
69 
37 
0 
10 
20 
30 
40 
50 
60 
70 
80 
90 
100 
1 2 3 
N
M
R
 c
o
n
ve
rs
io
n
 (
%
) 
Reaction cycle number 
9 
 
Table S1. The substrate scope of the biocatalytic oxidation of 1,4-dihydropyridines.  
Substrate Catalyst 
ABTS 
(mol%) 
CuSO4 
(mol%) 
Product 
yield (%) 
DHPy1
d
 BliLacc 1.5 – 94
a 
DHPy1 E. coli (BliLacc) 1.6 1.7 97
b 
DHPy1 E. coli TOP10 1.6 1.7 95
b 
DHPy2
d
 BliLacc 1.5 – 11
a 
DHPy2 E. coli (BliLacc) 1.6 1.7 0
b 
DHPy2 E. coli TOP10 1.6 1.7 0
b 
DHPy3
d
 BliLacc 1.5 – 15
a 
DHPy3 E. coli (BliLacc) 1.6 1.7 11
b 
DHPy3 E. coli TOP10 1.6 1.7 7
b 
DHPy4
d
 BliLacc 1.5 – 94
a 
DHPy4 E. coli (BliLacc) 1.6 1.7 >99
b  
DHPy4 E. coli TOP10 1.6 1.7 >99
b 
DHPy5
d
 BliLacc 1.5 – 16
a 
DHPy5 E. coli (BliLacc) 1.6 1.7 9
b 
DHPy5 E. coli TOP10 1.6 1.7 7
b 
DHPy6
d
 BliLacc 1.5 – 0
c 
DHPy6 E. coli (BliLacc) 1.6 1.7 0
b 
DHPy6 E. coli TOP10 1.6 1.7 0
b 
 
a 
Isolated yield 
b
 Judged by conversion of starting material (based on NMR analysis) 
c
 Judged by conversion of starting material (based on TLC analysis) 
d 
CuSO4 was added during the production of the recombinant protein  
 
References 
[1] H.T. Abdel-Mohsen, J. Conrad, U. Beifuss, Laccase-catalyzed oxidation of Hantzsch 1,4-
dihydropyridines to pyridines and a new one pot synthesis of pyridines. Green Chem. 14 
(2012) 2686-2690. 
[2] O. De Paolis, J. Baffoe, S.M. Landge, B. Toeroek, Multicomponent Domino Cyclization-
Oxidative Aromatization on a Bifunctional Pd/C/K-10 Catalyst: An Environmentally 
Benign Approach toward the Synthesis of Pyridines. Synthesis 21 (2008) 3423-3428. 
[3] V. Hegde, C.Y. Hung, P. Madhukar, R. Cunningham, T. Hopfner, R.P. Thummel, Design of 
receptors for urea derivatives based on the pyrido [3, 2-g] indole subunit.  J. Am. Chem. 
Soc. 115 (1993) 872-878. 


English

Supplementary data for article: Simić, S.; Jeremic, S.; Djokic, L.; Božić, N.; Vujčić, Z.; Lončar, N.; Senthamaraikannan, R.; Babu, R.; Opsenica, I. M.; Nikodinovic-Runic, J. Development of an Efficient Biocatalytic System Based on Bacterial Laccase for the Oxidation of Selected 1,4-Dihydropyridines. Enzyme and Microbial Technology 2020, 132. https://doi.org/10.1016/j.enzmictec.2019.109411

