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Enzymatic Oxidation of Methyl Groups on Aromatic Heterocycles A Versatile Method for the Preparation of Heteroaromatic Carboxylic Acids.

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L. Hough, A. C. Richardson in The Carbohydrares Vol. l A , (Eds.: W.
Pigman. D. Horton), 2nd ed., Academic Press, San Diego, 1972, p. 12713x.
A. S . Perlin in The Carbohydrates Vol. l B , (Eds.: W. Pigman, D. Horton).
2nd ed., Academic Press, New York. 1980, pp. 1167-1215.
Y. Nakahara, K. Beppu. T. Ogawa. Tetrahedron Lett. 1981,22, 3197-3200.
M. Kinoshira. A. Hagiwara, S . Aburaki, Bull. Chem. Suc. Japan 1975,48,
570. M. Kinoshitd, S . Mariyama. ibid. 1975, 48, 2081. S. Aburaki, N.
Konishi, M. Kinoshita, %id. 1975,48,1254.H. R. Schuler, K. Slessor, Can.
J. Chem. 1977,55,3280.J. F'. H. Verheyden, A. C. Richardson, R. S . Bhatt,
B. D. Grant, W. L. Fitch, J. G. Moffatt, Pure Appl. Chem 1978, 51, 1363.
B. Kaskar. G. L. Heise, R. S . Michalak, B. R. Vishnuvajjala, Synlhesis
1990. 1031-1032.
L. Chan. G. Just. Telrahedron 1990, 46, 151-162.
E.J. Corey. A. Marfat, G. Goto, F. Brion, J. An?. Chem. SOC.1980, 102,
7984-7985.
Enzymatic Oxidation of Methyl Groups
on Aromatic Heterocycles: A Versatile Method
for the Preparation of Heteroaromatic Carboxylic
Acids**
By Andreas Kiener*
Chemical oxidation reactions used for the industrial scale
preparation of heteroaromatic monocarboxylic acids from
heteroaromatic compounds bearing one or more methyl
groups are often nonspecific and lead to the formation of
undesired by-products. To overcome this problem we have
been interested in developing a biological oxidation method
for this type of reaction. Although enzymatic oxidations of
alkyl side chains on heteroarenes by bacteria and fungi have
been described,['] none of these biotransformations have
been used for the preparation of aromatic heterocyclic carboxylic acids or their hydroxymethyl derivatives. This communication describes a versatile enzymatic method capable
of selectively oxidizing a single methyl group on heteroarenes on a pilot plant scale.
The wild-type strain Pseudomonas putida ATCC 33015
was used as the biocatalyst in our investigations. This microorganism can grow on toluene, m-xylene, or p-xylene as
sole carbon and energy source.[z1Both the b i ~ c h e m i s t r y41~ ~ .
and geneticsr5*'I of the xylene degradative pathway have
been extensively studied. p-Xylene, for example, is oxidized
by xylene monooxygenase to 4-methylbenzylalcohol, which
is then further oxidized by benzylalcohol and benzylaldehyde dehydrogenase to 4-niethylbenzoic acid. The aromatic
carboxylic acid is converted by toluate dioxygenase and dihydroxycyclohexadienecarboxylate dehydrogenase into 4methylcatechol prior to the cleavage of the aromatic ring by
catechol dioxygenase. The cleavage product is then transformed into Krebs-cycle intermediates. Investigations on the
substrate specificity of key enzymes in the xylene degradative
pathway have focused mainly on substituted aromatic cdrbocycles.[7]Only a few publications concerning heterocycle oxidation have appeared. One example is the bacterial formation of indigo initiated by hydroxylation of indole by xylene
monooxygenase.[']
We have now demonstrated that P. putida grown on pxylene as sole carbon and energy source was capable of oxi["I
[**I
Dr. A. Kiener
Lonza AG
Biotechnology Research Department
CH-3930 Visp (Switzerland)
I thank R. Glockler and K. Heinzmann for technical assistance, and Dr.
M. Bokel and Dr. M. Hauck for analysis of oxidation products. Fermentations on pilot-plant scale were performed by Dr. M. Rohner.
