A Biocatalytic Synthesis of Heteroaromatic N-Oxides by Whole Cells of Escherichia coli Expressing the Multicomponent, Soluble Di-Iron Monooxygenase (SDIMO) PmlABCDEF

Article abstract of DOI:10.1002/adsc.201801491

Aromatic N-oxides (ArN?OX) are desirable biologically active compounds with a potential for application in pharmacy and agriculture industries. As biocatalysis is making a great impact in organic synthesis, there is still a lack of efficient and convenient enzyme-based techniques for the production of aromatic N-oxides. In this study, a recombinant soluble di-iron monooxygenase (SDIMO) PmlABCDEF overexpressed in Escherichia coli was showed to produce various aromatic N-oxides. Out of 98 tested N-heterocycles, seventy were converted to the corresponding N-oxides without any side oxidation products. This whole-cell biocatalyst showed a high activity towards pyridines, pyrazines, and pyrimidines. It was also capable of oxidizing bulky N-heterocycles with two or even three aromatic rings. Being entirely biocatalytic, our approach provides an environmentally friendly and mild method for the production of aromatic N-oxides avoiding the use of strong oxidants, organometallic catalysts, undesirable solvents, or other environment unfriendly reagents. (Figure presented.).

Full text of DOI:10.1002/adsc.201801491

Title: A Biocatalytic Synthesis of Heteroaromatic <i>N</i>-Oxides
by Whole Cells of <i>Escherichia coli</i> Expressing the
Multicomponent, Soluble Di-Iron Monooxygenase (SDIMO)
PmlABCDEF
Authors: Vytautas Petkevičius, Justas Vaitekūnas, Daiva Tauraitė,
Jonita Stankevičiūtė, Jonas Šarlauskas, Narimantas Čėnas,
and Rolandas Meškys
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201801491
Link to VoR: http://dx.doi.org/10.1002/adsc.201801491

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
2
3
A Biocatalytic Synthesis of Heteroaromatic N-Oxides by Whole Cells of
Escherichia coli Expressing the Multicomponent, Soluble Di-Iron Monooxygenase
(SDIMO) PmlABCDEF
4
a,
a
a
a
5
6
Vytautas Petkevičius *, Justas Vaitekūnas , Daiva Tauraitė , Jonita Stankevičiūtė ,
b
b
a
Jonas Šarlauskas , Narimantas Čėnas , Rolandas Meškys
a
7
8
Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry,
Life Sciences Center, Vilnius University, Saulėtekio 7, Vilnius LT-10257, Lithuania.
9
Tel: +37052234386; Fax +37052687009; e-mail: vytautas.petkevicius@bchi.vu.lt
b
1
1
0
1
Department of Xenobiotics Biochemistry Institute of Biochemistry, Life Sciences
Center, Vilnius University, Saulėtekio 7, Vilnius LT-10257, Lithuania.
1
1
1
2
3
4
ABSTRACT
1
1
1
1
1
2
2
2
2
2
2
2
2
5
6
7
8
9
0
1
2
3
4
5
6
7
Aromatic N-oxides (ArN-OX) are desirable biologically active compounds with a
potential for application in pharmacy and agriculture industries. As biocatalysis is
making a great impact in organic synthesis, there is still a lack of efficient and
convenient enzyme-based techniques for the production of aromatic N-oxides. In this
study, a recombinant soluble di-iron monooxygenase (SDIMO) PmlABCDEF
overexpressed in Escherichia coli was showed to produce various aromatic N-oxides.
Out of 98 tested N-heterocycles, seventy were converted to the corresponding N-oxides
without any side oxidation products. This whole-cell biocatalyst showed a high activity
towards pyridines, pyrazines, and pyrimidines. It was also capable of oxidizing bulky N-
heterocycles with two or even three aromatic rings. Being entirely biocatalytic, our
approach provides an environmentally friendly and mild method for the production of
aromatic N-oxides avoiding the use of strong oxidants, organometallic catalysts,
undesirable solvents, or other environment unfriendly reagents.
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
2
8
Keywords
2
3
9
0
Biocatalysis, chemoselectivity, nitrogen heterocycles, oxidation, regioselectivity, soluble
di-iron monoxygenase.
3
3
3
1
2
3
INTRODUCTION
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Various N-heteroaromatic compounds are widely used as starting materials in
diverse chemical syntheses.[1] However, unsubstituted N-heterocycles are rather inert
molecules that are challenging to modify chemically. One of the approaches for their
functionalization is N-oxidation, which increases their reactivity with either electrophilic
[
2]
and nucleophilic agents. In fact, the chemistry and applications of aromatic N-oxides
have been receiving increased attention over the last two decades due to their
usefulness as potential anticancer, antibacterial, antihypertensive, antiparasitic, anti-
HIV, anti-inflammatory, herbicidal, neuroprotective, and procognitive agents.[3,4] ArN-OX
are useful as protecting groups, auxiliary agents, oxidants, ligands in metal complexes
and catalysts.[5] Also, ArN-OX have been shown to be useful in energetic materials
applications such as propellants, explosives, and pyrotechnics.[6] The most common
[
7]
approach for the synthesis of these compounds employ oxidation by peroxy acids or
hydrogen peroxide.[8] Although these reagents are rather inexpensive, they are
dangerous to handle and produce significant amount of waste. Hydrogen peroxide-
mediated oxidation has been improved by using transition metal catalysts, which form
highly reactive oxo or peroxymetal species. Most commonly used metals include
rehnium,[9] ruthenium,[10] manganese,[11] and tungsten.[12] The main drawback of these
methods is a possible product contamination with potentially hazardous metals. The
alternatives include condensation reactions, various ring transformations,
rearrangements and cycloadditions.[13,14] These reactions, however, are relatively
unspecific and often complicated. Most recently, Mo/P-catalyzed oxidation[15] and 2,2,2-
trifluoroacetophenone/H
2
O
based oxidation[16] have been introduced as promising new
2
methods. They were shown to be chemoselective and operated under mild reaction
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
5
5
7
8
conditions. Nonetheless, a practically useful biocatalytic method to synthesize ArN-OX
has yet to be developed.
