An in-depth study of the biotransformation of nitriles into amides and/or acids using Rhodococcus rhodochrous AJ270

Article abstract of DOI:10.1039/a607977f

A variety of aliphatic, aromatic and heterocyclic nitriles have been readily hydrolysed into the corresponding amides and/or acids under very mild conditions using Rhodococcus sp. AJ270. The nitrile hydratase involved in this novel nitrile-hydrolysing microorganism efficiently hydrates most nitriles tested, irrespective of the electronic and steric effects of the substituents, to form the amides. Conversion of amides into acids catalysed by the associated amidase is rapid and efficient in most cases. Substrates bearing an adjacent substituent (which may be an ortho substituent on an aromatic nitrile, an adjacent heteroatom in a heterocyclic ring or a geminal substituent in an α,β-unsaturated nitrile) undergo slow hydrolysis of the amides allowing efficient amide isolation. The scope, limitations and reaction mechanism of this enzymatic process have been systematically studied. A molecular size of >7 A diameter and the presence of functions capable of metal complexation near to the nitrile inhibit hydrolysis.

Full text of DOI:10.1039/a607977f

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An in-depth study of the biotransformation of nitriles into amides
and/or acids using Rhodococcus rhodochrous AJ 2701
Otto Meth-Cohn* and Mei-Xiang Wang
Department of Chemistry, University of Sunderland, Sunderland SR1 3SD, UK
A variety of aliphatic, aromatic and heterocyclic nitriles have been readily hydrolysed into the
corresponding amides and/or acids under very mild conditions using Rhodococcus sp. AJ270. The nitrile
hydratase involved in this novel nitrile-hydrolysing microorganism efficiently hydrates most nitriles tested,
irrespective of the electronic and steric effects of the substituents, to form the amides. Conversion of
amides into acids catalysed by the associated amidase is rapid and efficient in most cases. Substrates
bearing an adjacent substituent (which may be an ortho substituent on an aromatic nitrile, an adjacent
heteroatom in a heterocyclic ring or a geminal substituent in an á,â-unsaturated nitrile) undergo slow
hydrolysis of the amides allowing efficient amide isolation. The scope, limitations and reaction mechanism
of this enzymatic process have been systematically studied. A molecular size of >7 Å diameter and the
presence of functions capable of metal complexation near to the nitrile inhibit hydrolysis.
atic hydrolyses of nitriles using nitrilase and nitrile hydratase
Introduction
have also been proposed 4b,13 and for those transition metal-
containing nitrile hydratases it is believed that the hydration
stage involves the complexion of the nitrile nitrogen to a transi-
tion metal (iron or cobalt) followed by hydration mediated by a
possible prosthetic group such as a pyrroloquinoline quinone.14
It should be noted here that for a long time it was mistakenly
assumed that aliphatic nitrile hydrolyses were mediated by the
hydratase enzyme, while aromatic and heterocyclic systems util-
ised a nitrilase enzyme.15
The organisms and their enzymes
There has been increasing interest in enzyme-catalysed organic
reactions due to their high chemo-, regio- and stereo-selectivity
and very mild conditions.2 Applications of hydrolytic enzymes
such as lipases and esterases have been well documented while,
surprisingly, the use of nitrile-hydrolysing enzymes in synthesis
has received scant attention until recently despite the fact that
the biotransformation of nitriles into the corresponding carb-
oxylic acids has been known for decades.2,3 Enzyme-catalysed
hydrolysis of nitriles has been shown to proceed by two distinct
routes; a nitrilase4 transforms the nitriles directly into acids, or
a nitrile hydratase forms the amide which is hydrolysed to the
acid by an amidase.5 (Scheme 1). So far a range of microorgan-
The transformations observed
Despite the fact that commercial production of acrylamide
through microbial hydrolysis of acrylonitrile has been operat-
ing successfully for some years, preparative biotransformations
of nitriles into amides and/or acids have remained largely
unexplored.15 No systematic study of substrate specificity or
stereoselectivity of the enzymes involved has appeared. The lit-
erature data is rather confused in terms of predicting effective
hydrolysis and the associated selectivity. This is partly due to
the variety of systems used and their mode of generation.
