Oxidation of secondary amines by molecular oxygen and cyclohexanone monooxygenase

Article abstract of DOI:10.1016/j.tet.2003.10.100

Cyclohexanone monooxygenase from Acinetobacter calcoaceticus catalyzed the oxidation of tertiary and secondary amines to N-oxides and nitrones, respectively. The formation of a hydroxylamine intermediate was involved with secondary amines as starting substrates.

Full text of DOI:10.1016/j.tet.2003.10.100

Tetrahedron 60 (2004) 569–575
Oxidation of secondary amines by molecular oxygen and
cyclohexanone monooxygenase
a
b
b
Stefano Colonna,a Vincenza Pironti, Giacomo Carrea, Piero Pasta
,
*
and Francesca Zambianchib
a
`
Istituto di Chimica Organica, ‘Alessandro Marchesini’, Facolta di Farmacia, via Venezian 21, 20133 Milano, Italy
bIstituto di Chimica del Riconoscimento Molecolare, CNR, via Mario Bianco 9, 20131 Milano, Italy
Received 20 June 2003; revised 23 July 2003; accepted 17 October 2003
Abstract—Cyclohexanone monooxygenase from Acinetobacter calcoaceticus catalyzed the oxidation of tertiary and secondary amines to
N-oxides and nitrones, respectively. The formation of a hydroxylamine intermediate was involved with secondary amines as starting
substrates.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
sequent hydrolysis leads to 6-hydroxyhexanoate, that is
oxidized to adipate, and then to the acetyl-CoA and
succinyl-CoA by b-oxidation.2 Mechanistic studies have
suggested that the reactive intermediate is the FAD-4a-
hydroperoxyflavin, that has a twofold activity since it can
react either as a nucleophilic species, as in the case of the
Baeyer–Villiger reaction and the boronic acid oxidation, or
as an electrophilic species, as in the sulfoxidation reaction.2a
More recent investigations would, however, indicate that
the oxygenating intermediate is the anionic 4a-peroxide and
not its protonated form, the 4a-hydroperoxyflavin.3
The increasing demand for enantiomerically pure com-
pounds for the pharmaceutical and agrochemical industries
implies a great incentive for the study of the biotransform-
ations. Oxidative biotransformations are amongst the most
useful of all identified biologically mediated conversions.1
They usually involve monooxygenases or dioxygenases,
that catalyze the insertion of one oxygen or two oxygen
atoms at a specific point of a molecule, often with high
stereo and/or regioselectivity. The cyclohexanone mono-
oxygenase from Acinetobacter calcoaceticus NCIMB 9871
(CHMO) (EC 1.14.13.22) is an interesting enzyme for its
application in the manufacture of fine chemicals and in
organic synthesis.2
Apart from cyclohexanone, CHMO is able to transform a
great variety of other substituted cyclic ketones, bicyclic
ketones and aldehydes.4 These biotransformations have
wide applications for the stereoselective oxidations of
prochiral ketones and constitute the only efficient method-
ology to resolve racemic ketones.5 Furthermore, CHMO can
oxidize a wide series of organic compounds containing
electron rich heteroatoms; the sulfides are converted to
sulfoxides,2b the sulfites to sulfates,6 the selenides to
selenoxides,7 tertiary amines to N-oxides8 and phosphines
to phosphinoxides.9 In many cases these reactions are highly
enantioselective. Recently, our group has shown that the
enzyme can epoxidize electron poor olefins but with severe
limitations; in fact, only two compounds are accepted as
substrates, namely, the dimethyl and the diethylvinyl-
phosphonate, compounds structurally related to Fosfo-
mycin. They are converted into the corresponding
epoxides with ee .98%.10
CHMO is a yellow FAD-containing enzyme of about
60,000 Da, it is active as a monomer and contains one
tightly but non-covalently bound FAD per monomer.2a This
enzyme catalyzes the Baeyer–Villiger oxidation of cyclo-
hexanone with the formation of the corresponding 1-capro-
lactone; the only reagents consumed are O2 and NADPH.
The enzyme shows an absolute specificity for the electron
donor (NADPH) and a wide specificity for the ketonic
substrates.
The bacterium NCIMB 9871 can grow on cyclohexanol or
cyclohexanone as sole carbon source; the key step is the ring
expansion with formation of 1-caprolactone. The sub-
Keywords: Cyclohexanone monooxygenase; Nitrones; Amines;
Hydroxylamines; N-Oxides.
