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Reagents with Hypervalent Iodine Formation of Convenient Chiral Synthetic Intermediates by Fragmentation of Carbohydrate Anomeric Alkoxy Radicals.

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c) R. J. Batchelor, H. B. Davis, F. W. B. Einstein, R. K. Pomeroy, J. A m .
Chrfn. SOC.1990. 112, 2036.
114) IR of 1: v(C0) 1890 c m - ‘ (CHJI,); 1892 e n - ’ (THF): 1R of 2: v(C0):
1925(s), 1946(s), 2006(w) cm-’. v(OICCF;): 1674cm-’ (CH,CI,);
r(C0): 1927(s). 1946(s). 2010(w) cm-’. v(0,CCF;): 1676cm-’ (THF).
[15] M . J. Therien. W. C. Trogler. J. AM. Ciimi. Sor. 1986. 108, 3697.
[16] The intensities of the absorbances of i(C0) at 1975 and 2045cm-’ and
v(0,CCF;) at 1694and 1644 em-’ increase in a c o n ~ t a nratio.
t
Compared
with v(0,CCF;) of 1676cm-’ in 2 and 1692cm-’ in [Bu,N](O,CCF,).
the hypsochromic shift ofone 0,CCF; group (1694 cm-’) corresponds to
an ionic 0,CCF; unit and the bathochromic shift of a second 0,CCF;
group (1644cm-’) is typical for the formation of a bidentate complex.
Therefore the absorbances are assigned to [Os(CO),(PPh,),(t$
O,CCF,)](O,CCF,) The other absorbance at 1892 cm-’ is identical to
that of 1.
1171 IR spectrum of the final product from thermal reation of 2 in CH,CI,:
r(C0): 1977(s) and 2046(s) c m - ’ , v(0,CCF;): 1691 cin-’. For coinparison the known [OsCI,(CO),(PPh,)J. prepared by the method of Collman
and Roper [6]. caused these absorptions to increase when added to the
same solution. The uncoordinated 0,CCF; group in [Bu,N](O2CCF3)
has an absorbance at 1691 cm-’ in CH,CI,.
Cleavage of the CI-C2 carbon bond in carbohydrate
chemistry can be performed by several methods such as
those of Ruff, Wohl, Weerman etc., but they suffer from
several drawbacks, for instance, the number of steps required, low yields, and inconveniently strong reaction conditions.[61Although sodium periodate and lead tetraacetate
are routinely used to test for the presence of vicinal diols,[’j
they have hardly been used in synthesis to cleave the C1 -C2
carbon bond.[81
The carbohydrate derivative was treated with (diacetoxyiodo)benzene (DIB) and iodine in cyclohexane under the conTahlr 1 IGFramnmtation o f c;irhohvdr:ite a n o m r r i r alknrv radical< lnl
Entry
Substrate [b]
Reagents with Hypervalent Iodine: Formation of
Convenient Chiral Synthetic Intermediates by
Fragmentation of Carbohydrate Anomeric Alkoxy
Radicals**
4
0.1
243
1.2
0
24
TBDMSo 4
iO
HOCO
A
c
36
84
no
reaction
0
2
B nBnO
o b OBn
O H
4
BnOHOCO
3
5
85
OBn
4
Bnot@PoH
BnO
1.2
1
8
91
4
5
6
QO h
Brio
OH
Bnp
1.2
B n O v o A C
HOCO OBn
1
OBn
6
7
8
‘ * P O H
AcO
g9
7
;:; ;
96
0.3 [cl
OAc
nco+
I
44
44
HOCO
8
9
9
10
11
BnO,
10
@OH
BnO
OBn
1.2
1
32
OBn OAc 81
B n O e O B n
HOCO OBn
ra~’]
-Ro4~~~~~S
RO
H
RO OR
RO OR
HOCO
1
13
14
1s
11
[*] Prof. E. Suirez. Dr. P. de Armas, Dr. C. G. Francisco
Instituto de Productos Natnrales y Agrobiolgia del C.S.I.C.