Nagasawa, Yamada and their co-workers16 have reported that a
few aromatic and heterocyclic nitriles give amides as the sole
product when reacted with R. rhodochrous J1 organism (a sys-
tem lacking significant amidase action) grown in a medium con-
taining cobalt ions and crotonamide or urea. Several groups17
showed that an immobilised whole cell Rhodococcus sp. (a
nitrile hydratase system) developed by Novo Industri of Den-
mark slowly hydrolysed para- and meta-substituted benzoni-
triles though with poor conversions. Using the same Novo
preparation, several heterocyclic nitriles were slowly converted
into amides and/or acids.18 When aliphatic nitriles were sub-
ject to nitrile hydratase systems, apart from the formation of
acrylamide from acrylonitrile, acids were produced in most
cases.17b,19 A Hammett-type linear free energy correlation was
observed between initial rates and the nature of para-
substituents in the Novo enzyme-catalysed hydrolysis of para-
substituted benzyl cyanides.20
O
R
C
NH2
nitrile
hydratase
1b–63b
H2O
NH3
H2O
amidase
OH
R
C
N
nitrilase
1a–63a
H2O
O
C
NH3
R
1c–63c
Scheme 1
isms, including Arthrobacter, Brevibacterium, Corynebacterium,
Nocardia, Pseudomonas and Rhodococcus, has been shown to
contain a nitrilase or nitrile hydratase–amidase, or both.3 For
examples, Nocardia rhodochrous NCIB 11216 is a long-used
nitrilase system,4,6 while Brevibacterium B312 is a well-studied
nitrile hydratase-containing organism.3b,3c,7 Rhodococcus sp. N-
7748 and Pseudomonas chlororaphis B239 were selected as the
first- and second-generation strains in the 1980s for the indus-
trial production of acrylamide from acrylonitrile. It has been
demonstrated more recently that both nitrilase and nitrile
hydratase–amidase pathways can occur in the same strains,
such as Nocardia rhodochrous LL100-2110 and Rhodococcus
rhodochrous J1.11 More importantly the three different enzymes
have been induced selectively by the growth of the organism
under optimum culture conditions. Rhodococcus rhodochrous J1
is currently being used in Japan to produce tens of thousands of
tonnes of acrylamide per annum.12 The mechanisms of enzym-
Synthetic applications of Rhodococcus sp. AJ 270
At Sunderland over a number of years a large number of soil-
derived nitrile hydrolysing organisms have been screened 21 and
we have currently focused on the most robust and versatile,
Rhodococcus sp. AJ270, a nitrile hydratase system. We herein
J. Chem. Soc., Perkin Trans. 1, 1997
1099

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Table 1 Hydrolysis of mono-substituted benzonitriles RC6H4CNa
conversions of nitriles to acids without isolation of amides. In
sharp contrast, ortho-substituted benzonitriles were rapidly and
efficiently transformed into amides while conversion of amides
to acids proceeded slowly. Amides or acids are thus both separ-
ately available from the same starting nitrile simply by control-
ling the reaction time (Table 1). This ortho-substituent effect,
which operates irrespective of the nature of the substituent,
suggests that while the first step, hydration of the linear nitrile
group, is not significantly hindered by steric and electronic fac-
tors, the second amidase-mediated step is very sensitive to steric
factors. In other words, in reactions of benzonitriles without an
ortho-substituent the amidase reaction is significantly faster
than the hydratase step, while for with ortho-substituted com-
pounds the reverse is true. To examine the generality of this
steric effect a number of other aromatic nitriles were studied.
The results (Table 2) further illustrate convincingly the general-
ity of the steric effect.
