Oxidation of amines catalyzed by cyclohexanone monooxygenase

Article abstract of DOI:10.1016/S0040-4039(02)02427-9

Cyclohexanone monooxygenase catalyzed the oxidation of tertiary, secondary and hydroxylamines to N-oxides, hydroxylamines and nitrones respectively.

Full text of DOI:10.1016/S0040-4039(02)02427-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 869–871
Oxidation of amines catalyzed by cyclohexanone monooxygenase
Stefano Colonna,a,* Vincenza Pironti,a Piero Pastab and Francesca Zambianchib
aIstituto di Chimica Organica, Facolta` di Farmacia, via Venezian 21, 20133 Milano, Italy
bIstituto di Chimica del Riconoscimento Molecolare, CNR, via Mario Bianco 9, 20131 Milano, Italy
Received 27 August 2002; accepted 22 October 2002
Abstract—Cyclohexanone monooxygenase catalyzed the oxidation of tertiary, secondary and hydroxylamines to N-oxides,
hydroxylamines and nitrones respectively. © 2003 Elsevier Science Ltd. All rights reserved.
Oxidative biotransformations are amongst the most
useful of all identified biologically mediated conver-
sions. They usually involve monooxygenases or diooxy-
genases that catalyze the insertion of one or two oxygen
atoms at a specific point of a molecule, often with high
stereo and/or regioselectivity. These very attractive fea-
tures are now increasingly exploited in the synthesis of
many pharmaceuticals and other highly valuable inter-
mediates. In the repertoire of monooxygenases, cyclo-
enzyme has been extended by our group to the asym-
metric epoxidation of electron-poor olefins.8
Another important point is that there are two enzyme
systems contained in the mammalian liver, responsible
for the oxidation of N-substituted amine drugs, in a
NADPH and O2 dependent manner; similar to CHMO,
one of them is a flavoprotein.9
hexanone
monooxygenase
from
Acinetobacter
Finally the nitrones, which are among the expected
reaction products of amine oxidation, are useful inter-
mediates for the synthesis of various nitrogen-contain-
ing biologically active compounds such as antibiotics,
alkaloids, aminosugars and b-lactams.10 They are excel-
lent spin traps in physiological media and protect the
central nervous system against oxidative damage;11 for
instance a-phenyl-tert-butyl nitrone exhibits antioxi-
dant and neuroprotective activity.12
calcoaceticus NCIMB 9871 (CHMO) (EC 1.14.13.22)
has been studied most intensively. CHMO is a flavoen-
zyme of about 60,000 Da, active as a monomer which
contains one firmly but non-covalently bound FAD
unit per enzyme molecule.1 It has wide potential for
application in the manufacture of fine chemicals and in
organic synthesis based on the Baeyer–Villiger oxida-
tion, which transforms racemic ketones into enan-
tiomerically pure esters. The only reagents consumed
are dioxygen, NADPH and the substrate ketone.2
Mechanistic studies have shown that the reactive inter-
mediate is 4a-hydroperoxyflavin which can act as an
electrophile towards trimethyl phosphite and iodide
ions, and as a nucleophile towards boronic acids and
ketones.1 The CHMO can catalyze the asymmetric sul-
foxidation of numerous alkyl aryl sulfides,3 dialkyl
sulfides,4 1,3-dithioacetals5 and organic cyclic sulfites.6
This enzyme also oxygenates heteroatoms such as N, Se
and P.7 Tertiary amines are converted into N-oxides,
selenides into selenoxides and phosphines into phosphi-
noxides. Recently, the synthetic potential of this
Keywords: cyclohexanone monooxygenase; nitrones; amines.
* Corresponding author. Tel.: +39 02 5031 4473; fax: +39 02 5031
4476; e-mail: stefano.colonna@unimi.it
Scheme 1. CHMO catalyzed oxidation of N-methylbenzyl-
amine with in situ coenzyme regeneration.
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02427-9