The main goal of this last work was to extend the synthetic
repertoire of CHMO; for this reason we have started
investigating the oxidation of secondary amines to nitrones,
*
Corresponding author. Tel.: þ39-02-5031-4473; fax: þ39-02-5031-4476;
e-mail address: stefano.colonna@unimi.it
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.100

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S. Colonna et al. / Tetrahedron 60 (2004) 569–575
a reaction previously studied with 4a-hydroperoxyflavin as
oxidizing species.11 It is known that mammalian liver
contains two enzymatic systems responsible for the
oxidation of N-substituted amine drugs, one containing a
flavoprotein (NADPH cytochrome P-450 reductase) and the
other a cytochrome P-450. The former catalyzes the
N-oxidation of secondary and tertiary amines in a NADPH
and O2-dependent process similar to CHMO and the latter
the C-oxidation of amines, that leads to N-dealkylation.12
second enzymatic reaction This has been achieved using
glucose-6-phosphate/glucose-6-phosphate dehydrogenase
(G6PDH) (Scheme 1). The conversions and the identities
of the products have been determined by HPLC analysis by
comparison of their elution order with nitrones and
hydroxylamines synthesized ad hoc. The first screening of
potential amine substrates for CHMO showed that the
reaction is quite sensitive to steric and electronic effects.
Indeed, aliphatic amines such as diisopropylamine and
pipecoline or sterically bulky amines such as dibenzylamine
were not substrates for CHMO.
Another point of interest is that the reaction products, the
nitrones, are highly versatile synthetic intermediates, in
particular as 1,3-dipoles and for the preparation of various
nitrogen-containing biologically active compounds such as
antibiotics, alkaloids, amino-sugars and b-lactams.13 They
have also pharmacological activity, e.g., the tert-butyl-
benzylnitrone (PBN) acts as a spin trap in physiological
buffer, and exhibits antioxidant and neuroprotective activity
against oxidative damage.
We have chosen N-methylbenzylamine (1a) as the model
substrate in order to investigate the mechanism involved in
this biooxidation. We have found that the formation of the
nitrones occurs through a double oxidation, i.e., of the
starting secondary amine and, then, of the hydroxylamine
intermediate (2a) which leads to the two regioisomeric
nitrones (Scheme 2). The occurrence of hydroxylamine was
proved by HPLC comparison with a standard of N-methyl-
benzylhydroxylamine (2a). Moreover, hydroxylamine 2a
was converted by CHMO faster than the starting amine (1a),
in agreement with its higher specificity constant value
(Vmax/Km) (Table 1). The ratio of the two regioisomeric
nitrones formed was independent of the enzymatic reaction
and in favour of the most stable nitrone, in agreement with
the results found by Bruice et al.11a in the oxidation with
4a-hydroperoxyflavin.
Nitrones are currently synthesized either by condensation of
carbonyl compounds with N-monosubstituted hydroxyl-
amines14 or by oxidation of secondary amines or of the
corresponding hydroxylamines.15 The oxidative approach
provides the most direct and general method for their
preparation. This methodology uses hydrogen peroxide as
primary oxidant in the presence of catalysts such as
16
Na2MoO4 or Na2WO4,15 MeReO3 or SeO2.17 Very
recently, alkyl hydroperoxides have been used as primary
oxidant and titanium alkoxide complexes as catalyst,18 and
protected from the water formed as a co-product by the
combined use of trialkanolamine ligands and molecular
sieves.18
We have tested other substrates structurally related to the
model 1a, considering, first of all, the influence of the alkyl
chain bound to the nitrogen. N-Ethylbenzylamine (1b),
N-iso-propylbenzylamine (1c) and N-n-butylbenzylamine
(1d) were all substrates, the specificity constant (Vmax/Km)
order being: N-ethylbenzylamine.N-methylbenzylamine.