More sterically-hindered compounds, such as 2,6-difluoro-
and 2,6-dichloro-benzonitriles, gave amides as the major or the
sole product, while cyanonaphthalenes yielded acids. The lower
conversion (or slower rate of conversion) of 4-phenoxybenzo-
nitrile 4a, 2,6-dichlorobenzonitrile 24a and 1-naphthonitrile
28a, and the total lack of reaction of 2-phenoxybenzonitrile
18a, 2,6-dimethylbenzonitrile 25a, 2,6-dimethoxybenzonitrile
26a, 9-cyanoanthracene 30a and 9-cyanophenanthrene 31a also
reflect the size limitation of the nitrile hydratase in Rhodococcus
AJ270. It would seem that the metal of the nitrile hydratase
enzyme complexes the nitrile nitrogen and performs the hydra-
tion of this complex in a pocket of limited size, or that there is a
‘bottle-neck’ en route to the active site. This restriction is of a
diameter no greater than ca. 7 Å. Therefore 2,6-disubstituted
benzonitriles (9-cyanoanthracene being viewed as a special
example of such a molecule) are excluded, with the exception of
2,6-difluoro- and 2,6-dichloro-benzonitrile. This size restriction
explains why, for example, other bulky nitriles reported else-
where are unaffected by nitrile hydratases, even when the nitrile
function is quite remote from the steric problem. For example,
the interesting 4-hydroxy-2,6-di-tert-butylphenyl-substituted
nitriles 32 are unaffected by nitrile hydratases even when a long
Product yield (%)
Substrate
a
Reaction
time/h
R
Amide b
Acid c
Nitrile a
1
2
3
4
5
6
7
8
9
H
24
24
28
72
48
24
44
24
22
20
9
—
—
—
—
—
—
—
—
—
—
63.7
30.1
—
—
—
—
92.4
—
97.5
82.2
86.6
59.3
89.4
93.3
71.5
99.3
96.5
28.5
9.7
37.5
91.7
91.3
56.0
78.5
6.6
—
—
—
40.5
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
100
85.0
—
—
—
—
—
—
—
—
—
p-Me
p-MeO
p-PhO
p-Cl
p-F
p-NO2
p-Ac
p-MeO2C
p-HO
p-H2N
10
11
48
28
96
7
12
13
14
15
16
m-NO2
m-MeO
m-HO
m-H2N
o-Me
7
5.5
120
4
18
96
24
72
6.5
168
4
18
90
3
28
1
98.9
4.9
27.7
94.8
—
17
o-MeO
93.1
57.5
—
18
19
20
o-PhO
o-Cl
—
—
—
94.1
—
89.5
60.2
—
80.1
—
97.7
—
4.4
79.9
10.0
29.5
85.7
5.0
69.0
—
62.3
o-NO2
21
22
o-HO
o-H2N
24
a
3 mmol of the nitriles were used and reaction conditions were not
optimised.
report the first stage of a systematic study of the applications of
this novel organism in organic synthesis.1 In order to examine
the scope and limitations of these biotransformations, the sub-
strate specificity of Rhodococcus AJ270 was tested against both
saturated and unsaturated aliphatic nitriles, aromatic nitriles
with different substituents and substitution patterns and hetero-
cyclic nitriles. We present for the first time a rationalisation of
the enzyme-catalysed hydrolysis of nitriles.
X = CH2
or (CH2)2
HO
X
CN
or (CH2)2CONH(CH2)2
It is generally more convenient and economical in preparative
biotransformation to use whole cell systems rather than the
purified or semi-purified enzymes. The biomass of Rhodococcus
AJ270 was prepared in bulk in a 20 l fermentor using acetamide
as both the carbon and nitrogen source. The cells, harvested by
continuous centrifuge at 4 ЊC, were kept at Ϫ20 ЊC without los-
ing activity for several months. The biotransformations were
simply effected by resuspending the cells in potassium phos-
phate buffer (pH 7.0) and incubating with the nitriles at 30 ЊC.
