Process for the production of carboxylic acid chlorides of aromatic nitrogen heterocycles

The process for the production of the carboxylic acid chlorides of the aromatic nitrogen heterocycles of the series of the compounds of the general formulas: ##STR1## The hydroxy carboxylic acids of the aromatic nitrogen heterocycles from the series of the compounds of the general formulas: ##STR2## In the general formulas I to VI, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are the same or different and each is a hydrogen atom or a halogen atom. The chlorination is performed with a solution of phosphorous pentachloride in phosphoroxy chloride. The excess phosphorous pentachloride is converted into phosphoroxy chloride either with a C.sub.1 -C.sub.4 -carboxylic acid or with a silane of the general formula: EQU (CH.sub.3).sub.3 --Si--OR.sub.9 VII Then the product according to one of the general formulas I to III is isolated.

BACKGROUND OF THE INVENTION 
1. Field Of The Invention 
The invention relates to a new process for the production of carboxylic 
acid chlorides of aromatic nitrogen heterocycles of the series of 
compounds of general formulas: 
##STR3## 
by chlorination of the corresponding hydroxy carboxylic acids of aromatic 
nitrogen heterocycles from the series of compounds of the general 
formulas: 
##STR4## 
In the above general formulas I to VI, R.sub.1, R.sub.2, R.sub.3, R.sub.4, 
R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are the same or different and each 
is a hydrogen atom or a halogen atom. 
These carboxylic acid chlorides of aromatic nitrogen heterocycles, such as, 
6-chloropicolinic acid chloride, are important intermediate products for 
the production of herbicides [European Published Patent Application No. 
447,004]. 
2. Background Art 
The direct reaction of 6-hydroxypicolinic acid to 6-chloropicolinic acid 
chloride has not yet been described in the literature. Only the 
chlorination of 6-hydroxypicolinic acid to 6-chloropicolinic acid with 
phosphorous pentachloride in phosphoroxy chloride is known [Ber. D. Chem. 
ges., 45, (1912), page 2461]. 6-Chloropicolinic acid chloride can then be 
produced only by another chlorination of 6-chloropicolinic acid with, for 
example, thionyl chloride. A drawback of this process is the fact that 
under these conditions the product can only be produced by a 2-stage 
synthesis and that large amounts of phosphoric acid accumulate in the 
first stage as waste product in the working-up of 6-chloropicolinic acid 
as the intermediate product. 
Also, the production of 6-chloropicolinic acid chloride, starting from 
6-chloro-2-methylpyridine, is known [European Published Patent Application 
No. 032516]. A great drawback of this process is the fact that the 
feedstock 6-chloro-2 -methylpyridine must first be oxidized in a first 
stage with potassium permanganate to 6-chloropicolinic acid, the latter 
compound is then converted with thionyl chloride in the second stage into 
6-chloropicolinic acid chloride, and the product is obtained in a moderate 
yield. 
BROAD DESCRIPTION OF THE INVENTION 
The main object of the invention is to provide a simple and economical 
process for the production of carboxylic acid chlorides of aromatic 
nitrogen heterocycles and where the products can be isolated in very good 
yields. Other objects and advantages of the invention are set out herein 
or are obvious herefrom to one skilled in the art. 
The objects and advantages of the invention are achieved by the process and 
compounds of the invention. 
The invention involves a process for the production of a carboxylic acid 
chloride of the aromatic nitrogen heterocycles of the series of the 
compounds of the general formula: 
##STR5## 
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and 
R.sub.8 are the same or different and each is a hydrogen atom or a halogen 
atom. A corresponding hydroxy carboxylic acid of the aromatic nitrogen 
heterocycles of the series of the compounds of the general formula: 
##STR6## 
wherein R.sub.1 to R.sub.8 have the above-mentioned meanings, are 
chlorinated. The chlorination is performed with a solution of phosphorous 
pentachloride in phosphoroxy chloride. The excess phosphorous 
pentachloride is converted into phosphoroxy chloride either with a C.sub.1 
-C.sub.4 -carboxylic acid or with a silane of the general formula: 
EQU (CH.sub.3).sub.3 --Si--OR.sub.9 VII 
wherein R.sub.9 is a C.sub.1 -C.sub.4 -alkyl group or a tri(C.sub.1 
-C.sub.4)alkylsilyl group. Then the product according to one of the 
general formulas I to III is isolated. 
Preferably the C.sub.1 -C.sub.4 -carboxylic acid is formic acid. Preferably 
methoxytrimethyl silane, in which R.sub.9 is a methyl group, or hexamethyl 
disiloxane, in which R.sub.9 is a trimethyl group, is used as the silane 
of the general formula VII. Preferably the hydroxy carboxylic acid of the 
aromatic nitrogen heterocycles of the general formula IV is 
6-hydroxypicolinic acid, wherein R.sub.1, R.sub.2 and R.sub.3 each is a 
hydrogen atom, or 6-hydroxy-3,5-dichloropicolinic acid, wherein R.sub.2 is 
a hydrogen atom, and R.sub.1 and R.sub.3 is a chloride atom, or the 
hydroxy carboxylic acid of the aromatic nitrogen heterocycles of the 
general formula VI is 5-hydroxypyrazine carboxylic acid, wherein R.sub.7 
and R.sub.8 each is a hydrogen atom. 