Faculty of Chemistry Repository - Cherry

                 Supplementary material for the article:   Simić, S.; Jeremic, S.; Djokic, L.; Božić, N.; Vujčić, Z.; Lončar, N.; Senthamaraikannan, R.; Babu, R.; Opsenica, I. M.; Nikodinovic-Runic, J. Development of an Efficient Biocatalytic System Based on Bacterial Laccase for the Oxidation of Selected 1,4-Dihydropyridines. Enzyme and Microbial Technology 2020, 132. https://doi.org/10.1016/j.enzmictec.2019.109411  1  Supporting information Development of an efficient biocatalytic system based on bacterial laccase for the oxidation of selected 1,4-dihydropyridines Stefan Simić1, Sanja Jeremic2, Lidija Djokic2, Nataša Božić3, Zoran Vujčić1, Nikola Lončar 4,5, Ramsankar Senthamaraikannan6, Ramesh Babu 6,7, Igor M. Opsenica1,*, Jasmina Nikodinović-Runić1,2*  1University of Belgrade, Faculty of Chemistry, Studentski trg 16, P.O. Box 51, Belgrade, 11158, Serbia 2Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, Belgrade, 11000, Serbia 3Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia 4Groningen Enzyme and Cofactor Collection (GECCO Biotech), Groningen, The Netherlands 5Molecular Enzymology Group, University of Groningen, Groningen, The Netherlands 6AMBER Centre, Trinity College Dublin, College Green, Dublin 2, Ireland 7BEACON SFI Bioeconomy Research Centre, O'Brien Science Centre, University College Dublin, Ireland          *Corresponding authors: Tel. +381 11 3336684; E-mail: igorop@chem.bg.ac.rs Tel. +381 11 3976034; E-mail: jasmina.nikodinovic@imgge.bg.ac.rs   Supplementary Material2  Synthesis and characterization of Py1- Py6 Diethyl 2,6-dimethylpyridine-3,5-dicarboxylate (Py1) [1]  Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 26 mM) was oxidized using TvLacc (final concentration 1.16 U/ml; activity unit determined against catechol) during 4 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py1 as a white powder (28.4 mg, 94%). Following the same procedure, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 U/ml; activity unit determined against catechol) during 4 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py1 likewise (28.3 mg, 94%).  Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed BNC-supported laccase, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 26 mM) was oxidized using TvLacc (5 U) immobilized on BNC (10.7 mg) during 20 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py1 (26.3 mg, 87%).  Following the same procedure, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 26 mM) was oxidized using BliLacc (4 U) immobilized on BNC (10.7 mg) during 20 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py1 (20.8 mg, 69%).  3  Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. coli whole-cell suspension, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 11 mM) was oxidized using E. coli (BliLacc) (0.2 ml, OD600 = 150) during 7 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane, hexane/ethyl acetate 9:1 v/v) yielded Py1 (14.6 mg, 97%).  Following the same procedure, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.2 ml, OD600 = 150) during 24 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane, hexane/ethyl acetate 9:1 v/v) yielded Py1 (14.3 mg, 95%).   Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the BNC-supported E. coli, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 11 mM) was oxidized using E. coli (BliLacc) (0.24 ml, OD600 = 150) immobilized on BNC (9.7 mg) during 24 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane, hexane/ethyl acetate 9:1 v/v) yielded Py1 (13.2 mg, 87%).   