N-iso-propylbenzylamine.N-n-butylbenzylamine
(Table 1). These results were in fairly good agreement with
the conversion data (Table 2). The conversions decreased
when the branching of the alkyl chain was increased; N-tert-
butylbenzylamine (1e) was not transformed by CHMO
(Table 2). This result demonstrates the strict steric
requirements of substrates. Also methyl-(1-phenyl-ethyl)-
amine (1f) was not transformed; the substitution of a
2. Results and discussion
We have examined more extensively the enzymatic
oxidation of secondary amines, hydroxylamines and tertiary
amines mediated by CHMO, and preliminary results have
been reported.19
For economic reasons it is essential to regenerate NADPH in
situ at the expenses of a cheap co-substrate, by means of a
Scheme 1. CHMO catalyzed oxidation of N-methylbenzylamine with in
situ coenzyme regeneration.
Scheme 2. Mechanism of oxidation of N-methylbenzylamine by CHMO.

S. Colonna et al. / Tetrahedron 60 (2004) 569–575
Table 1. Kinetic constants of CHMO with amine substratesa
571
Amines
Vmax (mM£min21
)
Km (mM)
Vmax/Km£106 (min21
)
N-Methylbenzylamine (1a)
N-Methylbenzylhydroxylamine (2a)
N-Ethylbenzylamine (1b)
N-iso-Propylbenzylamine (1c)
N-n-Butylbenzylamine (1d)
N-Methyl-p-methoxybenzylamine (1h)
N-Methyl-p-methylbenzylamine (1i)
N-Methyl-p-nitrobenzylamine (1j)
N-Methylaniline (1l)
N,N-Dimethylbenzylamine (1m)
Methyl-(4-methylthiobenzyl)-amine (1p)
0.21
0.24
0.20
0.25
0.30
0.27
0.22
0.21
0.22
0.24
0.40
2400
2300
2120
4000
5100
3170
2520
1830
2650
3060
1570
87.5
104.3
95.2
62.5
58.8
86.4
89.4
112.7
84.6
78.4
255.7
a
For conditions see Section 3.
Table 2. CHMO catalyzed oxidation of amines
Amines
Substrate conversion (%)
98
Product
Product formed (%)
N-Methylbenzylamine (1a)
C6H5CH2N(OH)CH3 (2a)
C6H5CHvN(O)CH3 (3a)
C6H5CH2N(O)vCH2 (4a)
C6H5CHvN(O)CH3 (3a)
C6H5CH2N(O)vCH2 (4a)
C6H5CHvN(O)CH2CH3 (3b)
C6H5CH2N(O)vCHCH3 (4b)
C6H5CHvN(O)CH(CH3)2 (3c)
C6H5CHvN(O)(CH2)3CH3 (3d)
C6H5CH2N(O)vCH(CH2)2CH3 (4d)
80
10
8
36
16
30
18
13
14
2
N-Methylbenzylhydroxylamine (2a)
N-Ethylbenzylamine (1b)
52
68
N-iso-Propylbenzylamine (1c)
N-n-Butylbenzylamine (1d)
49
57
N-tert-Butylbenzylamine (1e)
N.R.
N.R
N.R
98
68
34
Methyl-(1-phenylethyl)-amine (1f)
N-Methyl-o-methoxybenzylamine (1g)
N-Methyl-p-methoxybenzylamine (1h)
N-Methyl-p-methylbenzylamine (1i)
N-Methyl-p-nitrobenzylamine (1j)
N-Methyl-p-chlorobenzylamine (1k)
N-Methylaniline (1l)
p-MeO–C6H4CHvN(O)CH3 (3h)
p-Me–C6H4CHvN(O)CH3 (3i)
p-NO2–C6H4CHvN(O)CH3 (3j)
10
9
26
N.R
60
C6H5N(OH)CH3 (3l)
C6H5N(O)(CH3)2 (3m)
58
62
N,N-Dimethylbenzylamine (1m)
4-(Ethylaminomethyl)-pyridine (1n)
Methyl-thiophen-2-ylmethylamine (1o)
Methyl-(4-methylthiobenzyl)-amine (1p)
63
N.R
N.R
58
p-CH3SO–C6H4CH2NCH3 (5p)
55 ee¼66%
N.R.: no reaction.
benzylic hydrogen with a methyl group led to total loss
of reactivity (Table 2). This fact prevented the possibility
of carrying out the kinetic resolution of the starting
amine.
hydroxylamine 2i decomposed in the usual reaction
conditions. In the case of N-methyl-p-chlorobenzylamine
(1k), the starting amine was recovered unchanged. These
results indicate that also the electronic requirement plays an
important role. N-Methylaniline (1l) and N,N-dimethyl-
benzylamine (1m), structurally similar to the amine model
1a were substrates for CHMO; the former reacted to give the
corresponding hydroxylamine while the latter was trans-
formed into the N-oxide. A further confirmation of the fact
that the electronic factors are important was the total loss of
reactivity in the case of 4-(ethyl-amino-methyl)-pyridine
(1n) and of methyl-thiophen-2-yl methylamine (1o)
(Table 2).