Preferably the chlorination is performed at a temperature of 20.degree. to 
110.degree. C. Preferably the addition of the C.sub.1 -C.sub.4 -carboxylic 
acid or the silane of the general formula VII is performed at a 
temperature of 0.degree. to 60.degree. C. Preferably the phosphoroxy 
chloride is removed by distillation. 
The invention also involves 3,5,6-trichloro-picolinic acid chloride, which 
is a compound of the general Formula I wherein R.sub.1 and R.sub.3 each is 
a chlorine atom and R.sub.2 is a hydrogen atom.

DETAILED DESCRIPTION OF THE INVENTION 
The invention process provides for the production of the carboxylic acid 
chlorides of the aromatic nitrogen heterocycles of the series of compounds 
of the general formulas: 
##STR7## 
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and 
R.sub.8 are the same or different and each is a hydrogen atom or a halogen 
atom, respectively from the hydroxy carboxylic acids of the aromatic 
nitrogen heterocycles of the series of the compounds of the general 
formulas: 
##STR8## 
wherein R.sub.1 to R.sub.8 have the mentioned meanings, A compound of one 
of the general formulas IV to VI is chlorinated. The chlorination is 
performed with a solution of phosphorous pentachloride in phosphoroxy 
chloride. The excess phosphorous pentachloride is converted into 
phosphoroxy chloride either with a C.sub.1 -C.sub.4 -carboxylic acid or 
with a silane of the general formula: 
EQU (CH.sub.3).sub.3 --Si--OR.sub.9 VII 
wherein R.sub.9 is a C.sub.1 -C.sub.4 -alkyl group or a tri(C.sub.1 
-C.sub.4)alkylsilyl group. Then the product according to one of the 
general formulas I to III is isolated. 
A special advantage of the invention process is the fact that the 
carboxylic acid chlorides of aromatic nitrogen heterocycles can be 
isolated by the addition of easily available reagents, such as, formic 
acid. 
The feedstocks, the aromatic hydroxy carboxylic acids of the nitrogen 
heterocycles, can be microbiologically produced in a simple way, for 
example, according to Swiss Patent Application No. 3572/91 or U.S. patent 
application Ser. No. 07/984,450, filed on Dec. 2, 1992. Details of said 
microbiological process for preparing the feedstock aromatic hydroxy 
carboxylic acids of the nitrogen heterocycles of the general formulas V 
and VI are set out below. 
As feedstocks, the hydroxy carboxylic acids of the aromatic nitrogen 
heterocycles from the series of the compounds of the general formulas IV 
to VI are used. 
Preferably 6-hydroxypicolinic acid, wherein R.sub.1, R.sub.2 and R.sub.3 
each is a hydrogen atom or 6-hydroxy-3,5-dichloropicolinic acid, wherein 
R.sub.2 is a hydrogen atom and R.sub.1 and R.sub.3 is a chlorine atom, is 
used as the hydroxy carboxylic acid of the aromatic nitrogen heterocycles 
of the general formula IV. As the aromatic hydroxy carboxylic acid of the 
aromatic nitrogen heterocycles of the general formula VI preferably 
5-hydroxypyrazine carboxylic acid, wherein R.sub.7 and R.sub.8 each is a 
hydrogen atom, is used. 
The chlorination of the feedstocks with phosphorous pentachloride in 
phosphoroxy chloride usually takes place with an excess of the phosphorous 
pentachloride relative to the amount of the feedstock used. Suitably the 
phosphorous pentachloride is used in an amount of 1 to 3 mol, preferably 
from 2 to 2.5 mol, per mole of the feedstock (the hydroxy carboxylic acid 
of the aromatic nitrogen heterocycles). Suitably the chlorination is 
performed at a temperature of 20.degree. to 110.degree. C., preferably 
from 60.degree. to 110.degree. C. 
After a usual chlorination time of 2 to 15 hours, suitably until no more 
HCl-gas is formed, according to the invention the excess phosphorous 
pentachloride is then converted either with a C.sub.1 -C.sub.4 -carboxylic 
acid or with a silane of the general formula VII into phosphoroxy 
chloride. Formic acid, acetic acid or propionic acid can be used, for 
example, as the C.sub.1 -C.sub.4 -carboxylic acids. Preferably formic acid 
is used as the C.sub.1 -C.sub.4 -carboxylic acid, with carbon monoxide and 
HCl are formed. As the silanes of the general formula: 
EQU (CH.sub.3).sub.3 --Si--OR.sub.9 VII 
suitably methoxytrimethyl silane, wherein R.sub.9 is a methyl group, or 
hexamethyl disiloxane, wherein R.sub.9 is a trimethylsilyl group, is used. 