Following the same procedure, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy1) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.18 ml, OD600 = 150) immobilized on BNC (6.9 mg) during 24 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane, hexane/ethyl acetate 9:1 v/v) yielded Py1 (11.4 mg, 75%).   4  The product was obtained as a white solid. FT-IR (ATR): 2980m, 2932m, 2870w, 1722s, 1591m, 1551m, 1440m, 1369m, 1290m, 1258m, 1224m, 1110m, 1046m; 1H NMR (500 MHz, CDCl3): δ = 1.42 (t, J = 7.1 Hz, 6H), 2.85 (s, 6H), 4.40 (q, J = 7.1 Hz, 4H), 8.67 (s, 1H) ppm; 13C NMR (125 MHz, CDCl3): δ =  14.3, 24.9, 61.4, 123.1, 140.9, 162.2, 166.0 ppm.   Diethyl 2,6-dimethyl-4-phenylpyridine-3,5-dicarboxylate (Py2) [1]    Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, diethyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy2) (final concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 U/ml) during 4 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py2 (4.2 mg, 11%).   The product was obtained as a white solid. FT-IR (ATR): 3058w, 2981m, 2959m, 2927m, 2854w, 1727s, 1559m, 1495w, 1447w, 1410w, 1376w, 1292m, 1235s, 1211m, 1105m, 1043m; 1H NMR (500 MHz, CDCl3): δ = 0.90 (t, J = 7.1 Hz, 6H), 2.60 (s, 6H), 4.00 (q, J = 7.1 Hz, 4H), 7.23-7.26 (m, 2H), 7.34-7.37 (m, 3H) ppm; 13C NMR (125 MHz, CDCl3): δ = 13.5, 22.9, 61.3, 126.9, 128.0, 128.1, 128.3, 136.5, 155.4, 167.8 ppm.      5  Diethyl 2,6-dimethyl-4-propylpyridine-3,5-dicarboxylate (Py3) [2]  Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, diethyl 2,6-dimethyl-4-propyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy3) (final concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 U/ml) during 4 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v) yielded Py3 (5.4 mg, 15%).   Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. coli whole-cell suspension, 2,6-dimethyl-4-propyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy3) (final concentration 11 mM) was oxidized using E. coli (BliLacc) (0.2 ml, OD600 = 150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be 11% by NMR spectroscopy.   Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. coli whole-cell suspension, 2,6-dimethyl-4-propyl-1,4-dihydropyridine-3,5-dicarboxylate (DHPy3) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.2 ml, OD600 = 150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be 7% by NMR spectroscopy.  6  The product was obtained as a colorless solid. FT-IR (ATR): 2965m, 2932m, 2875w, 1728s, 1568m, 1449m, 1412w, 1380w, 1283m, 1236s, 1200m, 1104m, 1040m; 1H NMR (500 MHz, CDCl3): δ = 0.93 (t, J = 7.3 Hz, 3H), 1.39 (t, J = 7.1 Hz, 6H), 1.58 (m, 2H), 2.51 (s, 6H), 2.55 (m, 2H), 4.41 (q, J = 7.1 Hz, 4H) ppm; 13C NMR (125 MHz, CDCl3): δ = 14.1, 14.4, 22.9, 24.2, 33.4, 61.5, 127.2, 146.3, 155.0, 168.5 ppm.  1,1'-(2,6-dimethylpyridine-3,5-diyl)diethanone (Py4) [1]  Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, 1,1'-(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)diethanone (DHPy4) (final concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 U/ ml) during 4 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 8:2 v/v, hexane/ethyl acetate 7:3 v/v) yielded Py4 (21.7 mg, 94%).   Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. coli whole-cell suspension, 1,1'-(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)diethanone (DHPy4) (final concentration 11 mM) was oxidized using E.coli (BliLacc) (0.2 ml, OD600 = 150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be >99% by NMR spectroscopy.   7  Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. coli whole-cell suspension, 1,1'-(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)diethanone (DHPy4) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.2 ml, OD600 = 150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be >99% by NMR spectroscopy.   