Regioselective biotransformations of dinitriles using Rhodococcus sp. AJ270

Article abstract of DOI:10.1039/a703904b

A variety of dinitriles have been hydrolysed selectively under very mild conditions using Rhodococcus sp. AJ270. Aliphatic dinitriles NC[CH₂]_nCN 1 undergo regioselective hydrolysis to give the mono acids 2 with up to 4 methylenes between the nitrile functions while those with n > 4 give the diacids 3 in good yield. Dinitriles NC[CH₂]_nX[CH₂]_nCN 4 bearing an ether or sulfide linkage are efficiently transformed into the mono acids 5 when an oxygen is placed β, γ or δ to the cyano group or a β- or γ-sulfur is present. Hydrolysis of N,N-bis(2-cyanoethyl)anilines 4h-j takes place slowly affording exclusively the monoacids 5h-j while the monocyano amides 5o-p are obtained as the sole isolable product from rapid hydrolysis of the corresponding N,N-bis(2-cyanomethyl)butylamine 4o and N,N-bis(3-cyanopropyl)butylamine 4p. Higher homologues of arylimino- and butylimino-dinitriles are inert to enzymatic hydrolysis. A variety of other aliphatic dinitriles have been converted readily into mono acids in good to excellent yields except for o-phenylenediacetonitrile which gives o-phenylenediacetamide as the major product. The title organism also effects the hydrolysis of aromatic dinitriles with regiocontrol such as m- and p-dicyanobenzenes, but nct the ortho-substituted analogue. The scope and limitations of this enzymatic process have been systematically studied and the mechanism of regioselective hydrolysis has been discussed in terms of a chelation-deactivation effect.

Full text of DOI:10.1039/a703904b

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Regioselective biotransformations of dinitriles using Rhodococcus
sp. AJ2701
Otto Meth-Cohn* and Mei-Xiang Wang
Department of Chemistry, University of Sunderland, Sunderland, UK SR1 3SD
A variety of dinitriles have been hydrolysed selectively under very mild conditions using Rhodococcus sp.
AJ270. Aliphatic dinitriles NC[CH2]nCN 1 undergo regioselective hydrolysis to give the mono acids 2 with
up to 4 methylenes between the nitrile functions while those with n > 4 give the diacids 3 in good yield.
Dinitriles NC[CH2]nX[CH2]nCN 4 bearing an ether or sulfide linkage are efficiently transformed into the
mono acids 5 when an oxygen is placed â, ã or ä to the cyano group or a â- or ã-sulfur is present. Hydrolysis
of N,N-bis(2-cyanoethyl)anilines 4h–j takes place slowly affording exclusively the monoacids 5h–j while
the monocyano amides 5o–p are obtained as the sole isolable product from rapid hydrolysis of the
corresponding N,N-bis(2-cyanomethyl)butylamine 4o and N,N-bis(3-cyanopropyl)butylamine 4p. Higher
homologues of arylimino- and butylimino-dinitriles are inert to enzymatic hydrolysis. A variety of other
aliphatic dinitriles have been converted readily into mono acids in good to excellent yields except for
o-phenylenediacetonitrile which gives o-phenylenediacetamide as the major product. The title organism
also effects the hydrolysis of aromatic dinitriles with regiocontrol such as m- and p-dicyanobenzenes, but
not the ortho-substituted analogue. The scope and limitations of this enzymatic process have been
systematically studied and the mechanism of regioselective hydrolysis has been discussed in terms of a
chelation–deactivation effect.
using nitrilase10a,13 and nitrile hydratase11a,g systems have been
Introduction
proposed and for the latter the hydration stage is believed to
involve complexion of the nitrile nitrogen to a transition metal
(iron or cobalt) followed by hydration mediated by a possible
prosthetic group such as a pyrroloquinoline quinone.14
Biotransformations in organic synthesis using micro-organisms
and isolated enzymes have received tremendous attention dur-
ing last two decades.2 Since they are performed under very mild
conditions such as at ambient temperature and in aqueous
media at pH ca. 7, biotransformations are regarded as
environmentally benign processes.3 More importantly, most
enzyme-catalysed reactions proceed in a chemo-, regio- and
stereo-selective manner. Therefore, they provide powerful and
efficient synthetic routes to compounds which are sometimes
not easily accessible by ‘conventional’ chemical means. For
example, lipases, esterases and acylases have been widely used
for the kinetic resolution of racemic esters and for the prepar-
ation of homochiral molecules from prochiral and meso
diesters.2,4 In contrast to these extensively studied hydrolytic
enzymes, nitrile-hydrolysing enzymes in synthesis have
remained largely unexplored until recently.5 In fact, bioconver-
sion of nitriles into the corresponding carboxylic acids has been
known for decades.6 Enzyme-catalysed hydrolyses of nitriles
have been shown to proceed by two distinct routes: a nitrilase7
transforms the nitriles directly into acids while a nitrile
hydratase produces amides which are subsequently hydrolysed
to the acids by an amidase.8 (Scheme 1). So far a range of
In our previous studies9 it has been demonstrated that Rhodo-
coccus sp. AJ270 is a powerful and robust nitrile hydratase/
amidase-containing organism. It has very broad activity against
all types of nitriles including aromatic, heterocyclic and ali-
phatic ones. To further explore its potential in organic synthesis,
and also as a prelude to investigating the hydrolysis of prochiral
dinitriles we have made a systematic study of the effect of struc-
ture on the regioselectivity of hydrolysis of dinitriles.
In 1980 Yamada et al. observed that biodegradation of
glutaronitrile by Pseudomonas sp. K-9 proceeded through 4-
cyanobutyramide, 4-cyanobutyric acid and glutaric acid,15 but
stopped at the 4-cyanobutyric acid stage using the fungus
Fusarium merismoides TG-1.16 Recently it has been reported
that a series of dinitriles, NC(CH2)nCN [n = 3-5], were hydro-
lysed to the diacids with no selectivity using Rhodococcus rhodo-
chrous NCIB 1121617 while Rhodococcus rhodochrous K-22
catalysed selective monohydrolysis of glutaronitrile in quan-
titative yield,18 though both organisms are nitrilase-containing
strains. Using an immobilised whole cell Rhodococcus SP361,
a nitrile hydratase system developed by Novo Industri of
Denmark, Hönicke-Schmidt and Schneider19 observed ex-
clusive formation of 4-cyanobutyric acid from glutaronitrile.
Turner et al. showed that succinonitrile gave solely 3-cyano-
propionic acid while glutaronitrile gave mono- and di-acids,
the ratios of which depended upon the reaction.20 Biotrans-
formation of adiponitrile into adipic acid has been closely
studied by a French group utilising the wild type and mutants
of Brevibacterium sp. R31221 while efficient monohydrolysis of
cyclohexane-1,4-dicarbonitrile has been effected with organ-
isms such as Acremonium sp. D9K22 and Corynebacterium sp.