Suitably the C.sub.1 -C.sub.4 -carboxylic acid or the silane according to 
the general formula VII is used in an amount of 0.1 to 2 mol, preferably 
of 1 to 1.5 mol, per mole of the feedstock. The addition of the C.sub.1 
-C.sub.4 -carboxylic acid or the silane of the general formula VII 
suitably takes place at a temperature of 0.degree. to 60.degree. C., 
preferably of 20.degree. to 40.degree. C. 
During or after a usual addition time of 15 to 60 minutes, the thus-formed 
phosphoroxy chloride can be removed by distillation. Preferably the 
phosphoroxy chloride is removed by distillation and optionally is recycled 
into the reaction solution. In this way pure phosphoroxy chloride, which 
can be used again for the reaction, is produced in excess. 
3,5,6-Trichloropicolinic acid chloride, which is a compound of the general 
formula I wherein R.sub.1 and R.sub.3 each is a chlorine atom and R.sub.2 
is a hydrogen atom, has not yet been described in the prior art and is 
part of the invention. 
EXAMPLE 1 
6-Chloropicolinic Acid Chloride 
49.9 g (239.5 mmol) of phosphorous pentachloride was added at room 
temperature to a suspension of 6-hydroxypicolinic acid (71.9 mmol; 10 g) 
in phosphoroxy chloride (165.4 mmol; 25.3 g). An exothermic reaction was 
observed, and HCl-gas was formed. After completion of this exothermic 
reaction the mixture was heated within 1.5 hours to 90.degree. C. Then 
this temperature was maintained for 12 hours. The clear solution was 
cooled to 30.degree. C. Then formic acid (93.5 mmol; 4.3 g) was instilled, 
and HCl gas and CO gas was formed. After distilling-off of the phosphoroxy 
chloride, 6-chloropicolinic acid chloride was distilled at 90.degree. C. 
at a pressure of 0.026 mbar. 12 g of 6-chloropicolinic acid chloride (68.2 
mmol) was obtained as a white solid corresponding to a yield of 94.8 
percent. The melting point of the product was 73.1.degree. to 75.8.degree. 
C. Other data concerning the product was: 
.sup.1 H-NMR: (CDCl.sub.3) (360 MHz) .delta. 7.66 (d, 9.6 Hz); 7.90 (t, 9.6 
Hz); 8.07 (d, 9.6 Hz). 
EXAMPLE 2 
6-Chloropicolinic Acid Chloride 
Analogously to Example 1, 100 g of 6-hydroxypicolinic acid was chlorinated 
to 6-chloropicolinic acid chloride, which resulted in a yield of 97.3 
percent. 
EXAMPLE 3 
3,5,6-Trichloropicolinic Acid Chloride 
Corresponding to Example 1, 10 g (48 mmol) of 
3,5-dichloro-6-hydroxypicolinic acid was chlorinated with phosphorous 
pentachloride (158.6 mmol; 33.1 g) in phosphoroxy chloride (171 mmol; 26.3 
g) at a temperature of 110.degree. C. During the reaction 13 g of 
phosphoroxy chloride was already distilled by the addition of formic acid, 
as in Example 1. After the distillation, at 110.degree. C. and 0.065 mbar, 
7.8 g of 3,5,6-trichloropicolinic acid chloride corresponding to a yield 
of 66 percent was obtained. The melting point of the product was 
49.degree. to 52.degree. C. Other data concerning the product was: 
.sup.1 H-NMR: (CDCl.sub.3) (360 MHz) 8.00 ppm; (s). 
EXAMPLE 4 
5-Chloropyrazine Carboxylic Acid Chloride 
Corresponding to Example 1, 5-hydroxypyrazine-2-carboxylic acid (57.1 mmol; 
8 g) was reacted with phosphorous pentachloride (188.4 mmol; 39.3 g) in 
phosphoroxy chloride (131.3 mmol, 20.15 g) at a temperature of 85.degree. 
to 5-chloropyrazine-2-carboxylic acid chloride. After addition of formic 
acid, as in Example 1, and distillation at 85.degree. C., 1.3 mbar, 6.7 g 
of 5-chloropyrazine-2-carboxylic acid chloride was obtained in the form of 
white crystals corresponding to a yield of 63 percent. The melting point 
of the product was &gt;45.degree. C. Other data concerning the product was: 
.sup.1 H-NMR: (CDCl.sub.3) (360 MHz) .delta. 8.78 (s); 9.10 (s). 