5
6
6
6
6
6
6
6
6
6
9
0
1
2
3
4
5
6
7
8
There are several reports on biological N-oxidation of N-heterocycles. Among the
isolated enzymes, methane monooxygenase[17] and cytochrome P450[18] as well as
Rhodococcus jostii TMP1 cells[19] have been shown to oxidize N-heterocycles with a
presumed formation of N-oxides; however, further studies have not been elaborated.
Other efforts to exploit enzymes or cells for the synthesis of N-oxides involved
peroxygenase from Agrocybe aegerita,[20] cyclohexanone monooxygenase from
Lysobacter antibioticus,[21] and whole cells of Verticillium sp. GF39[22] and Burkholderia
sp. MAK1.[23] The main shortcomings of those biocatalysts include low productivity,
inconvenient atypical microorganisms, and narrow range of substrates. Thus a
practically useful biocatalytic method to synthesize ArN-OX has yet to be developed.
6
7
7
7
9
0
1
2
In this study, we report that monooxygenase PmlABCDEF recombinantly
expressed in E. coli is highly functional N-oxidizing biocatalyst. The optimized whole cell
bioconversions produced a controllable, productive and green method for the synthesis
of ArN-OX (Scheme 1). We demonstrated a broad substrate specificity of this enzyme
7
3
7
7
7
4
5
6
Scheme 1. Synthesis of aromatic N-oxides using PmlABCDEF monoxygenase producing E. coli
cells as biocatalyst. This process requires atmospheric oxygen as an oxidant. X = C or N, n = 1
or 2.
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
7
7
7
8
7
8
9
0
testing close to a hundred of different compounds, thus surpassing the scale of previous
studies. Our approach proved to be chemoselective as it can be used for the molecules
bearing oxidation-sensitive functional groups. Also, this method provides regioselectivity
as only specific mono N-oxides are formed.
8
8
8
8
8
1
2
3
4
5
RESULTS AND DISCUSSION
Selection of ArN-OX producing enzyme
8
8
8
8
9
9
9
9
9
9
9
9
9
9
6
7
8
9
0
1
2
3
4
5
6
7
8
9
SDIMOs are a group of enzymes that hydroxylate various aromatic compounds
enabling bacteria to assimilate them.[24] Because of their broad substrate range and
chemo-, stereo-, regioselectivity they are attractive enzymes for biocatalysis.[25] We
previously reported that the cells of Burkhoderia sp. MAK1 are capable of transforming
certain pyridine derivatives to the corresponding N-oxides.[23] Later, the genes
responsible for this activity were identified as a new type of SDIMOs. [26] However,
recombinant expression of this monooxygenase in different hosts did not produce active
enzyme, while wild-type Burkhoderia sp. MAK1 cells were a troublesome biocatalyst to
work with. Convinced that particular SDIMOs are able to catalyze N-oxidation, we
sought for ones functional in the E. coli cells. Thus, we explored the DNA sequences of
our collection of various oxygenases obtained from metagenomes looking for an
occurrence of SDIMOs. Hence, seven clones displaying sequence similarities to
SDIMOs were selected. Initial experiments showed that one clone designated as p577A
was capable of oxidation of pyridine. After the full DNA sequence of plasmid p577A was
obtained, a 4.5 kb gene cluster of six open reading frames was identified and entitled as
pmlABCDEF. PmlABCDEF shared overall ~80 % amino acid sequence identity with
butylphenol monooxygenase from Pseudomonas putida, an enzyme of the SDIMO
group (Table S1).
1
1
1
1
00
01
02
03
1
04
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
1
05
06
Optimization of PmlABCDEF production
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
To proceed with a bioconversion experiments, our initial goal was to maximize
the expression of PmlABCDEF as well as biomass production of the recombinant cells.
Thus, pmlABCDEF was amplified by PCR as a continuous DNA fragment and cloned
into a protein expression vector, creating the pET_PmlABCDEF plasmid. E. coli BL21
(DE3) and E. coli Rosetta(DE3)pLysS cells carrying aforementioned plasmid (PML and
PML_Ros respectively) were used for further studies. The recombinant cells were
grown in nutritionally rich medium (LB or BHI) with glucose at 37 °C. After addition of
IPTG bacteria strains were transferred to 30 °C overnight induction. However, SDS-
PAGE analysis revealed that PmlABCDEF was produced mainly as insoluble proteins.
Consequently, cultivation temperatures were lowered maintaining 30 °C for the growth
stage and 20 °C for protein expression. This process drastically increased production of
PmlABCDEF as soluble proteins (Fig. S1). Regarding the medium, there were no
significant differences between BHI or LB media in terms of protein or biomass
production. Also, final concentration of IPTG did not significantly enhanced
PmlABCDEF expression as 0.5 mM of IPTG seemed to reach the saturation. Since no
distinguished difference in an oxidation activity of PML and PML_Ros strains was
observed, for further studies PML was used as PML_Ros was vulnerable to cell lysis
during bioconversion.
1
1
1
25
26
27
Investigation on substrate scope of PML
1
1
1
1
1
28
29
30
31
32
The initial bioconversion experiments showed that PmlABCDEF strain was able
to oxidize pyridine and pyrazine. Using HPLC-MS the end products of the reactions
were identified as pyridine-1-oxide and pyrazine-1-oxide, accordingly, by comparison
with the analytical standards. Further, we tried to elucidate the substrate scope of PML
(Table 1). The experiments were carried out on an analytical scale in 1 mL reaction
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
1
1
1
33
34
35
36
volume using whole-cell PML and 1 mM substrate at 30 °C overnight. Among 98 tested
N-aromatic compounds, 70 were shown to be converted by various conversion degree
into products whose molecular mass increased by 16 Da (HPLC-MS data in Supporting
information).
1
1
37
38
Table 1. The compounds used as substrates by PML. The conversion (%) was estimated by the
consumption of the substrate (initial concentration – 1 mM).
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
39
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
40
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
1
1
1
1
1
1
1
1
1
1
41
42
43
44
45
46
47
48
49
50
51
The derivatives of pyridine comprised a significant part of the examined
compounds. Pyridines harboring small aliphatic groups (2a, 3a, 4a, 5a, 6a, 8a, 9a) were
fully converted by PML. The exceptions were 2,6-dimethylpyridine (7a) and 2,4,6-
trimethylpyridine (10a), as they possess two o-substituents with respect to nitrogen
which possibly hinders the attack. From known biocatalyst only Burkholderia sp.