CH.23 Two groups studied hydrolysis of prochiral dinitriles.
Ohta24 and Turner25 and their co-workers have shown that
some 3-hydroxyglutaronitrile derivatives, when treated with
Rhodococcus rhodochrous ATCC 21197 and Rhodococcus
SP361, respectively, were converted into the monocyano acids
O
nitrile
hydratase
R
NH2
H2O
NH3
H2O
R
C
N
amidase
nitrilase
H2O
O
NH3
R
OH
Scheme 1
nitrile-hydrolysing micro-organisms has been isolated9 and
some nitrilases,7a,10 nitrile hydratases11 and amidases12 have been
purified. The mechanisms of enzymatic hydrolysis of nitriles
J. Chem. Soc., Perkin Trans. 1, 1997
3197

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Table 1 Conversion of NC[CH2]nCN 1 into NC[CH2]nCO2H 2 and/or
HO2C[CH2]nCO2H 3
4d was used. The sulfide substrates 4e–g underwent similar
rapid hydrolysis; however, a γ-sulfur 4f allowed optimal regio-
control, while the β-analogue 4e showed selectivity but not the
δ-analogue 4g. 3,3Ј-Imino- and 3,3Ј-methylimino-dipropio-
nitriles proved to be unstable under the conditions used, even in
the absence of the organism. We therefore included the N-aryl
4h–n and N-butyl 4o–r analogues in the study. Probably because
of steric factors, all reactions utilising the N-aryl derivatives
proceeded slowly to give the monoacids 5h–j and with n > 2,
not at all, even with added co-solvents such as acetone and
methanol. Because of their limited reactivity, small steric
changes remote from the nitrile group resulted in a dramatic
difference in hydrolysis rate; the para-substituted substrate 4i
was hydrolysed after one week compared to 64 h of complete
conversion for the parent aniline 4h. Steric limitations of the
nitrile hydratase were further exemplified with the substrate
N,N-bis(cyanoalkyl)butylamines 4o–r (Fig. 1). The longer
homologues bearing four and five methylenes such as 4q and
4r remained intact and were recovered after a week of inter-
action with Rhodococcus sp. AJ270. In contrast, the butylamine
analogue with two methylenes 4o was hydrolysed rapidly, being
converted within 1 h, while that with three methylenes, 4p,
completely reacted within 8 h, low yields of monocyano amides
5o and 5p being the sole product; this suggested that the ami-
dase as well as the nitrile hydratase enzyme suffers similar steric
limitations in the amine series. Longer incubation of 4o and 4p
led to the total loss of products, indicating that the organism
rapidly metabolises the derived amino amides or, most likely,
amino acids utilising other enzymes. At this stage, we cannot
yet define the limits of regiocontrol with chain length in the
nitrogen series but good selectivity was shown with a β- or a γ-
nitrogen. Nevertheless, regioselectivity in the biotransformation
of simple aliphatic dinitriles with Rhodococcus sp. AJ270 is
principally dependent upon the placement of the heteroatom
rather than the overall chain length.
Substrate
Reaction conditionsa
Product yieldb (%)
1
n
Amount (mmol) Time (h)
2
3
1a
1b
1c
2
2
3
3
4
4
4
5
5
6
7
8
5
5
5
5
5
5
5
3
3
3
3
3
3
24
3
16
3
24
48
3
48
48
48
48
30
17
41
35
41
28
—
—
—
—
—
—
—
—
—
—
4
23
26
46
74
78
88
89
1d
1e
1f
1g
a Reaction conditions were not optimised. b Isolated yield.
(CH2)nCN
(CH2)nCN
Bu
N
Bu
N
(CH2)nCN
(CH2)nCONH2
4o, n = 2; 4p, n = 3
4q, n = 4; 4r, n = 5
5o, n = 2, 17%
5p, n = 3, 31.5%
Figure 1
in an enantioselective manner. Interestingly, R. rhodochrous
ATCC 21197 hydrolysed a prochiral malononitrile to afford
its monoacid monoamide derivative with high enantiomeric
excess.26 Some aromatic and unsaturated dinitriles were hydro-
lysed regioselectively with both nitrilase17,27 and nitrile
hydratase5 systems except for o-phthalonitrile which gave dif-
ferent products with different organisms.20,28
Results and discussion
M
M
The purpose of our research was to define the extent of regio-
selective control in the hydrolysis of a wide variety of dinitriles
and to try to understand the mechanism of this control. It is
probable that all nitrile hydratase/amidase systems differ more
in a quantitative than a qualitative manner, and that our find-
ings should be of wider generality. We first systematically exam-
ined the regioselectivity of hydrolysis of a series of aliphatic α,
ω-dinitriles 1, monitoring the reaction against time (Table 1).
Unless otherwise specified, all hydrolyses gave acids as prod-
ucts, the amides being occasionally seen by TLC but not isol-
ated since they were rapidly transformed into acids. Both suc-
cinonitrile 1a and glutaronitrile 1b were hydrolysed efficiently to
give solely 3-cyanopropionic acid 2a and 4-cyanobutyric acid
2b, respectively. Only the diacids 3d–g were obtained from
pimelo-, subero-, azelao- and sebaco-nitriles 1d–g while adipo-
nitrile 1c was selectively converted into 5-cyanovaleric acid 2c
or adipic acid 3c depending upon time of reaction. It was quite
clear that dinitriles with more than four methylenes separating
the functions gave the diacid as the sole product irrespective of
reaction time. With less than four methylenes, however, regio-
selective monohydrolysis occurred. It should be pointed out
that, as we noted in earlier work,9 low molecular weight nitriles
tend to be metabolised by other enzymes in the organism and
therefore overall chemical yields were limited and reduced with
increasing time of interaction.
N
C
R
X
N
hydration
hydration
C
X
Scheme 2
The regioselectivity of the hydrolysis of aliphatic dinitriles
was interpreted on the basis of chelation–deactivation of the
enzyme (Scheme 2). It is believed that hydrolysis occurs by
complexation of the nitrile nitrogen to a metal (iron or cobalt)
followed by hydration of the nitrile function by the presumed
co-factor present in such nitrile hydratase, pyrroloquinoline
quinone, as documented elsewhere.14 When a suitably placed
ligand atom is also present in the nitrile, bidentate complex-
ation to the metal occurs, which interferes with this hydration
Ϫ
process. This ligand may be a suitably positioned CO2 group
generated from a CN function, or a heteroatom. 4-Cyano-
butyric acid 2b, the hydrolysis product of glutaronitrile 1b
(n = 3), bears a δ-oxygen ligand as does the ether 5c obtained
from 4c (n = 4, X = O). The C᎐S bond is considerably longer
than the C᎐O bond (1.81 and 1.43 Å respectively) and, not
surprisingly, therefore a γ placement of sulfur lends optimal
regioselectively. Clearly, if this mechanism is correct, con-
straints in the chain between the nitrile functions such as
multiple bonds or trigonal groups will change the preferred
regioselective effect of heteroatoms.