EXAMPLE 5 
6-Chloropicolinic Acid Chloride 
(a) Corresponding to Example 1, 6-hydroxypicolinic acid was chlorinated 
with phosphorous pentachloride in phosphoroxy chloride. After completion 
of the reaction, 10.5 g (100 mmol) of methoxytrimethylsilane was added at 
50.degree. C., and the formation of chloromethane was observed. After 
distilling-off of phosphoroxy chloride, 12.05 g of product was obtained 
corresponding to a yield of 95 percent. 
(b) Corresponding to Example 5(a), 6-chloropicolinic acid chloride was 
produced with hexamethyldisiloxane (16.2 g) and 12.05 g of product was 
obtained corresponding to a yield of 95 percent. 
The following deals with the above-mentioned microbiological process for 
the production of hydroxy-heterocyclic carboxylic acids of the general 
formula: 
##STR9## 
wherein R.sub.9 is a hydrogen or a halogen atom and X is a nitrogen atom 
or a CR.sub.10 function, wherein R.sub.10 is a hydrogen or halogen atom. 
The aromatic hydroxy carboxylic acids of the nitrogen heterocycles of the 
general formulas V and VI are within the scope of the general formula VII. 
In the process, in step (a), an aerobic biomass which utilizes nicotinic 
acid or its soluble salts is cultivated with nicotinic acid or its soluble 
salts and a mineral acid in a molar ratio of nicotinic acid or its soluble 
salts to the mineral acid of 1 to 8. This ratio is assured over the entire 
cultivation phase. Then, in step (b), the hydroxylation of the 
corresponding heterocyclic carboxylic acid of the general formula: 
##STR10## 
wherein R.sub.9 and X have the above-mentioned meanings, or its soluble 
salts, is performed with the biomass. Preferably, in step (a), sulfuric 
acid in a molar ratio of nicotinic acid or its soluble salts to sulfuric 
acid of 3 to 5 is used as the mineral acid. Preferably, in the cultivation 
phase in step (a), microorganisms of species Pseudomonas acidovorans DSM 
7205, and/or species Pseudomonas acidovorans DSM 7203, and/or species 
Alcaligenes faecalis DSM 7204 and/or microorganisms with the designation 
DSM 7202 are concentrated and then, in step (b), the hydroxylation takes 
place with these microorganisms. Preferably, in step (b) as the 
heterocyclic carboxylic acid, nicotinic acid or its soluble salts is 
hydroxylated to 6-hydroxynicotinic acid. Preferably, in step (b) as the 
heterocyclic carboxylic acid, pyrazine carboxylic acid or its soluble 
salts is hydroxylated to 5-hydroxypyrazine carboxylic acid. Preferably the 
cultivation in step (a) and the hydroxylation in step (b) are performed at 
a temperature of 15.degree. to 50.degree. C. and a pH of 5 to 9. 
By the phrase "cultivating aerobic biomass which utilizes nicotinic acid or 
its salts", the following is meant: if biomass is cultivated, for example, 
from sewage sludge as an inoculum with the described molar nicotinic 
acid-mineral salt ratio under aerobic conditions, an aerobic biomass which 
utilizes nicotinic acid is obtained, i.e., a biomass that grows with 
nicotinic acid as sole (only) carbon, nitrogen and energy source in the 
presence of oxygen. As inoculum soil, samples from various countries can 
also be used, such as, soil from the city park in Stanander (Spain) or 
soil from the vineyard in Visperterminen near Visp (Switzerland). 
The process is performed not with homogeneous (biologically pure) cultures 
of microorganisms, but it is performed with a biomass consisting of mixed 
cultures. 
The molar ratio of nicotinic acid to the mineral acid, i.e., the addition 
of the mixture consisting of nicotinic acid and mineral acid to the cell 
suspension, takes place so that during the entire cultivation phase a 
molar ratio of nicotinic acid to mineral acid of 1 to 8 is assured. As 
mineral acids, for example, sulfuric acid, hydrochloric acid, nitric acid 
or phosphoric acid can be used, preferably sulfuric acid is used. Suitably 
the addition of the mixture takes place during the cultivation of the 
biomass [step (a)] so that a molar ratio of nicotinic acid to sulfuric 
acid of 3 to 5 is assured. That is, suitably 3 to 5 mol of nicotinic acid 
per mol of sulfuric acid is used for the cultivation. Preferably 4 to 5 
mol of nicotinic acid per mol of sulfuric acid is used for the 
cultivation. 
Usually the cultivation of the aerobic biomass which utilizes nicotinic 
acid, takes place in a mineral salt medium, preferably in the mineral salt 
medium whose composition is described in Table 1 below. The cultivation of 
the biomass takes place suitably at a pH of 5 to 9, preferably at pH 6 to 
8. Suitably the temperature during the cultivation of the biomass is 
between 15.degree. and 50.degree. C., preferably between 25.degree. and 
40.degree. C. Usually the cultivation of the biomass takes place over a 
period of 0.5 to 3 days. 