MAK1[23] appeared to possess a very similar preference for alkylated pyridines.
Peroxygenase from Agrocybe aegerita also acted on those compounds, however side-
products of methyl group oxidation were present.[20] Alkylated pyridines did not fall into
substrate scope of Verticillium sp. GF39 cells;[22] however, the N-oxides were proposed
to be formed when Rhodococcus jostii TMP1[19] was used for conversion of 3-methyl-
and 3-ethylpyridine, 2,3-dimethyl-, 3,5-dimethyl-, and 2,3,5-trimethylpyridine.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
Other favorable substrates for PML included those bearing hydroxymethyl (12a,
13a, 14a), methoxy (15a) and cyano moieties (30a, 31a). ). It appears that the electron-
donating or -accepting properties of the substituents in the 3rd and 4 positions of
pyridine ring did not affect the reaction yield. One of the factors which significantly
lowered the reaction yield might be a pyridine/pyridone tautomerism of 2-
hydroxypyridine and its derivatives (compounds 17a, 19a, 20a). The same factor,
including a possible 2-aminopyridine/pyridinimine tautomerism might be responsible for
the absence of reactivity in the case of 2-mercaptopyridine, 2,3-dihydroxypyridine, 2,4-
dihydroxypyridine, 4-hydroxypyridine, 2-amino-3-hydroxypyridine, and an decreased
activity towards 2-amino derivatives 21a, 22a, 26a, 27a, 28a. Another important factor
decreasing the reactivity might be the presence of a negative charge, because all
pyridine carboxylic acid as well as pyridine-2,6-dicarboxylic acid were not oxidized by
PML. In fact, pyridinecarboxylic acids were not transformed by peroxygenase from
Agrocybe aegerita, Verticillium sp. GF39 and Burkholderia sp. MAK1 cells as
well.[20,22,23]
th
1
1
1
1
67
68
69
70
The halogenated and especially polyhalogenated N-heteroaromatics are known
to be difficult to oxidize.[27] The electron withdrawing effect of halogens reduces the
nucleophilicity of the ring nitrogen as well as the reactivity towards m-CPBA, H
2
O
2
,
HOF·CH CN and other generated electrophilic oxygen species. Enzymes from the
3
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
1
1
1
71
72
73
74
SDIMOs group usually employ iron-peroxo compIex in the active center,[28] thus not
surprisingly PML does not catalyze N-oxidation of 2,6-dichloropyridine, 2,6-
difluoropyridine and 3,5-dibromopyridine. Only 2-chloropyridine was converted to a
corresponding N-oxide.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
We also investigated diazine compounds as potential substrates.
Methylpyrazines (34a, 35a, 36a) were shown to be excellent substrates with the
exception of 2,3,6-trimethylpyrazine (37a) which o-substituents might hinder
accessibility to nitrogen atom. PML also oxidized the molecules such as 5-methyl-6,7-
dihydro-5H-cyclopenta[b]pyrazine (38a) or acetylpyrazine (39a) containing complex
substituents. All four tested pyrimidines (43a, 44a, 45a, 46a) were converted in high
yields to the corresponding N-oxides. Interestingly, some of acceptable diazine
substrates possessed amino group, halogen atom or both, in contrast, these
substituents in pyridines impeded the catalysis. Pyridazine (40a) and its derivatives
(41a, 42a) were not as acceptable substrates as pyrazines and pyridines. A plausible
explanation for such behavior is the difference in the electron densities around the
nitrogen atoms. In fact, pyridazine possess the lowest basicity as well as nucleophilicity
of all tested diazines (pyrazine > pyrimidine > pyridazine) which makes it less
chemically reactive towards electrophilic interactions.[29] Compounds that were
completely
inactive
included
tetramethylpyrazine,
pyrido[2,3-b]pyrazine,
2-
hydroxypyrimidine and 2,3,6-tetrahydropyridazine-3,6-dione.
1
1
1
1
1
1
1
1
1
2
91
92
93
94
95
96
97
98
99
00
Regarding two-ring heterocycles several of them also fell into the range of PML
substrates. Unsubstituted compounds like quinoline (47a), isoquinoline (48a),
quinoxaline (52a), 1,5-naphtyridine (53a) and quinozaline (54a) were transformed at
high conversion yields. Their derivatives with one alicyclic ring (50a, 51a, 55a) or methyl
group (49a) also possessed good reactivity. However, PML did not catalyze N-oxidation
of 2-methylquinoline like of 2,6-dimethylpyridine or tetramethylpyrazine, as well as of
6,7-dihydro-5H-isoquinolin-8-one, 4-aminoquinoline, 11H-indeno[1,2-b]quinoxalin-11-
one. From the aforementioned biocatalysts only Verticillium sp. GF39 cells were shown
to utilize two-ring heterocycles. The three-ring containing N-heterocycles 57a, 58a, and
59a were poor substrates for PML, and only the trace amounts of putative monoxides
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
were detected by HPLC-MS. The exception was acridine (56a), which at 0.5 mM
concentration underwent a substantial conversion to a possible acridine-10-oxide. To
our best knowledge, only Lysobacter antibioticus[21] catalyzed the N-oxidation of three-
ring N-heterocycles (phenazine). As expected, PML did not transformed 1,10-
phenanthroline, the Fe-chelating agent, which rapidly colored reaction mixture in red
inhibiting the catalysis. The two-ring compounds with two or three nitrogen atoms (57a,
[
6]
60a, 62a) prominent starting materials for synthesis of anticancer 1,4-di-N-oxides were
poor substrates for PML, except compound 52a. A low bio-convertability was observed
in the case of compounds 63a and 64a as well. The derivatives not transformed at all
included nicotine, nicotinamide, 3-bromopyrazolo[1,5-a]pyrimidine, bis(pyridin-2-
yl)ethane-1,2-dione, 1-(4-pyridyl)piperidine, 2-(pyridin-2-yl)pyridine, pyrazolo[1,5-
a]pyrimidine. However, a group of complex substrates harboring two rings such as 4-(4-
nitrobenzyl)pyridine (65a), 4-(pyridin-4-ylsulfanyl)pyridine (66a), 3-(pyrrol-1-yl)pyridine
(67a), 4-(pyridin-4-yl)pyridine (68a), 4-(1,3-oxazol-5-yl)pyridine (69a) and 2-
phenylpyridine (70a) were converted at high yields.