If this mechanism is correct the above-mentioned ligands, i.e.
monocyano acids derived from dinitriles should behave as
competitive inhibitors in the hydrolysis of other easily hydro-
lysed nitriles. To clarify this issue, hydrolysis of benzonitrile was
studied in the presence and absence of 4-(3-cyanopropyl-
thio)butyric acid 5f. Using HPLC we were able to record the
Next examined were a range of α, ω-dinitriles NC[CH2]nX-
[CH2]nCN 4 containing a heteroatom X in the chain (Table 2).
Significant regioselectivity was observed. With the ether series
4a–d, hydrolysis was rapid and generally efficient. Regiocontrol
was optimally efficient when an oxygen atom was placed δ to
the cyano group in the chain. Hydrolysis was also effected
selectively with dinitriles 4a–b bearing a β- or γ-oxygen, while
regioselectively was lost entirely when bis(5-cyanopentyl) ether
3198
J. Chem. Soc., Perkin Trans. 1, 1997

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Table 2 Conversion of NC[CH2]nX[CH2]nCN 4 into NC[CH2]nX[CH2]nCO2H 5 and/or HO2C[CH2]nX[CH2]nCO2H 6
Substrate
Reaction conditionsa
Product yieldb (%)
4
n
X
Amount (mmol) Time (h)
5
6
4
4a
4b
2
3
3
4
4
5
5
2
2
3
3
4
4
2
2
2
3
3
4
5
O
O
O
O
O
O
O
S
S
S
S
S
3
5
3
3
3
3
3
5
3
3
3
3
3
2
2
2
48
1
72
2
120
2
48
1
48
2
96
2
96
64
168
72
168
168
168
168
61
35
—
73
75
—
—
45
—
52
83
—
—
93
71
91
—
—
—
—
—
Trace
70
—
5
—
35
—
—
—
60
—
10
—
29
—
Trace
—
—
26
—
95
92
94
99
4c
4d
4e
4f
25
97.5
—
60
—
—
83
86
—
—
—
—
—
—
—
4g
S
NPh
NC6H4Cl-p
NC6H4OMe-p
NPh
NC6H4OMe-p
NPh
4h
4i
4j
4k
4l
4m
4n
1.5
1.5
1.5
1.5
NPh
a Reaction conditions were not optimised. b Isolated yield.
This accessibility is even more true for the o-cyanophenylaceto-
nitrile 7a where the aliphatic nitrile moiety is hydrolysed while
aromatic nitrile remains intact, since an ortho substituent poses
further steric hindrance. m-Phenylenediacetonitrile 10a was
hydrolysed to give the monocyano acid 10b as sole product, as
was the para-analogue 11. Interestingly, however, hydrolysis of
o-phenylenediacetonitrile 9a afforded o-phenylenediacetamide
9c as the major product, with the monocyano acid 9b being
formed only in 11% yield. This is the first example of the
diamide formation from enzymatic dinitrile hydrolysis. The
reason for the accumulation of diamide 9c is not clear at this
stage though one may suggest that either hydration of the
second group is faster and non-selective or the amidase is less
effective towards the monocyano amide intermediate and o-
phenylenediacetamide 9c, or both. It is worth noting that
trans-cyclohexane-1,4-diyldiacetonitrile 12a was also converted
into the corresponding monocyano acid 12b in almost quan-
titative yield although the two cyano groups are separated by
six carbons. Compared with the outcome of the hydrolysis of
suberonitrile 1e, it is apparent that the conformation of the
cyclohexane moiety plays an important role in determining
regiospecificity. Rhodococcus sp. AJ270 was able to hydrolyse
unsaturated dinitriles such as fumaronitrile 13a and cis,cis-
mocononitrile 14a regioselectively in a manner similar to those
hydrolyses utilising other organisms.17b,19
Finally, the aromatic dinitriles 15–17 were subjected to the
hydrolysis (Table 4). The same reaction pattern as those
reported in the literature20,28 was found for meta- and para-
phthalonitriles, i.e., high chemical conversion with total regio-
control to give monocyano acids 16b and 17b, respectively.
However, hydrolysis of o-phthalonitrile 15a proceeded slug-
gishly, with more than half the starting material being
recovered after 70 h interaction with Rhodococcus sp. AJ270,
the products being o-cyanobenzoic acid 15b (12%) and
phthalimide 15c (12%). It has been shown previously that the
same substrate gave diacid or o-cyanobenzamide and phthalim-
ide with Rhodococcus SP36120 or Rhodococcus equi A4,28
respectively. Reaction probably involves the hydration of one
cyano group with a nitrile hydratase forming o-cyanobenz-
amide followed either by hydrolysis with amidase yielding o-
cyanobenzoic acid 15b or a non-enzymatic ring-closure afford-
ing phthalimide 15c during the time of reaction and/or work-
up. As we have noted earlier all ortho or adjacently substituted
aromatic and alkenic nitriles undergo a slow amidase mediated
hydrolysis, allowing isolation of the amide.9 It is tempting to
Fig. 2 Hydrolysis of benzonitrile in the absence (a) and presence (b)
of inhibitor 4-(3-cyanopropylthio)butyric acid 5f
course of the hydrolysis over time. As illustrated in Fig. 2, 5f
indeed significantly slowed the hydrolysis of benzonitrile; thus
after reaction for 5 min, about 12% of unchanged benzonitrile
remained with no added cyano acid, while in the presence of the
inhibitor almost 25% remained. After 10 min the figures were
ca. 1 and 15%. Furthermore, the rate of decrease of benzamide
and the rate of formation of benzoic acid were both signifi-
cantly slower in the presence of 5f. Rhodococcus sp. AJ270 also
effectively catalysed the hydrolysis of a variety of miscellaneous
aliphatic dinitriles (Table 3). Excellent regioselectivity was evi-
dent in almost all cases that we have examined. o-Cyanophenyl-
acetonitrile 7a was transformed into o-cyanophenylacetic acid
in good yield while p-cyanophenylacetonitrile gave a mixture of
primarily p-cyanophenylacetic acid 8b and p-cyanomethyl-
benzoic acid 8c (4:1 ratio) in a combined yield of 81%. These
results suggest a preference of Rhodococcus sp. AJ270 for the
hydrolysis of aliphatic nitriles over aromatic ones. The prefer-
ential attack on the aliphatic cyano group is most probably
attributable to the lower steric demand of the aliphatic sub-
strates. For example, the aliphatic cyano group in p-cyano-
phenylacetonitrile 8a is more accessible than the aromatic one.