870
S. Colonna et al. / Tetrahedron Letters 44 (2003) 869–871
The enzyme used was as a partially purified preparation
obtained from an Escherichia coli strain in which the
gene of CHMO was cloned and overexpressed.13 The
amines (15 mM) were reacted, at 25°C, under stirring,
in 0.85 ml of 0.05 M Tris–HCl buffer, pH 8.6, contain-
ing NADP+ (0.5 mM), 5 units of CHMO, glucose-6-
phosphate (50 mM) and 18 units of glucose-6-
phosphate dehydrogenase (G6PDH) that served to
regenerate the cofactor (see Scheme 1). After 24 h, the
reaction mixtures were extracted with ethyl acetate (3×1
ml) and organic extracts were dried and analyzed. The
degree of conversion was determined by HPLC analysis
on a Chiralcel OD column using n-hexane–propan-2-ol
(9:1) as the mobile phase. The products of the reaction
were identified by comparison of their eluition order in
HPLC with the nitrones and the hydroxylamines pre-
pared according to literature.10,14 In the case of the
tertiary amine the product formed was the amine N-
oxide.7
We have chosen N-methylbenzylamine as model sub-
strate in order to understand the mechanism involved
in this biooxidation. The enzyme reacts with amine to
form first hydroxylamine and then, by further N-oxida-
tion, two regioisomeric nitrones in agreement with the
study on 4a-hydroperoxyflavin oxidation described by
Ball and Bruice.15 The two nitrones originate by N-oxi-
dation of the parent hydroxylamine (see in Scheme 2);
their ratio is in favor of the most stable one. The degree
of conversion and the Vmax/Km values indicate that the
catalytic efficiency of CHMO is similar for tertiary and
secondary amines (see Table 1). In contrast the oxida-
tion rate of N-methylaniline by 4a-hydroperoxyflavin
was two orders of magnitude lower than that of N,N-
dimethybenzylamine.15
Scheme 2. Mechanism of oxidation of N-methylbenzylamine
by CHMO.
racemic starting material. Furthermore in the case of
iso-propyl derivative, replacement of the methyl group
with more sterically demanding substituents leads to
minor reactivity; the N-tert-butylbenzyl derivative did
not react. Not only steric but also electronic effects
influence the reaction rate; indeed N-methyl-p-
chlorobenzylamine and 4-(ethylamino-methyl)-pyridine
were recovered unchanged. Dialiphatic secondary
amines such as diisopropylamine and pipecoline were
not transformed by CHMO.
The N-oxidation of amines by CHMO resembles the
sulfur oxidation of organosulfur derivatives, since both
represent overall a nucleophilic displacement upon the
terminal oxygen of the 4a-hydroperoxyflavin.
The reaction is quite sensitive to steric effects; indeed
methyl-(1-phenyl-ethyl)-amine did not react, thus pre-
venting the possibility of the kinetic resolution of the
Table 1. CHMO catalyzed oxidation of amines
Amines
Products
Conversion (%)
V )
max/Km (min−1 a
N-Methylbenzylamine
C6H5CHꢀN(O)CH3
C6H5CH2N(O)ꢀCH2
C6H5CH2N(OH)CH3
C6H5CHꢀN(O)CH3
C6H5CH2N(O)ꢀCH2
C6H5CHꢀN(O)CH2CH3
C6H5CH2N(O)ꢀCHCH3
C6H5CHꢀN(O)CH(CH3)2
N.R.
C6H5CHꢀN(O)(CH2)3CH3
C6H5CH2N(O)ꢀCH(CH2)2CH3
N.R.
C6H5N(OH)CH3
N.R.
10
8
87.5
80
36
16
30
18
13
N-Methylbenzylhydroxylamine
N-Ethylbenzylamine
104.3
95.2
62.5
58.8
N-iso-Propylbenzylamine
N-tert-Buthylbenzylamine
N-n-Buthylbenzylamine
14
2
N-Methyl-p-chlorobenzylamine
N-Methylaniline
4-(Ethylaminomethyl)-pyridine
Methyl-(1-phenyl-ethyl)-amine
N,N-Dimethylbenzylamine
60
63
84.6
78.4
N.R.
C6H5N(O)(CH3)2
N.R.: no reaction.
a The kinetic constants of CHMO for substrates were determined in 50 mM Tris–HCl buffer at pH 8.6, 25°C, in 1 ml cuvettes, 1 cm path length.
The reaction mixture contained CHMO (30 milliunits), 0.1 mM NADPH and 0.4–4 mM substrate. The consumption of NADPH was
spectrophotometrically monitored at 340 nm.

S. Colonna et al. / Tetrahedron Letters 44 (2003) 869–871
871
In conclusion, we have shown that CHMO reacts with
tertiary, secondary and hydroxylamines. In spite of
their synthetic limits these biotransformations are very
interesting from a mechanistic point of view when
compared to the results obtained by 4a-hydroperoxy-
flavin or by hepatic flavoprotein microsomal oxidase.16
5. Colonna, S.; Gaggero, N.; Bertinotti, A.; Carrea, G.;
Pasta, P.; Bernardi, A. J. Chem. Soc., Chem. Commun.
1995, 1123–1124.
6. Colonna, S.; Gaggero, N.; Carrea, G.; Pasta, P. J. Chem.
Soc., Chem. Commun. 1998, 415–416.
7. (a) Branchaud, B. P.; Walsh, C. T. J. Am. Chem. Soc.
1995, 107, 2153 and references therein; (b) Alphand, V.;
Archelas, A.; Furstoss, R. Tetrahedron Lett. 1989, 28,
3663; (c) Ottolina, G.; Bianchi, S.; Belloni, B.; Carrea, G.;
Danieli, B. Tetrahedron Lett. 1999, 40, 8483–8485.
8. Colonna, S.; Gaggero, N.; Carrea, G.; Ottolina, G.;
Pasta, P.; Zambianchi, F. Tetrahedron Lett. 2002, 43,
1797–1799.
Acknowledgements
This work was partially supported by the COST Pro-
gramme, by the European Commission (QLK3-CT-
2001-00403) and by Murst (Programmi di Ricerca
Scientifica di Interesse Nazionale).
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