2
2
2
2
2
2
2
2
2
2
2
16
17
18
19
20
21
22
23
24
25
26
Overall, PML displayed an extraordinary wide substrate range towards N-
heterocyclic compounds that made it prominent among the previously reported
biocatalysts. Pyridines were among the most acceptable substrates. Pyrazines and
pyrimidines were also oxidized by PML in high conversion yields, while conversion rates
of pyridazines where considerably lower. Molecules featuring two six-membered rings
were moderate substrates, although mostly not as productive as compounds with a
single six-membered ring. The largest molecules that were oxidized by PML consisted
of three six-membered rings, albeit only traces of oxidation products were detected.
This observation suggested that PML biocatalytic activity declined as substrates size
and complexity increased. The particular features of undesirable substrates include two
ortho-substituents, carboxy-, hydroxy- and amino-groups or halogen atoms.
2
2
2
27
28
29
Biocatalytic synthesis of ArN-OX
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
2
2
2
2
2
2
2
2
2
2
30
31
32
33
34
35
36
37
38
39
For the preparative scale synthesis of N-oxides we selected 14 compounds
(Table 2) that reflect the elucidated substrate space of PML or are challenging to
produce by conventional methods. Pyridine (1a), pyrazine (33a) and their derivatives
represented the simplest single-ring heterocycles containing one and two nitrogen
atoms. These included 4-ethylpyridine (9a), 2-chloropyridine (29a), 4-cyanopyridine
(31a), 2- hydroxymethylpyridine (12a), and 2,6-dimethylpyrazine (36a). Other
compounds served as diverse two-ring systems, some of them possessing alicyclic or
heterocyclic ring. This group composed of quinoxaline (52a), 5,6,7,8-
tetrahydroquinoxaline (55a), isoquinoline (48a), 1-methylisoquinoline (49a), quinazoline
(54a), 4-pyridin-4-ylsulfanylpyridine (66a), and 4-(1,3-oxazol-5-yl)pyridine (69a).
2
40
Table 2 The list of synthesized and purified N-oxides.
Productivity
Yield
[%]
75
Substrate
Product
-1
-1
[
mg L h ]
39.6
Pyridine (1a)
Pyridine-1-oxide (1b)
4-Cyanopyridine-1-oxide (31b)
4-Ethylpyridine-1-oxide (9b)
4
2
-Cyanopyridine (31a)
15.0
15.4
4.3
68
64
43
78
82
48
66
45
64
42
81
75
4
-Ethylpyridine (9a)
-Chloropyridine (29a)
2-Chloropyridine-1-oxide (29b)
2-Hydroxymethylpyridine -1-oxide (12b)
Pyrazine-1-oxide (33b)
2
-Hydroxymethylpyridine (12a)
Pyrazine (33a)
10.4
40.0 and 480*
2
,6-Dimethylpyrazine (36a)
Quinoxaline (52a)
25.8
4.3
3,5-Dimethylpyrazine-1-oxide (36b)
Quinoxaline-1-oxide (52b)
5
,6,7,8-Tetrahydroquinoxaline (55a)
Isoquinoline (48a)
5.4
5,6,7,8-Tetrahydroquinoxaline-1-oxide (55b)
Isoquinoline-2-oxide (48b)
5.1
1
-Methylisoquinoline (49a)
-(Pyridin-4-ylsulfanyl)pyridine (66a)
-(1,3-Oxazol-5-yl)pyridine (69a)
5.1
1-Methylisoquinoline-2-oxide (49b)
4-(Pyridin-4-ylsulfanyl)pyridine-1-oxide (66b)
4-(1,3-Oxazol-5-yl)pyridine-1-oxide (69b)
4
17.0
6.5
4
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
Quinazoline (54a)
12.2
Quinazoline-3-oxide (54b)
67
2
41
*reaction was performed in 1 L bioreactor
2
2
2
2
2
2
2
2
2
2
2
2
2
42
43
44
45
46
47
48
49
50
51
52
53
54
The preparative scale synthesis was performed in 200 mL reaction volume for 24
h utilizing 1.0–10.0 mM of appropriate substrate. Pyrazine and pyridine were the most
productive substrates found so far. Both were completely converted to corresponding
monoxides after 24 h of exposure with PML using 10 mM as a final concentration. The
production yields of 75% for pyridine-1-oxide and 82% for pyrazine-1-oxide with
–1
–1
–1 –1
respective productivities of 39.6 mg L h and 40.0 mg L h , were achieved. This is
tenfold greater than productivities reached by using Burkholderia sp. MAK1 (3.8 mg L–1
–1
–1 –1
[23]
h for pyridine-1-oxide and 3.9 mg L h for pyrazine-1-oxide). On the other hand,
Verticillium sp. GF39 cells were reported to possess respectable productivities: 79.5 mg
–1
–1
–1 –1
–
L h for 1-methylisoquinoline-2-oxide, 10.5 mg L h for pyridine-1-oxide, 61.0 mg L
1
–1
–1 –1
–1
–
h for 2-methylpyridine-1-oxide, 76.9 mg L h for quinoline-1-xide and 5.1 mg L h
1
for isoquinoline-2-oxide.[22] However, the narrow substrate range of this biocatalyst
severely limits its application.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
55
56
57
58
59
60
61
62
63
64
65
66
67
68
Notwithstanding that the N-oxides of 9a, 29a, 31a can be synthesized chemically
in a variety of ways,[9,15,30] the PML-catalyzed synthesis is an attractive 'green'
alternative. The chemical synthesis of mono-N-oxides from compounds possessing two
nitrogen atoms (33a, 36a, 52a, 54a, 55a) and especially for pyrazine derivatives is
challenging as dioxides may form as well.[27, 31] Usually the side reactions are avoided
by adding an equimolar amount of the oxidizing agent and conducting the reaction at
low temperatures.[32] Besides aromatic mono-N-oxides preparation, our method is
suitable for a regioselective synthesis. Earlier reported N-oxidations of 2,6-
dimethylpyrazine (36a) resulted in a mixture of 3,5-dimethylpyrazine-1-oxide and 2,6-
dimethylpyrazine-1-oxide.[33,34] Our method yields a product, which was detected as a
single peak in HPLC-MS, and 1H NMR and 13C NMR analysis confirmed it as 3,5-
dimethylpyrazine-1-oxide (36b). An oxidation of quinazoline (54a) also gave only one
product which was identified as quinazoline-3-oxide (54a). Besides, we have
synthesized mono-N-oxides of 5-methyl-6,7-dihydro-5H-cyclopenta[b]pyrazine (38a)
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
13
2
2
69
70
and 2-amino-4-chloropyrimidine (44a). However, H NMR and C NMR analysis did not
revealed the exact structure of the final product.