J. Chem. Soc., Perkin Trans. 1, 1997
3199

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Table 3 Conversion of miscellaneous dinitriles into acids
Conditionsa
Substrate
mmol
Time (h)
24
Product(s) and yieldsb (%)
CN
CN
7b
7a
2
2
80
CO2H
CO2H
NC
NC
24
81c
8b/8c = 4/1d
8b
8c
8a
CO2H
CN
CO2H
HO2C
R1
R2
CN
CN
9a
3
24
R1 = R2 = CH2CN
13
11
9a
9b
R1 = CH2CN, R2 = CH2CONH2
R1 = R2 = CH2CONH2
CN
65
67
9c
CN
10b
10a
3
30
CO2H
CN
CN
NC
NC
11b
12b
11a
12a
69
99
3
30
39
CO2H
CO2H
CN
1.5
NC
NC
CO2H
CN
5
5
14
14
13b
14b
67
86
13a
14a
NC
NC
CO2H
CN
NC
NC
a Reaction conditions were not optimised. b Isolated yield. c Total isolated yield. d Ratio of 8b/8c (4:1) was determined on the basis of 1H NMR
measurements.
suggest that π-systems embedded in the molecule can also act as
effective ligands for the metal (iron or cobalt) when chelation
allows. Also included is trans-cyclohexane-1,4-diyldiacetonitrile
12a which shows total regioselective hydrolysis, presumably by
way of chelation of its axial or twist-boat conformer.
which would be difficult to obtain utilising other methods. For
simple aliphatic dinitriles and those bearing heteroatoms in-
cluding oxygen, sulfur and nitrogen, efficiency of regiocontrol
is dependent upon the distance between cyano function and
heteroatom; an oxygen placed β, γ or δ (optimally δ) to the
cyano group allows regiocontrol as does sulfur positioned β or
δ (optimally δ). Nitrogen substituents appear to behave as for
oxygen but suffer a steric limitation due to the size of the nitro-
gen substituent when it is trisubstituted. We explain the regio-
selectivity in the hydrolysis of these dinitriles by a chelation–
deactivation effect, i.e. a suitably placed heteroatom (which can
be derived from the first-formed CO2Ϫ group) also participates
in binding to the metalloenzyme and, therefore, interferes with
the nitrile hydration. This mechanism is consistent with the
fact that monocyano acids act as competitive inhibitors in the
hydrolysis of benzonitrile. The reason for the regioselective
Conclusion
Chemical hydrolysis of nitriles usually requires forceful condi-
tions such as strong acids or bases and proceeds without regio-
selectivity when a dinitrile is employed. Using a whole cell
Rhodococcus sp. AJ270 system we were able to hydrolyse vari-
ous types of dinitriles efficiently under very mild conditions.
Most significantly this enzymatic process showed excellent
regioselectivity against almost all the dinitriles we examined,
and it provided a powerful synthetic route to monocyano acids
3200
J. Chem. Soc., Perkin Trans. 1, 1997

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Table 4 Conversion of aromatic dinitriles into acids
Reaction conditionsa
cyanide to react with bis(chloroalkyl) ethers and sulfide,
respectively, according to a literature method.34 Compound 4o
was obtained through cyanoethylation of aqueous butyl-
amine35 while other amine derivatives 4m–n or 4p–r were pre-
pared by alkylation of aniline or butylamine with ω-iodoalkyl
cyanides following the methods reported in the literature.32,36
Amount Time
Substrate
(mmol) (h)
Product(s) and yieldsb (%)
CN
CN
CN
General procedure for the preparation of the bis(cyanoalkyl)
ethers 4b–d and the sulfide 4f34
3
70
52
15a
15a
15b
CN
CN
To a three-necked flask equipped with a mechanical stirrer was
added sodium cyanide (0.22 mol) and dry dimethyl sulfoxide
(90 cm3). The mixture was stirred and heated to 90 ЊC after
which bis(chloroalkyl) ether or sulfide (0.1 mol) was added
dropwise to it at such a rate that the temperature of the reaction
mixture was <110 ЊC. The reaction mixture was kept at ca.
100 ЊC for a further 30 min after the addition was complete, and
then allowed to cool to room temperature. After dilution with
dichloromethane (150 dm3), the mixture was poured into an
excess of saturated brine in a separatory funnel to which water
was then added to dissolve precipitated salts. After this, the
organic layer was separated and the aqueous layer was
extracted once with dichloromethane. The combined organic
phase and extract were washed with brine, dried (MgSO4) and
evaporated. The residue was then distilled in vacuo to give the
desired product.
12
12
CO2H
O
N
H
15c
O
CN
CN
3
3
24
20
71
81
16a
17a
16b
17b
CN
CO2H
CN
CN
Bis(3-cyanopropyl) ether 4c. Bis(3-chloropropyl) ether37 gave
the ether 4c as a colourless oil (12.6 g, 83%), bp 103 ЊC/0.02
mmHg (Found: Mϩ 152.0942. C8H12N2O requires M,
152.0949); νmax/cmϪ1 2247 (CN); δH 3.56 (4H, t, J 5.6), 2.48 (4H,
t, J 6.9) and 1.88–1.97 (4H, m); m/z (CI) 153 (Mϩ ϩ 1, 100%),
86 (92), 82 (26) and 68 (79).
CN
CO2H
a Reaction conditions were not optimised. b Isolated yield.
Bis(4-cyanobutyl) ether 4d. Bis(4-chlorobutyl) ether38 gave
the ether 4d as a colourless oil (16.2 g, 90%), bp 128 ЊC/0.03
mmHg (lit.,38 bp 75–80 ЊC/0.0001 mmHg);νmax/cmϪ1 2244 (CN);
δH 3.47 (4H, t, J 5.6), 2.39 (4H, t, J 6.9) and 1.69–1.80 (8H, m);
m/z (CI) 181 (Mϩ ϩ 1, 100%), 100 (55) and 82 (99).
action on other dinitriles including aromatic examples is not
known at this moment. However, a possible chelation inter-
action of π-system with the enzyme has been considered. The
extent of regiocontrol in dinitrile hydrolysis would be useful in
designing and conducting syntheses utilising prochiral and
chiral dinitriles, which we intend now to study.
Bis(5-cyanopentyl) ether 4e. Bis(5-chloropentyl) ether39 gave
the ether 4e as a pale yellow oil (17.9 g, 86%), bp 146–150 ЊC/
0.015 mmHg (Found: Mϩ 208.1579. C12H20N2O requires M,
208.1576); νmax/cmϪ1 2244 (CN); δH 3.41 (4H, t, J 5.9), 2.36 (4H,
t, J 6.9), 1.70 (4H, quin, J 6.9) and 1.47–1.67 (8H, m); m/z (CI)
209 (Mϩ ϩ 1, 92%) and 96 (100).
Experimental
Both melting points, which were determined using a Reichert
Kofler hot-stage apparatus, and boiling points are uncorrected.