In order to examine the stereoelectronic effects of
substituents on the aromatic ring, we have investigated
N-methyl-o-methoxybenzylamine (1g) and N-methyl-p-
methoxybenzylamine (1h). While the ortho-substituted
amine 1g was recovered unchanged, the p-substituted one
1h was totally transformed, giving the expected nitrone 3h
(10%) and a product deriving from the decomposition of
hydroxylamine 2h (Table 2). The instability of 2h was
demonstrated with a control experiment carried out under
the same reaction conditions in the absence of CHMO. For
shorter reaction times (15 min), the formation of hydroxyl-
amine was observed together with that of its decomposition
product.
Finally, we took in consideration the behaviour of a
substrate having two functions potentially able to undergo
oxidation by CHMO, namely, two different heteroatoms, i.e.
nitrogen and sulfur. Therefore, we prepared and tested
methyl-(4-methylthiobenzyl)-amine (1p). Since the kcat
values of CHMO for sulfur versus nitrogen are in the 8:1
ratio,2 a preferential attack at sulfur was expected. That was
indeed the case and the corresponding sulfoxide was formed
in 55% chemical yield and 66% ee (Table 2).
The research has been extended to other substrates
substituted in the para position of the aromatic ring;
N-methyl-p-methylbenzylamine (1i) and N-methyl-p-nitro-
benzylamine (1j) were substrates for CHMO. Again the

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S. Colonna et al. / Tetrahedron 60 (2004) 569–575
It should be stressed that the catalytic efficiencies of FAD-
4a-hydroperoxyflavin towards secondary and tertiary
amines are of the same order of magnitude (Table 1),
whereas in the case of 4a-hydroperoxyflavin as oxidant,
N,N-dimethyl aniline was reported to be twice as reactive
with respect to N-methyl aniline.12
3.1.1. N-Methyl-o-methoxybenzylamine (1g). Yellow
liquid, bp 104 8C (10 Torr), (lit.22 103 8C). 1H NMR
(200 MHz, CDCl3): d 2.41 (3H, s, NCH3), 3.74 (2H, s,
CH2N), 3.83 (3H, s, CH3O), 6.90 (1H, m, Ar-H), 7.23 (3H,
m, Ar-H). Yield¼85%.
3.1.2. N-Methyl-p-methoxybenzylamine (1h). Colourless
liquid, bp 106 8C (10 Torr), (lit.23 105 8C). 1H NMR
(300 MHz, CDCl3): d 1.68 (1H, bs, N–H), 2.44 (3H, s,
NCH3), 3.68 (2H, s, CH2N), 6.86 (2H, d, JH–H¼12.0 Hz,
Ar-H), 7.23 (2H, d, JH–H¼12.0 Hz, Ar-H). Yield¼95%.
In conclusion, we have shown that cyclohexanone mono-
oxygenase reacts with secondary amines, hydroxylamines
and tertiary amines. The reaction has a limited versatility
from the synthetic point of view since, basically, only
substituted benzylic amines are accepted as substrates by the
enzyme. On the other hand, we have shown that the
mechanism of CHMO oxidation at nitrogen in secondary
amines is similar to that of organosulfur derivatives, since
both heteroatoms undergo an electrophilic attack by the
terminal oxygen of the 4a-hydroperoxyflavin. In spite of the
synthetic limitations, these biotransformations have a
remarkable mechanistic interest if they are compared to
the 4a-hydroperoxyflavin system studied by Bruice and co-
workers and to hepatic flavoproteins involved in the
microsomial oxidation.12
3.1.3. N-Methyl-p-methylbenzylamine (1i). Orange liquid,
bp 84 8C (10 Torr), (lit.24 83 8C, 11 Torr). 1H NMR
(300 MHz, CDCl3): d 1.63 (1H, bs, N–H), 2.34 (3H, s,
Ar-CH3), 2.44 (3H, s, NCH3), 3.71 (2H, s, CH2), 7.18 (4H,
m, Ar). Yield¼95%.