Carretera de la Esperanza 2. 38206 La Laguna. Tenerife (Spain)
~
[**I This work was supported by the Investigation Programme no. PB87-0406
of the Direccion General de Tnvestigacion Cientifica y Tecnica.
r()
12
OR
Scheme 1 . DIB =(diacetoxyiodo)benzene.
772
,.
1.2
BnO
Carbohydrates owe their present importance in part to
their use as chiral template precursors for a variety of complex non-carbohydrate substances.[” In this context many
efforts have been made in recent years to develop a new and
efficient methodology to create or destroy asymmetric centers by chemical manipulation. The proposed reactions must
be compatible with the temporary protective groups that
might be employed in this type of synthesis. Special attention
has been devoted to the preparation of four-carbon chiral
building blocks of the threose and erythrose series,”] which
have been used in the synthesis of several polyhydroxylated
natural products.[3]
During the past few years, we have examined several compounds with hypervalent iodine that can generate alkoxy
radicals, in order to promote 8-fragmentation reactions.14’ A
new synthesis of medium-sized lactones and spirolactones
was developed by use of these reagents on appropriate oxabicyclic hemia~etals.[~’
We now report a new method for the
synthesis of four-carbon chiral building blocks of the D- and
L-erythrose types by p-fragmentation of the C1 -C2 carbon
bond of suitably protected D- and c-arabinose, D-ribose, and
2-deoxy-~-ribose(Scheme 1). The reaction has also been extended to the synthesis of five-carbon chiral building blocks
of the arabinose type by 8-fragmentation of D-mannose and
D-glucose derivatives.
O
Yield
[“/.I
L
1
By Pedro de Armus, Cosme G. Francisco,
and Ernest0 Suarez*
b
Product
-
TBDMSO,
1
2
3
Conditions
I,
time
DIB
[nimol] [inmol] [h]
VCH VeriugsjieseiIscha/t nihH,W-6940 Wch?l?ettn,I992
[a] 0.3 mmol scale at 20 ”C in cyclohexane (30 mL mmol-’). [b] TBDMS
methylsilyl, Bn = benzyl. [c] Under reflux.
X 3.50 + .25/0
0570-0833/92~0606-0772
=
tert-bntyldi-
Angev.. Chem. Inr. Ed. Engl. 31 (1992) No. 6
ditions specified in Table 1. The reaction conditions were determined with the readily available 5-0-(~~t-butyldimethylsi~y~)-2.3-~-isopropyhdene-D-ribofuranose
(1),191 which
gave a major stereoisomer a t C 1 (ca. 95: 5 ) of the erythrose
derivative 2 (entry 1). The proposed stereochemistry at C I is
based on the observed trans coupling constant between H1
and H2 (JH,,HZ
= 1.96 Hz). The presence of iodine was necessary for the reaction to take place (entry 3), and lower yields
were observed when catalytic amounts were used (entry 2).
The reaction mechanism seems to involve the initial formation of the anomeric alkoxy radical followed by fi-fragmentation and subsequent trapping of the carbon radical formed
at C 1 by an acetoxy radical from the reagent. At this point
the anomeric carbon is a part of a formate group that forms
an ester with the alcohol at C 3 (Scheme 1).
A differently protected erythrose building block 4 was
obtained from 2,3,5-tri-O-benzyl-~-ribofuranose
(3)['01(entry 4). Substrate 3, which lacks the steric bias of the previous
example. was found to give a mixture (ca. 1 : l ) of stereoisomers a t C 1.
The reaction course does not seem to be dependent on the
configuration of the alcohol at C 2 , since D- and ~-2,3,5-tri0-benzylarabinofuranose (5 and 6, respectively) were fragmented analogously to substrate 3 to give D- and L-erythrose
building blocks 4 and 7, respectively, in similar yields (compare entry 4 with entries 5 o r 6).