IR spectra were obtained on a Unicam Research Series 1 FTIR
instrument as liquid films of KBr discs. 1H and 13C NMR spec-
tra were recorded in CDCl3 or [2H6]-DMSO solution with
SiMe4 as internal standard on a JEOL 270 spectrometer. Chem-
ical shifts are reported in ppm and coupling constants are given
in Hz. Mass spectra were measured on a Kratos MS8ORF mass
spectrometer using EI or CI. Exact mass was measured on a
Kratos MSED mass spectrometer and microanalyses were car-
ried out on a Carlo Erba 1106 Elemental Analyser, both at
Newcastle University. High performance liquid chroma-
tography (HPLC) was performed using a Lichrosorb RP-18 10
micron column (250 × 4.6 mm) (Alltech UK) with a Gilson
Gradient System and a LDC/Milton Roy spectroMonitorR
3000 UV detector at 204 nm. Potassium phosphate buffer (pH
2.8, 0.01 ) and acetonitrile were used as mobile phase and
furan-2-carboxamide was used as an internal standard through-
out the analyses. Thin layer chromatography (TLC) was per-
formed with Merck silica 60F254 plates, and for flash chroma-
tography Janssen silica (35–70 mm) was used.
Bis(3-cyanopropyl) sulfide 4f. Bis(3-chloropropyl) sulfide40
gave the sulfide 4f as a colourless oil (13.2 g, 79%), bp 128–
133 ЊC/0.018 mmHg (Found: Mϩ 168.0719. C8H12N2S requires
M, 168.0721); νmax/cmϪ1 2246 (CN); δH 2.67 (4H, t, J 7.0), 2.53
(4H, t, J 7.0) and 1.96 (4H, quin, J 7.0); m/z (CI) 169 (Mϩ ϩ 1,
100%) and 102 (69).
Preparation of N,N-bis(4-cyanobutyl)aniline 4m32
A mixture of aniline (4.6 g, 50 mmol), 5-iodovaleronitrile (20.9
g, 100 mmol) and potassium carbonate (6.9 g, 50 mmol) in
water (70 dmϪ3) was refluxed for 24 h. After cooling, further
potassium carbonate (1 g) was added to the mixture which was
then extracted with dichloromethane. The extract was dried
(MgSO4) and evaporated in vacuo, to give the title compound as
a yellow solid, recrystallisation of which from light petroleum–
ethyl acetate gave pale yellow prisms (7.1 g, 56%), mp 48.5–
49 ЊC (Found: C, 75.70; H, 8.64; N, 16.75. C16H21N3 requires C,
75.26; H, 8.29; N, 16.46%); νmax(KBr)/cmϪ1 2246 (CN); δH 6.68–
7.26 (5H, m), 3.32 (4H, t, J 7.0), 2.40 (4H, t, J 6.5) and 1.71–
1.74 (8H, m); δC 147.6, 129.4, 119.4, 116.7, 112.6, 50.3, 26.5,
23.1 and 17.2; m/z (CI) 256 (Mϩ ϩ 1, 100%), 213 (33) and 187
(44).
Starting materials
The dinitriles 1a–g, 4a, 7a, 9a–11a, 13a and 14a, and the aro-
matic dinitriles 15a–17a were purchased from commercial
sources. The sulfides 4e29 and 4g,30 the anilines 4h–j31 and 4k–
l32 and trans-cyclohexane-1,4-dicarbonitrile 12a33 were pre-
pared according to the literature. The bis(cyanoalkyl) ethers
4b–d and the sulfide 4f were synthesized by allowing sodium
Preparation of N,N-bis(5-cyanopentyl)aniline 4n
Following the same synthetic procedure as for 4m, the title
compound 4n was obtained from distillation (Kugelrohr) at
225 ЊC/0.07 mmHg as pale yellow oil (8.6 g, 61%) (Found: Mϩ
283.2050. C18H25N3 requires M, 283.2048); νmax/cmϪ1 2246
J. Chem. Soc., Perkin Trans. 1, 1997
3201

View Article Online
(CN); δH 6.62–7.25 (5H, m), 3.28 (4H, t, J 7.0), 2.34 (4H, t, J
7.0) and 1.42–1.75 (12H, m); δC 147.6, 129.2, 119.5, 115.7,
111.9, 50.7, 26.4, 26.1, 25.2 and 17.1; m/z (CI) 284 (Mϩ ϩ 1,
100%), 201 (36) and 96 (64).
65.5, 34.6 and 18.6; m/z (CI) 144 (Mϩ ϩ 1, 15%), 126 (100) and
84 (33).
4-(3-Cyanopropoxy)butyric acid 5b. Colourless oil (Found:
Mϩ Ϫ OH, 154.0867. C8H13NO3 Ϫ OH requires 154.0868);
νmax/cmϪ1 2678–3444 (CO H), 2250 (CN), 1712 (C᎐O) and 1117
᎐
2
General procedure for the preparation of N,N-bis(2-cyanoalkyl)-
butylamines 4p–r36
(C᎐O᎐C); δH 6.59 (1H, s), 3.52 (2H, t, J 5.6), 3.49 (2H, t, J 6.3),
2.46 (2H, t, J 7.3), 2.45 (2H, t, J 7.0), 1.92 (2H, quin, J 6.0) and
1.90 (2H, t, J 6.1); δC 179.2, 119.4, 69.7, 68.0, 30.7, 25.6, 24.6
and 14.0; m/z (CI) 172 (Mϩ ϩ 1, 18%), 154 (100) and 87 (97).
5-(4-Cyanobutoxy)valeric acid 5c. Colourless oil (Found:
Mϩ Ϫ OH, 182.1183. C10H17NO3 Ϫ OH requires 182.1181);
νmax/cmϪ1 2678–3410 (CO H), 2246 (CN), 1709 (C᎐O) and 1116
(C᎐O᎐C); δH 6.87 (1H, s), 3.45 (2H, t, J 5.6), 3.43 (2H, t, J 5.9),
2.39 (2H, t, J 7.6), 2.38 (2H, t, J 7.2) and 1.57–1.82 8H, m); δC
179.4, 119.6, 69.5, 69.4, 33.6, 28.9, 28.5, 22.5, 21.4 and 16.9;
m/z (CI) 200 (Mϩ ϩ 1, 38%), 182 (100) and 101 (91).
A mixture of butylamine (2.2 g, 30 mmol), ω-iodoalkyl cyanide
(60 mmol) and potassium carbonate (8.3 g, 60 mmol) in ethanol
(50 dmϪ1) was refluxed for 48 h after which it was concentrated
by removal of ethanol in vacuo and then diluted with water (ca.
50 cm3); the aqueous phase was separated and extracted with
ether. The extract was dried (MgSO4) and concentrated, and the
residue was distilled under reduced pressure to give the product.