3.1.4. N-Methyl-p-nitrobenzylamine (1j). Orange liquid,
147 8C (10 Torr), (lit.25 156–158, 15 Torr). 1H NMR
(300 MHz, CDCl3): d 1.43 (1H, bs, N–H), 2.46 (3H, s,
NCH3), 3.85 (2H, s, CH2), 7.90 (2H, d, Ar-H, JH–H
9.2 Hz), 8.18 (2H, d, Ar-H, JH–H¼9.2 Hz). Yield¼96%.
¼
3.1.5. N-Methyl-p-chlorobenzylamine(1k). Orange liquid,
1
3. Experimental
bp 119 8C (10 Torr), (lit.25 120–123, 15 Torr). H NMR
(300 MHz, CDCl3): d 1.74 (1H, bs, N–H), 2.38 (3H, s,
NCH3), 3.66 (2H, s, CH2), 7.22 (4H, m, Ar-H). Yield¼76%.
1
The H NMR spectra were recorded on a Bruker AC 300
apparatus with CDCl3 as solvent. The kinetic measurements
were carried out with a Jasco V-530 UV spectrophometer.
Low-resolution mass spectra were run with a Fisons MD
800 spectrometer using the EI method. HPLC analyses were
performed on Chiralcel OD column on a Jasco HPLC
instrument (model 980-PU pump, model 975-UV detector)
using n-hexane/2-propanol (9:1) for compounds 1a–f and
1l–n and n-hexane/2-propanol (8:2) for compounds 1g–k
and 1o–p as the mobile phase; the flow rate was 1 ml/min
and readings were made at 254 nm. Melting points were
recorded on a Stuart Scientific apparatus. Chemical
reactions were monitored by analytical TLC, performed
on Merck silica gel 60 F254 plates and visualized by UV
irradiation or by iodine. Columns were packed using Merck
silica gel 60 (230–400 mesh) as the stationary phase and
eluted using the flash chromatographic technique. CHMO
was as a partially purified preparation obtained from an
E. coli strain in which the gene of the enzyme was cloned
and overexpressed.20 Glucose-6-phosphate, NADPþ and
glucose-6-phosphate dehydrogenase were purchased from
Sigma-Aldrich. N-Methylbenzylamine, N-ethylbenzyl-
amine, N-iso-propylbenzylamine, N-tert-butylbenzyl-
amine, N-n-butylbenzylamine, N-methylaniline, 4-(ethyl-
amino-methyl)-pyridine, N,N-dimethyl-benzylamine were
purchased from Sigma-Aldrich and used without further
purification.
3.1.6. Methyl-thiophen-2-ylmethylamine (1o). Yellow
liquid, 79 8C (10 Torr), (lit.26 75–80 8C, 14 Torr). 1H
NMR (300 MHz, CDCl3): d 1.56 (1H, bs, N–H), 2.48
(3H, s, NCH3), 3.95 (2H, s, CH2), 6.96–7.23 (4H, m, Ar).
Yield¼99%.
3.1.7. Methyl-(4-methylthiobenzyl)-amine (1p). Yellow
1
liquid, 108 8C (10 Torr), H NMR (200 MHz, CDCl3): d
1.52 (1H, bs, N–H), 2.44 (3H, s, SCH3), 2.47 (3H, s, NCH3),
3.70 (2H, s, CH2), 7.24 (4H, m, Ar-H). Yield¼94%.
3.2. Preparation of N-methyl-N-(1-phenylethyl)amine 1f
Amine 1l was prepared from the corresponding primary
amine by condensation with ethyl-chloroformate, followed
by reduction with LiAlH4.27
The amine was characterized as follows.
3.2.1. N-Methyl-N-(1-phenylethyl)amine 1f. Pale yellow
1
liquid, bp 82 8C (10 Torr), (lit.28 83–85 8C, 15 Torr). H
NMR (300 MHz, CDCl3): d 1.35 (1H, d, CH3, JH–H
9.9 Hz), 1.57 (1H, bs, N–H), 2.30 (3H, s, NCH3), 3.62 (1H,
¼
q, CHN, JH–H¼9.9 Hz), 7.30 (5H, m, Ar). Yield¼57%.