2
2
2
2
2
2
2
2
2
2
2
71
72
73
74
75
76
77
78
79
80
81
For compounds containing reactive substituents, e.g., 12a, 66a, 69a exposure to
typical oxidizing agents (m-CPBA, H
2
O
2
, trifluoroperoxyacetic acid, HOF·CH CN) may
3
modify the substituent as well. Although recently chemoselective methods[15,16] have
been introduced, they are not entirely environment-friendly. PML-catalyzed approach
also was shown to be chemoselective, hence 2-hydroxymethylpyridine was converted to
a single product (12b) without traces of possible pyridine-2-carbaldehyde or pyridine-2-
carboxylic acid. Moreover, 4-(pyridin-4-ylsulfanyl)pyridine was transformed to the
corresponding mono-N-oxide 66b avoiding the formation of a sulfoxide. Finally,
oxidation of 4-(1,3-oxazol-5-yl)pyridine resulted in N-oxide in the pyridine ring (69b),
while attack on a substituent oxazole ring was not observed. Undoubtedly, such
conversions are challenging for the standard chemical approaches.
2
2
2
2
2
2
2
2
2
2
2
2
82
83
84
85
86
87
88
89
90
91
92
93
The biocatalytic synthesis of pyrazine-1-oxide was also performed on a multi-
gram scale in 1 L bioreactor. The supplementation of pyrazine (800 mg, 10 mmol)
together with glucose (1.8 g, 10 mmol) owas executed periodically, once in two hours.
In total, 2.4 g of pyrazine was utilized in 6 h. Compared with the conversion in flasks,
–1
–1
–1 –1
this technique increased the productivity tenfold, from 40.0 mg L h to 480 mg L h .
It also allowed reaching gram-scale synthesis of 33b in a few hours (Fig. S2). We
assumed that the observed increase in productivity was caused by enhanced
supplementation of oxygen, which was crucial for the PmlABCDEF catalytic activity. It
was previously demonstrated that a proper level of oxygen saturation was essential for
various oxygenase-catalyzed processes conducted in bioreactors.[35] Indeed, this
example suggested that conducting PML-catalyzed N-oxidation in bioreactor should
increase productivity in the case of other substrates as well.
2
2
2
94
95
96
CONCLUSIONS
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
2
2
2
3
3
3
3
3
3
3
97
98
99
00
01
02
03
04
05
06
PmlABCDEF, belonging to the SDIMO group, serves as a novel biocatalyst for
synthesis of aromatic N-oxides with high conversion yields (70–100 %). Furthermore,
most of those N-oxides have not been previously prepared using a biocatalytic
approach. In addition, to best of our knowledge, the synthesis of 4-(pyridin-4-
ylsulfanyl)pyridine-1-oxide and 4-(1,3-oxazol-5-yl)pyridine-1-oxide has not been
documented neither by biocatalytic nor chemical approach. The presented method is
efficient and scalable making a gram-scale synthesis feasible in a few hours. Our
method stands out as being biocatalytic, environmentally friendly, productive as well as
being regio- and chemoselective. Moreover, further improvements of the biocatalyst are
possible via a directed evolution of the PmlABCDEF oxygenase.
3
3
3
3
3
07
08
09
10
11
EXPERIMENTAL SECTION
Materials
3
3
3
3
3
3
3
3
3
3
3
3
3
12
13
14
15
16
17
18
19
20
21
22
23
24
2-Hydroxypyridine, 2-hydroxypyrimidine, 2-aminopyrimidine, 2-methylpyridine, 3-
methylpyridine, 4-methylpyridine, 2-hydroxymethylpyridine, 2-mercaptopyridine, 2-
hydroxy-3-bromopyridine, 2-hydroxy-3-methylpyridine, 2,3-dihydroxypyridine, 2,4-
dihydroxypyridine, 2,3-dimethylpyridine, 2,4-dimethylpyridine, pyridine-2-carboxylic acid,
pyridine-4-carboxylic acid, pyridine-2,6-dicarboxylic acid, 2,6-dimethylpyridine, 3,5-
dimethylpyridine, 2,3,5-trimethylpyridine, acetylpyrazine, 2,3-dimethylpyrazine, 2,5-
dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, tetramethylpyrazine, 2-
(2-hydroxyethyl)pyridine,
pyridazine,
3-methylpyridazine,
6,7-dihydro-5H-
cyclopenta[b]pyridine, 5-methyl-6,7-dihydro-5H-cyclopenta[b]pyrazine, quinoxaline,
5,6,7,8-tetrahydroquinoxaline, isoquinoline,1-methylisoquinoline, 2-methylquinoline,
5,6,7,8-tetrahydroisoquinoline, 1,2,3,6-tetrahydropyridazine-3,6-dione, 2,4-diamino-6-
methyl-1,3,5-triazine, 2,4,6-triamino-1,3,5-triazine, 1H-pyrrolo[2,3-b]pyridine, 1H,2H,3H-
pyrrolo[2,3-b]pyridine, 8-acetyl-5,6,7,8-tetrahydroquinoline, nicotine, nicotinamide, 1,5-