The reactions were monitored by thin layer chromatography
(TLC) or high performance liquid chromatography (HPLC)
and the products obtained either through continuous extraction
of the whole reaction mass or, in sensitive cases, by extraction
with organic solvent after removal of the cells by filtration with
the aid of Celite.
32
OH
O
O
N
MeO
NH
NMe2 OH
O
HO
O
O
N
C
P
HO
O
OH
OH
OH
OMe
Hydrolysis of aromatic nitriles
When reacted with Rhodococcus AJ270, para- or meta-
substituted benzonitriles were hydrolysed efficiently to give the
corresponding acids irrespective of the nature of the substitu-
ents. Only in one case, 4-aminobenzonitrile, were both amide
and acid isolated, indicating that the amide-to-acid transform-
ation was slower than the nitrile-to-amide step (Table 1). Traces
of the amide are occasionally visible by TLC after a few hours
action. However, shortening the reaction time only led to low
33 (–)-Calyculin A
chain interposes between the aryl unit and the nitrile.22 Also we
find that remote nitrile functions attached to large molecules
suffer similar non-reactivity as exemplified by calyculin A 33,
though the AJ270 remains viable after several days attempted
hydrolysis.23
1100
J. Chem. Soc., Perkin Trans. 1, 1997

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Hydrolysis of heterocyclic nitriles
matic nitriles was also encountered in the cases of heterocyclic
Rhodococcus AJ270 also shows high nitrile hydratase activity
against heterocyclic nitriles (Table 3). However, six-membered
nitriles. The adjacent ring heteroatoms, including oxygen, sulfur
and nitrogen, behave like an ortho-substituent allowing selective
heterocyclic amide or acid formation. Isolation of isonicotin-
amide 40b, as with that of 4-aminobenzamide 11b, shows that
the presence of an amino group or a nitrogen in the heterocyclic
ring at the para-position also lowers the rate of amide
hydrolysis. This effect, which totally differs from that of
adjacent substituent, may result from competitive interaction
of the nucleophilic nitrogen atom with the active site of the
amidase.
heterocycles bearing a C᎐O or C᎐N group adjacent to the
᎐
᎐
nitrile are poor substrates.18 Thus, after six days’ interaction
with the biomass cyanopyridones 43a and 44a and pyridazi-
none 45a were recovered intact, while the hydrolyses of 2-
cyanopyridine 38a and 1-cyanoisoquinoline 42a proceeded
sluggishly with low conversion. This is in contrast with the
hydrolysis of 2-amino- and 2-hydroxy-benzonitriles. It would
appear that coordination of the adjacent group with the metal
could interfere with nitrile hydrolysis. To eliminate inefficient
diffusion of the substrates through the cell wall, the reactions
were repeated using a crude extract of the enzymes instead of
whole cells; no hydrolysis was observed. Interestingly, the
adjacent substituent effect discovered in the hydrolysis of aro-
Hydrolysis of aliphatic nitriles
Almost all of the aliphatic nitriles tested, whether saturated or
unsaturated, were efficiently hydrolysed to the corresponding
acids (Table 4). It is worth noting that acrylonitrile and cin-
namonitrile were transformed into the acids without significant
formation of the amide while methacrylamide was isolated
after short action with Rhodococcus AJ270. This shows again
that an adjacent substituent slows down the second hydrolytic
step. However, when the pivalo- 51a and 1-adamantylcarbo-
nitriles 52a having a very bulky group attached to the CN were
hydrolysed, no amide was found and only acid was isolated with
high conversion. The rate of the amide hydrolysis by amidase
here is greater than that of nitrile hydration. Apparently the
amidase activity is not only affected by the size but also by the
orientation of the substituents. The difference between aro-
matic or α,β-unsaturated amides bearing an adjacent group and
1-adamantylcarboxamide or pivalamide is that only in the non-
aliphatic molecules are adjacent substituents locked in the same
plane as the carbonyl group. This suggests that a coplanar
adjacent substituent hinders the attack of an amino acid resi-
due of the amidase on the amide carbonyl. Surprisingly, little2
has been reported on the mechanism of amidase action.