/&Fragmentation of 2-deoxy-~-ribofuranose (8) and
-pyranose derivatives (lo)[' 'I gave rise to two potentially
interesting synthetic intermediates, the I-iodo-2,3,4-trihydroxybutane derivatives 9 and 11, respectively; although
yields were lower (entries 7-9), the generation of the formyl
Table 2. Spectroscopic data for2,9.11. and 15. IR (CHCI,): 'H and
NMR
at 200 and 50.3 MHz. respectively, in CDCI, with internal TMS standard
(assignments by DEPT. 'H-IH COSY. and 'H-"C HETCOR).
2: [XI,, = - 33 (c = 0.lX. CHCI,): ' H N M R : 6 = 0.05(s, 6H. Si (CH,)J, 0.88
(a. 9 H , SiC(CH,),), 1.45 ( 5 , 3 H, isopropylidene-CH,). 1.48 (s, 3 H . isopropylidene-CH,). 2.09 (s. 3 H , CO-CH,), 3.78 (dd, J = 4.67. 13.32 Hz. 1 H , C4- Ha).
3.85 (dd. J = 4 . 0 7 . 11.35Hz. 1H.C4-Hb).4.42(dd, J = 2 . 3 1 , 5.30Hz, I H ,
C2-H). 5.09 (pseudo-q. 1 H, C3-H). 6.31 (d, J =1.96Hz. 1 H, C1-H). 8.10 (s.
1 H. OCOH). I3C NMR: 6 = -5.57 and -5.49 (Si(CH,)J. 18.23 (SiC(CH,),).
21.18 (CO-CH,). 25.82 (SiC(CH,),), 26.59 (isopropylidene-CH,). 27.22 (isopropylidene-CH,). 61.35 (C4). 72.75 (C3). 80.51 (C2). 95.90 (Cl), 112.77 (isopropylidene-CJ. 159.99 (OCOH), 169.95 (CO-CH,). IR: i. [cm-'1 = 1729.
FABMS ( N a l ) : nr:: 385 ([M+NaJe. 15%).
9: [ r ] , = - 25 (c = 0.24. CHCI,); ' H N M R : 6 = 2.07 (s, 3 H, CO-CH,), 2.13 (s,
3H. CO-CH,). 3.31 (dd, J = 6.29. 11.17 Hz. 1 H. C1-HA),3.44 (dd. J = 4.25.
Il.19Hz. 1 H . Cl-H,). 4.22 (dd. J = 5 . 8 4 . 12.4Hz. l H , C4-H,), 4.37 (dd,
J = 3.24. 12.45 Hz. 1 H, C4-H,), 5.01 (m.1 H. C2-H), 5.35 (m, 1 H, C3-H), 8.08
(s. 1 H, OCOH). I3C N M R . d = 2.25 (Cl). 20.54 (CO-CH,). 20.69 (CO-CH3).
61.34 (C4). 69.98 (C2). 71.18 (C3). 159.26 (OCOH): 169.41 (CO-CH,), 170.24
(C'O-CH,). I R . i,[cm-'] =1737. FABMS (glycerol. NaCI. NaI): in/; 345
([M+H]@. 24%).
11: [%ID = - 2 2 (c = 0.16. CHCI,): 'H N M R : 6 ~ 1 . 3 (s.
9 3H. isopropylideneCH,). 1.50 (s. 3 H, isopropylidene-CH,), 3.18 (dd, J = 6.37. 10.6 Hz. 1 H. C1HJ. 3.25 (dd.J=7.15. 10.22Hz. l H , CI-H,),4.16-4.55(m. 4H, C2-H, C3-H,
C4-HJ. 8.1 I (s. 1 H. OCOH). ' 3 CN M R : 6 = 0.67 (Cl), 25.43 (isopropylideneCH,). 27.97 (isopropylidene-CH,), 61.71 (C4). 74.77 (C3), 77.55 (C2), 109.95
(isopropylidene-C). 160.38 (OCOH). IR: I' [cm-'1 =1725. FABMS (NaI): nrjz
323 ( [ M + N a ] ' * , 40%).