174
(c) VCH Verlugsgesellschufi mbH, W-6940 Weinheim, I992
Table 1. Formation of heteroaroinatic carhoxylic acids from methylated heterocycles
with cells of Pseudomonas putida ATCC 33015 grown on p-xylene
Starting material
Product
Yield
["/.I
2,5-dimethylpyrrole
5-methylpyrrole-2-carboxylic
acid
3.5-dimethylpyrazole
5-methylpyrazole-3-carboxylicacid
2.5-dimethylfuran
5-methylfuran-2-carhoxylic acid
3-methylthiophene
thiophene-3-carboxylic acid
4-methylthiazole
thiazole-4-carboxylic acid
3-methylp yridine
pyridine-3-carboxylic acid
2-chloro-6-methylpyridine
6-chloropyridine-2-carboxylic acid
2-chloro-3-ethyl-6-methylpyridine
,6-chloro-5-ethylpyridine-2-carboxylic
acid
2-methylpyridine-4-carboxylic acid (90 %)
2,4-dimethylpyridine
4-methylpyridine-2-carboxylicacid (10 Yo)
2.5-dimethylpyrazine
5-methylpyrdzine-2-carboxylic
acid
2,3,6-trimethylpyrazine
5,6-dimethylpyrazine-2-carhoxylic
acid
3-chloro-2,5-dimethylpyrazine 6-chloro-5-methylpyrazine-2-carboxylic
acid
i
40
80
40
70
80
50
90
10
40
90
90
90
dizing many methylated heteroaromatic five- and six-membered rings to the corresponding monocarboxylic acids
(Table 1). The carboxylic acids accumulated in the fermentation broth and were isolated for analysis. In most cases the
generated heteroaromatic carboxylic acids were not further
degraded, which indicates that aromatic heterocycles are
poor substrates for toluate dioxygenase (none of the heterocycles supported the growth of P. putida). As shown in
Table 1, biotransformations of 2,3,6-trimethylpyrazine and
3-chloro-2,5-dimethylpyrazinewere also regiospecific; this
was expected since substituents ortho to a methyl group prevent hydroxylation by xylene monooxygenase.['] The oxidation product of 2,4-dimethylpyridine, however, contained a
mixture of 2-methylpyridine-4- and 4-methylpyridine-2-carboxylic acid. The formation of 6-chloro-5-ethylpyridine-2carboxylic acid from 2-chloro-3-ethyl-6-methylpyridine
showed that the enzymatic oxidation was specific for the
methyl group. In no experiment did we detect the formation
of dicarboxylic acids or the direct hydroxylation of the
heteroaromatic ring.
The performance of this biocatalyst system was studied in
more detail on 2,5-dimethylpyrazine (DMP). The oxidation
product, 5-methylpyrazine-2-carboxylic acid (MPCA), is an
intermediate for the production of 5-methylpyrazine-2-carboxylic acid 4-oxide, a drug with antilipolytic activity.['] The
chemical oxidation of DMP to MPCA along any routes still
remains inefficient.['01 Resting cell suspensions furnished
with more than 30 mM (3.2 gL-') DMP showed a strong
accumulation of 2-hydroxymethyl-5-methylpyrazine,
which
was only partially oxidized to MPCA. However, high product
concentrations and high yields were achieved by performing
the biotransformation with growing cells. For this reason a
mixture of 75% (v/v) p-xylene and 25% (v/v) DMP was
supplied as growth substrate in large-scale fermentations.
Figure 1 shows the accumulation of up to 20 g MPCA L-'
during 54 h bacterial growth on a 20 L scale. 2-Hydroxymethyl-5-methylpyrazine was detected in the medium during
early stages of fermentation. The biotransformation was terminated when bacterial growth entered the stationary phase
(MPCA concentrations > 15 gL-' inhibited the growth of
P. putida). At the end of the fermentation, MPCA was the
only oxidation product of DMP detected in the fermentation
broth by HPLC analysis.
Biotransformations up to a volume of 1000 L following a
slightly modified fermentation procedure were performed to
assess the potential of this biological oxidation method for
industrial application: The maximal product concentration
was 24 g MPCA L-I (at a yield >95?0); the concentration
of DMP at the end of the biotransformation was below
0570-0833/92/0606-0774$3.50+.25/0
Angew. Chem. Int. Ed. Engl. 31 ( 1 9 9 2 ) N o . 6
__
25
30 20
2515
20 -
DMP.MPCA
[g L-ll
10
5
0
10
20
30
flhl
LO
-
50
0
60
Fig. 1. Microbial oxidation of 2,5-dimethylpyrazine (DMP) to 5-methylpyrazine-2-carboxylic acid (MPCA). Absorption of the cell suspension at
650 nm (m); concentration of DMP (A) and MPCA ( 0 ) .After 52 h of fermentation the growth substrate was changed to 100% p-xylene.