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
naphtyridine, 2,6-difluoropyridine, 3-bromopyrazolo[1,5-a]pyrimidine, bis(pyridin-2-
yl)ethane-1,2-dione, 4,7-phenanthroline, 1-(4-pyridyl)piperidine, imidazo[1,2-a]pyrazine,
norharmane, quinazoline, 4-phenylpyrimidine,
3,5-dibromopyridine, 2-amino-3-
hydroxypyridine, 2-phenylpyridine, 2-(pyridin-2-yl)pyridine, 4-(pyridin-4-yl)pyridine,
acridine, 1,10-phenanthroline, phenazine were obtained from Sigma-Aldrich (Munich,
Germany). 2-Chloropyridine, 2,6-dichloropyridine, pyridine-1-oxide, 3-hydroxypyridine,
2-amino-3-nitropyridine, 2-aminopyridine, pyrazine, quinoline were purchased from
Merck (Darmstadt, Germany). Indole, 4-ethylpyridine, 4-(4-nitrobenzyl)pyridine, 4-
cyanopyridine, 3-cyanopyridine, 2,4,6-trimethylpyridine, 3-hydroxymethylpyridine, 4-
hydroxymethylpyridine, 4-hydroxypyridine were from Fluka (Buchs, Switzerland). The
compounds including 2-hydroxy-3-fluoropyridine, 2-amino-3-chloropyridine, 2-amino-4-
chloropyrimidine were the products of Combi Blocks Inc (San Diego,USA), 4-(4-
pyridinylsulfanyl)pyridine, 3-(pyrrol-1-ylmethyl)pyridine, 4-(1,3-oxazol-5-yl)pyridine, 6,7-
dihydro-5H-isoquinolin-8-one were purchased from FluoroChem (Hadfield, UK), 4-
methoxypyridine was ordered from Bide Pharmatech (Shanghai, China), and 6-amino-3-
cyanopyridine,
2-amino-5-chloropyridine,
2-amino-5-bromopyridine,
2-amino-6-
methylpyridine, 3-aminopyridazine, 2-amino-4,6-dimethylpyrimidine, 2,4-diamino-1,3,5-
triazine, 3-amino-5,6-dimethyl-1,2,4-triazine were from TCI EUROPE N.V. (Belgium).
3
3
3
43
44
45
Bacterial strains and growth conditions
3
3
3
3
3
3
3
3
46
47
48
49
50
51
52
53
E. coli DH5α (Thermo Fischer Scientific, Lithuania) was used as a host for gene
cloning. E. coli BL21 (DE3) and E. coli Rosetta(DE3)pLysS (Novagen, Germany) were
chosen as hosts for recombinant protein production. All bacterial strains were routinely
−1
grown in Luria-Bertani (LB) broth at 37 °C, supplemented with kanamycin (100 μg mL )
−1
or chloramphenicol (20 μg mL ) if necessary. Composition of media was as follows: LB
(g/L) – tryptone (Formedium, UK) 10.0, yeast extract (Merck, Germany) 5.0, NaCl
(Fluka, Switzerland) 5.0; brain heart infusion broth (BHI), (Oxoid, UK) – 37.0. All media
were adjusted to pH = 7.2 and autoclaved at 1 atm for 30 min.
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
3
3
3
54
55
56
Metagenomic libraries
3
3
3
3
3
3
57
58
59
60
61
62
DNA from soil and sediment samples were isolated by using ”ZR Soil Microbe
DNA MidiPrep™” (Zymo Research, Germany). Metagenomic libraries were constructed
using a pUC19 vector as described.[36] The oxygenases producing hits forming blue
colonies were screened by the spreading transformed E. coli cells on LB agar
supplemented with 1 mM of indole. The plasmid DNA was isolated from each positive
clone and the cloned fragment was partially sequenced from both ends.
3
3
3
63
64
65
Construction of pET_PmlABCDEF
3
3
3
3
3
3
3
3
3
3
3
3
3
66
67
68
69
70
71
72
73
74
75
76
77
78
The oligonucleotide primers phe_xba_F (5'-actatctagagcttcctgaccgtggtg-3') and
phe_hind_R (5'-aattaagctttcagatggccttgaacagc-3') were ordered from Metabion,
Germany. The primers were designed to amplify monoxygenase gene and its ribosomal
binding site on p577A plasmid. The amplification was performed using a Phusion Green
Hot Start II High-Fidelity PCR Master Mix (Thermo Fischer Scientific, Lithuania)
according to the manufacturer’s instructions. The PCR amplicon was hydrolyzed with
XbaI and HindIII (Thermo Fischer Scientific, Lithuania) and ligated to the properly
digested protein expression vector pET-28b (Novagen, Germany). Standard methods
and techniques were employed for DNA manipulations (plasmid transformation,
screening, isolation). In the final plasmid (designated pET_PmlABCDEF), the cloned
gene was checked by sequencing (Macrogen, Netherlands). The new strains harboring
pET_PmlABCDEF plasmid were named PML (E. coli BL21 (DE3)) and PML_Ros (E.
coli Rosetta(DE3)pLysS) (Table 3).
3
3
79
80
Table 3 Plasmids and strains used in this study.