The product was obtained as a white solid. FT-IR (ATR): 2970w, 2925w, 1682s, 1593m, 1531m, 1434m, 1358m, 1266s, 1206m, 1072m, 1025m, 952m, 928m; 1H NMR (500 MHz, CDCl3): δ = 2.63 (s, 6H), 2.77 (s, 6H), 8.23 (s, 1H) ppm; 13C NMR (125 MHz, CDCl3): δ = 24.9, 29.3, 130.1, 137.7, 160.2, 199.2 ppm.  3,3,6,6-tetramethyl-3,4,6,7-tetrahydroacridine-1,8(2H,5H)-dione (Py5) [3]  Following the general procedure for the laccase-catalyzed oxidation of 1,4-dihydropyridines, 3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (DHPy5) (final concentration 26 mM) was oxidized using BliLacc (final concentration 0.87 U/ml) during 4 h at 50 ˚C. Purification by dry-flash chromatography (SiO2; hexane/ethyl acetate 9:1 v/v, hexane/ethyl acetate 7:3 v/v) yielded Py5 (5.1 mg, 16%).  Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. coli whole-cell suspension, 3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (DHPy5) (final concentration 11 mM) was oxidized using E. coli (BliLacc) (0.2 ml, 8  OD600 = 150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be 9% by NMR spectroscopy.   Following the general procedure for the oxidation of 1,4-dihydropyridines catalyzed by the E. coli whole-cell suspension, 3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (DHPy5) (final concentration 11 mM) was oxidized using E. coli TOP10 (0.2 ml, OD600 = 150) during 24 h at 50 ˚C. The conversion of the starting material was determined to be 7% by NMR spectroscopy. The product was obtained as a white solid. FT-IR (ATR): 2957s, 2928s, 2871m, 1699s, 1588s, 1563w, 1465m, 1417m, 1389w, 1371w, 1335w, 1296w, 1260w, 1234m, 1120w; 1H NMR (500 MHz, CDCl3): δ = 1.12 (s, 12H), 2.57 (s, 4H), 3.05 (s, 4H), 8.81 (s, 1H) ppm.  Fig. S1. Recycling of the E. coli (BliLacc)/BNC catalyst in the oxidation of 1a. The reactions were run with 0.03 mmol of the substrate, according to general procedure. Substrate amount/CDW = 8.55.   >99 69 37 0 10 20 30 40 50 60 70 80 90 100 1 2 3 NMR conversion (%) Reaction cycle number 9  Table S1. The substrate scope of the biocatalytic oxidation of 1,4-dihydropyridines.  Substrate Catalyst ABTS (mol%) CuSO4 (mol%) Product yield (%) DHPy1d BliLacc 1.5 – 94a DHPy1 E. coli (BliLacc) 1.6 1.7 97b DHPy1 E. coli TOP10 1.6 1.7 95b DHPy2d BliLacc 1.5 – 11a DHPy2 E. coli (BliLacc) 1.6 1.7 0b DHPy2 E. coli TOP10 1.6 1.7 0b DHPy3d BliLacc 1.5 – 15a DHPy3 E. coli (BliLacc) 1.6 1.7 11b DHPy3 E. coli TOP10 1.6 1.7 7b DHPy4d BliLacc 1.5 – 94a DHPy4 E. coli (BliLacc) 1.6 1.7 >99b  DHPy4 E. coli TOP10 1.6 1.7 >99b DHPy5d BliLacc 1.5 – 16a DHPy5 E. coli (BliLacc) 1.6 1.7 9b DHPy5 E. coli TOP10 1.6 1.7 7b DHPy6d BliLacc 1.5 – 0c DHPy6 E. coli (BliLacc) 1.6 1.7 0b DHPy6 E. coli TOP10 1.6 1.7 0b  a Isolated yield b Judged by conversion of starting material (based on NMR analysis) c Judged by conversion of starting material (based on TLC analysis) d CuSO4 was added during the production of the recombinant protein   References [1] H.T. Abdel-Mohsen, J. Conrad, U. Beifuss, Laccase-catalyzed oxidation of Hantzsch 1,4-dihydropyridines to pyridines and a new one pot synthesis of pyridines. Green Chem. 14 (2012) 2686-2690. [2] O. De Paolis, J. Baffoe, S.M. Landge, B. Toeroek, Multicomponent Domino Cyclization-Oxidative Aromatization on a Bifunctional Pd/C/K-10 Catalyst: An Environmentally Benign Approach toward the Synthesis of Pyridines. Synthesis 21 (2008) 3423-3428. [3] V. Hegde, C.Y. Hung, P. Madhukar, R. Cunningham, T. Hopfner, R.P. Thummel, Design of receptors for urea derivatives based on the pyrido [3, 2-g] indole subunit.  J. Am. Chem. Soc. 115 (1993) 872-878. 

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