0.1 g L- '. No bacterial metabolites fromp-xylene which could
hamper product purification were detected in the broth.
In summary, our experiments showed that P. putida is a
useful biocatalyst for the selective oxidation of a methyl
group on aromatic heterocycles to the corresponding monocarboxylic acid. The yield and conditions of the oxidation
reactions described here have been optimized only for 2,5dimethylpyrazine. Nevertheless, in order to develop a general method for the biological oxidation of a methyl group on
aromatic heterocycles, we will now focus on the isolation of
various mutants defective in the xylene-degradative pathway
so that further degradation of certain heteroaromatic carboxylic acids can be prevented or benzylalcohol dehydrogenase inactivated to permit the production of hydroxymethylated compounds.
Experimental Procedure
Starter cultures of P. puridu were grown in 300 mL Erlenmeyer flasks containing 100 mL of a mineral salt medium (H. G. Kulla, F. Klausener, U. Meyer, B.
Ludeke, T. Leisinger, Arch. Microhiol. 1983, 135, 1-7) at pH 7. A lOmL
polypropylene tube containing 1 mL p-xylene as carbon source was inserted
into the Erlenmeyer flask. The xylene vapor formed during incubation at 30 "C
o n a rotatory shaker was sufficient for cell growth. For large-scale fermentations (20- 1000 L) xylene was added directly to the growth medium. The xylene
concentration in the fermenter was regulated by measuring the absorbance of
fermenter exhaust air at 214 nm with an ISCO UA-5 absorbance detector (ISCO, P.O. Box 5347. Lincoln, NE 68505, USA). The electronic signal of the
detector was coupled with a dosage pump for xylene in such a way that the
concentration of the growth substrate was about 0.1 mM. Best transformation
rates were obtained under substrate-limited growth.
For small-scale biotransfonnations, methylated heterocycles (10 mM were
added to 20 mL suspensions ofcells grown onp-xylene (A,,,,, = 10) in mineral
salt medium in 300 mL sealed flasks to prevent the loss of volatile compounds
and incubated for 16 h at 30°C on a rotatory shaker. Xylene was omitted
during biotransformation. The concentrations of oxidation products in cell-free
solution were analyzed by thin-layer chromatography. For incomplete conversions the babdnce of the yield was, in most cases, unoxidized starting material.
41-7; 2-methylpyridine-4-carboxylic acid, 4021-1 1-8; 4-methylpyridine-2-carhoxylic acid, 4021-OX-3; 5-methylpyrazine-2-carboxylicacid, 5521-55-1 ; 5.6dimethylpyrazine-2-carboxylicacid, 13515-06-5; 6-chloro-5-methylpyrarine2-carboxyhc acid, 138538-39-3.
[l] K. Kieslich, Microbiul Trun~formurions,Thieme. Stuttgart, 1976, p. 292523.
121 D. T. Gibson, Microhiul Degrudufion of' Organic Compounds, Marcel
Dekker. New York, 1984.
[3] S. Harayama, M. Rekik, M. Wubbolts, K . Rose, R. A. Leppik, K. N.
Timmis, J. Bucteriol. 1989, 171, 5048-5055.
(41 J. P. Shaw, S. Harayama, Eur. J. Biockem. 1990, i91, 705-714.
(51 S. J. Assinder, P. A. Williams, Adu. Microh. Phy~iot.1990, 31, 1-69
[61 M:A. Abril. C. Michan, K. N. Timmis, J. L. Ramos. J. Bucferiot. 1989,
/7i,6782-6790.
[7] M. G . Wuhbolts, K. N. Timmis, Appl. Environ. Microhiol. 1990, 56, 569571
[XI N. Mermod, S. Harayama, K. N. Timmis, Bioterhnofogy. 1986, 4, 321 324.
[9] P. P. Lovisolo, G. Briatico-Vangosa, G. Orsini. R. Ronchi, R. Angelucci,
Pharmucol. Rrs. Cammun. 1981, 13, 151- 161.