N,N-Bis(3-cyanopropyl)butylamine 4p. The butylamine 4p
was obtained as a colourless oil (4.8 g, 77%), bp 136–139 ЊC/
0.015 mmHg (Found: Mϩ 207.1727. C12H21N3 requires M,
207.1735); νmax/cmϪ1 2246 (CN); δH 2.53 (4H, t, J 6.6), 2.42 (4H,
t, J 6.9), 2.40 (2H, t, J 5.9), 1.78 (4H, quin, J 6.6), 1.18–1.47
(4H, m) and 0.95 (3H, t, J 7.2); δC 119.7, 53.3, 52.1, 28.9, 23.3,
20.6, 14.7 and 14.0; m/z (CI) 208 (Mϩ ϩ 1, 100%), 164 (82) and
153 (39).
᎐
2
3-(2-Cyanoethylthio)propionic acid 5e. Pale yellow oil (Found:
Mϩ, 158.9805. C6H9NO2S requires 159.0354); νmax/cmϪ1 2586–
3446 (CO H), 2252 (CN) and 1712 (C᎐O); δ 8.50 (1H, s) and
᎐
2
H
2.62–2.89 (4H, m); δC 177.1, 118.2, 34.4, 27.7, 26.6 and 18.8; m/z
(CI) 160 (Mϩ ϩ 1, 3%), 156 (11), 142 (80), 133 (13) and 114
(100).
N,N-Bis(4-cyanobutyl)butylamine 4q. The butylamine 4q was
4-(3-Cyanopropylthio)butyric acid 5f. Pale yellow oil (Found:
obtained as a colourless oil (5.6 g, 79%), bp 164 ЊC/0.02 mmHg
Mϩ, 187.0431. C8H13NO2S requires 187.0667); νmax/cmϪ1 2668–
(Found: Mϩ 235.2054. C14H25N3 requires M, 235. 2048); νmax
/
3440 (CO H), 2248 (CN) and 1708 (C᎐O); δ 8.45 (1H, s), 2.65
᎐
2
H
cmϪ1 2245 (CN); δH 2.34–2.44 (10H, m), 1.51–1.75 (8H, m),
1.20–1.44 (4H, m) and 0.94 (3H, t, J 7.3); δC 119.7, 53.6, 52.9,
29.2, 26.2, 23.2, 20.6, 17.1 and 14.0; m/z (CI) 236 (Mϩ ϩ 1,
100%), 193 (65) and 167 (23).
(2H, t, J 6.9), 2.59 (2H, t, J 7.3), 2.51 (2H, t, J 6.9), 2.50 (2H, t, J
7.3), 1.94 (2H, quin, J 6.9) and 1.93 (2H, quin, J 6.9); δC 178.9,
119.0, 32.5, 31.0, 30.3, 25.0, 24.2 and 15.9; m/z (CI) 188
(Mϩ ϩ 1, 7%), 187 (28), 171 (27), 170 (100), 142 (14), 128 (12)
and 87 (31).
3-(N-Cyanoethyl-N-phenylamino)propionic acid 5h. Colour-
less crystalline solid, mp 56.5–56.8 ЊC (Found: C, 66.25; H,
6.45; N, 12.72. C12H14N2O2 requires C, 66.04; H, 6.47; N,
12.84%); νmax/cmϪ1 2903–3549 (CO2H), 2258 (CN) and 1732
N,N-Bis(5-cyanopentyl)butylamine 4r. The butylamine 4r was
obtained as a colourless oil (6.9 g, 87%), bp 155–158 ЊC/0.01
mmHg (Found: Mϩ 263.2353. C16H29N3 requires M, 263.2361);
νmax/cmϪ1 2245 (CN); δH 2.32–2.41 (10H, m), 1.63–1.74 (4H, m),
1.25–1.50 (12H, m) and 0.91 (3H, t, J 6.9); δC 119.64, 53.75,
53.64, 29.16, 26.55, 26.45, 25.29, 20.61, 17.08 and 14.00; m/z
(CI) 264 (Mϩ ϩ 1, 100), 220 (58), 181 (80) and 169 (34).
(C᎐O); δ 9.50 (1H, s), 6.72–7.32 (5H, m), 3.72 (4H, t, J 6.9),
᎐
H
2.68 (2H, t, J 6.6) and 2.59 (2H, t, J 6.9); δC 177.9, 145.6, 129.7,
118.5, 118.2, 113.4, 47.9, 47.0, 32.4 and 15.9; m/z (EI) 218 (Mϩ,
14%), 178 (99), 159 (46), 136 (53), 106 (100), 104 (45) and 77
(55).
Preparation of Rhodococcus sp. AJ270 resting cells
Rhodococcus sp. AJ270 was cultured in a 20-dm3 fermentor as
we described in our previous paper.9 The biomass was stored at
Ϫ20 ЊC in a freezer.
3-(N-p-Chlorophenyl-N-cyanoethylamino)propionic acid 5i.
Colourless crystalline solid, mp 89.5–90 ЊC (Found: C, 57.26;
H, 5.33; N, 11.23. C12H13ClN2O2 requires C, 57.04; H, 5.19; N,
11.09%); νmax/cmϪ1 3042–3550 (CO2H), 2248 (CN) and 1720
(C᎐O); δ 9.86 (1H, s), 7.22 (2H, d, J 9.2), 6.66 (2H, d, J 9.2),
Biotransformation of dinitriles
An Erlenmeyer flask (250 cm3) with a screw cap was charged
with Rhodococcus sp. AJ270 cells (2 g wet weight) and the
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 shak-
ing. The dinitrile (1.5–5 mmol, see Tables 1–4 for each sub-
strate) was added in one portion to the flask 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 individual substrate). Biomass was
removed by filtration through a Celite pad and the aqueous
solution was basified to pH 12 with aqueous NaOH (2 ).
Extraction with diethyl ether gave, after drying (MgSO4) and
concentration, the amide and unchanged nitrile. Separation of
nitrile and amide was effected by flash chromatography. The
aqueous solution was then acidified using aqueous HCl (2 ) to
pH 2 and extracted with diethyl ether. Mono- and/or di-acid
were obtained after evaporation of the extracts in vacuo. Solid
and oily products were purified by recrystallisation and flash
chromatography, respectively. 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.
᎐
H
3.70 (4H, t, J 6.6), 3.69 (2H, t, J 6.6), 2.66 (2H, t, J 6.9) and 2.58
(2H, t, J 6.6); δC177.8, 144.3, 129.5, 123.5, 118.0, 114.6, 47.9,
47.0, 32.3 and 16.0; m/z (CI) 255 (7), 254 (9), 253 (Mϩ ϩ 1, 22%),
252 (20), 214 (32), 212 (100), 195 (18) and 193 (58).
3-(N-Cyanoethyl-N-p-methoxyphenylamino)propionic acid 5j.