Suitably, under these conditions microorganisms of species Pseudomonas 
acidovorans DSM 7205, or species Pseudomonas acidovorans DSM 7203, or 
species Alcaligenes faecalis DSM 7204 or microorganisms with the 
designation DSM 7202, or their mixtures, are concentrated in the 
cultivation phase. 
The microorganisms DSM 7205, DSM 7203, DSM 7204 and DSM 7202 were deposited 
on Aug. 13, 1992 in the Deutsche Sammlung von Mikroorganismen und 
Zellkulturen GmbH [German Collection for Microorganisms and Cell Cultures 
GmbH], Mascheroderweg 1b, D-3300 Braunschweig, Germany, according to the 
Budapest Treaty. These microorganisms are not yet known from the 
literature and accordingly are also a component of the invention. It was 
not yet possible to identify the microorganism with the designation DSM 
7202 taxonomically nor to assign it to a genus. 
The taxonomy of microorganisms Pseudomonas acidovorans DSM 7205, 
Pseudomonas acidovorans DSM 7203, and Alcaligenes faecalis DSM 7204 is 
described below. 
Taxonomic description of Pseudomonas acidovorans DSM 7205 is: 
______________________________________ 
Properties of the strain: 
______________________________________ 
cell shape rods 
width, micron 0.8-0.9 
length, micron 1.5-9.0 
mobility + 
flagella polar 1 
gram reaction - 
lysis by 3% KOH + 
aminopeptidase (Cerny) 
+ 
spores - 
oxidase + 
catalase + 
growth 
anaerobic - 
37.degree./41.degree. C. 
+/- 
pH 5.7 - 
MacConkey's broth + 
SS agar + 
Cetrimide agar + 
testosterone - 
pigments 
nondiffusing - 
diffusing - 
fluorescent - 
pyocyanine - 
acid from (OF test) 
aerobic glucose ? 
anaerobic glucose - 
alkaline glucose + 
gas from glucose - 
acid from 
glucose - 
fructose + 
xylose - 
adonite - 
L-arabinose - 
cellobiose - 
dulcitol - 
glycerol + 
m-inositol - 
lactose - 
maltose - 
raffinose - 
L-rhamnose - 
salicin - 
D-sorbitol - 
saccharose - 
trehalose - 
ethanol + 
dulcitol - 
ONPG/PNPG - 
ADH - 
VP - 
indole - 
NO.sub.2 from NO.sub.3 
+ 
phenylalanine desaminase 
w 
levan from saccharose 
- 
lecithinase - 
urease - 
hydrolysis of 
starch - 
gelatin - 
casein w 
DNA - 
Tween 80 + 
aesculin - 
PHB - 
tyrosine catabolism + 
use of substrate 
acetate + 
adipate + 
caprate + 
citrate + 
glycolate + 
laevulinate + 
malate + 
malonate + 
phenyl acetate + 
L-arabinose - 
fructose + 
glucose - 
mannose - 
maltose - 
D-xylose - 
mannitol + 
gluconate + 
2-ketogluconate + 
N-acetylglucosamine - 
L-serine - 
quinate + 
D,L-tryptophan + 
L-tartrate + 
acetamide + 
.alpha.-aminobutyrate 
+ 
ethanol w 
______________________________________ 
Taxonomic description of Pesudomonas acidovorans DSM 7203 is: 
______________________________________ 
Properties of the strain: 
______________________________________ 
cell shape rods 
width, micron 0.8-1.0 
length, micron 2.6-6.0 
mobility + 
flagella polar 1 
gram reaction - 
lysis by 3% KOH + 
aminopeptidase (Cerny) 
+ 
spores - 
oxidase + 
catalase + 
growth 
anaerobic - 
37.degree./41.degree. C. 
+/- 
pH 5.7 - 
MacConkey's broth + 
SS agar + 
Cetrimide agar + 
testosterone - 
pigments 
nondiffusing - 
diffusing - 
fluorescent - 
pyocyanine - 
acid from (OF test) 
aerobic glucose ? 
anaerobic glucose - 
alkaline glucose + 
gas from glucose - 
acid from 
glucose - 
fructose + 
xylose - 
adonite - 
L-arabinose - 
cellobiose - 
dulcitol - 
glycerol + 
m-inositol + 
lactose - 
maltose - 
raffinose - 
L-rhamnose - 
salicin - 
D-sorbitol - 
saccharose - 
trehalose - 
ethanol -? 