Table 2 Hydrolysis of other aromatic nitriles RCNa
Product yield (%)
Substrate
Reaction Amide Acid Nitrile
a
R
time/h
b
c
c
23
24
25
26
27
28
29
30
31
2,6-F2C6H3
2,6-Cl2C6H3
2,6-Me2C6H3
2,6-(MeO)2C6H3
3,4-(OCH2O)C6H3
1-Naphthyl
2-Naphthyl
9-Anthracenyl
9-Phenanthrenyl
24
192
120
216
24
168
120
168
168
80.0
23.1
—
—
—
—
—
—
—
14.9
—
—
—
69.6
87.5
80.1
—
16.3
—
—
88.9
80.3
69.3
—
96.1
92.7
—
a
3 Mmol of the nitriles were used and reaction conditions were not
optimised.
Table 3 Hydrolysis of heterocyclic nitriles RCNa
Product yield (%)
Reaction
Amount/
Substrate a
R
mmol
time/h
Amide b
Acid c
Nitrile a
34
35
36
2-Furyl
3
5
3
5
3
3
2
3
5
3
3
3
3
1
7
1.5
48
2
72
144
144
24
2
72
55
120
82.2
—
72.8
—
81.3
87.1
—
—
—
58.9
—
—
12.5
83.1
28.1
85.8
—
—
—
—
—
16.1
11.1
29.3
24.6
—
—
—
—
62.5
2-Thienyl
1,5-Dimethylpyrrolyl
—
37
38
39
40
3-Indolyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
50.4
46.3
61.8
—
50.1
57.7
—
41
42
3-Quinolyl
1-Isoquinolyl
12.0
43
44
3
144
144
—
—
—
93.2
96.4
Me
Me
N
H
O
O
Me
3
—
N
H
45
2
144
—
—
98.9
Ph
Ph
O
H
N
N
a
Reaction conditions were not optimised.
J. Chem. Soc., Perkin Trans. 1, 1997
1101

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Table 4 Hydrolysis of aliphatic nitriles RCNa
Product yield (%)
Amount/
mmol
Reaction
time/h
Substrate a
R
Amide b
Acid c
Nitrile a
46
Vinyl
10
10
3
10
10
10
5
0.5
16
16
0.5
16
17
19
2.5
19
26
72
16
12
24
17
17
19
24
19
19
96
72
—
—
—
43.4
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
98.1
68.6
91.5
44.5
98.0
85.8
90.2
75.4
83.9
60.3
94.4
71.9
99.5
89.2
98.3
91.3
89.8
99.4
82.6
85.5
91.3
93.9
—
—
—
—
—
—
—
trace
—
30.6
—
—
—
—
—
—
—
—
47
48
trans-Styryl
Propen-2-yl
49
50
51
Butyl
Isobutyl
tert-Butyl
5
5
52
1-Adamantyl
1.5
1
10
10
5
5
5
3.6
5
5.1
5
53
54
55
56
57
58
59
60
61
62
63
Allyl
trans-But-2-enyl
3-Phenylpropyl
2-Phenylethyl
Benzyl
p-Chlorobenzyl
p-Nitrobenzyl
p-Methylbenzyl
p-Methoxybenzyl
1-Naphthylmethyl
2-Naphthylmethyl
—
—
trace
—
3
3
a
Reaction conditions were not optimised.
In conclusion, it is clear that Rhodococcus AJ270 tolerates a
of the concentration of the substrates and careful monitoring
of the reaction process is undertaken to achieve optimal con-
version and chemical yield.