Colourless needles, mp 86–86.5 ЊC (Found: C, 63.01; H, 6.55;
N, 11.20. C13H16N2O3 requires C, 62.87; H, 6.50; N, 11.28%);
νmax/cmϪ1 2832–3548 (CO H), 2248 (CN), 1714, 1701 (C᎐O)
᎐
2
and 1244 (C᎐O᎐C); δH 9.45 (1H, s), 6.88 (4H, s), 3.52 (2H, t, J
6.6), 3.51 (2H, t, J 6.9), 2.55 (2H, t, J 6.6) and 2.49 (2H, t, J 6.9);
δC 177.3, 154.4,139.8, 119.0, 118.3, 115.1, 55.6, 49.7, 48.7, 32.5
and 16.2; m/z (EI) 248 (Mϩ, 16%), 208 (100), 189 (29), 166 (24),
136 (60), 135 (28), 134 (16) and 120 (40).
3-(N-Butyl-N-cyanoethylamino)propionamide 5o. Pale yellow
oil (Found: Mϩ, 197.1535. C10H19N3O requires M, 197.1528);
νmax/cmϪ1 3355, 3196 (NH2), 2247 (CN) and 1666, 1621
(C᎐O); δ 7.13 (1H, s), 5.51 (1H, s), 2.81 (2H, t, J 6.6), 2.76 (2H,
᎐
H
t, J 6.3), 2.48–2.54 (4H, m), 2.38 (2H, t, J 6.3), 1.79–1.53 (4H,
m) and 0.93 (2H, t, J 7.2); δC 177.3, 118.9, 53.1, 50.1, 49.4, 33.5,
28.8, 20.3, 16.6 and 13.9; m/z (CI) 198 (Mϩ ϩ 1, 46%), 157
(100), 154 (63) and 139 (98).
3-(2-Cyanoethoxy)propionic acid 5a. Pale yellow oil (Found:
4-(N-Butyl-N-cyanopropylamino)butyramide 5p. Pale yellow
oil (Found: Mϩ, 225.1846. C12H23N3O requires M, 225.1841);
νmax/cmϪ1 3354, 3197 (NH ), 2246 (CN) and 1667, 1622 (C᎐O);
Mϩ Ϫ OH, 126.0558. C6H9NO3 Ϫ OH requires 126.0555); νmax
/
cmϪ1 2657–3442 (CO H), 2252 (CN), 1720 (C᎐O) and 1116
᎐
᎐
2
2
(C᎐O᎐C); δH 6.39 (1H, s), 3.79 (2H, t, J 6.0), 3.70 (2H, t, J 6.8),
2.65 (2H, t, J 6.2) and 2.60 (2H, t, J 6.5); δC 176.1, 117.7, 66.2,
δH 5.82 (1H, s), 5.38 (1H, s), 2.52 (2H, t, J 6.6) 2.44 (2H, t, J
6.6), 2.42 (2H, t, J 6.9), 2.37 (2H, t, J 6.7), 2.27 (2H, t, J 7.6),
3202
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
1992; (c) Preparative Biotransformations: Whole Cell and Isolated
1.72–1.83 (4H, m), 1.17–1.45 (4H, m) and 0.91 (3H, t, J 7.3); δC
175.4, 120.1, 53.4, 53.2, 51.9, 33.6, 29.0, 23.3, 23.1, 20.6, 14.8
and 14.1; m/z (CI) 226 (Mϩ ϩ 1, 100%), 208 (86), 182 (93), 164
(79), 153 (93), 111 (67) and 86 (93).
p-Cyanophenylacetic acid 8b and p-cyanomethylbenzoic acid
8c. A mixture of 8b and 8c was obtained as a colourless crystal-
line solid, mp 138–139.5 ЊC; νmax/cmϪ1 2562–3424 (CO2H), 2252
Enzymes in Organic Chemistry, ed. S. M. Roberts, Wiley, New York,
1993; (d) C.-H. Wong and G. M. Whitesides, Enzymes in Synthetic
Organic Chemistry, Pergamon, 1994; (e) Enzyme Catalysts in
Organic Chemistry, ed. K. Drauz and H. Waldmann, VCH,
Weinheim, 1995.
3 For recent reviews, see (a) Benign by Design (ACS Symposium Series
577), ed. P. T. Anastas and C. A. Farris, American Chemistry
Society, Washington, DC, 1996; (b) T. Lester, Chem. Br., 1996, 45; (c)
R. A. Sheldon, Chem. Ind. (London), 1997, 12; (d) Green Chemistry:
Designing Chemistry for the Environment (ACS Symposium Series
626), ed. P. T. Anastas and T. C. Williamson, American Chemical
Society, Washington, DC, 1996.
4 (a) J. B. Jones, Aldrichimica Acta, 1993, 26, 105; (b) E. Schoffers,
A. Golebiowski and C. R. Johnson, Tetrahedron, 1996, 52, 3769.
5 For an overview, see J. Crosby, J. Moilliet, J. S. Parratt and N. J.
Turner, J. Chem. Soc., Perkin 1, 1994, 1679.
6 For reviews of nitrile biodegradations, see (a) Cyanide Compounds in
Biology (Ciba Foundation Symposium 140), ed. D. Evgred and S.
Harnett, Wiley, Chichester, 1988; (b) J.-C. Jallegeas, A. Arnaud and
P. Galzy, Adv. Biochem. Eng., 1980, 12, 1; (c) J. L. Legras, G. Chuzel,
A. Arnaud and P. Galzy, World J. Microbiol. Biotechnol., 1990, 6,
83.
7 (a) D. B. Harper, Biochem. Soc. Trans., 1976, 4, 502; (b) D. B.
Harper, Biochem. J., 1977, 165, 309; (c) M. Kobayashi and S.
Shimizu, FEMS Microbiol. Lett., 1994, 120, 217.
8 (a) Y. Asano, Y. Tani and H. Yamada, Agric. Biol. Chem., 1980, 44,
2251; (b) Y. Asano, K. Fujishiro, Y. Tani and H. Yamada, Agric.
Biol. Chem., 1982, 46, 1165; (c) Y. Asano, M. Tachibana, Y. Tani and
H. Yamada, Agric. Biol. Chem., 1982, 46, 1175.
9 For a useful overview of nitrile-hydrolysing micro-organisms, see
O. Meth-Cohn and M.-X. Wang, Tetrahedron Lett., 1995, 36, 9561;
O. Meth-Cohn and M.-X. Wang, J. Chem. Soc., Perkin Trans. 1,
1997, 1099 and references cited therein.
10 (a) R. H. Hook and W. G. Robinson, J. Biol. Chem., 1964, 234, 4263;
(b) A. K. Bandyopadhyay, T. Nagasawa, Y. Asano, K. Fujishiro,
Y. Tani and H. Yamada, Appl. Environ. Microbiol., 1986, 51, 302;
(c) M. Kobayashi, T. Nagasawa and H. Yamada, Eur. J. Biochem.,
1989, 182, 349; (d) D. E. Stevenson, R. Feng and A. C. Storer, FEBS
Lett., 1990, 277, 112; (e) M. Kobayashi, N. Yaanaka, T. Nagasawa
and H. Yamada, J. Bacteriol., 1990, 172, 4807; (f) T. Nagasawa,
J. Mauger and H. Yamada, Eur. J. Biochem., 1990, 194, 765.
11 (a) Y. Asano, K. Fujishiro, Y. Tani and H. Yamada, Agric. Biol.