3.3. General procedure for the preparation of
hydroxylamines 2a and 2h–i
3.1. Preparation of non-commercially available
secondary amines 1g–k and 1o–p
NaBH4 (2.0 mmol) was added to a solution of the
corresponding nitrone (1 mmol) in EtOH (5 ml) and the
mixture was stirred at room temperature for 5 h. The EtOH
was removed under reduced pressure. H2O was added (5 ml)
and the reaction mixture extracted with CH2Cl2 (3£15 ml).
The organic layer was dried with Na2SO4, filtered and the
Amines 1g–k and 1o–p were prepared by condensation of
the corresponding aldehydes with methylamine (30% in
aqueous solution), followed by reduction with NaBH4
according to known methods.21 The amines were charac-
terized as follows.

S. Colonna et al. / Tetrahedron 60 (2004) 569–575
573
solvent was removed under reduced pressure. Chromato-
graphy on silica gel with CH2Cl2/MeOH (95:5) gave
hydroxylamines 2a, 2h, 2i.
yellow solid, mp 72–73 8C (lit.33 73 8C). 1H NMR
(200 MHz, CDCl3): d 0.94 (3H, t, CH3, JH–H¼7.0 Hz),
1.44 (2H, sest., CH2, JH–H¼7.0 Hz), 1.96 (2H, quint., CH2,
JH–H¼7.0 Hz), 3.97 (2H, t, NCH2, JH–H¼7.0 Hz), 7.33
(4H, m, Ar-H and CHN), 8.24 (2H, m, Ar-H). Yield¼28%.
3.3.1. N-Benzyl-N-methylhydroxylamine 2a. Colourless
solid, mp 39 8C (lit.29 40–41 8C). 1H NMR (300 MHz,
CDCl3): d 2.58 (1H, s, NCH3), 3.72 (1H, s, NCH2), 7.27–
7.44 (5H, m, Ar-H), OH proton signal was not observed.
Yield¼84%.
3.4.6. N-Butylidenbenzylamine N-oxide 4d. Pale yellow
solid, mp 74–75 8C (lit.34 76 8C). 1H NMR (200 MHz,
CDCl3): d 0.86 (3H, t, CH3, JH–H¼7.3 Hz), 1.44 (2H, sest.,
CH2, JH–H¼7.3 Hz), 2.37 (2H, q, CH2, JH–H¼7.0 Hz), 4.80
(2H, s, Ar-CH2), 6.63 (1H, t, CHN, JH–H¼7.3 Hz), 8.24
(2H, m, Ar-H). Yield¼42%.
3.3.2. N-p-Methoxybenzyl-N-methylhydroxylamine 2h.
White solid, mp 53–54 8C (lit.29 56–58 8C). 1H NMR
(300 MHz, CDCl3): d 2.57 (1H, s, NCH3), 3.67 (2H, s,
NCH2), 3.78 (3H, s, OCH3), 7.27–7.44 (5H, m, Ar-H), OH
proton signal was not observed. Yield¼84%.
3.4.7. N-Benzylidene-tert-buthylamine N-oxide 3e. White
solid, mp 72–74 8C (lit.35 75–76 8C). 1H NMR (300 MHz,
CDCl3): d 1.61 (9H, s, CH3,) 7.41 (3H, m, Ar-H) 7.54 (1H,
s, CHN), 8.29 (2H, m, Ar-H). Yield¼91%.
3.3.3. N-p-Methylbenzyl-N-methylhydroxylamine 2i.
White solid, mp 52 8C (lit.29 52–54 8C). 1H NMR
(300 MHz, CDCl3): d 2.32 (1H, s, Ar-CH3), 2.57 (3H, s,
NCH3), 3.70 (2H, s, NCH2), 7.10–7.21 (5H, m, Ar-H), OH
proton signal was not observed. Yield¼85%.
3.4.8. N-Methyl-(1-phenylethylidene) amine N-oxide 3f.
Pale yellow solid, mp 114–115 8C (lit.36 115 8C). 1H NMR
(300 MHz, CDCl3): d 2.43 (1H, s, CH3), 3.63 (1H, s,
NCH3), 7.23–7.44 (5H, m, Ar-H). Yield¼54%.