broad spectrum of nitrile substrates, in contradistinction to
some earlier reports of related organisms.3,7–10 Both the nitrile
hydratase and amidase activities involved in Rhodococcus
AJ270 are mainly influenced by steric and coordinating factors,
in particular the presence of α- or ortho-substituents. For effect-
ive nitrile hydrolysis, a molecular diameter of the substrate of
no more than ca. 7 Å appears the limit. The amidase is more
sensitive than the nitrile hydratase to the geometry of the
molecule. Without adjacent substituents such as an ortho-
substituent in benzonitriles, an adjacent heteroatom in hetero-
cycles or a geminal substituent in an α,β-unsaturated nitrile, the
amidase catalyses the conversion of amides to acids rapidly and
efficiently. When such adjacent substituents are present in the
molecule, on the contrary, biotransformation of an amide into
an acid proceeds sluggishly. The presence of an amino group at
the para-position but not the meta-position of a benzamide (or
an amino group in a 6-membered heterocycle γ to the amide
group) also has a deleterious effect on amidase efficiency.
It is worth noting that the adjacent-substituent effect is also
generally observed in most of the published examples using the
nitrile hydratase–amidase systems. Thus, the combination of
electronic and steric insensitivity of the nitrile hydratase and the
steric sensitivity of the amidase accounts for the formation of
amides and the poor regioselectivity observed in the hydrolysis
of perfluoro and unsymmetrical aromatic dinitriles.15 The out-
comes of the published hydrolyses of heterocyclic nitriles also
fit this generalisation.18
There is one last factor that causes low yields in the biotrans-
formation of nitriles using whole cell systems. Small aliphatic
nitriles and those bearing amino and hydroxy groups appear to
be metabolised in a different manner by the organism (presum-
ably by other enzymes), leading to lowered yields of acids and
amides. Thus aminobenzoic acids 10c, 14c and 21c and
hydroxybenzoic acids 11c, 15c and 21c as well as aceto- and
propio-nitriles give lower yields. Also, acrylonitrile 46a and allyl
cyanide 53a gave about a 70% yield of the acid in 16 h while
cinnamonitrile 47a and methacrylonitrile 48a gave the corres-
ponding acid almost quantitatively in the same reaction period.
Longer reaction times with the smaller nitriles lead consistently
to lower yields. Similar problems were also encountered in the
hydrolysis of dinitriles24 which will be the subject of a further
full study. It is strongly recommended, therefore, that variation
Experimental
Melting points, which are uncorrected, were determined using a
Reichert Kofler hot stage apparatus. Infrared spectra were
obtained on a Unicam Research Series 1 FTIR instrument as
liquid films or KBr discs. NMR Spectra were recorded in
CDCl3 or [2H6]DMSO solution with SiMe4 as internal standard
on a JEOL 270 spectrometer. Mass spectra were measured on a
Kratos MS8ORF mass spectrometer and microanalyses were
carried out at Newcastle University on a Carlo Erba 1106
Elemental Analyser. Thin layer chromatography (TLC) was
performed with Merk silica 60 F254 plates, and for flash chroma-
tography Janssen silica (35–70 mm) was used.
Preparation of biocatalyst
The medium used was the chelate mineral medium (CMM) plus
vitamins.25 Rhodococcus sp. AJ270, previously isolated from a
soil sample, was subcultured at 30 ЊC in a conical flask (1 dm3)
containing 300 cm3 of the medium omitting ammonium sulfate.
Acetonitrile (3 cm3) was used as the carbon and nitrogen source.
After 24 h the culture was transferred into a 20 dm3 fermentor
containing the same medium (15 dm3) and an antifoam agent
[antifoam 204 (Sigma)] (1 cm3). Acetamide (44.3 g, 50 m) was
used instead of acetonitrile as the carbon and nitrogen source.
After inoculation at 30 ЊC for about 14 h (late log phase), cells
were collected by continuous centrifugation at 4 ЊC. Cells were
washed twice with potassium phosphate buffer (0.1 , pH 7.0)
and were stored at Ϫ20 ЊC in a freezer for further use.