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
Source or
reference
Plasmid or strain
Relevant characteristics
Plasmid
p577A
pUC19/HindIII + 9 kb metagenomic DNA insert containing
pmlABCDEF gene
This study
r
pET-28b
pBR322-derived ColE1, T7 lac promoter, Km , protein
expression vector
Recombinant pET-28b containing pmlABCDEF gene
Novagen,
Germany
This study
pET_pmlABCDEF
Strain
E. coli DH5α
F– endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG
Thermo Fischer
Scientific,
–
purB20 φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(r
+), λ–
F– ompT gal dcm lon hsdS
T7p07 ind1 sam7 nin5]) [malB+]K-12(λS)
K
m
K
Lithuania
E. coli BL21 (DE3)
B
(r
(r
B
–m
B
–) λ(DE3 [lacI lacUV5-
Novagen,
Germany
E. coli
Rosetta(DE3)pLysS
F– ompT gal dcm lon hsdS
T7p07 ind1 sam7 nin5]) [malB+]K-12(λS)
B
B
–m
B
–) λ(DE3 [lacI lacUV5-
Novagen,
Germany
pLysSRARE[T7p20 ileX argU thrU tyrU glyT thrT argW
metT leuW proL orip15A](CmR)
PML
E. coli BL21 (DE3) harboring pET_PmlABCDEF
This study
This study
PML_Ros
E. coli Rosetta(DE3)pLysS harboring pET_PmlABCDEF
3
3
3
3
81
82
83
84
Induction conditions of PmlABCDEF monooxygenase
3
3
3
3
3
3
3
85
86
87
88
89
90
91
PML and PML_Ros strains were cultivated at 30 °C on a rotary shaker at 180
rpm in 1 L Erlenmeyer flasks containing either 200 mL LB, or BHI medium
supplemented with corresponding antibiotic. The broth was additionally supplemented
with glucose (to a final concentration of 1.0%) to prevent basal recombinant protein
expression. The incubation continued until OD600 reached 0.5–0.8. Then the culture
was supplemented with isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final
concentration of 0.5 mM and placed at 20 °C on a rotary shaker at 180 rpm overnight.
3
3
3
92
93
94
Bioconversions of N-heterocycles using whole cells of PML
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
3
3
3
3
3
4
4
4
95
96
97
98
99
00
01
02
After induction, cells were separated from the medium by centrifugation (4000 g
for 5 min). Then the biomass (3.0–4.0 g of wet weight) was washed twice with 10 mM
potassium phosphate buffer (pH = 7.0) and transferred to 1 L Erlenmeyer flasks
containing 200 mL of the same buffer and 1.0–10.0 mM of the appropriate substrate.
The reaction was carried out at 30 °C on a rotary shaker at 180 rpm. After 8 h of
incubation, reaction mixture was supplemented with glucose (5 mmol) and the
conversion was continued overnight for a total of 24 h or until 15–50 mg of the selected
substrate was completely consumed.
4
4
4
4
4
4
4
4
4
4
03
04
05
06
07
08
09
10
11
12
The conversion in a Biostat® B plus bioreactor was accomplished utilizing
PML biomass from collected from 2 L of growth medium. The reaction was carried out in
1 L of 10 mM potassium phosphate buffer (pH = 7.0) and started with 10 mmol of
pyrazine. The bioconversion was monitored by TLC, and portions of 10 mmol of
pyrazine were periodically added after observing significant depletion of the substrate.
The temperature was kept at 30 °C, and pH was maintained at 6.8–7.0 by auto-
equilibration with 0.2 M NaOH. Oxygen saturation (pO ) was kept at 80–95% level by
2
passing air (3 atm) through the reaction mixture and vigorous stirring (400 rpm).
Glucose (portions of 10 mmol) was added periodically alongside substrate. The
conversion process was controlled using the BioPAT® MFCS/ DA 3.0 software.
4
4
4
13
14
15
Isolation of N-oxides
4
4
4
4
4
4
4
16
17
18
19
20
21
22
The reaction mixture was separated from biomass by centrifugation (4000 g for
30 min). The volume of the supernatant was reduced under vacuum to a volume of 10–
15 mL and it was transferred to the separation funnel. Then water phase was washed
with equivalent volume of ethyl acetate at least three-four times. The organic phases
were combined and dried by anhydrous Na
2
SO . The resulting solution was evaporated
4
under reduced pressure to give a final product, N-oxide. All isolation stages were
monitored by TLC. The purity of the final product was verified by high-performance
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
4
4
23
24
liquid chromatography-mass spectrometry (HPLC-MS) and the chemical structure was
confirmed by nuclear magnetic resonance (NMR) spectroscopy.
4
4
4
4
4
25
26
27
28
29
The reaction mixture from bioreactor was also separated from biomass by
centrifugation (4000 g for 60 min). Then the supernatant was completely dried out under
reduced pressure. The resulting debris was several times gently washed with 5 mL of
warm ethyl acetate. Then organic phases were combined and dried by anhydrous
Na
2
SO , the solution was evaporated under vacuum to give a final product.
4
4
4
4
30
31
32
Calculation of bioconversion parameters
4
4
4
4
4
4
33
34
35
36
37
38
A conversion is expressed as ((I
a
– F
a
)/I
a
)
ͯ
100 %, where I = initial amount of a
a
substrate before the conversion, F – final amount of a substrate after conversion. The
a
substrate amount was determined by integrating the absorbance area (254 nm) of a
particular peak in HPLC chromatogram. If no substrate was left, the conversion is
defined as complete (>99%). Productivity is an expected amount of N-oxide (mg)
produced in 1 L reaction mixture under optimum conditions per 1 h.
4
4
39
40
HPLC-MS analysis
4
4
4
4
4
4
4
41
42
43
44
45
46
47
Before the analysis the cells were separated from the reaction mixture by
centrifugation. The resulting supernatant was mixed with an equal part of acetonitrile,
centrifuged and analyzed using a high performance liquid chromatography system.
HPLC-MS analysis was performed using a high performance liquid chromatography
system (Shimadzu, Japan) equipped with a photo diode array (PDA) detector and a
mass spectrometer (LCMS-2020, Shimadzu, Japan) equipped with an ESI source. The
data was analyzed using the LabSolutions LCMS software.
4
4
48
49
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
1
13
4
50
H NMR and C NMR
1
4
4
4
4
4
51
52
53
54
55
NMR spectra were recorded in DMSO-d
6
on an Ascend 400: H NMR – 400
1
3
MHz, C NMR – 100 MHz (Bruker, USA). Chemical shifts (δ) are reported in ppm
relative to the solvent resonance signal as an internal standard.
GenBank accession number(s)
4
56
4
4
4
57
58
59
The sequence of the p577A clone containing pmlABCDEF gene cluster was
deposited in GenBank under the accession number MK037457.
4
4
4
4
60
61
62
63
Conflict of interest
There are no conflicts to declare.
ACKNOWLEDGEMENTS
4
4
4
4
4
64
65
66
67
68
This research was funded by the European Social Fund according to the activity
‘Improvement of researchers’ qualification by implementing world-class R&D projects’ of
Measure No. 09.3.3-LMT-K-712 (grant No. DOTSUT-34(09.3.3-LMT-K712-01-
0058/LSS-600000-58)). This research was performed in cooperation with the INMARE
consortium.