3.4. General procedure for the preparation of nitrones
3a–i, 3k, 3n–o, 4b, 4d and 4n
3.4.9. 2-Methoxybenzylidenemethylamine N-oxide 3g.
1
White solid, mp 84 8C (lit.30 85 8C). H NMR (200 MHz,
Aqueous H2O2 (3.06 g, 27 mmol) was added dropwise to a
stirred solution of the amines 1a–i, 1k and 1n–o
(9.0 mmol) and Na2WO4·2H2O (0.148 mg, 0.45 mmol) in
MeOH (20 ml) with ice cooling The reaction mixture was
stirred at room temperature for 3–12 h until TLC (CH2Cl2/
MeOH, 95:5) revealed the complete disappearance of the
amine. MeOH was removed under reduced pressure. The
reaction mixture was washed with a saturated solution of
Na2SO3 (15 ml) and extracted with CH2Cl2 (3£30 ml). The
organic layer was dried over anhydrous Na2SO4, filtered and
the solvent was removed under reduced pressure. Chroma-
tography on silica gel with CH2Cl2/MeOH (95:5) gave the
nitrones 3a–i, 3k, 3n–o, 4b, 4d and 4n.
CDCl3): d 3.77 (3H, s, NCH3), 3.82 (3H, s, OCH3), 6.81–
6.97 (2H, m, Ar-H), 7.20 (1H, dt, Ar-H, JH–H¼9.9, 2.7 Hz)
7.78 (1H, s, CHN), 9.20 (1H, dd, Ar-H, JH–H¼11.9,
2.7 Hz). Yield¼78%.
3.4.10. 4-Methoxybenzylidenemethylamine N-oxide 3h.
1
White solid, mp 75 8C (lit.30 76 8C). H NMR (200 MHz,
CDCl3): d 3.81 (6H, bs, CH3), 6.90 (2H, d, Ar-H, JH–H
9.0 Hz) 7.27 (1H, s, CHN), 8.18 (2H, d, Ar-H, JH–H
9.0 Hz). Yield¼91%.
¼
¼
3.4.11. 4-Methylbenzylidenemethylamine N-oxide 3i.
White solid, mp 117 8C (lit.37 119 8C). 1H NMR
(300 MHz, CDCl3): d 2.42 (3H, s, Ar-CH3), 3.83 (3H, s,
NCH3), 6.92 (2H, d, Ar-H, JH–H¼9.0 Hz), 7.27 (1H, s,
CHN), 8.18 (2H, d, Ar-H, JH–H¼9.0 Hz). Yield¼68%.
3.4.1. N-Benzylidenemethylamine N-oxide 3a. White
solid, mp 82–83 8C (lit.30 84 8C). 1H NMR (300 MHz,
CDCl3): d 3.88 (1H, s, CH3), 7.37 (4H, m, Ar-H and CHN),
8.22 (2H, m, Ar-H). Yield¼53%.
3.4.12. 4-Chlorobenzylidenemethylamine N-oxide 3k.
White solid, mp 126 8C (lit.30 128 8C). 1H NMR
(300 MHz, CDCl3): d 3.83 (3H, s, NCH3), 7.37 (3H, m,
Ar-H and CHN), 8.18 (2H, d, Ar-H, JH–H¼11.0 Hz).
Yield¼64%.
3.4.2. N-Benzylideneethylamine N-oxide 3b. Pale yellow
1
liquid, bp 118 8C (0.8 Torr), (lit.31 116 8C, 0.8 Torr). H
NMR (200 MHz, CDCl3): d 1.53 (3H, t, CH3, JH–H
7.0 Hz), 3.93 (2H, q, NCH2, JH–H¼7.0 Hz) 7.33 (4H, m,
¼
Ar-H and CHN), 8.22 (2H, m, Ar-H). Yield¼56%.
3.4.13. N-Ethyl-4-pyridylmethylidene N-oxide 3n.
1
Orange liquid, bp 125 8C, 10 Torr. H NMR (300 MHz,
3.4.3. N-Ethylidenebenzylamine N-oxide 4b. Pale yellow
solid, mp 76 8C (lit.32 77 8C). 1H NMR (200 MHz, CDCl3):
d 1.95 (3H, d, CH3, JH–H¼7.0 Hz), 4.89 (2H, s, NCH2), 6.72
(2H, q, CHN, JH–H¼7.0 Hz), 7.37 (5H, m, Ar-H).
