Rationalisation of the regioselective hydrolysis of aliphatic dinitriles with Rhodococcus rhodochrous AJ270

Article abstract of DOI:10.1039/a700859g

Aliphatic dinitriles undergo regioselective hydrolysis with the title organism to give monoacids with up to four methylenes between the nitrile functions (optimally 2-3) or when either an oxygen is placed β, γ or δ to the nitrile (δ-placement being optimal) or β or γ (optimally γ) but not δ sulfur substituents are present; nitrogen substituents appear to behave as for oxygen but suffer a steric limitation of the size of the nitrogen substituent.

Full text of DOI:10.1039/a700859g

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Rationalisation of the regioselective hydrolysis of aliphatic dinitriles with
Rhodococcus rhodochrous AJ270
Otto Meth-Cohn* and Mei-Xiang Wang
Chemistry Department, Sunderland University, Sunderland, UK SR1 3SD
Aliphatic dinitriles undergo regioselective hydrolysis with
the title organism to give monoacids with up to four
methylenes between the nitrile functions (optimally 2–3) or
when either an oxygen is placed b, g or d to the nitrile
(d-placement being optimal) or b or g (optimally g) but not
d sulfur substituents are present; nitrogen substituents
appear to behave as for oxygen but suffer a steric limitation
of the size of the nitrogen substituent.
However a g-sulfur allows optimal regiocontrol, the b-analogue
showing selectivity but not the d-analogue. In the nitrogen
series we cannot yet define the limits of regiocontrol with
chainlength but good selectivity is shown with a b-nitrogen.
Interestingly, in this slowly hydrolysed series, a much greater
sensitivity to p-substituents is evident than in the hydrolysis of
mononitriles.
We explain the regioselectivity on the basis of chelation-
deactivation of the enzyme. We believe 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
We have demonstrated1 that the title organism is a powerful,
general nitrile hydrolysing organism, involving two enzymes
effecting nitrile-to-amide and amide-to-acid conversions re-
spectively. As a prelude to studying the hydrolysis of prochiral
dinitriles we have made a broad study of the effect of structure
on the regioselectivity of hydrolysis of aliphatic dinitriles. The
literature contains a number of papers on this topic but the
overall background picture is decidedly unclear.
Table 1 Conversion of NC[CH2]nCN 1 into NC[CH2]nCO2H 2 and/or
HO2C[CH2]nCO2H 3
Conditions
Sub-
strate 1
n
Product yields
Conc./
mmol
In 1980 Yamada showed that a Fusarium strain transformed
glutaronitrile to 4-cyanobutyric acid2 and 1,3,6-tricyanohexane
to a mixture of diacids,3 the former transformation also being
accomplished by a Pseudomonas organism. Gutman4 observed
that the series NC[CH2]nCN with n = 3–5 hydrolysed with no
selectivity to the diacids with Rhodococcus rhodochrous NCIB
11216 grown on propionitrile while Yamada5 observed se-
lective monohydrolysis of glutaronitrile (n = 3) using R.
rhodochrous K22 in quantitative yield. Schneider6 made similar
observations to Yamada with another R. rhodochrous strain
while Turner7 showed that succinonitrile gave solely 3-cyano-
propionic acid while glutaronitrile gave both mono- and di-
acids, ratios depending upon time of reaction. Prochiral
glutaronitriles were hydrolysed by Kakeya8 and by Turner9 to
give enantio-efficient monohydrolysis in some cases, although
not in others. A chiral malononitrile has been converted10 into
its monoacid monoamide derivative utilising an R. rhodochrous
strain. Other organisms have been used for the efficient
monohydrolysis of 1,4-cycohexanedicarbonitrile,11 while the
conversion of adiponitrile to adipic acid has been closely
studied by Moreau12 utilising a Brevibacterium.
Entry
t/h
2 (%)
3 (%)
1
2
3
4
5
6
7
8
9
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
10
11
12
Table 2 Conversion of NC[CH2]nX[CH2]nCN 4 into NC[CH2]nX[CH2]n-
CO2H 5 and/or HO2C[CH2]nX[CH2]nCO2H 6
Conditions
Substrate 4
Yield (%)
Conc./
Entry
n
X
mmol t/h
5
6
4
We first examined the selectivity of hydrolysis in a series of
a,w-dinitriles, monitoring the reaction against time (Table 1).
As we noted in earlier work,1 the low molecular weight nitriles
tended to be metabolised by other enzymes in the organism,
limiting overall yields. However, a clear pattern emerged:
dinitriles with more than four methylenes separating the
functions gave solely diacids irrespective of reaction time. With
less than four methylenes, regioselective monohydrolysis
occurred.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
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
1.5
48
61
35
—
73
75
—
—
45
—
52
83
—
—
93
71
91
—
—
—
—
—
tr
70
—
5
—
35
—
—
—
60
—
10
—
29
—
tr
—
—
26
—
95
92
94
99
1
72
2
120
2
48
1
48
2
25
97.5
—
60
—
—
83
86
—
—
—
—
—
—
—
We next examined
a
series of a,w-dinitriles
NC[CH2]nX[CH2]nCN 4 containing a heteroatom X in the chain
(Table 2). With the oxygen or sulfur series, reactions were rapid
and generally efficient, whereas with NH or NMe the nitriles
proved unstable. We therefore included the NAr series which,
probably for steric reasons, reacted slowly and with n > 2, not
at all, even with added co-solvents such as acetone or methanol.
A remarkable feature emerges: regioselectivity is principally
dependent upon the placement of the heteroatom, not the overall
chain length. Thus a d-oxygen is optimally efficient, with
regiocontrol being effective with a b- or g-oxygen also.
2
2
S
NPh
NC6H4Cl-p
NC6H4OMe-p
NPh
NC6H4OMe-p 1.5
NPh
NPh
96
64
168
72
168
168
168
168
1.5
1.5
Chem. Commun., 1997
1041