4
4
4
69
70
71
REFERENCES
4
4
4
4
72
73
74
75
[1] J. Alcazar, D. Oehlrich, Future Med. Chem. 2010, 2, 169–176.
[2] J. Clayden, N. Greeves, S. G. Warren, Organic Chemistry, Oxford University
Press, 2012, 2, 730–731.
[3] A. M. Mfuh, O. V Larionov, Curr. Med. Chem. 2015, 22, 2819–2857.
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
[4] J. Balzarini, M. Stevens, E. De Clercq, D. Schols, C. Pannecouque, J.
Antimicrob. Chemother. 2005, 55, 2, 135–138.
[5] Y. Shaker, Arkivoc. 2001,1, 242–248.
[6] D. E. Chavez, in: Heterocyclic N-oxides (Eds.: O. V. Larionov, B. Maes, J. Cossy,
S. Polanc), Springer, 2017, 1–29.
[7] H. P. Kokatha, P. F. Thomson, S. Bae, V. R. Doddi, M. K. Lakshman, J. Org.
Chem. 2011, 76, 7842–7848.
[8] H. S. Mosher, L. Turner, A. Carlsmith, Org. Synth. 1963, 4, 828-830.
[9] C. Coperet, C. H. Adolfsson, V. T. Khovong, K. A. Yudin, B. K. Sharpless, J. Org.
Chem.1998, 63, 1740–1741.
[10] P. Veerakumar, S. Balakumar, M. Velayudham, K. L. Lu, S. Rajagopal, Catal.
Sci. Technol. 2012, 2, 1140–1145.
[11] A. Thellend, P. Battioni, W. Sanderson, D. Mansuy, Synthesis. 1997, 12, 1387–
1388.
[12] D. Sloboda-Rozner, P. Witte, P. L. Alsters, R. Neumann, Adv. Synth. Catal.
2004, 346, 339–345.
[13] A. W. Chucholowski, S. Uhlendorf, Tetrahedron Lett. 1990, 31, 1949–1952.
[14] R. Nesi, D. Giomi, S. Papaleo, S. Turchi, J. Org. Chem. 1992, 57, 3713–3716.
[15] O. V. Larionov, D. Stephens, A. M. Mfuh, H. D. Arman, A. S. Naumova, G.
Chavez and B. Skenderi, Org. Biomol. Chem. 2014, 12, 3026–3036.
[16] D. Limnios, C. G. Kokotos, Chem. Eur. J. 2014, 20, 559–563.
[17] J. Colby, D. I. Stirling, H. Dalton, Biochem. J. 1977, 165, 395–402.
[18] L. A. Damani, P. A. Crooks, M. S. Shaker, J. Caldwell, J. D'souza, R. L. Smith,
Xenobiotica. 1982, 12, 527–534.
[19] S. Kutanovas, R. Rutkienė, D. Tauraitė, R. Meškys, Chemija. 2013, 24, 67–73.
[20] R. Ullrich, C. Dolge, M. Kluge, M. Hofrichter, FEBS. Lett. 2008, 582, 4100–4106.
[21] Y. Zhao, G. Qian, Y. Ye, S. Wright, H. Chen, Y. Shen, F. Liu, L. Du, Org. Lett.
2016, 18, 2495–2498.
[22] K. Mitsukura, M. Hayashi, T. Yoshida, T. Nagasawa, J. Biosci. Bioeng. 2013,
115, 651–653.
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
[23] J. Stankevičiūtė, J. Vaitekūnas, V. Petkevičius, R. Gasparavičiūtė, D. Tauraitė, R.
Meškys. Sci. Rep. 2016, 6, 39129.
[24] J. G. Leahy, P. J. Batchelor, S. M. Morcomb, FEMS Microbiol Rev. 2003, 27,
449–479.
[25] D. E. Torres Pazmiño, M. Winkler, A. Glieder, M. W. Fraaije, J Biotechnol. 2010,
146, 9–24.
[26] V. Petkevičius, J. Vaitekūnas, J. Stankevičiūtė, R. Gasparavičiūtė, R. Meškys,
Appl. Environ. Microbiol. 2018, 84, 00387–18.
[27] M. Carmeli, S. Rozen, J. Org. Chem. 2006, 71, 5761–5765.
[28] C. E. Tinberg, S. J. Lippard, Biochemistry. 2009, 48, 12145–12158.
[29] H. J.Soscún, M. A. Hinchliffe, J. Mol. Struct. 1994, 304, 109–120.
[30] R. T. Shuman, P. L. Ornstein, J. W. Paschal, P. D. Gesellchen, J. Org. Chem.
1990, 55, 738–741.
[31] S. Rozen, A. Shaffer, Org. Lett., 2017, 19, 4707–4709.
[32] F. Roudesly, L. F. Veiros, J. Oble, G. Poli, Org. Lett. 2018, 20, 2346–2350.
[33] B. Klein, J. Berkowitz, J. Am. Chem. Soc. 1959, 81, 5160–5166.
[34] J. S. Dickschat, H. Reichenbach, I. Wagner-Dobler, S. S. Novel, Eur. J. Org.
Chem. 2005, 18, 4141–4153.
[35] W. A. Deutz, J. B. van Beilen, B. Witholt, Curr Opin Biotechnol. 2001, 12, 419–
425.
[36] N. Urbelienė, S. Kutanovas, R. Meškienė, R. Gasparavičiūtė, D. Tauraitė, M.
Koplūnaitė, R. Meškys. Microb. Biotechnol. 2018. doi: 10.1111/1751-7915.13316.
5
5
5
5
5
5
28
29
30
31
32
33
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201801491
VERY IMPORTANT PUBLICATION
FULL PAPER
5
5
5
34
35
36
5
5
5
37
38
39
A Biocatalytic Synthesis of Heteroaromatic N-Oxides by Whole Cells of
Escherichia coli Expressing the Multicomponent, Soluble Di-Iron Monooxygenase
(SDIMO) PmlABCDEF
5
40
5
41
This article is protected by copyright. All rights reserved.