Yield¼15%.
CDCl3) d 1.97 (3H, t, CH3, JH–H¼7.3 Hz), 3.97 (2H, q,
NCH2, JH–H¼7.3 Hz), 7.41 (1H, s, NCH), 7.96 (2H, d,
Ar-H, JH–H¼8.0 Hz), 8.63 (2H, d, Ar-H, JH–H¼8.0 Hz). EI
MS: m/z (%)¼150.1 (100) [Mþ]. Yield¼44%.
3.4.14. N-Ethylidenepyridyl-4-methylamine N-oxide 4n.
Orange solid, mp 82 8C. 1H NMR (300 MHz, CDCl3) d 1.96
(3H, d, CH3, JH–H¼6.0 Hz), 4.83 (2H, s, NCH2), 6.87 (1H,
q, NCH, JH–H¼6.0 Hz), 7.25 (2H, d, Ar-H, JH–H¼7.5 Hz),
8.52 (2H, d, Ar-H, JH–H¼7.5 Hz). EI MS: m/z (%)¼150.1
(100) [Mþ]. Yield¼22%.
3.4.4. N-Benzylideneisopropylamine N-oxide 3c. Yellow
1
liquid, bp 119 8C (0.8 Torr), (lit.31 118 8C, 0.8 Torr). H
NMR (200 MHz, CDCl3): d 1.50 (6H, d, CH3, JH–H
6.7 Hz), 4.17 (1H, q, NCH, JH–H¼6.7 Hz) 7.37 (4H, m,
¼
Ar-H and CHN), 8.24 (2H, m, Ar-H). Yield¼48%.
3.4.5. N-Benzylidene-n-butylamine N-oxide 3d. Pale
3.4.15. N-Methyl-thienyl-2-methyleneamine N-oxide 3o.

574
S. Colonna et al. / Tetrahedron 60 (2004) 569–575
1
Pale yellow solid, mp 118–119 8C. H NMR (300 MHz,
CDCl3): 3.82 (3H, s, CH3), 7.12 (1H, dd, Ar-H, JH–H¼4.8,
4.0 Hz), 7.38 (1H, d, Ar-H, JH–H¼4.0 Hz), 7.42 (1H, d,
Ar-H, JH–H¼4.8 Hz), 7.83 (1H, s, CHN). EI MS: m/z
(%)¼141.1 (100) [Mþ]. Yield¼94%.
3.8. General procedure to determine the kinetic
constants
The experiments were carried out in 50 mM Tris–HCl
buffer at pH 8.6, 25 8C, in 1 ml cuvettes, 1 cm path length.
The reaction mixture contained CHMO (30 milliunits),
0.1 mM NADPH and 0.4–5 mM substrate. The consump-
tion of NADPH was spectrophotometrically monitored at
340 nm.
3.5. General procedure for the preparation of nitrones 3j
and 3p
The appropriate aldehyde (1.0 mmol) was added to a
solution of N-methylhydroxylamine hydrochloride
(1.0 mmol) and sodium acetate (1.1 mmol) in EtOH
(10 ml). The reaction mixture was stirred at 50–60 8C for
3 h. EtOH was removed under reduced pressure, water was
added (5.0 ml) and the reaction mixture extracted with
CH2Cl2 (3£10 ml). The organic layer was dried over
anhydrous Na2SO4, filtered and the solvent was removed
under reduced pressure. Chromatography on silica gel with
CH2Cl2/MeOH (95:5) gave nitrones 3j and 3p.
Acknowledgements
We thank the Biotechnology Programme of the European
Commission (QLK3-CT-2001-00403) and the Murst
(Programmi di Ricerca Scientifica di Interesse Nazionale)
for financial support.
3.5.1. 4-Nitrobenzylidenemethylamine N-oxide 3j.
Yellow solid, mp 205 8C (lit.30 208 8C). 1H NMR
(300 MHz, CDCl3): d 2.42 (3H, s, Ar-CH3), 3.83 (3H, s,
NCH3), 6.92 (2H, d, Ar-H, JH–H¼9.0 Hz), 7.27 (1H, s,
CHN), 8.18 (2H, d, Ar-H, JH–H¼9.0 Hz). Yield¼68%.
References and notes
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3.5.2. 4-Methylthiobenzylidenemethylamine N-oxide 3p.
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