dulcitol - 
ONPG/PNPG - 
ADH - 
VP - 
indole - 
NO.sub.2 from NO.sub.3 
+ 
phenylalanine desaminase 
w 
levan from saccharose 
- 
lecithinase - 
urease - 
hydrolysis of 
starch - 
gelatin - 
casein + 
DNA - 
Tween 80 + 
aesculin - 
PHB - 
tyrosine catabolism + 
use of substrate 
acetate + 
adipate + 
caprate + 
citrate + 
glycolate + 
laevulinate + 
malate + 
malonate + 
phenyl acetate + 
L-arabinose - 
fructose + 
glucose - 
mannose - 
maltose - 
D-xylose - 
mannitol + 
gluconate + 
2-ketogluconate + 
N-acetylglucosamine - 
L-serine - 
quinate + 
D,L-tryptophan + 
L-tartrate + 
acetamide + 
.alpha.-aminobutyrate 
w 
ethanol + 
______________________________________ 
Taxonomic description Alcaligenes faecalis DSM 7204 is: 
______________________________________ 
Properties of the strain: 
______________________________________ 
cell shape rods 
width, micron 0.6-0.8 
length, micron 1.0-2.0 
mobility + 
flagella peritrichous 
gram reaction - 
lysis by 3% KOH + 
aminopeptidase (Cerny) 
+ 
oxidase + 
catalase + 
growth 
anaerobic - 
37.degree./41.degree. C. 
+/+ 
pH 5.7 - 
MacConkey's broth + 
SS agar + 
Cetrimide agar + 
pigments 
nondiffusing - 
diffusing - 
fluorescent - 
pyocyanine - 
acid from (OF test) 
aerobic glucose ? 
anaerobic glucose - 
alkaline glucose + 
gas from glucose - 
acid from 
D-glucose - 
D-fructose + 
D-xylose - 
ONPG/PNPG - 
ADH - 
VP - 
indole - 
NO.sub.2 from NO.sub.3 
+ 
dentrification - 
phenylalanine desaminase 
- 
levan from saccharose 
- 
lecithinase - 
urease - 
hydrolysis of 
starch - 
gelatin - 
casein - 
DNA - 
Tween 80 - 
aesculin - 
tyrosine catabolism 
+ 
use of substrate 
acetate + 
adipate - 
caprate + 
citrate + 
glycolate + 
laevulinate - 
D-malate + 
malonate + 
phenyl acetate + 
L-arabinose - 
D-fructose - 
D-glucose - 
D-mannose - 
maltose - 
D-xylose - 
mannitol - 
gluconate - 
2-ketogluconate + 
N-acetylglucosamine 
- 
L-serine - 
______________________________________ 
Herein, the phrase "heterocyclic carboxylic acid (substrate) to be 
hydroxylated" is meant to also include its salts, such as, its water 
soluble alkaline salts. 
After the cultivation, the biomass can then be separated for the actual 
biotransformation (hydroxylation) either in a way usual to one skilled in 
the art or the heterocyclic carboxylic acid (general formula VIII) to be 
hydroxylated is directly added to the cultivated biomass. 
The actual hydroxylation of the heterocyclic carboxylic acid (substrate) 
takes place in a way known to one skilled in the art with nongrowing 
cells. Preferably the actual hydroxylation of the heterocyclic carboxylic 
acid takes place with the microorganisms concentrated in the cultivation 
phase of the species Pseudomonas acidovorans DSM 7205, species Pseudomonas 
acidovorans DSM 7203, species Alcaligenes faecalis DSM 7204 or 
microorganisms with the designation DSM 7202, or with mixtures of these. 
As the substrate, for example, nicotinic acid, pyrazine carboxylic acid or 
their halogenated derivatives can be used. As the halogenated derivatives 
of nicotinic acid or pyrazine carboxylic acid, for example, 
5-chloronicotinic acid, 4-chloronicotinic acid or 6-chloropyrazine 
carboxylic acid can be used. Preferably nicotinic acid is hydroxylated to 
6-hydroxynicotinic acid or pyrazine carboxylic acid is hydroxylated to 
5-hydroxypyrazine carboxylic acid. The substrate for the biotransformation 
can be added continuously or batchwise. Suitably the substrate addition 
takes place so that the substrate amount in the fermenter does not exceed 
20 percent by weight, preferably 15 percent by weight. 
As medium for the hydroxylation those usual to one skilled in art can be 
used, preferably either the mineral salt medium described in Table 1 or 
the A-N medium described in Table 4. Usually the biotransformation is 
performed with cells that have an optical density at 550 nm (OD.sub.550) 
or at 650 nm (OD.sub.650) of 5 to 100. Suitably the biotransformation is 
performed at a pH of 5 to 9, preferably of 6.5 to 7.5 and at a suitable 
temperature of 15.degree. to 50.degree. C., preferably of 25.degree. to 
35.degree. C. 
After a usual reaction time of 5 to 24 hours the hydroxylated heterocyclic 
carboxylic acid, according to general formula VII, can be isolated by 
methods usual to one skilled in the art, e.g., by acidification of the 
cell-free fermentation solution or by precipitation in the form of poorly 
soluble salts. Preferably as the hydroxylated heterocyclic carboxylic 
acid, 6-hydroxynicotinic acid or 5-hydroxypyrazine carboxylic acid is 
isolated. 