15: I,,= -47 ( c = 0.1X, CHCI,): 'H N M R : 6 = I 2 5 (s. 3 H , isopropylideneCH,). 1.35 (s. 3 H , isopropylidene-OH,). 1 .SO (s. 6H. 2 x isopropylidene-CH,).
2.09(CO-CH,).3.X6(dd,J=5.79,8.76Hz,1H,Cj-H,),4.06(dd,J=6.15,
8.73 Hz. 1 H. CS-H,). 4.27 (pseudo-q, C4-H), 4.48 (dd. J = 2.82, 2.55 Hz, 1 H,
C2-H). 5.29 (dd. J = 3.22.6.92 Hz. 1 H. C3-H), 6.1 1 (d, J = 2.1 Hz. 1 H. CI-H),
8.15 (s, 1 H, OCOH). I3C N M R : d = 21.08 (CO-CH,), 25.21 (isopropylideneCH,). 26.43 (isopropylidene-CH,). 26.70 (isopropylidene-CH,). 27.03 (isopropylidene-CH,). 66.15 (CS). 71.05 (C3),74.04 (C4). 81.29 (C2). 96.67 (Cl).
109.72 (isopropylidene-CJ. 113.58 (isopropylidene-C). 159.81 (OCOH), 170.06
(CO-CH,) IR: ~ ' [ c m - ' ]=1732. FABMS (glycerol. NaCI): tniz 317 ([M-H]@.
8 %)
An,qew. C'henr. h i t . Ed. Engl. 31 (1992) No. 6
group allows subsequent selective modifications of these
building blocks to be carried out.
The reaction was also extended to hexoses (entries 10
and 2 1 ) ; thus the 2,3,4,6-tetra-U-benzyl-~-glucopyranose
(12) and 2,3 :5,6-di-U-isopropylidene-~-mannofuranose
(14)
were transformed into the D-arabinose derivatives 13 and 15.
In the glucopyranose a mixture (ca. 1 :1) of C 1 stereoisomers
of 13 is observed, where the formyl group forms an ester with
the hydroxyl group a t C4, whereas in the mannofuranose a
single stereoisomer is obtained, the formate group being at
C 3 (the observed frans coupling constant between H i and
H, was JH1,H2
= 2.1 Hz). The spectroscopic data of compounds 2, 9, 11, and 15 are summarized in Table 2.
The fragmentation reaction is independent of ring size;
thus, similar yields were obtained for furanose and pyranose
derivatives (compare entries 7-8 with 9, and 10 with 11).
We consider that some features of the reported procedure
are remarkable: for example, the mild reaction conditions,
compatible with the protective groups most widely used in
carbohydrate chemistry; the regioselectivity observed during
the fragmentation; and also the fact that the reaction outcome does not depend on C 2 configuration, the protective
groups, or ring size. The generation of the formate group and
of the masked aldehyde that permits a flexible use of the
resulting building blocks is very valuable. This reaction can
also be considered a convenient method to descend the aldose series of carbohydrates and compares well with those
reported.[61
Experimental Procedure
A solution of 5-O-(rert-butyldimethylsilyl)-2.3-O-isopropylidene-u-ribofuranose (1) [9] (100 mg, 0.33 mmol) in dry cyclohexane (10 mL) containing (diacetoxyiodo)benzene (127 mg, 0.39 mmol) and iodine (84 mg, 0.33 mmol) was
stirred at room temperature (20'C) for 3 h. The reaction mixture was then
poured into water and extracted with ether. The organic layer was washed with
aqueous sodium thiosulfate and water. Chromatography of the residue (Chromatotron. Merck silica gel 60 P F 254. n-hexanelethyl acetate 9: 1 vlv) gave 2
(99 mg. 84%).