Biotransformation of nitriles
To an Erlenmeyer flask (250 cm3) with a screw cap was added
Rhodococcus sp. AJ270 cells (2 g wet weight) and potassium
phosphate buffer (0.1 , pH 7.0, 50 cm3) and the resting cells were
activated at 30 ЊC for 0.5 h with orbital shaking. Nitrile (1–10
mmol, see Tables 1–4 for each substrate) was added in one por-
tion and the mixture was incubated at 30 ЊC using an orbital
shaker (200 rpm). The reaction, monitored by TLC, was
stopped after a period of time (see Tables 1–4 for each indi-
vidual substrate) and worked up following one of two methods.
1102
J. Chem. Soc., Perkin Trans. 1, 1997

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Method A, mainly used for the aliphatic nitriles, involved
filtration through a Celite pad to remove the biomass followed
by basifying the aqueous solution to pH 11 with aqueous
NaOH (2 ). Extraction with diethyl ether or ethyl acetate
(2 × 30 cm3) gave, after drying over magnesium sulfate and con-
centration, the amide and unreacted nitrile. Separation of
nitrile and amide was effected by flash chromatography. The
aqueous solution was then acidified using concentrated HCl to
pH 2 and extracted with diethyl ether or ethyl acetate (2 × 30
cm3). The acid was obtained after the solvent had been removed
in vacuo.
33–35 ЊC (lit.,65 34–35 ЊC); 52c, mp 176 ЊC (lit.,66 175–176.5 ЊC);
53c, oil (lit.,67 bp 163–165 ЊC); 54c, oil (lit.,68 bp 97–98 ЊC/20
mmHg); 55c, mp 51–52 ЊC (lit.,69 52 ЊC); 56c, mp 46.5 ЊC (lit.,60
46.4–46.8 ЊC); 57c, mp 77–78 ЊC (lit.,60 76.7–76.9 ЊC); 58c, mp
103–105 ЊC (lit.,70 105 ЊC); 59c, mp 151–153 ЊC (lit.,70 152 ЊC);
60c, mp 93.5–95 ЊC (lit.,71 94 ЊC); 61c, mp 84–86 ЊC (lit.,72
82 ЊC); 62c, mp 132–132.5 ЊC (lit.,47 132 ЊC); 63c, mp 143–
144 ЊC (lit.,47 144 ЊC).
Acknowledgements
Method B was used mainly for aromatic and heterocyclic
nitriles. The reaction mixture was adjusted to pH 11 with aque-
ous NaOH (2 ) and was extracted continuously with dichloro-
methane for 12–24 h. The aqueous phase was acidified with
concentrated HClto pH 2and wasextracted again with dichloro-
methane for another 12–24 h. The amide and/or nitrile and
acid were obtained respectively from the two extractions. For
pyridinecarboxylic acids, the pH was adjusted to their iso-
electric point (isonicotinic acid 3.2; nicotinic acid 3.4; picolinic
acid 3.6) and extracted continuously with dichloromethane.
Method A was used for the isolation of aminobenzoic acids
and after addition of glacial acetic acid and concentration of
the solution in vacuo, anthranilic acid was obtained as a crystal-
line product. 3- And 4-aminobenzoic acids were obtained when
the aqueous solution was concentrated to dryness and the resi-
due was extracted with ethanol.
We thank the BBSRC for a post-doctoral grant (M.-X. W.) and
generous funding that made this work possible.
References
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All products were characterised by their spectral data and
comparison of the melting points with the known compounds,
which are listed below, or by full characterisation.
1,5-Dimethyl-1H-pyrrole-2-carboxamide 36b
Colourless crystalline solid, mp 152.5 ЊC; νmax/cmϪ1 3370, 3185,
1637 and 1612; δH 6.54 (1 H, d, J 3.8), 5.89 (1 H, dd, J 3.8 and
0.8), 5.44 (2 H, br s, NH2), 3.85 (3 H, Me) and 2.24 (3 H, Me);
m/z (El) 138 (Mϩ, 100%), 122 (76) and 94 (26) (Found: C,
60.75; H, 7.24; N, 20.28. C6H10N2O requires C, 60.83; H,
7.30; N, 20.28%).