EXAMPLE A 
(a) Cultivation of the Biomass 
The fermentation was performed in an unsterile mineral salt medium (Table 
1) with 1 g of nicotinic acid per liter, in a fermenter with a working 
volume of 15 l at pH 7.0, at a temperature of 30.degree. C. and an 
aeration rate between 5 to 20 l/min. For pH regulation, only acid in the 
form of an aqueous suspension consisting of 307 g of nicotinic acid (2.5 
mol) and 49 g (0.5 mol) of H.sub.2 SO.sub.4 and 1 l water from a vessel 
with a stirrer, which was fastened to the cover of the fermenter by a 
pneumatically controlled ball valve, was added to the medium. The 
fermenter was inoculated with 500 ml of sewage sludge from the waste water 
purification plant, Visp, Switzerland (Table 2). After 36 hours the 
fermenter was emptied except for one liter and filled with fresh medium. 
This procedure was repeated after another 24 hours, 48 hours and 72 hours. 
(b) Hydroxylation (production of 6-Hydroxynicotinic Acid) 
When the optical density at 550 nm had reached a value between 5 to 20, the 
biomass was used to spectrophotometrically measure the specific 
6-hydroxynicotinic acid formation rate. For this purpose, first the 
biomass was washed once with 0.9 percent (w/v) of NaCl solution. Then 10 
.mu.l of this cell suspension was added to a quartz cuvette (1 cm light 
path) preheated to 30.degree. C., that contained 2990 .mu.l of a solution 
consisting of 6.5 g of nicotinic acid/l, 10.1 g of K.sub.2 HPO.sub.4 /l 
and 4.0 g of KH.sub.2 PO.sub.4 /l, pH 7.0. The absorption of the cuvette 
at 550 nm was measured and then, from the same vessel, the linear increase 
of the absorption at 295 nm per minute was calculated. The specific 
activity (U) was determined according to the formula below: 
##EQU1## 
The fermentations were repeated with a sludge sample from the Zermatt 
(Switzerland) sewage treatment plant, soil samples from Visperterminen, 
Switzerland, and soil samples from Lac de Joux, Switzerland and a soil 
sample from Santander, Spain (Table 2). 
EXAMPLE B 
Production of 5-Hydroxypyrazine Carboxylic Acid 
The biomass from fermentations 4, 5 and 6 (Table 2) were centrifuged off 
and washed once in 0.9 percent (w/v) NaCl solution. Then the cells were 
resuspended in a liter of solution containing 0.5 mol (70 g) of pyrazine 
carboxylic acid ammonia salt, pH 7.0. The optical density at 650 nm was 
then 20. After an incubation time of 16 hours under aerobic conditions at 
pH 7.0 and a temperature of 30.degree. C., a quantitative conversion from 
pyrazine carboxylic acid to 5-hydroxypyrazine carboxylic acid could be 
determined by UV spectroscopy. The formed 5-hydroxypyrazine carboxylic 
acid was not catabolized from the microorganisms. 
As a control test Pseudomonas acidovorans D3 (DSM 4746), which is 
especially suitable for the industrial production of 5-hydroxypyrazine 
carboxylic acid cultured as was described above, was used for the reaction 
according to the process described in European Patent Application 
92110425.3. The results are summarized in Table 2. 
TABLE 1 
______________________________________ 
Composition of the mineral salt medium 
MgCl.sub.2.6H.sub.2 O 0.8 g/l 
CaCl.sub.2 0.16 g/l 
Na.sub.2 SO.sub.4 0.25 g/l 
K.sub.3 PO.sub.4.2H.sub.2 O 
0.7 g/l 
Na.sub.3 PO.sub.4.12H.sub.2 O 
2.4 g/l 
SLF 1.0 ml/l 
FeEDTA 15.0 ml/l 
Composition of the trace elements (SLF) 
in the mineral salt medium: 
KOH 15.0 g/l 
EDTANa.sub.2.2H.sub.2 O 100.0 g/l 
ZnSO.sub.4.7H.sub.2 O 9.0 g/l 
MnCl.sub.2.4H.sub.2 O 4.0 g/l 
H.sub.3 BO.sub.3 2.7 g/l 
CoCl.sub.2.6H.sub.2 O 1.8 g/l 
CuCl.sub.2.2H.sub.2 O 1.5 g/l 
NiCl.sub.2.6H.sub.2 O 0.18 g/l 
Na.sub.2 MoO.sub.4.2H.sub.2 O 
0.2 g/l 
Composition of FeEDTA: 
EDTA Na.sub.2.2H.sub.2 O 5.0 g/l 
FeSO.sub.4.7H.sub.2 O 2.0 g/l 
(The pH of the solution was adjusted to 7.0) 
______________________________________ 
TABLE 2 
__________________________________________________________________________ 
OD.sub.550 before 
activity Special activity 
Inoculum measurement 
(A.sub.295 .multidot. OD.sub.550.sup.-1) 
__________________________________________________________________________ 
(1) ARA 5.3 35.5 
LONZA 
(2) Lac de 14 20 
Joux 
(3) ARA 9.6 26 
Zermatt 
(4) Vineyard 
12 44 
V'terminen 
(5) soil 10 32 
Spain 
(6) 
control 
3.5 35.1 
double determination 
Pseudomonas 
8 39 
acidovorans 
__________________________________________________________________________ 
Note: 
Places where the microorganisms were found: 
(1) Sewage sludge from the sewage treatment plant of the LONZA company in 
Visp, Switzerland. 