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and rate of formation of acid are both significantly slower in the
presence of the cyano acid.
Table 3 Conversion of miscellaneous dinitriles into acids
Conditions
When the aliphatic a,w-dinitrile chain is interrupted by vinyl
or aryl substituents, regiocontrol is efficiently observed in
almost all cases that we have examined except for
o-phenylenediacetonitrile (Table 2, entry 3). Although the
regiocontrol here (and in the case of aromatic dinitriles14) may
derive from a different basis, it is tempting to suggest that the
p-systems can also act as effective ligands for the iron when
chelation allows (Table 3). Also included in Table 3 is trans-
cyclohexane-1,4-diacetonitrile (entry 6) which shows total
regioselective hydrolysis, presumably by way of chelation of its
axial or twist–boat conformer.
In conclusion, we have defined the extent to which
regiocontrol can be expected in the hydrolysis of dinitriles
bearing O, S and to a limited extent N substituents, and
considered the control with p-functionalised dinitriles. The
application of these ideas to prochiral and chiral systems is now
being actively examined.
Conc./
Entry Substrate
mmol t/h
Product(s) and yields
CN
CN
CN
1
2
24
CO2H
80%
NC
2
R1
2
24
CN
R2
R1 = CN, R2 = CO2H 65%
R
1 = CO2H, R2 = CN 16%
R1
R2
CN
CN
3
3
24
R1 = R2 = CN 13%
We thank the BBSRC for a generous research grant that made
this work possible.
R
R
1 = CN, R2 = CO2H 11%
1 = R2 = CONH2 65%
Footnotes
CN
CN
* E-mail: otto.meth-cohn@sunderland.ac.uk
† We thank a referee for this suggestion.
4
5
3
3
30
30
CN
CO2H
67%
References
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1.5 39
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99%
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CO2H
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14
14
NC
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CN
CO2H
8
NC
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86%
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function or a heteroatom. The glutaronitrile 1 (n = 3) bears a
d-oxygen ligand as does the ether 4 (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 g placement
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benzonitrile disappearance is dramatically slower in the former
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benzonitrile remained with no added cyano acid. In the presence
of the inhibitor almost 40% remained. After 10 min the figures
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Received in Cambridge, UK, 6th February 1997; Com.
7/00859G
1042
Chem. Commun., 1997