[lo] G. P. Borsotti, M. Foa, N. Gatti, ,Synthesis, 1990. 207-208.
One-Pot Syntheses of Novel Arene Tricarbonyl
Chromium Macrocycles and Macrobicycle:
Structure of a Pentanuclear Trichromium(o)Dicopper(1) Cryptate**
By Marie- Ther2se Youinou,* Jean Sujfert,*
and Raymond ZiesseP
Polyaza receptor molecules with large cavities have recently received a great deal of attention as hosts for metal ions
which form binuclear or polynuclear complexes,['. 21 in view
of their potential as supramolecular catalysts.[31Schiff-base
condensation of the tris(2-aminoethy1)amine (tren) with suitable dicarbonyl compounds provides N, cryptands in good
~ie1d.l~'
Although macrocycles synthesized from difunctiona1 metal complexes were reported p r e v i ~ u s l y / such
~ . ~ ~macrobicycles have not yet been prepared. The use of such subunits may provide new photoactive, electroactive, and chiral
cryptates. We have recently reported new molecular tweezer
ligands based on chiral arene tricarbonyl chromium subunits. Their complexation properties with rhodium(~)['Iand
copper(I)[*I as well as their photochemical*g1and electrochemical" O1 behavior were studied.
Pursuing this work, we now report a very efficient route,
through condensation of various amines with a terephthalaldehyde tricarbonyl chromium complex, to macrocyclic ligands 1 and 2, macrobicyclic ligands 3, and a novel trichromium(o)-dicopper(1) cryptate 4.
The reaction of equimolecular amounts of 1,3-bis(aminomethy1)benzene and chromium(0) complex 6 (synthesized
from its precursor 5["]) in chloroform gave pure macrocycle
[*I
Received: December 4, 1991 [Z 5057 IE]
German version: A n g a r . Chem. 1992, 104, 748
CAS Registry numbers:
2.5-dimethylpyrrole, 625-84-3; 3,5-dimethylpyrazole, 67-51-6; 2,Sdimethylfuran, 625-86-5; )-methylthiophene, 616-44-4; 4-methylthiazol, 693-95-8;
3-methylpyridine, 108-99-6; 2-chloro-6-methylpyridine, 18368-63-3; 2-chloro3-ethyl-6-methylpyridine, 138538-40-6;2,4-dimethylpyridine, 108-47-4; 23-di14667-55-1; 3-chloro-2.5methylpyrazine, 123-32-0; 2,3,6-trimethylpyrazine,
dimethylpyrazine, 95-89-6; 5-methylpyrrole-2-carboxylic acid, 3757-53-7;
5-methylpyrazole-3-carboxylicacid, 402-61-9; 5-methylfuran-2-carboxylic
acid, 1917.1 5-3; thiophene-3-carboxylic acid. 88-1 3-1 ; thiazole-4-carboxylic
acid, 3973-08-8: pyridine-3-carboxylic acid, 59-67-6; 6-chloropyridine-2acid, 138538carboxylic acid, 4684-94-0; 6-chloro-5-ethylpyridine-2-carboxylic
Angen'. Chi'ni. I n t . Ed. Engl. 31 (1992) No. 6
8
I**)
Dr. M.-T. Youinou
Laboratoire de Chimie des Metaux de Transition et de Catalyse
UA au CNRS no 424, Institut Le Be1
Universite Louis Pasteur
4, rue Blaise Pascal, F-67000 Strasbourg (France)
Dr. J. Suffert
Laboratoire de Phannacologie Moleculaire
UPR au CNRS no 421, Centre de Neurochimie
5, rue Blaise Pascal, F-67084 Strasbourg Cedex (France).
Dr. R. Ziessel
Ecole Europeenne des Hautes Etudes des Industries Chimiques de Strasbourg
IPCMS/GMI, URM au CNRS no 46
I , rue Blake Pascal, F-67008 Strasbourg Cedex (France)
This work was supported by the Centre National de la Recherche Scientifique. RZ thanks Prof. J. M. Lehn for research facilities.
VCH KwlugsgeselI.~chaftm b H , W-6940 Weinheim, 1992
0570-0833~92~0606-077S
$3.50+ .25/0
175
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