Chem., 1982, 46, 1165; (b) T. Nagasawa, H. Nanba, K. Ryuno,
K. Takeuchi and H. Yamada, Eur. J. Biochem., 1987, 162, 691;
(c) T. Nagamune, H. Kurata, M. Hirata, J. Honda, H. Koike,
M. Ikeuchi, Y. Inoue, A. Hirata and I. Endo, Biochem. Biophys. Res.
Commun., 1990, 168, 437; (d) T. Nagasawa, K. Takeuchi and
H. Yamada, Eur. J. Biochem., 1991, 196, 581; (e) M. Kobagashi,
M. Nishiyama, T. Nagasawa, S. Horinouchi, T. Beppu and
H. Yamada, Biochem. Biophys. Acta, 1991, 1129, 23; ( f ) W.-Z. Li,
Y.-Q. Zhang and H.-F. Yang, Appl. Biochem. Biotechnol., 1992, 36,
171, (g) J. Honda, T. Nagasawa, S. Horionouchi, T. Beppu and
H. Yamada, Ann. N. Y. Acad. Sci., 1992, 672, 29; (h) T. Nagasawa,
K. Ryuno and H. Yamada, Biochem. Biophys. Res. Commun., 1986,
139, 1305.
12 (a) Y. Asano, M. Tachibana, Y. Tani and H. Yamada, Agric. Biol.
Chem., 1982, 46, 1175; (b) M. Kobayashi, H. Komeda, T. Nagasawa,
M. Nishiyama, S. Horinouchi, T. Beppu, H. Yamada and S.
Shimizu, Eur. J. Biochem., 1993, 217, 327.
13 (a) S. Mahadevan and K. V. Hhimann, Arch. Biochem. Biophys.,
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and references cited therein; (b) H. Yamada and M. Kobayashi,
Biosci. Biotech. Biochem., 1996, 60, 1391 and references cited
therein.
and 2229 (CN) and 1695 (C᎐O); δ 8.07 (0.5H, d, J 8.4), 7.61
᎐
H
(2H, d, J 8.4), 7.42 (2.5H, d, J 8.4), 3.82 (0.5H, s) and 3.66 (2H,
s).
m-Cyanomethylphenylacetic acid 10b. Colourless crystalline
solid,20 mp 45–47 ЊC (Found: C, 68.72; H, 5.14; N, 7.65.
C10H9NO2 requires C, 68.56; H, 5.18; N, 8.00%); νmax/cmϪ1
2540–3432 (CO H), 2252 (CN) and 1702 (C᎐O); δ 7.25–7.36
᎐
2
H
(4H, m), 6.49 (1H, s), 3.75 (2H, s) and 3.67 (2H, s); δC 176.8,
134.4, 130.3, 129.4, 129.1, 128.9, 126.9, 117.6, 40.7 and 23.4;
m/z (EI) 175 (Mϩ, 35%), 130 (100) and 104 (25).
trans-4-Cyanomethylcyclohexylacetic acid 12b. Colourless
needles, mp 89.5–90 ЊC (Found: C, 66.34; H, 8.20; N, 7.64.
C10H15NO2 requires C, 66.27; H, 8.34; N, 7.73%); νmax/cmϪ1
2632–3400 (CO H), 2250 (CN) and 1694 (C᎐O); δ 2.26 (4H,
᎐
2
H
d, J 6.5), 1.89 (4H, d, J 9.7), 1.76 (1H, s), 1.65 (1H, s), 0.98–1.28
(4H, m); δC 178.8, 118.6, 41.3, 34.4, 33.9, 32.0, 31.9 and 24.5;
m/z (CI) 182 (Mϩ ϩ 1, 1%), 164 (100) and 136 (12).
Melting points
2a, mp 50 ЊC (lit.,41 50–51 ЊC); 2b, oil (lit.,42 bp 110–115 ЊC/3
mmHg); 2c (lit.,42 bp 133–137 ЊC/3 mmHg); 3c, mp 147–148 ЊC
(lit.,43 146–147 ЊC); 3d, mp 103–104 ЊC (lit.,44 103–105); 3e, mp
139–141 ЊC (lit.,45 140 ЊC); 3f, mp 105–106 ЊC (lit.,46 105–
106 ЊC); 3g, mp 134–135 ЊC (lit.,47 133.5–134 ЊC); 6b, mp 81–
82 ЊC (lit.,48 81 ЊC); 6c, mp 87–89 ЊC (lit.,38 88.5–89.5 ЊC); 6d,
mp 90–91 ЊC (lit.,48 90–91 ЊC); 6e, mp 127–130 ЊC (lit.,49 128–
130 ЊC); 6g, mp 97–97.5 ЊC (lit.,50 96.5–97.5 ЊC); 7b, mp 123–
124 ЊC (lit.,51 122–123 ЊC); 9b, mp 99–100 ЊC (lit.,52 101 ЊC); 9c,
mp 197–198 ЊC (lit.,53 198 ЊC); 11b, mp 122 ЊC (lit.,54 120–
121.5 ЊC); 13b, mp 75–77 ЊC (lit.,17b 73–77 ЊC); 14b, 137.5–
138.5 ЊC (lit.,55 136–138 ЊC); 15b, 180–183 ЊC (lit.,56 178–
183 ЊC); 15c, mp 227–228 ЊC (lit.,57 227 ЊC); 16b, mp 220 ЊC
(lit.,58 220–221 ЊC); 17b, mp 218–221 ЊC (lit.,59 219–220 ЊC).
Hydrolysis of benzonitrile
The same biotransformation procedure as described above was
employed in the study of the hydrolysis of benzonitrile over
time. Two experiments were conducted in parallel, one with
added 4-(3-cyanopropylthio)butyric acid 5f (99.5 mg, 0.53
mmol) and the other without. After incubation for 30 min at
30 ЊC with shaking, benzonitrile (206.2 mg, 2 mmol) was added
in one portion to the mixture which was then incubated at 30 ЊC
using an orbital shaker (200 rpm). A sample (0.1 cm3) was
taken from the mixture at different time intervals and the bio-
mass was removed using a microcentrifuge (30 000 rpm). The
supernatant (0.08 cm3) was removed by pipette and mixed with
furan-2-carboxamide solution (1 m in methanol; 0.04 cm3).
HPLC analysis of the mixture (0.01 cm3), after being calibrated
with calibration curves, gave the concentrations of benzonitrile,
benzamide and benzoic acid (Fig. 2).
15 H. Yamada, Y. Asano and Y. Tani, J. Ferment. Technol., 1980, 58,
495.
Acknowledgements
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We are grateful to the BBSRC for a post-doctoral grant
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Paper 7/03904B
Received 4th June 1997
Accepted 11th July 1997
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