(2) Soil from the banks of Lac de Joux, Le Sentier, Switzerland. 
(3) Sewage sludge from the sewage treatment plant in Zermatt, Switzerland 
(4) Soil from the vineyard in Visperterminen in Visp, Switzerland. 
(5) Soil from the city park in Santander, Spain 
EXAMPLES C TO F 
From the cultivated biomass according to Example A (a), the following 
microorganisms were able to be concentrated: 
Pseudomonas acidovorans DSM 7205 
Pseudomonas acidovorans DSM 7203 
Alcaligenes faecalis DSM 7204 
microorganisms with the designation DSM 7202. 
These microorganisms were cultivated under the following conditions and 
used for the hydroxylation of nicotinic acid from 6-hydroxynicotinic acid. 
The results are summarized in Table 3. 
In this connection the microorganisms were cultivated in a 7 l fermenter 
containing 5 l of A-N medium (Table 4) with 2 g of sodium nicotinate per l 
at a temperature of 30.degree. C. and a pH of 7.0. For pH regulation, 5N 
NaOH and 8.5 percent (v/v) H.sub.3 PO.sub.4 were used. After 18 hours of 
growth, an additional 2 g of sodium nicotinate per l was added to the 
fermentation solution. As soon as the cells were in the exponential growth 
phase, the fermentation was interrupted and the microorganisms separated 
from the medium by centrifugation. Then the cells were resuspended in 500 
ml of a solution containing 0.27 mol (40 g) of sodium nicotinate, pH 7.0. 
The optical density was then 20. The hydroxylation of nicotinic acid to 
6-hydroxynicotinic acid was tracked spectrophotometrically (Table 3). 
TABLE 3 
______________________________________ 
Isolated amount 
Time necessary for 
of 6-hydro- 
the hydroxylation 
xynicotinic acid 
Yield in 
of 0.27 mol after acidification 
% relative 
nicotinic acid 
of the cell- to nicoti- 
Examples 
in 500 ml free solution 
nic acid 
______________________________________ 
Example 3: 
22 hours 15.7 g (0.11 mol) 
41 
DSM 7202 
Example 4: 
9 hours 30.1 g (0.22 mol) 
80 
DSM 7203 
Example 5: 
10 hours 28.1 g (0.2 mol) 
73 
DSM 7204 
Example 6: 
5 hours 32.0 g (0.23 mol) 
85 
DSM 7205 
______________________________________ 
TABLE 4 
______________________________________ 
A + N medium 
Composition Concentration (mg/l) 
______________________________________ 
Na.sub.2 HPO.sub.4 2000 
KH.sub.2 PO.sub.4 1000 
NaCl 3000 
MgCl.sub.2.6H.sub.2 O 400 
CaCl.sub.2.2H.sub.2 O 14.5 
FeCl.sub.3.6H.sub.2 O 0.8 
pyridoxal hydrochloride 
10 .multidot. 10.sup.-3 
riboflavin 5 .multidot. 10.sup.-3 
nicotinic acid amide 5 .multidot. 10.sup.-3 
thiamine hydrochloride 
2 .multidot. 10.sup.-3 
biotin 2 .multidot. 10.sup.-3 
pantothenic acid 5 .multidot. 10.sup.-3 
p-aminobenzoate 5 .multidot. 10.sup.-3 
folic acid 2 .multidot. 10.sup.-3 
vitamin B12 5 .multidot. 10.sup.-3 
ZnSO.sub.4.7H.sub.2 O 100 .multidot. 10.sup.-3 
MnCl.sub.2.4H.sub.2 O 90 .multidot. 10.sup.-3 
H.sub.3 BO.sub.3 300 .multidot. 10.sup.-3 
CoCl.sub.2.6H.sub.2 O 200 .multidot. 10.sup.-3 
CuCl.sub.2.2H.sub.2 O 10 .multidot. 10.sup.-3 
NiCl.sub.2.6H.sub.2 O 20 .multidot. 10.sup. -3 
Na.sub.2 MoO.sub.4.2H.sub.2 O 
30 .multidot. 10.sup.-3 
EDTANa.sub.2.2H.sub.2 O 
5 .multidot. 10.sup.-3 
FeSO.sub.4.7H.sub.2 O 2 .multidot. 10.sup.-3 
(pH of the solution was adjusted to 7.0) 
______________________________________