Dioxygenase-catalysed formation of dihydrodiol metabolites of N-methyl- 2-pyridone

Article abstract of DOI:10.1016/S0040-4039(00)00541-4

IPTG-induced cells of a recombinant strain of Escherichia coli JM109(DE3)pDTG141 expressing naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 oxidized specifically N-methyl-2-pyridone to give the corresponding cis-5,6-dihydro-5,6-dihydroxy derivative as a major product. A small amount of the cis-3,4-dihydro-3,4-dihydroxy isomer was simultaneously formed. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00541-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 3865–3869
Dioxygenase-catalysed formation of dihydrodiol metabolites of
N-methyl-2-pyridone
Ludmila Modyanova and Robert Azerad
†
∗
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS et Université René Descartes-Paris V,
4
5 rue des Saints-Pères, 75270-Paris Cedex 06, France
Received 18 February 2000; accepted 29 March 2000
Abstract
IPTG-induced cells of a recombinant strain of Escherichia coli JM109(DE3)pDTG141 expressing naphthalene
dioxygenase from Pseudomonas sp. NCIB 9816-4 oxidized specifically N-methyl-2-pyridone to give the corres-
ponding cis-5,6-dihydro-5,6-dihydroxy derivative as a major product. A small amount of the cis-3,4-dihydro-3,4-
dihydroxy isomer was simultaneously formed. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: dihydroxylation; biocatalysis; heterocyclic nitrogen compounds.
The microbial metabolism of pyridine derivatives (unlike that of benzene) does not involve a cis-
dihydroxylation reaction as the initial step of their biodegradation.1 Instead, the metabolism of such
heterocycles usually starts with hydroxylation in an adjacent position to the nitrogen atom, leading
to the formation of monohydroxy derivatives. While working with whole microorganisms which are
,2
3
known to contain dioxygenase enzymes (Pseudomonas wild strains or mutants for example), only
monohydroxylation in the 2(6)-position of the pyridine ring or N-oxidation are observed, masking any
possible dihydroxylation reaction.
However, a dihydroxylation reaction (and especially a stereospecific one) should be quite a useful tool
for the functionalization of pyridine derivatives to the corresponding dihydropyridines, in view of their
4
5
6
potential synthetic use as precursors of azasugars, alkaloids, unnatural amino acids, etc.
We have thus undertaken a systematic investigation of the biotransformation of several substituted
pyridine derivatives by available recombinant strains of Escherichia coli expressing various dioxygenases
from Pseudomonas species.
As shown in Scheme 1, N-methyl substituted 2-pyridone (1) was specifically and completely
7
transformed by Escherichia coli JM109(DE3)(pDTG141), a recombinant strain which expresses naph-
thalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4,8 to give in good yield (about 50%)
,9
∗
Corresponding author. Tel: +33 (0)1 42862171; fax:+33 (0)1 42868387; e-mail: robert.azerad@biomedicale.univ-paris5.fr
(
R. Azerad)
†
Present address: 677bis, Avenue de la République, Appt.12, 59000-Lille, France.
0
040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00541-4
tetl 16820