Melting points
1c, mp 122 ЊC (lit.,26 120–122 ЊC); 2c, mp 181 ЊC (lit.,27 181 ЊC);
3c, mp 184 ЊC (lit.,28 185 ЊC); 4c, mp 159 ЊC (lit.,29 158–160 ЊC);
5c, mp 243 ЊC (lit.,30 240 ЊC); 6c, mp 185 ЊC (lit.,31 182 ЊC); 7c,
mp 241.5 ЊC (lit.,27 241 ЊC); 8c, 208 ЊC (lit.,32 207.5–209.5 ЊC);
9c, mp 230 ЊC (lit.,33 218–230 ЊC); 10c, mp 213–214 ЊC (lit.,29
212–213 ЊC); 11b, mp 183–183.5 ЊC (lit.,34 175–179 ЊC); 11c, mp
186–187 ЊC (lit.,35 184 ЊC); 12c, mp 140–141 ЊC (lit.,36 139–
140 ЊC); 13c, mp 110 ЊC (lit.,27 110.5 ЊC); 14c, mp 200.8 ЊC
(lit.,29 197–201 ЊC); 15c, mp 174 ЊC (lit.,35 173 ЊC); 16b, mp
143 ЊC (lit.,37 144–145 ЊC); 16c, mp 105.5 ЊC (lit.,38 104.8–
105.4 ЊC); 17b, mp 130 ЊC (lit.,39 130–131 ЊC); 19b, mp 142–
143 ЊC (lit.,40 142–143.5 ЊC); 19c, mp 141–143 ЊC (lit.,31 142 ЊC);
20b, mp 177–178 ЊC (lit.,41 174–175 ЊC); 20c, mp 146–147 ЊC
(lit.,42 148 ЊC); 21b, mp 142 ЊC (lit.,40 136.5–138.5 ЊC); 21c, mp
160–162 ЊC (lit.,29 156–158 ЊC); 22b, mp 111.5–112 ЊC (lit.,43
108.5–111 ЊC); 22c, mp 147.5–148 ЊC (lit.,35 144–145 ЊC); 23b,
mp 146.5–147 ЊC (lit.,44 144.5–145.5 ЊC); 23c, mp 156.5–157 ЊC
(lit.,44 159–160 ЊC); 24b, mp 200–201 ЊC (lit.,45 202 ЊC); 27c, mp
230.5–232 ЊC (lit.,46 229–230 ЊC); 28c, mp 162–163 ЊC (lit.,47
162 ЊC); 29c, mp 187 ЊC (lit.,47 187.5 ЊC); 34b, mp 143–144 ЊC
(lit.,48 141–142 ЊC); 34c, mp 134 ЊC (lit.,49 132–133 ЊC); 35b, mp
183–184 ЊC (lit.,50 179–180 ЊC); 35c, mp 132.5–133 ЊC (lit.,51
130 ЊC); 37c, mp 221 ЊC (lit.,52 222 ЊC); 38c, 135–137 ЊC (lit.,53
135–136 ЊC); 39c, mp 235–236 ЊC (lit.,54 235 ЊC); 40b, mp 156–
156.5 ЊC (lit.,55 154–155 ЊC); 40c, mp 315–316 ЊC (lit.,56 317–
318 ЊC); 41c, mp 276–277 ЊC (lit.,57 274–275 ЊC); 42b, mp 171–
172 ЊC (lit.,58 170–171 ЊC); 46c, oil (lit.,59 bp 141–142 ЊC); 47c,
mp 133–134 ЊC (lit.,60 132.4–132.7 ЊC); 48b, mp 109 ЊC (lit.,61
108–109 ЊC); 48c, oil (lit.,62 bp 65 ЊC/11 mmHg); 49c, oil (lit.,63
bp 80–83 ЊC/10 mmHg); 50c, oil (lit.,64 bp 175–177 ЊC); 51, mp
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Paper 6/07977F
Received 25th November 1996
Accepted 17th December 1996
1104
J. Chem. Soc., Perkin Trans. 1, 1997