Received: December2. 1991 [Z 5049 IE]
German version: A n p i . Chem. 1992, 104, 746
CAS Registry numbers:
1, 68703-51-5; 2,141062-52-4; 3 ( a anomer), 8961 5-45-2; 3 (fl anomer). 8936152-4: 4, 141062-53-5: 5, 37776-25-3; 6, 77870-89-4; 8, 141062-54-6; 9, 14106255-7: 10, 65236-75-1; 11, 141062-56-8: 12, 38768-81-9; 13, 141062-57-9; 14,
40036-82-6; 15,141062-58-0; (diacetoxyiodo)benzene, 3240-34-4; iodine. 755356-2.
[ l ] S. Hanessian, Total Synrhesi.s of Nuturul Products. The Chiron Approu'h,
Pergamon Press. Oxford 1983. S. Hanessian, G . Rauncourt, Piwe Appl.
Chem. 1977. 49. 1201. 9 . Fraser-Reid, T. F. Tom, K. M. Sun in Orgunic
Synthesis. Toduy and Tomorrow (Eds.: B. M. Trost. C. R. Hutchinson),
Pergamon Press, Oxford. 1981. T. D. Inch, Tetrahedron 1984, 40. 3161 3213. K. L. Bhat, S.-Y Chen. M . M. Joullie, Heterocrcies 1985, 23, 691 729. Trends in Syntheric Curhohrdrute Cherni.?trT (Eds.: D. Horton. L. D.
Hawkins, G. J. McGarvey), American Chemical Society. Washington.
1989.
[2] T. Hudlicky, H. Luna. J. D. Price. F. Rulin, EJtruhedron Lerr. 1989, 30.
4053-4054. B. R. Baker, K. Hewson. J. Org. Chem. 1957, 22. 966. A. S.
Perlin, Methods Carbohydr. Chem. Vbl. I , 1962, p. 67 M . Kiso. A.
Hasegawa. Curbohjdrutr Res. 1976, 52, 95. C. E. Ballow. J A m . Clrmi.
Soc. 1957, 79. 165. T. Mukaiyama, K. Suzuki, T. Yamada. F. Tabusa.
E,tralredron 1990. 46, 265- 276. T. Mukaiyama, K. Suzuki, T. Yamada.
Chmr. Liw. 1982. 929-932.
(31 D. R. Williams. F. D. Klingler. J. Orz. Cheni. 1988. 53. 2134 -2136. G.
Stork, S. Raucher. J. Am. Chem. Soc. 1976.96.1583-1584, P. T. Ha. (bn.
J. Chem. 1980. 88. 858-860.
[4] R. Freire. R. Hernhndez. M. S. Rodriquez, E. Suhrez, A. Perales, E,iruhe&on Lett. 1987,28,981- 984. C. G. Francisco, R. Freire, M. S. Rodriguez.
E. Suarez, ;bid 1987. 28, 3397- 3400. R. Hernindez, J. J. Marrero. E.
Suirez. ibrd. 1988.29. 5979-5982. R. Hernindez, J. J. Marrero, D. Melihn.
E. Suarez, ibid. 1988. 29, 6661-6664. R. Hernandez. J. J. Marrero, E.
Suirez, ibrd. 1989, 30, 5501 - 5504.
[5] R. Freire, J. J. Marrero. M. S. Rodriguez, E. Suirez. Terruhedron Lerl.
1986, 27. 383-387. M T. Arencibia. R. Freire. A. Perales. M. S. Rodriguez. E. Suarez, J Chem. Soc., Perkiri Truns. I , 1991, 3349-3360.
VCH ~~rluys~esi,ll.?chali
mbH, W-6940 Weinherm, 1992
0570-0833/92~0606-0773$3.50+ .25/0
773
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
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reagents, formation, intermediate, iodine, fragmentation, convenient, carbohydrate, radical, alkoxy, synthetic, chiral, anomeric, hypervalent
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