3
866
one major dihydroxylated product, isolated by silica gel chromatography, and identified as the cis-5,6-
dihydro-5,6-dihydroxy-1-methyl-2-pyridone (2).
Scheme 1.
In addition, a minor metabolite, identified as the cis-3,4-dihydro-3,4-dihydroxy isomer 3 was also
isolated in minute amount (about 3%). Moreover, depending on the work-up conditions, a variable
amount of trans-5,6-dihydro-5,6-dihydroxy-1-methyl-2-pyridone 4 (2–10%) could be isolated. The
1
13
structures of all three compounds were established by NMR ( H, C, 2D COSY, HMQC, HMBC,
1
0
NOESY) and MS studies.
When analyzed by GC/EI-MS, pure dihydroxy isomers 2 and 4 exhibited molecular ions at m/z 143
corresponding to dihydro dihydroxy-derivatives) and major fragment ions at m/z 115, 86 and 85.11
(
A
small molecular ion at m/z 143 was also found for isomer 3, together with a base peak corresponding
to a dehydration fragment ion at m/z 125. In NMR spectra, characteristic coupling constants were found
for the vinylic protons in 3,4-position (2, J=10 Hz; 4, J=9.6 Hz) compared to those in 5,6-position (3,
4
a
J=7.8 Hz), clearly demontrating 5,6- or 3,4-dihydroxylation patterns. Moreover, the HMBC spectrum
3
of 2 exhibited J-coupling between the N-methyl protons and the nearest CH(OH) carbon, without any
significant coupling with a double-bonded carbon atom. The reverse situation was found in the HMBC
spectrum of 3. The assignment of cis- and trans-relative configurations of 2 and 4 was based upon the
3
measured J-coupling constants between H-5 and H-6 vicinal protons, correlated with dihedral angle
values calculated from molecular modelisation after energy minimization12 (Fig. 1). As a result, the
vicinal coupling constants for the cis-isomer 2 are larger (J =4.4 Hz) compared to the trans-isomer 4
5,6
(
J =2.0 Hz), and they are in agreement with the calculated values of dihedral angles in the model (55
5,6
and 71°, respectively). On the other hand, the observed H-4/H-5 coupling constants similarly fit with
the calculated dihedral angles: 2.0 Hz for the cis-isomer (95°), compared to 5.2 Hz for the trans-isomer
(30°).
In case of the 3,4-dihydroxy isomer (3), the characteristic H-3/H-4 and H-4/H-5 coupling constants
(
(
4.7 and 6.2 Hz respectively) were in full agreement with the calculated corresponding dihedral angles
51 and 27°) for a cis-compound (Fig. 2).
When kept at room temperature, cis-dihydrodiols 2 and 3 were shown to be relatively stable. However,
even mild heating of 2 (∼50°C) led to isomerisation to the corresponding trans-isomer 4. The formation
of trans-dihydrodiols along with the cis-isomers has been previously described in the dihydroxylation
1
3
13,14
13
of thiophene, benzothiophene,
and benzofuran by the toluene dioxygenase of P. putida UV4 and
attributed to the properties of the resulting cis-hemi(thio)acetal ring, which equilibrates with its anomer
15
(
trans-isomer) via an open chain aldehyde intermediate. The slight formation of the trans-dihydro-diol
4
which we observed could be attributed to a similar isomerisation, during incubation and work-up of the
cis-hemiaminal 2 (major product) initially formed in the NDO-catalysed dihydroxylation reaction.
However, no mutarotation was observed at room temperature, indicating a higher stability of the

3
867
Fig. 1. Energy minimized conformations of cis-5,6-dihydroxylated 1-methyl-2-pyridone (2) and its trans-isomer 4 and vicinal
coupling constants calculated from the corresponding dihedral angles H-5/H-6 and H-4/H-5
Fig. 2. Energy minimized conformations of cis- and trans-3,4-dihydro-3,4-dihydroxy-1-methyl-2-pyridone and vicinal coupling
constants calculated from the dihedral angles H-3/H-4 and H-4/H-5
hemiaminal compounds. As expected, the cis-3,4-dihydro dihydroxy-derivative 3 did not suffer such
an isomerisation reaction.
Heating above 60°C or a mild acidic treatment converted both 5,6-dihydroxy isomers 2 and 4 into
a dehydration product, 5-hydroxy-2-pyridone 5.16 Consequently, all attempts to prepare mono- (or
di-)ester derivatives with activated acylating reagents were unsuccessful due to the easy aromatization
of the diols in the reaction conditions. Such features prevented the use of diastereomeric esters of
optically active acids (such as MTPA or camphanic acid) to estimate the optical purity of the diols
and eventually determine their absolute configuration. It has been shown15 that the dihydroxylation of
aromatic compounds by toluene dioxygenase or naphthalene dioxygenase generally yields enantiopure
dihydro dihydroxylated products with a single absolute configuration. However, in a few examples, the
formation of cis-dihydrodiols of lower enantiomeric excess has been described.15
No reaction was observed with unsubstituted 2-pyridone or N-methyl-4-pyridone. In control experi-

3
868
ments with E. coli JM109(DE3)(pT7-5), a strain which does not contain the structural genes for NDO,
no oxidation of 1-methyl-2-pyridone was observed. Until now, the NDO system had not been reported
to catalyse the cis-dihydroxylation of monocyclic arenes or heteroarenes at any bond.15,17 It is also
remarkable that another recombinant strain, E. coli JM109(pDTG601) expressing toluene dioxygenase
from P. putida F1, is not able to oxidize 1-methyl-2-pyridone.
The reported results appear to be an unprecedented example of dihydrodiol formation from a mono-
heterocyclic nitrogen compound, resulting from a dioxygenase-catalysed oxidation. To our knowledge,
there is only one reported example of a comparable dioxygenase-catalysed cis-dihydroxylation in the
pyridine ring of a bicyclic heteroarene compound: the oxidation of 2-chloroquinoline by toluene dioxy-
genase of P. putida UV-4 has been recently described to give, as a minor product, a cis-3,4-dihydro-3,4-
dihydroxy-2-quinolone, which probably derives from an intermediate 2-quinolone.15,18
Work is in progress to determine the absolute configuration of the dihydroxylated products, and to
extend the scope of this reaction to a series of unsubstituted and substituted monocyclic and bicyclic
heteroaromatic systems.
18
Acknowledgements
We thank Professor D. T. Gibson for providing all E. coli strains and the Centre National de la
Recherche Scientifique, France, for financial support.
References
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. For recent references about amino acids obtained from dihydropyridine derivatives, see: (a) Rutjes, F. P. J. T.; Shoemaker,
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M.; Schoenfelder, A.; Didier, B.; Mann, A.; Wermuth, C.-G. Chem. Commun. 1999, 683–684.
. Typical experimental procedure: IPTG-induced cells of E. coli JM109(DE3)pDTG141, grown at 37°C in 1 L of minimal
medium (see Ref. 8), were harvested at the end of exponential growth, washed and resuspended in 0.3 vol. of 0.05 M
Na/K phosphate buffer pH 7.2 containing 0.2% glucose. After addition of N-methyl-2-pyridone (60 mg), incubation was
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column chromatography and preparative TLC on silica gel to give the dihydro dihydroxy derivatives.
. Resnick, S. M.; Gibson, D. T. FEMS Microbiol. Lett. 1993, 113, 297–302.
8
9
. Resnick, S. M.; Gibson, D. T. Appl. Environ. Microbiol. 1996, 62, 3355–3359.
1
1
0. Compound 2: [α]
D
+41 (c 0.345, MeOH). H NMR (CD
6
-acetone), δ=6.37 (1H, dt, J4,3=10, J4,5=2, J4,6=1.7, H-4), 5.70
(
1H, dd, J3,4=10, J3,5=2.0, H-3), 4.92 (1H, dd, J6,5=4.4, J6,4=1.7, H-6), 4.57 (1H, br.dd, J5,6=4.4, J5,4=J5,3=2, H-5), 2.94 (3H,

3
869
s, CH
3
). 13C NMR (CD
3
OD), δ=168.1 (CO), 146.7 and 125.1 (−CH_), 86.7 and 69.6 (CHOH), 35.0 (CH
3
). EI-MS, see
1
Ref. 11. Compound 3: [α]
D
+25 (c 0.16, MeOH). H NMR (CD
3
OD), δ=6.28 (1H, dd, J6,5=7.8, J6,4=0.8, H-6), 5.38 (1H,
dd, J5,6=7.8, J5,4=6.2, H-5), 4.26 (1H, d, J3,4=4.7, H-3), 4.17 (1H, ddd, J4,3=4.7, J4,5=6.1, J4,6=0.8, H-4), 3.06 (3H, s, CH
3
).
13
C NMR (CD
3
OD), δ=172.1 (CO), 134.0 and 106.8 (−CH_), 72.1 and 65.6 (CHOH), 34.2 (CH
3
). EI-MS: m/z 143 (4),
+207 (c 0.285, MeOH). H NMR (CD
1
1
25 (100), 97 (30), 96 (30), 82 (8), 68 (36), 55 (23). Compound 4: [α]
D
6
-acetone),
δ=6.57 (1H, ddd, J4,3=9.6, J4,5=5.2, J4,6=1.5, H-4), 5.86 (1H, d, J3,4=9.6, H-3), 4.90 (1H, br.t, H-6), 4.10 (1H, dd, J5,6=2,
5,4=5.2, H-5), 2.96 (3H, s, CH ). EI-MS, see Ref. 11.
J
3
1
1. Both pure 5,6-dihydroxy isomers 2 and 4 exhibited two distinct chromatographic peaks, with retention time at 7.5 and 9.4
min, in a constant 1:1 ratio, with very small differences in their fragmentation pattern (M at m/z 143; base peak at m/z 86
+
or 85, respectively). This chromatographic splitting and the resulting fragmentation pattern probably originates from the
isomerisation of the remaining double bond under the GC conditions.
1
1
2. Molecular modelization and energy minimization performed with the CS Chem3D Pro package (Cambridge Soft Corp.)
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1
1
5. Boyd, D. R.; Sheldrake, G. N. Nat. Prod. Rep. 1998, 309–324.
1
6. Compound 5: H NMR (CD
3
OD), δ=7.28 (1H, dd, J4,3=9.6, J4,6=3, H-4), 7.05 (1H, d, J6,4=3, H-6), 6.46 (1H, d, J3,4=9.6,
H-3), 3.15 (3H, s, CH
3
). EI-MS: m/z 125 (100), 97 (35), 96 (16), 82 (8), 68 (29), 55 (16).
1
1
7. Resnick, S. M.; Lee, K.; Gibson, D. T. J. Ind. Microbiol. 1996, 17, 438–457.
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83–684.